C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo.

We previously demonstrated that a synthetic monomer peptide derived from the C-terminus of p53 (aa 361−382) induced preferential apoptosis in mutant p53 malignant cells, but not normal cells. The major problem with the peptide was its short half-life (half-life < 10 min.) due to a random coil topology found in 3D proton NMR spectroscopy studies. To induce secondary/tertiary structures to produce more stability, we developed a peptide modelled after the tetrameric structure of p53 essential for activation of target genes. Starting with the above monomer peptide (aa 361−382), we added the nuclear localization sequence of p53 (aa 353−360) and the end of the C-terminal sequence (aa 383−393), resulting in a monomer spanning aa 353−393. Four monomers were linked by glycine to maximize flexibility and in a palindromic order that mimics p53 tetramer formation with four orthogonal alpha helices, which is required for p53 transactivation of target genes. This is now known as the 4 repeat-palindromic-p53 peptide or (4R-Pal-p53p). We explored two methods for testing the activity of the palindromic tetrapeptide: (1) exogenous peptide with a truncated antennapedia carrier (Ant) and (2) a doxycycline (Dox) inducer for endogenous expression. The exogenous peptide, 4R-Pal-p53p-Ant, contained a His tag at the N-terminal and a truncated 17aa Ant at the C-terminal. Exposure of human breast cancer MB-468 cells and human skin squamous cell cancer cells (both with mutant p53, 273 Arg->His) with purified peptide at 7 µM and 15 µM produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361−382, termed p53p-Ant), at 15 µM and 30 µM induced 15% and 24% cell death, respectively. Compared to the p53p-Ant, the exogenous 4R-pal-p53p-Ant was over five-fold more potent for inducing apoptosis at an equimolar concentration (15 µM). Endogenous 4R-Pal-p53p expression (without Ant), induced by Dox, resulted in 43% cell death in an engineered MB468 breast cancer stable cell line, while endogenous p53 C-terminal monomeric peptide expression produced no cell death due to rapid peptide degradation. The mechanism of apoptosis from 4R-Pal-p53p involved the extrinsic and intrinsic pathways (FAS, caspase-8, Bax, PUMA) for apoptosis, as well as increasing reactive oxygen species (ROS). All three death pathways were induced from transcriptional/translational activation of pro-apoptotic genes. Additionally, mRNA of p53 target genes (Bax and Fas) increased 14-fold and 18-fold, respectively, implying that the 4R-Pal-p53p restored full apoptotic potential to mutant p53. Monomeric p53p only increased Fas expression without a transcriptional or translational increase in Fas, and other genes and human marrow stem cell studies revealed no toxicity to normal stem cells for granulocytes, erythrocytes, monocytes, and macrophages (CFU-GEMM). Additionally, the peptide specifically targeted pre-malignant and malignant cells with mutant p53 and was not toxic to normal cells with basal levels of WT p53.

Deep brain stimulation for movement disorders after stroke: a systematic review of the literature.

OBJECTIVE: Stroke remains the leading cause of disability in the United States. Even as acute care for strokes advances, there are limited options for improving function once the patient reaches the subacute and chronic stages. Identification of new therapeutic approaches is critical. Deep brain stimulation (DBS) holds promise for these patients. A number of case reports and small case series have reported improvement in movement disorders after strokes in patients treated with DBS. In this systematic review, the authors have summarized the patient characteristics, anatomical targets, stimulation parameters, and outcomes of patients who have undergone DBS treatment for poststroke movement disorders. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. The PubMed, Scopus, and SpringerLink databases were searched for the keywords "DBS," "stroke," "movement," and "recovery" to identify patients treated with DBS for movement disorders after a stroke. The Joanna Briggs Institute Critical Appraisal checklists for case reports and case series were used to systematically analyze the quality of the included studies. Data collected from each study included patient demographic characteristics, stroke diagnosis, movement disorder, DBS target, stimulation parameters, complications, and outcomes. RESULTS: The authors included 29 studies that described 53 patients who underwent placement of 82 total electrodes. Movement disorders included tremor (n = 18), dystonia (n = 18), hemiballism (n = 6), spastic hemiparesis (n = 1), chorea (n = 1), and mixed disorders (n = 9). The most common DBS targets were the globus pallidus internus (n = 32), ventral intermediate nucleus of thalamus (n = 25), and subthalamic area/subthalamic nucleus (n = 7). Monopolar stimulation was reported in 43 leads and bipolar stimulation in 13. High-frequency stimulation was used in 57 leads and low-frequency stimulation in 6. All patients but 1 had improvement in their movement disorders. Two complications were reported: speech impairment in 1 patient and hardware infection in another. The median (interquartile range) duration between stroke and DBS treatment was 6.5 (2.1-15.8) years. CONCLUSIONS: This is the first systematic review of DBS for poststroke movement disorders. Overall, most studies to date have been case reports and small series reporting heterogeneous patients and surgical strategies. This review suggests that DBS for movement disorders after a stroke has the potential to be effective and safe for diverse patients, and DBS may be a feasible option to improve function even years after a stroke.

