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Background: Conventional approaches to the thoracic spine can require extensive tissue dissection, bony disruption, and instability that may warrant the need for instrumentation and fusion. Furthermore, anterior approaches may require the involvement of various surgeons from multiple disciplines to ensure a successful operation and mitigate complications. Currently, available minimally invasive approaches still require bony removal and usually rely heavily on computed tomography (CT)-guided imaging without direct gross visualization. Endoscopic spinal procedures have provided an ultra–minimally invasive alternative to access many areas in and around the spinal column. Methods: We present a 12-year-old boy with a right-sided 2.0 × 3.2-cm paravertebral lesion at the level of T5. The patient successfully underwent an endoscopic approach to the lesion with minimal tissue and bony disruption for tissue diagnosis and tumor resection. Results: At initial and 6-month follow-up, the patient remained asymptomatic and without issues. Conclusions: We demonstrate here the feasibility and suggest the safety of a posterior ultra–minimally invasive endoscopic spinal approach to obtain a tissue biopsy of an incidentally found ventrolateral paraspinal tumor in the thoracic region in a pediatric patient. This minimal approach can prove to achieve similar results as other approaches that may otherwise necessitate more extensive or transthoracic procedures.

Sacroiliac joint (SIJ) dysfunction is an underdiagnosed condition. Several published cohorts have reported favorable midterm outcomes after SIJ fusion using titanium implants placed across the SIJ. Herein, we report 12-month follow-up from SIJ fusion in a standard clinic setting.A carefully selected group of 160 consecutive patients with painful SIJ dysfunction were diagnosed at a single center and underwent unilateral or staged bilateral SIJ fusion using triangular titanium implants. Patients were routinely seen in clinic for follow-up every 3 months where they completed visual analog scale (0-10 range) pain ratings and Oswestry Disability Index (ODI). Follow-up CT scan was performed at 1 year.Mean patient age was 58 years, and 68% were women. 30% underwent staged bilateral SIJ fusion. By month 12, SIJ pain decreased from 8.0 to 2.5 (p 0.0001) and disability (ODI) decreased from 45.3 to 16.4 (p 0.0001). The proportion with clinically significant improvements in SIJ pain and ODI was high ( 95%). Perioperative adverse events were mild and decreased with increasing surgical experience with the procedure. Subgroup analysis showed slightly smaller improvements in those undergoing bilateral surgery and those with a spinal cord stimulator in place. CT scan at 1 year showed reabsorption along one or more implants in 16% of cases, but there were no breakages or implant removals.In standard clinical practice, SIJ fusion with triangular titanium implants produces significant improvement in pain and disability related to SIJ dysfunction. These slides can be retrieved under Electronic Supplementary Material.

Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy. We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines. Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy—events that could be prevented by silencing ABCB6 via siRNA treatment. Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.

Advances in medical and surgical treatments in the last decades have resulted in quantum leaps in the overall survival of patients with many types of malignant disease, while survival of patients with malignant gliomas (WHO histological grades 3 and 4) has been only moderately improved. Maximum surgical resection, external fractionated radiotherapy, and oral chemotherapy during and after irradiation currently represent the pillars of malignant glioma therapy. Novel and experimental modalities aimed at a more selective and more effective treatment are however being increasingly developed and tested in clinical studies. Improved understanding of the fundamental mechanisms of glioma growth, resistance, and recurrence has resulted in the introduction of biologically and molecularly targeted therapies such as virus-mediated gene therapy, often in combination with spatially defined delivery methods specifically designed to be used in the local environment of the brain, such as convection-enhanced delivery. This review summarizes the key findings of the most important phase I and II clinical studies employing gene therapy with naturally occurring or genetically modified non-replicating or conditionally replicating (oncolytic) viruses, such as retrovirus, adenovirus, herpes-simplex-virus, Newcastle disease virus, or reovirus, in patients with primary or recurrent malignant gliomas. In addition, the two phase III gene therapy studies carried out to date in glioma patients and employing retrovirus or adenovirus vectors are presented in detail and critically discussed. Areas of necessary improvements and possible future developments of viruses and delivery methods are outlined.

MicroRNAs are single-stranded small non-coding RNA molecules which regulate mammalian cell growth, differentiation, and apoptosis by altering the expression of other genes and play a role in tumor genesis and progression. MiR-106a is upregulated in several types of malignancies and provides a pro-tumorigenic effect. However, its role in glioma is largely unknown. Our findings demonstrate that the low expression of miR-106a in human glioma specimens is significantly correlated with high levels of E2F1 protein and high-grade glioma. Here, we present the first evidence that miR-106a provides a tumor-suppressive effect via suppressing proliferation of and inducing apoptosis in human glioma cells. We further show that E2F1 is a direct functional target of miR-106a, suggesting that the effect of miR-106a on the glioma suppressive effect may result from inhibition of E2F1 via post-transcriptional regulation. In addition, our results reveal that miR-106a can increase p53 expression via E2F1 inhibition, whereas the effect of miR-106a on the proliferation of glioma cells is independent of p53 status. Further investigations will focus on the therapeutic use of miR-106a-mediated antitumor effects in glioma.

Randomized phase III trials have shown significant improvement of survival 1, 2, and 3 years after implantation of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) wafers for patients with newly diagnosed malignant glioma. But these studies and subsequent non-phase III studies have also shown risks associated with local chemotherapy within the central nervous system. The introduction of concomitant radiochemotherapy with temozolomide (TMZ) has later demonstrated a survival benefit in a phase III trial and has become the current treatment standard for newly diagnosed malignant glioma patients. Lately, this has resulted in clinical protocols combining local chemotherapy with BCNU wafers and concomitant radiochemotherapy with TMZ although this may carry the risk of increased toxicity. We have compiled the treatment experience of seven neurosurgical centers using implantation of carmustine wafers at primary surgery followed by 6 weeks of radiation therapy (59–60 Gy) and 75 mg/m2/day TMZ in patients with newly diagnosed glioblastoma followed by TMZ monochemotherapy. We have retrospectively analyzed the postoperative clinical course, occurrence and severity of adverse events, progression-free interval, and overall survival in 44 patients with newly diagnosed glioblastoma multiforme. All patients received multimodal treatment including tumor resection, BCNU wafer implantation, and concomitant radiochemotherapy. Of 44 patients (mean age 59 ± 10.8 years) with glioblastoma who received Gliadel wafer at primary surgery, 28 patients (64%) had died, 16 patients (36%) were alive, and 15 patients showed no evidence of clinical or radiographic progression after a median follow-up of 15.6 months. At time of analysis of adverse events in this patient population, the median overall survival was 12.7 months and median progression-free survival was 7.0 months. Surgical, neurological, and medical adverse events were analyzed. Twenty-three patients (52%) experienced adverse events of any kind including complications that did not require treatment. Nineteen patients (43%) experienced grade 3 or grade 4 adverse events. Surgical complications included cerebral edema, healing abnormalities, cerebral spinal fluid leakage, meningitis, intracranial abscess, and hydrocephalus. Neurological adverse events included newly diagnosed seizures, alteration of mental status, and new neurological deficits. Medical complications were thromboembolic events (thrombosis, pulmonary embolism) and hematotoxicity. Combination of both treatment strategies, local chemotherapy with BCNU wafer and concomitant radiochemotherapy, appears attractive in aggressive multimodal treatment schedules and may utilize the sensitizing effect of TMZ and carmustine on MGMT and AGT on their respective drug resistance genes. Our data demonstrate that combination of local chemotherapy and concomitant radiochemotherapy carries a significant risk of toxicity that currently appears underestimated. Adverse events observed in this study appear similar to complication rates published in the phase III trials for BCNU wafer implantation followed by radiation therapy alone, but further add the toxicity of concomitant radiochemotherapy with systemic TMZ. Save use of a combined approach will require specific prevention strategies for multimodal treatments. peerReviewed

