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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.

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.

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.

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.

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.

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.

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.

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.

Purpose: To test the in vitro efficacy of the chemotherapeutic alkylating agent prodrug 4-ipomeanol (4-IM) combined with ionizing radiation (IR) in cells transduced with an adenoviral vector carrying a chimeric fusion protein of rabbit cytochrome CYP4B1 and enhanced green fluorescent protein (EGFP) under the control of the radiation-inducible EGR1 promoter. Materials and Methods: Cassette effect was tested by comparing survival fractions determined by low-input MTT assay for HEK293 cells stably transfected with the prodrug-activating fusion protein CYP4B1-EGFP under the control of the EGR1 promoter with that of control HEK293 cells stably transfected with EGFP alone, 8 days after treatment with 4-IM and IR. Radiosensitization by activated 4-IM was tested by comparing survival fractions from clonogenic assays of 9L cells transfected with the CYP4B1-EGFP cassette or EGFP alone, both under a constituitive CMV promoter, after 24 hours of 4-IM exposure. The EGR-4B1-EGFP cassette was inserted into the E1A region of an adenoviral vector, and efficacy compared to adenoviral vector carrying CMV-EGFP (MOI = 100), was tested in U87-MG cells using MTT assay 3 days after treatment with 4-IM and IR. In each case, cells were irradiated at doses of 0, 2, and 4 Gy on day 1, following 4-IM treatment at concentrations of 0, 2.5 and 5.0 μg/ml. Results: MTT assays demonstrated a decrease in the relative survival fractions (survival with 4-IM/survival without 4-IM) of EGR1-CYP4B1-EGFP transfected cells with increasing radiation dosage, but not of the control cells: 88%, 57%, 39% at 0, 2 and 4 Gy for the EGR1-CYP4B1-EGFP cells, and 96%, 87%, 96% at 0, 2 and 4 Gy for the control cells (2.5 μg/ml 4-IM). Clonogenic assays for radiosensitization demonstrated that survival fractions decreased with increasing radiation doses and 4-IM concentrations (for the CYP4B1 bearing cells), but no significant differences in D0 were seen with 4-IM. Of relevance, a similar lack of sensitization was seen in the same model using rat cytochrome P450 2B1 (CYP2B1) to activate cyclophosphamide, but a pronounced effect was seen when this combination was used in a replication competent HSV-1 vector (rRp450). 4-IM (5.0 μg/ml) reduced the relative survival of cells treated with adenovirus carrying EGR-4B1-EGFP to 66 +/− 11% with the addition of 2 Gy IR and to 57 +/− 10% with 4 Gy IR. No effect was seen in cells treated with adenovirus carrying CMV-EGFP. Conclusion: IR potentiates the cytotoxic activity of the EGR1/CYP4B1/4-IM transgene system. Cytochrome P450 activated alkylating agent prodrugs do not appear to themselves sensitize cells to radiation in the models tested, but significant augmentation of IR efficacy can be obtained when these are incorporated into appropriate transgene and vector systems. There may be additional variability between agents and cell lines, as has been reported for temozolomide. We are proceeding to test these gene therapy vectors in murine models.