Deep Brain Stimulation and Motor Cortex Stimulation for Chronic Pain.

Deep brain stimulation (DBS) and Motor Cortex stimulation (MCS) have been used for control of chronic pain. Chronic pain of any origin is complex and difficult to treat. Stimulation of various areas in brain-like sensory thalamus, medial nuclei of thalamus including centro-lateral nucleus of thalamus (CL), periaqueductal gray, periventricular gray, nucleus accumbence and motor cortex provides partial relief in properly selected patients. This article reviews the pain pathways, theories of pain, targets for DBS and rationale of DBS and MCS. It also discusses the patient selection, technical details of each target.

Deep brain stimulation of the subthalamic nucleus reverses oral tremor in pharmacological models of parkinsonism: interaction with the effects of adenosine A2A antagonism.

Deep brain stimulation (DBS) of the subthalamic nucleus is increasingly being employed as a treatment for parkinsonian symptoms, including tremor. The present studies used tremulous jaw movements, a pharmacological model of tremor in rodents, to investigate the tremorolytic effects of subthalamic DBS in rats. Subthalamic DBS reduced the tremulous jaw movements induced by the dopamine D2 family antagonist pimozide and the D1 family antagonist ecopipam, as well as the cholinomimetics pilocarpine and galantamine. The ability of DBS to suppress tremulous jaw movements was dependent on the neuroanatomical locus being stimulated (subthalamic nucleus vs. a striatal control site), as well as the frequency and intensity of stimulation used. Importantly, administration of the adenosine A2A receptor antagonist MSX-3 reduced the frequency and intensity parameters needed to attenuate tremulous jaw movements. These results have implications for the clinical use of DBS, and future studies should determine whether adenosine A2A antagonism could be used to enhance the tremorolytic efficacy of subthalamic DBS at low frequencies and intensities in human patients.

Biomechanical evaluation of a novel posterior integrated clamp that attaches to an existing posterior instrumentation for use in thoracolumbar revision.

STUDY DESIGN: An in vitro biomechanical study. PURPOSE: To evaluate the biomechanics of a novel posterior integrated clamp (IC) that extends on an already implanted construct in comparison to single long continuous bilateral pedicle screw (BPS) and rod stabilization system. OVERVIEW OF LITERATURE: Revision surgery in the thoracolumbar spine often necessitates further instrumentation following a failed previous back surgery. Stability of these reconstructed constructs is not known. METHODS: Six osteoligamentous T12-L5 calf spines were tested on a spine motion simulator in the following configurations: intact, four level constructs (T13-L4), three level constructs (L1-L4), and two level constructs (L2-L4), by varying the ratio between BPS and IC. A load control protocol of 8 Nm moments was applied at a rate of 1°/sec to establish the range of motion value for each construct in flexion-extension, lateral bending, and axial rotation. Statistical analysis was performed on raw data using repeated measures analysis of variance and significance was set at p<0.05. RESULTS: On an average, the reduction in motion for the four level continuous pedicle screw and rod construct (67%) was similar to those extended with integrated clamps (64%). Furthermore, for three level and two level constructs, no significant difference was observed between continuous pedicle screw constructs and those revised with the integrated clamps (regardless of the ratio between BPS and IC). CONCLUSIONS: The novel posterior IC showed equivalent biomechanical rigidity to continuous pedicle screw rod constructs in revision scenarios. Clinical studies on posterior rod adjunct systems are necessary to confirm these results.

