A clinical trial report of autologous bone marrow-derived mesenchymal stem cell transplantation in patients with spinal cord injury.

Spinal cord injury (SCI) is a severe neurological disease. An effective strategy for the treatment of SCI is urgently required. Stem cell transplantation has emerged as a viable therapeutic option with great potential for restoring neurological function lost following SCI. From 2009 to 2010, a total of 20 SCI patients were enrolled in a clinical trial by Wuhan Hongqiao Brain Hospital; all patients completed and signed informed consent prior to autologous bone marrow-derived mesenchymal stem cell transplantation. Analysis of subsequent treatment results indicated significant improvements in sensory, motor and autonomic nerve function as assessed by the American Spinal Injury Association's impairment scale. Thirty days after transplantation, a total of 15 patients (75%) demonstrated improvement, including four of the eight patients (50%) with grade A SCI, three of the four patients (75%) with grade B injury and all eight patients (100%) with grade C injury. The most common adverse events, fever and headache, disappeared within 24-48 h without treatment.

Exosomes from murine-derived GL26 cells promote glioblastoma tumor growth by reducing number and function of CD8+T cells.

AIM: Brain tumors almost universally have fatal outcomes; new therapeutics are desperately needed and will only come from improved understandins of glioma biology. METHODS: Exosomes are endosomally derived 30~100 nm membranous vesicles released from many cell types. Examples from GL26 cells were here purified using density gradient ultracentrifugation and monitored for effects on GL26 tumor growth in C57BL/6j mice (H-2b). Lactate dehydrogenase release assays were used to detect the cytotoxic activity of CD8+T and NK cells. Percentages of immune cells producing intracellular cytokines were analyzed by FACS. RESULTS: In this study, exosomes from murine-derived GL26 cells significantly promoted in vivo tumor growth in GL26-bearing B6 mice. Then we further analyzed the effects of the GL26 cells-derived exosomes on immune cells including CD8+T, CD4+T and NK cells. Inhibition of CD8+T cell cytotoxic activity was demonstrated by CD8+T cell depletion assays in vivo and LDH release assays in vitro. The treatment of mice with exosomes also led to a reduction in the percentages of CD8+T cells in splenocytes as determined by FACS analysis. Key features of CD8+T cell activity were inhibited, including release of IFN-gamma and granzyme B. There were no effects of exosomes on CD4+T cells and NK cells. CONCLUSION: Based on our data, for the first time we demonstrated that exosomes from murine derived GL26 cells promote the tumor growth by inhibition of CD8+T cells in vivo and thus may be a potential therapeutic target.

Cryosurgery and rhTNF-α play synergistic effects on a rat cortex C6 glioma model.

Glioma, a type of brain tumor originating from glioma cells, varies widely in aggressiveness and causes serious symptoms, but the treatments are limited. Studies have shown that cryosurgery has multiple effects on tumor treatments, and administration of human tumor necrosis factor-alpha (rhTNF-α) arguments the anti-tumor effect of cryotherapy in breast and prostate cancers. To test the hypothesis that cryosurgery and rhTNF-α play synergistic effects against brain tumors, we established a brain glioma model on rat cortex regions following different treatments: the G1 group was sham-operated; the G2 group was treated with cryosurgery; the G3 group was treated with rhTNF-α; and G4 group received combined treatment with cryosurgery and rhTNF-α. Tumor sizes were measured by magnetic resonance imaging; DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21(WAF1/CIP1) and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining. G2 and G4 rats had significantly longer survival time than did G1 rats. Tumor sizes in each group were significantly decreased as compared with those in G1 rats. PCNA-positive cells were significantly decreased in G2, G3 and G4 rats as compared with G1 rats. In contrast, DNA fragmentation and P21(WAF1/CIP1)-positive cells were significantly increased in each treatment group. Importantly, a combined treatment enhanced the effects of cryosurgery. Combined treatment with cryosurgery and rhTNF-α may have a synergistic effect on glioma tumor therapy, enhancing the inhibition of proliferation and the induction of apoptosis.

c-Met antisense oligodeoxynucleotides as a novel therapeutic agent for glioma: in vitro and in vivo studies of uptake, effects, and toxicity.