Imatinib mesylate (STI571, Gleevec) is a signal transduction inhibitor and novel anti-cancer agent. It selectively inhibits aberrantly activated tyrosine kinases in malignant cells, for example, bcr-abl in leukaemia, platelet-derived growth factor receptor and stem cell factor receptor (c-Kit) in solid cancers including malignant glioma. However, recently published clinical studies with imatinib monotherapy in patients with malignant glioma demonstrated only very modest anti-tumour activity. The aim of this study was to investigate the biological activity of imatinib, its cellular mechanisms of action and its synergism with other chemotherapeutic agents in human malignant glioma cells in culture. Expression of PDGF/R and c-Kit was analyzed by RT-PCR. Proliferation was measured by MTT assays and drug synergy was assessed by the Chou-Talalay method. Cell cycle and apoptosis were analyzed by flow cytometry and migration by monolayer migration assays. Multi-immunoblot was performed on imatinib-treated and control malignant glioma cells. Results indicate that imatinib is more effective in inhibiting cell colony formation and migration rather than proliferation. Imatinib treatment caused cell cycle arrest of glioma cells in G0-G1 or G2/M, with significant elevation of a few cyclin-dependent kinases. Furthermore, imatinib acted synergistically with chemotherapy agents, such as the DNA alkylating agent, temozolomide, and riboneucleotide reductase inhibitors, for example, hydroxyurea at varied effective dose levels. In conclusion, imatinib exerts varied biological effects on malignant glioma cells in culture. Synergistic interaction of imatinib with chemotherapy agents may be related to cell cycle control mechanisms and could be potentially important in a clinical setting.

Malignant gliomas have retained their dismal prognosis despite aggressive multimodal conventional therapeutic approaches, illustrating the need for novel therapeutic strategies. Recent advances in the cellular and molecular biology of gliomas have enhanced our understanding of the role of receptor tyrosine kinases (RTK) and RTK-mediated signal transduction pathways in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Special attention has been focused on targets such as epidermal growth factor receptors (EGFR), platelet-derived growth factor receptors (PDGFR), vascular endothelial growth factor receptors (VEGFR), and on pathways such as the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Novel targeted drugs known as small molecule inhibitors have been shown to modify the activity of these receptors and signaling pathways. Thus far, however, small molecule RTK inhibitor development has concentrated on a few RTK only, and drug activity has been comprehensively evaluated only in a limited number of different malignancies. One of the limiting factors for novel drug design and development is the incomplete knowledge of RTK functions in malignant glioma. This review summarizes current basic and clinical knowledge on the role of RTK in malignant glioma and on their importance as targets for new forms of therapy.

Primary central nervous system lymphomas (PCNSL) are rare tumours occurring in the brain. Their biology and the factors predicting survival are not well known. This study investigated expression of the antiapoptotic protein survivin and platelet-derived growth factor A (PDGF-A) and receptor (PDGFRalpha) in PCNSL.A total of 44 patients with histologically confirmed PCNSL treated between 1992 and 2004 were included in this study, and tumour specimens were investigated immunohistochemically for expression of survivin, PDGF-A and PDGFRalpha. Protein expression and clinical variables were analyzed statistically.Of the 44 tumours 43(98%) were diffuse large B-cell non-Hodgkin's lymphomas (NHL) and one was a T-cell NHL. Around 37 (84%) of the examined PCNSL specimens showed expression of survivin, 16 (36%) of PDGF-A and 34 (77%) of PDGFRalpha. Tumours expressing surviving co-expressed PDGFRalpha frequently and PDGF-A occasionally. Expression of the above proteins was not predictive for survival in this patient group. Except for age and therapy, no other clinical variables correlated significantly with overall survival.PCNSL express survivin and PDGFRalpha in the majority of investigated cases. PDGF-A is expressed less frequently. Immunohistochemical detection of these proteins does not correlate with overall survival and cannot be used as a prognostic factor.

Targeted toxins represent a new class of agents with high specificity for tumor cells. Toxins in current clinical use for the treatment of brain tumors are mostly recombinant polypeptides consisting of a tumor-selective ligand coupled to a peptide toxin of bacterial origin. Targeted toxins are highly potent - one single molecule of toxin is enough to cause cell death. Toxins are able to kill tumor cells independent of any malignancy-associated genetic alterations and/or mutations. The blood-brain barrier has been a major obstacle for using targeted toxins for treatment of malignant glioma. Convection-enhanced delivery (CED), a method for delivery of large molecules to brain tissue via continuous interstitial microinfusion, has permitted direct administration of toxins to brain tumors or to surrounding brain tissue infiltrated by tumor cells. Four targeted toxins advanced to at least phase II clinical trials and are being used for treatment of adult or pediatric patients with recurrent or progressive malignant glioma. These are IL4-P. aeruginosa exotoxin (IL4-PE, NBI-3001), tumor growth factor (TGF)alpha-P. aeruginosa exotoxin (TP-38), IL13-P. aeruginosa exotoxin (IL13-PE38), and transferrin-C. diphtheriae toxin (TransMID(trade mark), Tf-CRM107). All of these toxins have shown an acceptable profile of toxicity and safety in phase I and II clinical studies and have demonstrated some evidence for tumor response. Current phase I and II clinical protocols are exploring several parameters, such as placement of catheters for CED either intratumorally or in the brain tissue surrounding a tumor, surgical resection of tumor before or after toxin infusion, and single vs. repeated infusion. Two large randomized and controlled phase III multicenter studies using IL13-PE38 or TransMID(trade mark) are currently enrolling patients. This review summarizes the study protocols and key findings of all previously completed and currently ongoing clinical studies with targeted toxins for malignant glioma. It offers in addition an outlook into future areas of development of targeted toxins, such as improved delivery modes and non-invasive in vivo imaging of intracerebral and intratumoral distribution of toxin in patients.

Suicide gene transfer using thymidine kinase (TK) and ganciclovir (GCV) treatment or the cytosine deaminase (CD)/5-fluorocytosine (5-FC) system represents the most widely used approach for gene therapy of cancer. However, molecular pathways and resistance mechanisms remain controversial for GCV-mediated cytotoxicity, and are virtually unknown for the CD/5-FC system. Here, we elucidated some of the cellular pathways in glioma cell lines that were transduced to express the TK or CD gene. In wild-type p53-expressing U87 cells, exposure to GCV and 5-FC resulted in a weak p53 response, although apoptosis was efficiently induced. Cell death triggered by GCV and 5-FC was independent of death receptors, but accompanied by mitochondrial alterations. Whereas expression of Bax remained unaffected, in particular, GCV and also 5-FC caused a decline in the level of Bcl-2. Similar findings were obtained in 9L and T98G glioma cells that express mutant p53, and also underwent mitochondrial apoptosis in both the TK/GCV and CD/5-FC system. Upon treatment of 9L cells with 5-FC, Bcl-xL expression slowly declined, whereas exposure to GCV resulted in the rapid proapoptotic phosphorylation of Bcl-xL. These data suggest that TK/GCV- and CD/5-FC-induced apoptosis does neither require p53 nor death receptors, but converges at a mitochondrial pathway triggered by different mechanisms of modulation of Bcl-2 proteins.

The remarkable migratory and tumor-tropic capacities of neural stem cells (NSCs and/or neuroprogenitor cells) represent a potentially powerful approach to the treatment of invasive brain tumors, such as malignant gliomas. We have previously shown that whether implanted directly into or at distant sites from an experimental intracranial glioma, NSCs distributed efficiently throughout the main tumor mass and also tracked advancing tumor cells, while stably expressing a reporter transgene. As therapeutic proof-of-concept, NSCs genetically modified to produce the prodrug activating enzyme cytosine deaminase (CD), effected an 80% reduction in the resultant tumor mass, when tumor animals were treated with the systemic prodrug, 5-fluorocytosine. We now extend our findings of the tumor-tropic properties of NSCs (using a well-characterized, clonal NSC line C17.2), by investigating their capacity to target both intracranial and extracranial tumors, when administered into the peripheral vasculature. We furthermore demonstrate their capacity to target extracranial non-neural tumors such as prostate cancer and malignant melanoma. Well-characterized NSC lines (lacZ and/or CD-positive) were injected into the tail vein of adult nude mice with established experimental intracranial and/or subcutaneous flank tumors of neural and non-neural origin. The time course and distribution of NSCs within the tumor and internal organs was assessed in various models. Resulting data suggest that NSCs can localize to various tumor sites when injected via the peripheral vasculature, with little accumulation in normal tissues. Our findings suggest the novel use of intravascularly administered NSCs as an effective delivery vehicle to target and disseminate therapeutic agents to invasive tumors of neural and nonneural origin, both within and outside of the brain.