Intracranial hemorrhage from undetected aneurysmal rupture complicating transphenoidal pituitary adenoma resection.

We report a case of a 39-year-old man who presented with a nonfunctioning pituitary macroadenoma which extended into the suprasellar region. He underwent a transcranial resection of the tumor followed eight months later by transsphenoidal surgery for the residual tumor. Postoperatively he developed massive subarachnoid and intraventricular hemorrhage. A cerebral angiogram revealed a leaking anterior communicating artery aneurysm which was not seen on the computed tomography angiography and magnetic resonance angiography before the surgery. Complications of transsphenoidal surgery, particularly vascular hemorrhagic complications, and risk of rupture of undetected aneurysms are discussed.

Pharmacological and physiological characterization of the tremulous jaw movement model of parkinsonian tremor: potential insights into the pathophysiology of tremor.

Tremor is a cardinal symptom of parkinsonism, occurring early on in the disease course and affecting more than 70% of patients. Parkinsonian resting tremor occurs in a frequency range of 3-7 Hz and can be resistant to available pharmacotherapy. Despite its prevalence, and the significant decrease in quality of life associated with it, the pathophysiology of parkinsonian tremor is poorly understood. The tremulous jaw movement (TJM) model is an extensively validated rodent model of tremor. TJMs are induced by conditions that also lead to parkinsonism in humans (i.e., striatal DA depletion, DA antagonism, and cholinomimetic activity) and reversed by several antiparkinsonian drugs (i.e., DA precursors, DA agonists, anticholinergics, and adenosine A(2A) antagonists). TJMs occur in the same 3-7 Hz frequency range seen in parkinsonian resting tremor, a range distinct from that of dyskinesia (1-2 Hz), and postural tremor (8-14 Hz). Overall, these drug-induced TJMs share many characteristics with human parkinsonian tremor, but do not closely resemble tardive dyskinesia. The current review discusses recent advances in the validation of the TJM model, and illustrates how this model is being used to develop novel therapeutic strategies, both surgical and pharmacological, for the treatment of parkinsonian resting tremor.

Typical variations of subthalamic electrode location do not predict limb motor function improvement in Parkinson's disease.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective treatment for patients with medically refractory Parkinson's disease (PD). The degree to which the anatomic location of the DBS electrode tip determines the improvement of contralateral limb movement function has not been defined. This retrospective study was performed to address this issue. Forty-two DBS electrode tips in 21 bilaterally implanted patients were localized on postoperative MRI. The postoperative and preoperative planning MRIs were merged with the Stealth FrameLink 4.0 stereotactic planning workstation (Medtronic Inc., Minneapolis, MN, USA) to determine the DBS tip coordinates. Stimulation settings were postoperatively optimized for maximal clinical effect. Patients were videotaped 1 year postoperatively and assessed by a movement disorder neurologist blinded to electrode tip locations. The nine limb-related components of the Unified PD Rating Scale Part III were tabulated to obtain a limb score, and the electrode tip locations associated with the 15 least and 15 greatest limb scores were evaluated. Two-tailed t-tests revealed no significant difference in electrode tip location between the two groups in three-dimensional distance (p=0.759), lateral-medial (x) axis (p=0.983), anterior-posterior (y) axis (p=0.949) or superior-inferior (z) axis (p=0.894) from the intended anatomical target. The range of difference in tip location and limb scores was extensive. Our results suggest that anatomic targeting alone may provide the same clinical efficacy as is achieved by "fine-tuning" DBS placement with microelectrode recording to a specific target.

Staged bilateral thalamic electrode implantation utilizing frameless stereotactic guidance.