BACKGROUND: c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility, and invasion and are known to be overexpressed in gliomas. The aim of our study was to investigate the uptake and effects of c-Met antisense oligodeoxynucleotides (ASODNs) on rat and human glioma cells in vitro and the uptake and toxicity of these nucleotides in rat carcinomatosis and brain tumor models. MATERIALS AND METHODS: The three human cell lines (U87, BT325, SHG44) and the C6 rat glioma cell line were cultured. To study the uptake of oligodeoxynucleotides (ODNs) by glioma cells in vitro, cultured glioma cells readily incorporated caroboxyfluorescein-5-succimidyl ester (FAM) labeled phosphorothioate oligodeoxynucleotides, as demonstrated by immunofluorescence microscopy and flow cytometry. To study the effect of ASODNs treatment on c-Met expression in vitro, Expression of c-Met was assessed by immunofluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. For animal studies of ODNs toxicity and uptake, eight rats underwent placement of cisternal catheters, under general anesthesia. Four rats were given 24 mug FAM-labeled ASODNs while the others were given a saline control injection. After a 24 h observation period, rats were sacrificed by barbiturate overdose, and their brains were studied. RESULTS: For all cell lines, fluorescence was seen to increase with increasing ASODNs concentration. Cells treated in similar fashion were also analyzed by flow cytometry to graphically illustrate the differing fluorescence. Multiple glioma cell lines were tested, with similar results. c-Met ASODNs was found to be successfully incorporated from the media into cultured human glioma cells, even at concentrations as low as 2 muM. In addition, maintenance of the pH-dependent green fluorescence color, as seen by immunofluorescence microscopy and by using flow cytometry, indicated that the FAM was not contained within lysosomes. Immunofluorescence microscopy and RT-PCR analysis showed decreases in c-Met expression with oligodeoxynucleotides treatment. Uptake into tumor cells was also demonstrated in vivo, with no detectable toxicity at concentrations exceeding expected therapeutic levels. CONCLUSION: These data are encouraging for further study of c-Met antisense oligodeoxynucleotides as a therapeutic modality for glioma.

Hepatocyte growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells.

Hepatocyte growth factor (HGF) is a pleiotrophic cytokine that stimulates motility and invasion of several cancer cell types and induces angiogenesis, which is known to be expressed in several malignancies including glioma. The effect of transforming growth factor-beta (TGF-beta) isoforrns as well as gangliosides on HGF production was investigated in human glioma cell lines. TGF-beta isoforms and gangliosides were found to differentially stimulate HGF production by these cells. The ganglioside GD3 enhanced this release to the greatest extent and the stimulation was more marked in a glioblastoma cell line than in the two other anaplastic astrocytoma cell lines. These results suggest that both TGF-betas and gangliosides may act as indirect angiogenic factors by stimulating HGF secretion.

Stereotactic cyst/ventricular-peritoneal shunting for the treatment of prepontine arachnoid cyst: case report.

BACKGROUND: Prepontine (suprasellar) arachnoid cysts are uncommon in clinical practice, so experiences in their management are limited and the best method of treatment for them remains unclear. Here we report our experience in using stereotactic cyst/ventricular-peritoneal shunting for the treatment of prepontine arachnoid cyst. CASE DESCRIPTION: A 42-year-old woman with prepontine arachnoid cyst was treated with cyst/ventricular-peritoneal shunting: the ventricular catheter was precisely inserted at a point where it could drain from the cyst and the ventricle at the same time. The postoperation CT scan showed that the cyst and the enlarged ventricle shrunk markedly. During a 1-year follow-up period, she remained symptom-free and had returned to full-time work. CONCLUSION: Stereotactic cyst/ventricular-peritoneal shunting appears to be an effective method for treating prepontine arachnoid cyst.

Forskolin and 8-cyclopentyltheophylline synergistically facilitate the neuronal activity in the CA2 area of rat hippocampus via cAMP and non-cAMP cascades.