Retrovirus (RV) has been one of the earliest recombinant vectors to be investigated in the context of cancer gene therapy. Experiments in cell culture and in animal brain tumor models have demonstrated the feasibility of RV mediated gene transduction and killing of glioma cells by toxicity generating transgenes. Phase I and II clinical studies in patients with recurrent malignant glioma have shown a favorable safety profile and some efficacy of RV mediated gene therapy. On the other hand, a prospective randomized phase III clinical study of RV gene therapy in primary malignant glioma failed to demonstrate significant extension of the progression-free or overall survival times in RV treated patients. The failure of this RV gene therapy study may be due to the low tumor cell transduction rate observed in vivo. The biological effects of the treatment may also heavily depend on the choice of transgene/prodrug system and on the vector delivery methods. Retrovirus clinical trials in malignant glioma have nevertheless produced a substantial amount of data and have contributed toward the identification of serious shortcomings of the non-replicating virus vector gene therapy strategy. Novel types of therapeutic virus vector systems are currently being designed and new clinical protocols are being created based on the lessons learned from the RV gene therapy trials in patients with malignant brain tumors.

Metastatic lesions are the most common spinal extradural tumours. Significant advances in their neurosurgical management have been made in the last two decades. This retrospective study was undertaken to summarise the long-term results of surgery and the outcome of patients with cervical spine metastases. Sixty-two patients with cervical spine metastases who underwent instrumented spinal surgery at a single centre in an 12-year period (1989-2000) were analysed. All patients presented with local pain and with either neurological deficits, spinal instability, or a combination of both. A standard anterior approach to the cervical spine was chosen, and a partial or total vertebrectomy and vertebral body replacement with subsequent anterior instrumented fusion were carried out in all cases. General and neurological status was evaluated at baseline and in regular intervals thereafter. Plain X-rays, CT, and MRI were used for preoperative planning. Postoperative follow-up was done by X-rays. The mean follow-up time for all patients was 1.5 years. A stable bony fusion of the cervical spine was achieved in 60 patients (96.8%), with two additional patients needing a further procedure for maintaining the mechanical stability of the spine. There was mild early surgery-related morbidity, and no mortality. The most frequent temporary surgery-related side effect was reversible vocal cord paresis in 5 cases (8.0%). There were 3 cases (4.8%) of early instrumentation failure. One of these was symptomatic and underwent second-look surgery. No late complications occurred due to instrumentation hardware failure. The 1-year survival rate of all patients after surgery was 58%, and the 2-year survival rate was 21%. Our results demonstrate that surgical removal of extradural metastases with subsequent instrumented fusion is a low-morbidity and low-complications procedure with high rates of permanent stabilisation of the compromised cervical spine. In addition, it improves the neurological deficits and relieves the local pain in a significant proportion of patients. Excellent local control of malignant disease can be achieved by the surgical procedure aided by subsequent local and systemic adjuvant therapy. Overall survival time and prognosis of the patients, however, are mainly depending on the type and the stage of the primary malignancy.

Recombinant retroviruses (RV) have been widely used as vectors for clinical gene therapy of malignant brain tumors. Because of the very limited stability of these vectors in vivo, RV producing cells (VPC) are routinely used for intratumoral RV release. The host immune system, however, recognizes the intratumorally grafted allogeneic or xenogeneic VPC, and mounts an immune response against them. Humoral and cellular immune responses eventually result in reduction of VPC numbers and in limited success of RV mediated gene therapy approaches. This study presents a non-pharmacological and spatially limited approach for protection of VPC grafted in the CNS against destruction by host immune responses. Murine fibroblast-derived VPC expressing herpes-simplex-virus type I thymidine kinase (HSV-tk) were genetically modified to co-express a human Fas ligand (CD95L) deletion mutant (DeltaFasL) resistant to enzymatic cleavage and shedding. Direct interactions between Fas (CD95) on lymphocytes and DeltaFasL on VPC upon cell-cell contact rapidly caused apoptosis in lymphocytes. In addition, cultured malignant brain tumor cells (U87, LN18, LN229) transduced with DeltaFasL-RV were rendered apoptotic by Fas/DeltaFasL interaction. DeltaFasL-expressing VPC grafted in a 9L rat brain tumor model survived in significantly higher numbers compared with control VPC, and did not cause an increase in neutrophil infiltration of tumors. Gene therapy of tumor bearing animals grafted with the modified DeltaFasL-VPC and given the prodrug Ganciclovir resulted in significantly increased survival rates compared to treatment with control VPC and Ganciclovir. In conclusion, prolonged intratumoral presence of DeltaFasL-VPC seems to be a direct consequence of the expression of the membrane-bound mutant FasL, and may result in increased total RV output and improved tumor transduction with RV.

Purpose: This was an open-label, dose-escalation trial of intratumoral administration of IL-4Pseudomonas exotoxin (NBI-3001) in patients with recurrent malignant glioma. Patients and methods: A total of 31 patients with histologically verified supratentorial grades 3 and 4 astrocytoma were studied. Of these, 25 patients were diagnosed with glioblastoma multiforme (GBM) while six were diagnosed with anaplastic astrocytoma. Patients were over 18 years of age and had Karnofsky performance scores ≥60. Patients were assigned to one of four dose groups in a dose-escalation fashion: 6 µg/ml × 40 ml, 9 µg/ml × 40 ml, 15 µg/ml × 40 ml, or 9 µg/ml × 100 ml of NBI-3001 administered via convection-enhanced delivery intratumorally using stereotactically placed catheters. Patients were followed with serial MRI scans and clinical assessments every four weeks for the first 16 weeks and then every eight weeks until week 26. Results: No drug-related systemic toxicity, as evident by lack of hematological or serum chemical changes, was apparent in any patients; treatment-related adverse effects were limited to the central nervous system. No deaths were attributable to treatment. Drug-related grade 3 or 4 toxicity was seen in 39% of patients in all dose groups and 22% of patients at the maximum tolerated dose of 6 µg/ml × 40 ml. The overall median survival was 8.2 months with a median survival of 5.8 months for the GBM patients. Six-month survival was 52% and 48%, respectively. Gadolinium-enhanced magnetic resonance imaging of the brain showed areas of decreased signal intensity within the tumor consistent with tumor necrosis following treatment in many patients. Conclusions: NBI-3001 appears to have an acceptable safety and toxicity profile when administered intratumorally in patients with recurrent malignant glioma.

Glioblastoma multiforme (GBM) is an incurable brain tumor resistant to standard treatment modalities such as surgery, radiation, and chemotherapy. Since recurrent GBM tends to develop predominantly within the infiltrative rim surrounding the primary tumor focus, novel therapy strategies need in addition to focal tumor destruction to target this somewhat diffuse area. This is a phase I/II clinical study in adult patients with recurrent GBM which is aimed at evaluating biological safety, maximum tolerated dose, and antitumor efficacy of a genetically modified replication-disabled Semliki forest virus vector (SFV) carrying the human interleukin 12 (IL-12) gene and encapsulated in cationic liposomes (LSFV-IL12). The vector will be administered in doses of 1 x 10(7)-1 x 10(9) infectious particles by continuous intratumoral infusion, thus exploiting the advantages of convection-enhanced drug delivery in the brain. The present protocol is also designed to investigate systemic and local immune response and to identify factors predicting tumor response to LSFV-IL12 therapy, such as volume of extracellular space of the tumor, volume of contrast enhancing lesion, and immune status of the patients. SFV, an insect alphavirus, infects mitotic and non-mitotic cells and triggers apoptosis in tumor cells within 48-72 h. Preclinical work with the LSFV-IL12 vector in breast and prostate cancer animal models demonstrated its biosafety and some antitumor efficacy. An ongoing phase I clinical study in patients with melanoma and renal cell carcinoma seems also to confirm the biosafety of intravenously administered vectors. This protocol will be the first study of SFV-IL12 therapy of human recurrent GBM.

Die obere Wirbelsaule, bestehend aus dem okzipito-zervikalen Ubergang und dem atlanto-axialen Komplex, ist ein Ubergangsbereich, der die Wirbelsaule mit dem Kranium verbindet. Wirbel und Gelenke unterscheiden sich von denen im subaxialen Ruckgrat, diese Unterschiede ermoglichen eine besondere Bewegungsfreiheit. Nach einem Trauma ist die Wiederherstellung der strukturellen Integritat von Bedeutung. Isolierte Atlasfrakturen werden entsprechend des spezifischen Atlasfrakturtyps behandelt: Ist die Fraktur stabil oder die Instabilitat durch einen Knochenbruch bedingt, kann mit externer Immobilisation behandelt werden, z. B. mit einem steifen Kragen, Minerva-Korsett oder Halo-Weste. Ist die Instabilitat ligamentar bedingt, ist die operative Therapie indiziert, um eine korrekte Fusion der hypermobilen und dislozierten Bewegungssegmente und ausreichenden Schutz fur das Ruckenmark zu gewahrleisten. Eine direkte C1-C2-Schrauben-Fixation (Magerl-Technik) kann eine sofortige Stabilisierung gewahrleisten und dadurch die Knochen- und Fragmentheilung optimieren.

The human egr - 1 gene encodes a zinc finger transcription factor induced by endogenous and exogenous stimuli such as growth factors, cytokines, and mitogens. Egr - 1 regulates other genes involved in growth and differentiation. The present study investigated the influence of genotoxic agents, such as chemotherapy drugs and other DNA damaging agents, on egr - 1 expression in normal and neoplastic cells. A transcriptional fusion between the human egr - 1 promoter and the enhanced green fluorescent protein ( EGFP ) gene was used for direct visualization of intracellular Egr-1 regulation. The transcriptional activity of the egr - 1 promoter in this reporter system faithfully reflects intrinsic egr - 1 expression and induction, as demonstrated by FACS analysis of fluorescence and by RT–PCR for egr - 1 . EGFP was expressed under the control of the egr - 1 promoter in stably transfected immortalized cell lines, such as HEK293, T98G, LNZ308, and 9L, which were then treated with genotoxic agents. A multitude of DNA damaging agents and therapeutic drugs caused significant upregulation of egr - 1 transcription. Furthermore, cytotoxic compounds without a direct DNA damaging effect, such as resveratrol and vincristine, which interfere with DNA replication and cell division, were also able to activate egr - 1 transcription. This suggests that cell cycle arrest rather than DNA damage seems to be the condition triggering egr - 1 transcription. Moreover, treatment with the MAP kinase (MAPK) inhibitor SB203580, which specifically blocks the stress inducible p38/SAPK2 pathway, did not alter egr - 1 induction. On the other hand, treatment with the inhibitor PD98059, which specifically blocks the MAPK/ERK pathway, partially suppressed the induction effect. In addition, the egr - 1 induction effect caused by genotoxic stress was found to be at least in part independent from the cellular p53 status, as it was observed in p53-deficient as well as in wild type p53 cell lines. These results suggest that induction of egr - 1 , a gene to which until now no relation to DNA repair has been assigned, may belong to the fundamental cellular responses elicited by genotoxic and mitotic stress in normal as well as in neoplastic cells, and that enhanced levels of Egr-1 protein may be needed to regulate genes involved in DNA repair, cell survival, and apoptosis.

Facial neuralgia is the last common pathway for a variety of pathological conditions with different etiology. Neuropathic facial pain is often refractory to routine medical or surgical treatments. We present here a long-term follow-up of two patients with unilateral facial neuropathic pain due to idiopathic trigeminal neuropathy or to surgical trauma to the glossopharyngeal nerve, respectively. These patients have been treated by other modalities for several years without obtaining satisfactory pain relief. Electrical stimulation of the motor cortex (MCS) with a quadripolar electrode contralateral to the painful area of the face was attempted in both cases for control of the facial pain, and resulted in immediate analgesia with more than 50% pain reduction. During a follow-up period of 72 months, a sufficient (> 50%) and stable analgesic effect of MCS was observed. These cases are discussed and the recent literature on MCS is reviewed in an attempt to identify indications for MCS as well as key structures in the brain for mediating the MCS effect.

Gene therapy employing the suicide gene/prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) is effective in killing malignant tumor cells. Labeling of the HSV-TK enzyme with fluorescent proteins makes possible the non-invasive imaging of transduction efficiency, enzyme localization and activity in cell culture and in animal models of human cancers. Here we report the expression of HSV-TK tagged with different fluorescent proteins (EGFP, DSRed1, DsRed2, dsdrFP616) and show that intracellular localization of the fusion products depends on the nature of the fluorescent tag despite the presence of several nuclear targeting signals within the enzyme itself. Coexpression of red fluorescent HSV-TK fusion proteins with TK-EGFP or untagged HSV-TK allowed these proteins to enter the nucleus by inhibiting formation of red fluorescent protein oligomers. As enzyme localization may influence HSV-TK activity, this observation is of potential importance to gene therapy studies.

This paper reviews data supporting the existence of individual, predictable, and unpredictable fluctuations in the severity of chronic pain and spasticity. It also evaluates what is known on the use of implantable programmable drug delivery systems for the management of predictable fluctuations in pain and spasticity. In addition to fixed rate infusion pumps, programmable drug delivery systems have been developed over the past 20 years for the management of predictable pain or spasticity fluctuations. The published literature on experimental and clinical studies of those topics is reviewed and evaluated. Programmable drug delivery systems can tailor dosing to a patient's individual pattern of symptoms, providing more medication during peak intensity of symptoms and less medication when symptoms are reduced. Fluctuations in either pain or spasticity are difficult to predict precisely, and therefore even programmable pumps cannot administer the appropriate amount of medication at any particular time. Ideally, the patient should be able to treat unpredictable fluctuations in symptoms, and a combination of patient controlled analgesia (PCA) with programmable drug delivery systems is currently in development. The future management of unpredictable fluctuations in the intensity of chronic pain and spasticity was subjected to critical evaluation. There seems to be a general agreement on the clinical importance of these phenomena, but stronger evidence is needed for a widespread change in the current management of most chronic pain patients.