High level of adenosine A1 receptor-like immunoreactivity has been found in the CA2/CA3a region of adult rat hippocampus, but its roles in the neuronal activity or signal propagation in hippocampus and its intracellular cascade remain to be studied. In this study, we examined the relation between adenosine-3',5'-cyclic monophosphate (cAMP) cascade and suppression of synaptic transmission by endogenous adenosine through adenosine A1 receptor in the CA2 area. In transverse hippocampal slice, maximal electrical stimulation of the hilus region (0.6 mA) only evoked small population spikes (PSs) in the CA2 area (0.5 mV). In the presence of forskolin (20 micromol/L), a direct adenylate cyclase activator, PSs in CA2 were increased to 1.1 mV. When 8-cyclopentyltheophylline (8CPT, 2 micromol/L), an adenosine A1 receptor antagonist, was added in the presence of 20 micromol/L forskolin, PSs with an average amplitude of 4.7 mV were recorded in the CA2 area, much higher than the sum of the amplitude of PSs in the presence of forskolin and 8CPT alone. To test whether this synergistic potentiation results from the additive activation of cAMP cascade, the cAMP content in hippocampal slices was measured with enzyme immunoassay (EIA). Results showed that 8CPT did not increase the cAMP content in CA2 with or without forskolin. Co-application of forskolin and Ro 20-1724, a cAMP-specific phosphodiesterase-IV inhibitor, only increased PSs in CA2 to 1.3 mV but increased cAMP content by 4.4 times. On the other hand, co-application of 8CPT and 1, 9-dideoxyforskolin, a forskolin analog which has no effect on adenylate cyclase, did not mimic the synergistic effect of 8CPT and forskolin on PSs in CA2. These results indicate that up-regulation of adenylate cyclase activity and inhibition of adenosine A1 receptor activity synergistically facilitate the neuronal activity in the CA2 area and the effect of adenosine A1 receptor antagonist is via non-cAMP cascade. These data also suggest that acting on adenosine A1 receptors, endogenous and extragenous adenosine/adenosine A1 agonist(s) inhibit neuronal activity through different pathways.

In vitro and in vivo potentiating the cytotoxic effect of radiation on human U251 gliomas by the c-Met antisense oligodeoxynucleotides.

C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, and invasion, and are known to be overexpressed in gliomas, which are related to the repair of damaged DNA. In this study, we investigated both in vitro and in vivo whether inhibition of the c-Met gene by antisense oligonucleotides (ODNs) enhances the cytotoxic effect of radiation on human U251 gliomas. A volume of 100 nM of c-Met antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of human U251 glioma cells with 100 nM of c-Met antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with nonsense ODNs. When the glioma cells were implanted in the cisterna magna of nude mice followed by treatment with c-Met antisense ODNs, the survival time of the nude mice was markedly prolonged compared to that of the untreated group (P < 0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing nude mice much longer than could be achieved with radiation alone (P < 0.0001, logrank test). These results suggest that inhibition of c-Met can be expected to serve as a novel potentiator for radiation therapy in human U251 gliomas.

Antisense oligonucleodes targeting the focal adhesion kinase inhibit proliferation, induce apoptosis and cooperate with cytotoxic drugs in human glioma cells.

To examine the role of focal adhesion kinase in human glioma cells, we studied its effects on proliferation and apoptosis using FAK antisense oligonucleotide. U251 MG cells were transfected with ODNs, sense FAK, mismatch FAK and antisense-FAK, respectively. Expression of FAK proteins were detected by Western blots and Immnofluoressence. Cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry. Caspase-3 activity was measured by spectrofluorometer. MTT assay was used to examine changes in cell proliferation. The protein expression of FAK in U251 MG cells decreased in antisense-FAK ODNs group significantly. Caspase-3 activity increased in cells treated with antisense-FAK and down-regulated when treated with caspase-3 inhibitor. The level of cell apoptosis and loss of mitochondrial membrane potential in antisense-FAK group was higher than in the mismatch sense group. Cells proliferation was inhibited by antisense-FAK, and the effects were clearly additive when antisense oligonuceotides were added to cells treated with the anticancer agents. The results suggest that antisense-FAK ODNs inhibit U251 MG cells proliferation and induce their apoptosis. It is possible that FAK via mitochondrial and caspase-3 inhibits U251 MG cells apoptosis. And antisense oligonucleotide treatment enhances U251 MG cells sensitivity to chemotherapy.

[Effect of FK506 on axonal regeneration of rat sciatic nerve in regeneration chamber: an experimental study].