The time course of cytotoxicity induction and the bystander effect of the rabbit cytochrome P450 4B1 (cyp4B1)/4-ipomeanol (4-IM) or 2-aminoanthracene (2-AA) pharmacogene therapy systems were investigated and compared with the herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV-tk/GCV) system. Experiments were performed in rat 9L gliosarcoma cells stably expressing cyp4B1 (9L-4B1), HSV-tk (9L-tk), or their egfp (enhanced green fluorescent protein) fusion genes. Cyp4B1-mediated activation of 2-AA showed a high cell killing efficiency within only 48 hours with an onset after already 15 minutes of prodrug exposure. Residual 9L-4B1 cells were mostly damaged sublethally upon 2-AA treatment showing an S phase arrest by cell cycle analysis. 4-IM treatment of 9L-4B1 cells generated an overall weaker cell killing, especially after prodrug exposure times of less than 48 hours. Residual cells surviving 4-IM treatment showed a G2/M arrest and restarted proliferation after prodrug treatment was stopped. HSV-tk/GCV pharmacogene therapy resulted in a slower cytotoxicity induction than cyp4B1/2-AA treatment with a significantly lower cell killing efficiency after 24 and 48 hours. HSV-tk/GCV-mediated cytotoxicity was widely similar to the cytotoxicity induced by cyp4B1/4-IM with the exception of a continuous 48-hour prodrug exposure where 4-IM treatment showed a significantly higher cell killing rate. Cells surviving HSV-tk/GCV suicide gene therapy were not viable and showed an S-phase arrest. Whereas HSV-tk/GCV induced a strong bystander effect, only moderate bystander cell death depending on cell-to-cell contact was demonstrated in 9L/9L-4B1 cocultures upon 2-AA treatment and was even absent with 4-IM, thereby contrasting with earlier reports. The absence of a strong bystander effect may limit, on one hand, the overall utility of the cyp4B1 systems for cancer gene therapy. On the other hand, the weak bystander effect together with the fast induction of cytotoxicity may provide marked advantages for the use of the cyp4B1 systems as biosafety enhancers for gene marking or replacement studies and donor lymphocyte infusions after allogeneic bone marrow transplantation.

During ganciclovir treatment of an adolescent ependymoma patient two weeks after intracranial implantation of HSVtk retroviral vector producer cells, increasing numbers of peripheral T- and B-cells were found as well as enhanced T-cell activation and elevated serum levels of interleukin 12 and soluble Fas ligand. These findings suggest the systemic activation of the immune system during ganciclovir treatment in our patient. The induction of an immune response by HSVtk/ganciclovir supports the concept of an anti-tumor vaccination effect by prodrug activating gene therapy systems and may open new promising perspectives for enhancing therapeutic efficiency by combined prodrug activating and immunological gene therapy strategies.

The JetCutter technology originally developed for high-throughput encapsulation of particles and substances into small beads was applied in this study for the entrapment of mammalian cells in alginate beads. In contrast to other established techniques such as the air jet droplet generation or laminar jet break-up, the JetCutter is capable of working with highly viscous fluids necessary for the production of stable beads based on hydrogels. A 1.5% (w/v) sodium alginate solution containing 2.0 x 106 murine fibroblasts/ml was processed under good manufacturing practice (GMP) conditions to beads with a mean diameter of 320 microm. The production capacity of the JetCutter technology was 5200 beads/s or to approximately 330 ml bead suspension per h. Beads were coated with poly-L-lysine and with an additional alginate layer to produce hollow microcapsules containing living cells. The influence of this method of encapsulation on the cell viability and morphology was investigated by light microscopic techniques. Encapsulated cells showed unchanged rates of proliferation and preserved morphology. They were able to survive in culture for extended periods of time. In conclusion, the JetCutter technology seems to be well suitable for alginate bead encapsulation of living mammalian cells.

Efficient virus and non-virus vector systems for gene transfer to tumor cells have been developed and tested in cell culture and in animal experiments. With some of the earliest and most comprehensively evaluated vectors, such as retroviruses, advanced clinical trials were performed in tumor patients. Malignant primary brain tumors (gliomas) have been chosen for the first clinical studies on novel gene therapy approaches because these tumors are non-metastatic and develop on the largely postmitotic background of normal glial and neuronal tissue. However, the human cancer gene therapy studies performed so far were not as successful as preclinical animal experiments. Furthermore, the clinical studies did not address major limiting factors for in vivo gene therapy, such as insufficient gene transfer rates to the tumor with the used local delivery modalities, and the resulting inability of a particular transgene-prodrug system to confer permanently eradicating cytotoxicity to the whole neoplasm. Critical evaluation of gene transfer and therapy studies has led to the conclusion that, even using identical vectors, the anatomical route of vector administration can dramatically affect both the efficiency of tumor transduction and its spatial distribution, as well as the extent of intratumoral and intracerebral transgene expression. This review concentrates on different physical methods for vector delivery to malignant primary brain tumors in experimental or clinical settings: stereotactic or direct intratumoral injection or convection-enhanced bulk-flow interstitial delivery; intrathecal and intraventricular injection; and intravascular infusion with or without modification of the blood-tumor-barrier. The advantages and drawbacks of the different modes and delivery routes of in vivo vector application, and the possibilities for tumor targeting by modifications of the native tropism of virus vectors or by using tissue-specific or inducible transgene expression are summarized.

Continuous intrathecal infusion of analgesic drugs by implantable pumps is recognized as an established treatment option for patients with chronic pain resistant to oral or parenteral medication. Polyanalgesia, the simultaneous use of more than one intrathecal analgesic drug, is practiced relatively often, but there are only a few published clinical studies on intrathecal polyanalgesia for chronic nonmalignant pain. This pilot study represents a long-term evaluation of a treatment regimen consisting of intrathecal morphine admixed with bupivacaine, clonidine, or midazolam in patients with chronic nonmalignant back and leg pain due to degenerative lumbar spinal disease. Twenty-six adult patients have been treated by intrathecal programmable pump-controlled infusion of analgesic drugs and followed for up to 3.5 years (27 +/- 11 months). Combination of morphine with a second drug was used in 10 cases, morphine with 2 additional drugs in 12 cases, and morphine with 3 additional drugs in 4 cases. Mean daily doses at 24 months after pump implantation were 6.2 +/- 2.8 mg for morphine, 2.5 +/- 1.5 mg for bupivacaine, 0.06 +/- 0.03 mg for clonidine, and 0.8 +/- 0.4 mg for midazolam. Nineteen patients reported excellent or good long-term treatment results, 6 patients had sufficient results, and only 1 patient complained of poor therapeutic efficacy. No long-term clinical side effects of intrathecal polyanalgesia were noted. Mean morphine dose had to be increased from 1.2 mg at baseline to 5.1 mg at 24 months due to tolerance development and disease progression. This experience suggests that intrathecal polyanalgesia employing morphine combined with additional nonopioid drugs can have a favorable analgesic efficacy in patients with complex chronic pain of spinal origin, and lacks major drug-related complications.

Although current treatment modalities for malignant gliomas, such as surgery, radiation and chemotherapy, have been improved markedly in the past two decades, the prognosis of these neoplasms remains poor, the two year survival rate being approximately 5%. Therefore, alternative treatment options, such as gene therapy and immunotherapy are rapidly gaining momentum. One of the most promising immunotherapeutic approaches for the treatment of cancer is the vaccination of cancer patients with dendritic cells (DC) pulsed with tumor antigens. Immunotherapy with DC seems to be able to overcome, at least partially, the immunosuppressive state associated with primary malignant gliomas. DC therapy proved to be safe in both animal models and clinical trials. No serious side effects and no evidence of autoimmune toxicity occurred. Most studies used DC pulsed with an array of tumor-associated antigens rather than single peptides, to allow for presentation of unknown tumor-specific antigens to DC. Routes of administration either were subcutaneous, intradermal or intraperitoneal, with multiple injections of DC to enhance antitumor immunity. DC therapy as an adjuvant treatment for patients with malignant glioma seems to be biologically safe. Further clinical studies are warranted.

Expression of anti-apoptotic or neurotrophic transgene proteins in hypoxic neurons may provide a novel therapeutic strategy for neuroprotection and alleviation of damage to ischemic brain areas. NT2, a human neoplastic cell line which terminally differentiates into postmitotic neurons (NT2N) by treatment with retinoic acid was used in this study as a cell culture model for human neuronal cells. The hypoxia-inducible VEGF promoter in plasmid vectors was employed to drive the expression of marker genes (luciferase) and therapeutic genes (bcl2) in hypoxic NT2 cells and NT2N neurons in culture. Cationic liposomes complexed with plasmid DNA were used for transfection of vectors with the constitutive cytomegalovirus promoter (pCMV) or the hypoxia-inducible VEGF promoter (pHRE). Hypoxic or normoxic control NT2 cells transfected with pCMV-luciferase showed high transgene expression (2.4 x 10(8) relative light units (RLU)/mg protein). Control NT2 cells transfected with pHRE-luciferase had a rather low activity (4.9 x 10(6) RLU/mg protein), which was induced 34-fold under hypoxic conditions. Four-fold induction of luciferase expression was obtained in hypoxic NT2N neurons transfected with pHRE compared with normoxic controls. The hypoxia-induced luciferase expression in NT2N cells was 34% of the activity of pCMV-luciferase under the same conditions. Transfection of NT2N neurons with pCMV-bcl2 or pHRE-bcl2 was demonstrated to reduce significantly the numbers of apoptotic cells after hypoxia. These results demonstrate efficient VEGF promoter-mediated induction of transgene expression in hypoxic human neurons. This cell culture model may be employed for the further investigation of therapeutic proteins against ischemic brain damage due to neuronal loss.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.

“Refraktare” Angina pectoris beschreibt ein chronisch viszerales Schmerzsyndrom, welches die Lebensqualitat im besonderen Mase beeintrachtigt. Nach meist extensiver medikamentoser, interventioneller und operativer Vorbehandlung kann die Symptomatik durch Intensivierung der medikamentosen Therapie, operative Myokardrevaskularisation oder Koronarangioplastie nicht weiter gebessert werden. Als weitere Behandlungskonzepte kommen daher verschiedene Therapieansatze zur Anwendung, insbesondere die Neuromodulation, die Opiatapplikation, die zervikale bzw. zervikothorakale Sympathektomie, die myokardiale Laserbehandlung sowie die intermittierende Urokinasetherapie. Diese Fallbeschreibung beschreibt exemplarisch die epidurale Elektrostimulation des Ruckenmarks (SCS) als effektive und minimal-invasive Behandlungsoption bei einem Patienten mit therapieresistenter Angina pectoris.

Expression of human leukocyte antigens (HLA) is important for the immune response against infectious agents and malignant cells. Association of single HLA antigens or HLA haplotypes with disease has been investigated previously, and positive correlations between HLA and some cancers, such as cervical or nasopharyngeal carcinomas have been reported. In the present study, HLA antigen frequencies of 65 adult Caucasian patients with low-grade, anaplastic, or malignant astrocytic glioma (WHO grades II–IV) were compared with 157 racially similar, asymptomatic control individuals. Both standard serologic and PCR techniques for HLA typing were employed for all patients and controls.

This study was conducted to investigate immunological components of somatic gene therapy for primary glioblastoma multiforme (GBM) in adults. It involved 13 patients treated by surgical resection of tumor with subsequent radiation therapy. Seven of them received additional herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy by direct intracerebral injection of retrovirus (RV) vector producing cells (VPC) during tumor surgery and subsequent systemic administration of GCV. Peripheral blood for FACS immunophenotyping, isolation of peripheral mononuclear cells (PMNC), and serum ELISA assays for IL-12 and soluble Fas ligand (sFasL) was collected daily during the first 4 post-operative weeks. Tumor specimens were obtained at primary or recurrent surgery and at autopsy. Tumors from gene therapy patients showed varying degrees of peritumoral necrosis around the former tumor resection cavity. Numbers of tumor-infiltrating lymphocytes found weeks after gene therapy were not significantly increased compared with primary tumors. Mitotic tumor cells were sparse close to the VPC injection sites, but abundant in brain areas somewhat distant from these sites. Serum ELISA revealed significantly increased sFasL and IL-12 levels in the gene therapy group compared with controls. Immunophenotyping of PMNC did not show a significant activation of T cells or NK cells during gene therapy. Interferon gamma secretion was evaluated by ELISPOT assays employing PMNC cocultivated with autologous tumor cells. It demonstrated an antitumor immune response in the gene therapy group, but not in the control group. These findings support the concept of in vivo induction of a systemic immune response by local intracerebral HSV-tk/GCV pharmacogene therapy for primary human GBM.

A number of different viral vectors have been used for gene therapy of tumors, with many more under construction, ultimately designed to improve tumor targeting and transduction efficiency. It has become apparent that insufficient viral delivery can be a key limitation to treatment efficacy. We have studied the in vivo mass distribution of a herpes simplex virus type 1 (HSV) vector, hrR3, by radiolabeling it with 111in-oxine. the virus was administered to intracerebral 9l glioma bearing fisher (f-344) rats by intracarotid and intratumoral injection. the blood half-life of the virus was 1 min (fast component, 10% contribution) and 180 min (slow component, 90% contribution). approximately 20% of activity had been excreted by 24 h. with intracarotid injection, the total amount of virus that accumulated in tumor was 0.10 ± 0.07% of the injected dose (id)/g at 1 h and 0.19 ± 0.01% id/g at 24 h. by comparison, co-injection of rmp-7, a synthetic bradykinin analog, with the virus, resulted in slightly increased tumor delivery of 0.17 ± 0.10% id/g (p 0.05) at 1 h. the 1 h organ distribution after intra-arterial injection (%id/organ) was as follows: liver 27.3 ± 2.86%, lung 2.10 ± 0.68% and kidney 1.78 ± 1.60% with lesser amounts in other organs. when virus was injected directly into the tumor, 71% of virus remained in tumor at 24 h (590 ± 212 %id/g, consistent with the small tumor mass containing most of the virus) with the following distribution regions: tumor > border zone > normal brain (99:40:1). These studies are the first quantitative mass distribution studies of HSV vectors in an experimental brain tumor model. Localization and quantitation of viral accumulation in vivo will enable detailed analysis of viral and organ interactions critical for advancing the therapeutic use of vectors.

Subcutaneous vaccination therapy with glioma cells, which are retrovirally transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), has previously proven effective in C57BL/6 mice harboring intracerebral GL261 gliomas. However, clinical ex vivo gene therapy for human gliomas would be difficult, as transgene delivery via retroviral vectors occurs only in dividing cells and ex vivo glioma cells have a low growth fraction. To circumvent this problem, a helper virus-free herpes simplex virus type 1 (HSV-1) amplicon vector was used. When primary cultures of human glioblastoma cells were infected with HSV-1 amplicon vectors at an MOI of 1, more than 90% of both dividing and nondividing cells were transduced. When cells were infected with an amplicon vector, HSVGM, bearing the GM-CSF cDNA in the presence of Polybrene, GM-CSF secretion into the medium during the first 24 hr after infection was 1026 ng/10(6) cells, whereas mock-infected cells did not secrete detectable GM-CSF. Subcutaneous vaccination of C57BL/6 mice with 5 x 10(5) irradiated HSVGM-transduced GL261 cells 7 days prior to intracerebral implantation of 10(6) wild-type GL261 cells yielded 60% long-term survivors (80 days), similar to the 50% long-term survivors obtained by vaccination with retrovirally GM-CSF-transduced GL261 cells. In contrast, animals vaccinated with the same number of nontranduced GL261 cells or with GL261 cells infected with helper virus-free packaged HSV-1 amplicon vectors carrying no transgene showed only 10% long-term survivors. In conclusion, helper virus-free HSV-1 amplicon vectors appear to be effective for cytokine-enhanced vaccination therapy of glioma, with the advantages that both dividing and nondividing tumor cells can be infected, no viral proteins are expressed, and these vectors are safe and compatible with clinical use.