OBJECTIVE: To evaluate the effect of FK506 on expediting nerve regeneration of rat Sciatic Nerve in regeneration chamber and to look for a proper way of its administration to treat peripheral nerve injuries. METHODS: Sixty adult male SD rats which were randamizely divided into 3 groups received a neurotomy to bilateral sciatic nerves, then we reconnected the broken nerves with silicon tube to make regeneration chambers. The regeneration chambers were filled with either normal saline (group A and group B) or 1 microg/ml FK506 (group C). The rats of group B also received daily injection of FK506 (1 mg/kg) at the back of the neck for 14 days. Local immunoreaction, weight of fresh gastrocnemius muscles, histological changes and electrophysiology were observed at designate time after neurotomy. RESULTS: At 6 weeks postoperation the extent of local lymphocytes infiltration in group B and C were less than that in group A, all results in group B were much better than that of group A. Results of group C were better than that of group A without significance. CONCLUSIONS: (1) Systemic administration of FK506 (1 mg/kg) showed neuroprotective and neurotrophic effect, which can facilitate nerve regeneration and promote functional recovery. (2) Local administration of FK506 (1 microg/ml) showed some extent of neuroprotective effect at early period of nerve injury, but the neurotrophic function is uncertain and still needs to be studied further.

[An experimental study of the neuroprotective effect of FK506 on acute spinal cord injury in dogs].

OBJECTIVE: To explore the neuroprotective effect of FK506 on acute spinal cord injury in dogs. METHODS: Acute spinal cord injury model was made with the Allen technique. Animals were randomly divided into 3 groups. Group A (n = 8) was the control group and received operation but no therapy, while group B and C (n = 8) received a single dose of FK506 (0.18 mg/kg and 0.3 mg/kg, respectively) administered with an arterial duct 2 h after spinal cord injury (SCI). Spine MRI, neurological function, histopathological examination of injured spinal cord and immunohistochemical examination of expression of NF(200) in neurons and GFAP in astrocytes were assessed at certain time after injury. RESULTS: Neurological function score of group C and B was better than that of group A (P < 0.05), with significance between group C and A, while no significance between group B and A statistically. The signal scope of spinal cord injury on MRI in group C was the smallest among all the groups, and the signal scope in group B was smaller than that in group A, which was directively associated with the neurological outcome. The expression of NF and GFAP was significantly higher in group C than in group A (P < 0.05), but without statistical significance between group B and A. CONCLUSION: Local administration of FK506 (0.3 mg/kg) possesses neuroprotective effect on acute spinal cord injury, which can improve neurological function recovery and attenuate secondary spinal cord injury. Local administration of FK506 possesses a dosage-effect relation.

[The expression of hepatocyte growth factor and its receptor in brain astrocytomas].

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF) mRNA and its receptor (c-Met) mRNA in brain astrocytomas and their relationships with tumor proliferation, angiogenesis, clinical pathology and prognosis. METHODS: The expression of HGF mRNA, c-Met mRNA in the resected tumor tissues of 76 patients with brain astrocytomas, 43 males and 33 females, aged 20 - 71, were detected by in situ hybridization. Immunohistochemistry technique was used to test the expression of proliferating cell nuclear antigen (PCNA) protein and the microvessel density (MVD) was determined by immunohistochemistry with monoclonal antibody against CD34. RESULTS: The positive rates of expression of HGF, c-Met and PCNA in low pathologic grades of brain astrocytoma were 34.5%, 44.8% and 15% +/- 9% respectively, and in high pathologic grades of brain astrocytoma were 34.5%, 44.8% and 48% +/- 12% respectively (P < 0.05). MVD in low and high pathologic grades of brain astrocytoma were 17 +/- 7 and 31 +/- 13 respectively (P < 0.05). The expression of HGF, c-Met, PCNA and CD34 was not related to sex, age, position of tumor and diameter of tumor. The expression of c-Met was related to the expression of HGF, PCNA and the MVD in the tumor tissues of these patients. The pathological grade, position of tumor, HGF, c-Met, PCNA, MVD had a significant influence on the survival time. CONCLUSION: HGF/c-Met plays an important role in the formation and progression of the brain astrocytoma and can promote tumor proliferation and intratumoral microvascular formation, and is closely related to the prognosis of the patients.