A large animal tumor model for anaplastic glioma has been recently developed using immunotolerant allogeneic Beagle dogs and an established canine glioma cell line, J3T. This model offers advantages in terms of tumor morphology and similarity to human anaplastic glioma. The present study was aimed at evaluating the biological characteristics of the J3T canine glioma cell line as related to experimental gene therapy studies. Furthermore, development and morphology of canine brain tumors in a xenogeneic immunodeficient SCID mouse model was investigated. It was demonstrated that cultured J3T cells can be efficiently infected by adenovirus (AV), herpes-simplex type I (HSV), or retrovirus (RV) vectors, as well as by non-virus vectors such as cationic liposome/DNA complexes. Thus, in terms of infectability and transfectability, J3T cells seem to be closer to human glioma than the 9L rodent gliosarcoma. Cytotoxicity of selection antibiotics such as G418, puromycin, and hygromycin on J3T cells essentially resemble cytotoxicity seen with other established glioma lines, for example, 9L, U87, or U343. RV-mediated HSV-TK/GCV gene therapy demonstrated comparable LD50 for TK-expressing and control (non-expressing) J3T and 9L cells treated with Ganciclovir. Further, it was proven that J3T cells are tumorigenic and may grow heterotopically and orthotopically in a xenogeneic immunodeficient host, the SCID mouse, although morphology and growth pattern of these xenogeneic tumors differ from the demonstrated invasive phenotype in the Beagle dog.

Cationic liposomes (CL) are widely used vectors for gene transfer. Recently, ultrasound (US) was reported to enhance liposome-mediated gene transfer to eucaryotic cells in culture. The present study was aimed at studying the effects of 2-MHz pulsed Doppler US on malignant brain tumor cells transfection by cationic liposome/plasmid-DNA complexes (lipoplexes). Cationic liposomes consisting of DOSPA/DOPE were complexed with a plasmid carrying the cDNA encoding green autofluorescent protein (EGFP). Rodent (9L) and canine (J3T) glioma cells were exposed to pulsed US in the presence of EGFP-lipoplexes. A diagnostic transcranial Doppler device (MultiDop L) was used for insonation for 30, 60, and 90 s at 2 MHz/0.5 W/cm(2). To eliminate US reflection and cavitation, a custom-made absorption chamber was designed, where US is applied through a water tank before interacting with the cells and is fully absorbed after passing through the cell layer. Expression of the marker gene EGFP was quantified by FACS analysis and intravital fluorescent microscopy. Cell viability was accessed by Trypan Blue staining. US treatment of tumor cells on microplates for 60 s yielded a significant increase in transfection rates without damaging the cells, but 90-s treatment killed most of the cells. In the absorption chamber, no significant effects of US on transfection were noted. Additional experiments employed US contrast agent (Levovist, Schering) which was able to significantly increase tumor cell transfection rate by enhancing cavitation effects, and also severely damaged most cells when applied at a concentration of 200 mg/mL. In conclusion, our results support the assumption that US effects on lipoplex transfection rates in brain tumor cells in culture are mediated by cavitation effects.

Cellular delivery of a replication-conditional herpes simplex virus type 1 (HSV-1) vector provides a means for gene therapy of invasive tumor cells. LacZ -bearing neural precursor cells, which can migrate and differentiate in the brain, were infected with a ribonucleotide reductase-deficient HSV-1 mutant virus (rRp450) that replicates only in dividing cells. Replication of rRp450 in neural precursor cells was blocked prior to implantation into the tumor by growth arrest in late G 1 phase through treatment with mimosine. Viral titers in the medium of mimosine-treated, rRp450-infected neural precursor cells were below detection levels 3 days after infection. In culture, after removal of mimosine and passaging, cells resumed growth and replication of rRp450 so that, 7 days later, virus was present in the medium and cell death was evident. Mimosine-treated neural precursor cells injected into established intracerebral CNS-1 gliomas in nude mice migrated extensively throughout the tumor and into the surrounding parenchyma beyond the tumor over 3 days. Mimosine-treated neural precursor cells, infected with rRp450 and injected into intracerebral CNS-1 tumors, also migrated within the tumor with the appearance of foci of HSV–thymidine kinase-positive (TK + ) cells, presumably including tumor cells, distributed throughout the tumor and in the surrounding parenchyma over a similar period. This migratory cell delivery method has the potential to expand the range of delivery of HSV-1 vectors to tumor cells in the brain.

¶ The Cloward ventral interbody fusion is often employed for treatment of cervical degenerative disease. The present study was aimed at evaluating results and complications in this classical type of autologous bone graft procedure in a cohort of patients with radiculopathy (RP) or myeloradiculopathy (MRP). Indications for and limitations of the technique were investigated by retrospective data analysis in a series of 106 patients (30 females and 76 males). These underwent single or multiple level Cloward fusion in a total of 145 levels. Neuroradiological investigations included lateral and antero-posterior cervical spine X-rays, axial CT scans, and MRI. The presence of postoperative ossification and stable bony fusion in the fused segments was confirmed by X-rays and, when necessary, by CT. The median postoperative follow-up period was 6.5 years (range 4–10.5 years). Short term outcome in RP patients was good in 26 cases (92.9%) and fair in 2 cases (7.1%). A good short term outcome was seen in 55 MRP patients (70.5%), a fair outcome in 20 patients (25.6%), and a poor outcome in 3 patients (3.8%). Patients having myelopathy signs for less than 1 year had a significantly better outcome than those with clinical signs for more than 1 year (p

Background This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. Methods Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. Results The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14HD3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. Conclusion These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression. Copyright # 2000 John Wiley & Sons, Ltd.

Bcl-2 protein plays an important role in inhibiting apoptosis and protecting normal and neoplastic cells from toxicity. Bcl-2 overexpression in malignant tumors, on the other hand, may cause resistance against adjuvant treatment. Since there are subpopulations of patients with glioma that differ considerably in their treatment benefit, it is important to identify prognostic factors for outcome and to tailor adjuvant protocols in accordance with specific biological features of the respective tumor. The present study aimed at investigating the role of bcl-2 expression in higher-grade glioma (WHO grade III and IV). Bcl-2 expression was correlated with clinical and paraclinical parameters, and evaluated in univariate and multivariate statistical models. In addition, bcl-2-overexpressing human glioma cells in culture were used for modeling the in vivo findings and for investigating the importance of bcl-2 for tumor resistance against cytotoxic treatment. A group of 86 patients with higher-grade glioma were investigated. Anaplastic astrocytoma (AA; WHO G III, n = 29) showed bcl-2 expression in 48% of the cases, and immunohistochemical positivity was associated with a significantly shorter survival time (p = 0.0068). In glioblastoma patients (GBM; WHO G IV, n = 57), 51% of tumors were bcl-2 positive, but bcl-2 expression did not correlate significantly with survival (p = 0.39). In a Cox proportional hazards regression model, bcl-2 positivity was confirmed as a negative prognostic parameter in AA, but not in GBM. Bcl-2 overexpressing and control human glioma cell clones (T98MG line) were treated in culture with the cytotoxic drugs carmustine (BCNU), paclitaxel, vincristine, and doxorubicin. In addition, bcl-2-overexpressing and control cells were infected with a retrovirus carrying the herpes-simplex-virus thymidine kinase gene (HSV-tk), and then treated with ganciclovir (GCV). Bcl-2 overexpression significantly increased tumor cell resistance against all of the above cytotoxic drugs, and also against HSV-TK/GCV mediated gene therapy.

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ activated memory T cells. In an effort to elucidate the biological effects of IL-17 in glial cells, we investigated the ability of this cytokine in order to activate nuclear factor (NF)-kappaB, which is being discussed as one of the most important transcription factors in the regulation of neuronal and glial cell function. Activation of NF-kappaB involves the degradation of its cytoplasmatic inhibitor IkappaB-alpha, which allows the nuclear translocation of NF-kappaB, and ensures transcriptional activation of genes including IkappaB-alpha itself. Using a competitive RT-PCR, we examined the IL-17-induced IkappaB-alpha mRNA expression in glioblastoma cells, and we examined IL-17 up-regulated IkappaB-alpha mRNA expression in a dose- and time-dependent fashion with a maximum time between 1 and 3 h. This induction could be inhibited by Calphostin C (protein kinase C inhibitor) and genistein (tyrosine kinase inhibitor). After 60 min of IL-17 stimulation, a degradation of the IkappaB-alpha protein was detectable. Furthermore, IL-17 stimulated the secretion of IL-6 and IL-8 in glial cells, and IL-17 and IL-1beta in combination showed a superadditive effect. We suggest IL-17 to play a role as an immune factor, possibly involved in complex pathophysiological interactions of neurodegenerative diseases.

Previous studies with animal models of supratentorial ICP elevation have demonstrated a pressure gradient between the supratentorial and the infratentorial compartments. The present study was designed to investigate the possible presence of such a gradient in the case of infratentorial ICP elevation. An inflatable infratentorial balloon catheter was implanted in seven domestic pigs. The infratentorial ICP (ICPi) was measured in the left cerebellar hemisphere, and the supratentorial ICP (ICPs) was measured in the left cerebral hemisphere. The corresponding pulse amplitudes (ICPi-PA, ICPs-PA) were recorded in both compartments, and the cerebral perfusion pressure (CPP) was calculated. ICPi and ICPs values prior to balloon inflation were 4.4 (SD 2.2) and 4.1 (SD 2.3) mm Hg, respectively, and increased to 63.1 (SD 32.6) and 62.3 (SD 28.1) mmHg after balloon inflation. ICPi-PA rose from 3.1 (SD 0.43) to 12.8 (SD 8.0) mmHg, and ICPs-PA rose from 3.2 (SD 0.63) to 13.0 (SD 7.1) mmHg. CPP decreased from 86.1 (SD 1...

From our panel of expert Editorial Board members, an update on the developments and activities ongoing in their laboratories and institutions, as well as recommendations of other groups whose work is of considerable interest to the readers of EOBT.

STUDY DESIGN This study was conducted to evaluate an anterior cervical fusion plate system, the Orion locking plate, regarding its surgical handling, hardware-related failures, and short-term and long-term results. OBJECTIVES A comprehensive evaluation of the implant in a broad range of patients with cervical spine diseases. SUMMARY OF BACKGROUND DATA Locking plates are the most recent devices for achieving anterior cervical spinal fusion and offer considerable advantages such as faster and easier implantation and fewer implant-related failures than older plate systems. METHODS Ninety-six patients were investigated. All underwent anterior cervical plate fusion as a component of the surgical treatment for symptomatic degenerative cervical spinal disease or for vertebral destruction caused by trauma, tumor, or inflammation. Besides plate fixation, 6 of the 96 patients had a combined ventrodorsal fusion. In 28 cases, one or more vertebral bodies were removed and replaced with titanium place-holders. The remaining 62 patients were first treated by intervertebral inlay placement, and the fused segments were subsequently plated. Neurologic signs and symptoms were evaluated before and after surgery and during a follow-up period of at least 1 year. RESULTS The rate of neurologic improvement was highest in radiculopathy patients and lowest in patients with severe myelopathy. In all cases, control radiographs demonstrated a solid bony fusion. Clinical deterioration after surgery was seen in four cases of severe myelopathy in which considerable neurologic deficits existed before surgery, possible because of rapid decompression of the cord and associated microvascular alterations. In two of these cases, there was long-term improvement. Additional general complications caused by surgical retraction included temporary swallowing disturbance in seven patients and a large wound hematoma in one. Hardware failures were encountered in three cases, all of them caused by improper implantation technique and not material failure, per se. CONCLUSION In the study group, the Orion locking plate was easy to use, failure-free if properly implanted, safe for the patient and supported solid bony fusion in every case.

Gene therapy offers significant advantages to the field of oncology with the addition of specifically and uniquely engineered mechanisms of halting malignant proliferation through cytotoxicity or reproductive arrest. To confer a true benefit to the therapeutic ratio (the relative toxicity to tumor compared to normal tissue) a vector or the transgene it carries must selectively affect or access tumor cells. Beyond the selective toxicities of many transgene products, which frequently parallel that of contemporary chemotherapeutic agents, lies the potential utility of targeting the vector. This review presents an overview of current and potential methods for designing vectors targeted to CNS malignancies through selective delivery, cell entry, transport or transcriptional regulation. The topic of delivery encompasses physical and pharmaceutic means of increasing the relative exposure of tumors to vector. Cell entry based methodologies are founded on increasing relative uptake of vector through the chemical or recombinant addition of ligand and antibody domains which selectively bind receptors expressed on target cells. Targeted transport involves the potential for using cells to selectively carry vectors or transgenes into tumors. Finally, promoter and enhancer systems are discussed which have potential for selectivity activating transcription to produce targeted transgene expression or vector propagation.

This paper describes a modified and less traumatic approach to the thoracolumbar spine and compares it with standard techniques for instrumented spinal fusion. Ten patients with unstable fractures of the thoracolumbar spine were included in the open prospective investigation, and were treated by a surgical technique consisting of a dorsolateral approach to the injured segment, filling the disk space and the fractured vertebra with autologous bone, and transpedicular fixation with an AO internal fixator. All patients were followed for 6 to 12 months after surgery by clinical tests and spinal X-rays. Excellent short-term and long-term results were obtained. A stable bony fusion was achieved in all cases, and a minimal mean decrease of 2 degrees in the kyphosis angle was found at late follow-up. No major complications related to the procedure were encountered, and no worsening of neurological deficits occurred after surgery. In conclusion, the far-lateral approach to the thoracolumbar spine yields results which are equivalent or better than those of standard techniques. Major advantages of our procedure, as evaluated in this rather small group of patients, are selective immobilization of the injured segment without involvement of functionally intact spinal levels, no manipulations within the spinal canal boundaries, and relatively limited exposure of the spine.

A second-generation replication-conditional herpes simplex virus type 1 (HSV) vector defective for both ribonucleotide reductase (RR) and the neurovirulence factor gamma34.5 was generated and tested for therapeutic safety and efficiency in two different experimental brain tumor models. In culture, cytotoxic activity of this double mutant HSV vector, MGH-1, for 9L gliosarcoma cells was similar to that of the HSV mutant, R3616, which is defective only for gamma34.5, but was significantly weaker than that of the HSV mutant hrR3, which is defective only for RR. The diminished tumoricidal effect of the gamma34.5 mutants could be accounted for by their reduced ability to replicate in 9L cells. The MGH-1 vector did not achieve significant prolongation of survival in vivo in the syngeneic 9L rat gliosarcoma model for either single brain tumor focus or multiple intracerebral and leptomeningeal tumors, when the vector was applied intratumorally or intrathecally, respectively, and with or without subsequent ganciclovir (GCV) treatment. In identical 9L brain tumor models with single and multiple foci, application of hrR3 with or without GCV was previously shown to result in marked long-term survival. Contrary to the findings with intrathecal injection of hrR3, no vector-related mortality was observed in any animals treated with MGH-1. Thus, in these rat brain tumor models, the double mutant, replication-conditional HSV vector MGH-1 showed a higher therapeutic safety than the RR-minus vector, hrR3, but had clearly decreased therapeutic efficiency compared to hrR3. The development of new HSV vectors for brain tumor gene therapy will require a balance between maximizing therapeutic efficacy and minimizing toxicity to the brain. Standardized application in brain tumor models as presented here will help to screen new HSV vectors for these requirements.

Chronic facial neuralgias often do not respond sufficiently to standard treatment methods. Alternative modalities are needed for long-term reduction of pain in such cases. The present preliminary report describes two patients with trigeminal and glossopharyngeal neuralgia, respectively, treated with standard methods without obtaining satisfactory pain relief. Electrical stimulation of the motor cortex contralateral to the pain area was employed in both cases and proved able to produce a long-term facial pain reduction. Alleviation of pain occurred after activation of the flat quadripolar electrode placed epidurally on the precentral cortical area and lasted as long as the stimulator was working. By changing the polarity of the electrodes, it was possible to induce tingling sensations and muscle activation not only contralaterally to the stimulated motor cortex, but also in the ipsilateral part of the face. No stimulator-independent pain reduction resulted from long-term use of the stimulation device. During a follow-up period of 18 months, a sufficient and relatively stable analgesic effect of electrostimulation was observed. One major complication of motor cortex stimulation during the follow-up period was a single generalized epileptic seizure in one of the patients.