Deep brain stimulation (DBS) for medically intractable Parkinson's disease (PD) is well established, but carries the inconveniences of frame-based neurosurgery. Previous reports have demonstrated that ventricular shunt placement and some functional procedures can be accurately performed using frameless stereotaxy. We present a report indicating that staged deep brain electrode placement can be accurate and efficacious using a frameless skull-mounted guide.

A technique for minimally altering anatomically based subthalamic electrode targeting by microelectrode recording.

OBJECT: Implantation of a subthalamic nucleus (STN) deep brain stimulation (DBS) electrode is increasingly recognized as an effective treatment for advanced Parkinson disease (PD). Despite widespread use of microelectrode recording (MER) to delineate the boundaries of the STN prior to stimulator implantation, it remains unclear to what extent MER improves the clinical efficacy of this procedure. In this report, the authors analyze a series of patients who were treated at one surgical center to determine to what degree final electrode placement was altered, based on readings obtained with MER, from the calculated anatomical target. METHODS: Subthalamic DBS devices were placed bilaterally in nine patients with advanced PD. Frame-based volumetric magnetic resonance images were acquired and then transferred to a stereotactic workstation to determine the anterior and posterior commissure coordinates and plane. The initial anatomical target was 4 mm anterior, 4 mm deep, and 12 mm lateral to the midcommissural point. The MERs defined the STN boundaries along one or more parallel tracks, refining the final electrode placement by comparison of results with illustrations in a stereotactic atlas. In eight of 18 electrodes, the MER results did not prompt an alteration in the anatomically derived target. In another eight placements, MER altered the target by less than 1 mm and two of 18 electrode positions differed by less than 2 mm. The anterior-posterior difference was 0.53 +/- 0.65 mm, whereas the medial-lateral direction differed by 0.25 +/- 0.43 mm. The ventral boundary of the STN defined by MER was 2 +/- 0.72 mm below the calculated target (all values are the means +/- standard deviation). All patients attained clinical improvement, similar to previous reports. CONCLUSIONS: In this series of patients, microelectrode mapping of the STN altered the anatomically based target only slightly. Because it is not clear whether such minor adjustments improve clinical efficacy, a prospective clinical comparison of MER-refined and anatomical targeting may be warranted.

Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide.

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382, p53p), linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction, induced rapid apoptosis in human premalignant and malignant cell lines. Here, we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas, which express sufficient quantities of endogenous mutant or WT p53, may be restored or activated, respectively, by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system, which circumvents the blood-brain barrier.

The transformation of pediatric gliomatosis cerebri to cerebellar glioblastoma multiforme presenting as supra- and infratentorial acute disseminated encephalomyelitis. Case report.

Cerebellar glioblastoma multiforme (GBM) is a rare entity in adults and an extremely rare entity in children. Approximately 30 cases have been reported in the literature. The authors report the case of a histologically confirmed cerebellar GBM presenting initially as supra- and infratentorial gliomatosis cerebri. Acute disseminated encephalomyelitis had been diagnosed in the patient and that diagnosis remained until near the end of his treatment. This case underscores the need for recognizing the clinical presentation of gliomatosis cerebri and multifocal GBM in the pediatric subpopulation thought to harbor demyelinating disease.

Subthalamic stimulation for Parkinson disease: determination of electrode location necessary for clinical efficacy.

OBJECT: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) performed using intraoperative microelectrode recording (MER) to adjust electrode placement has become a widely used treatment for patients with advanced Parkinson disease (PD). Few studies have been conducted to examine the location of implanted electrodes relative to the intended target, and even fewer have been undertaken to investigate the degree to which variations in the location of these electrodes impacts their clinical efficacy. This study was performed to examine these issues. METHODS: The authors located 52 bilaterally implanted DBS electrode tips on postoperative magnetic resonance (MR) images obtained in 26 consecutive patients. Postoperative and preoperative planning MR images were merged to determine the DBS electrode tip coordinates relative to the midcommissural point. Surgical records listed the intended target coordinates for each DBS electrode tip. Clinical outcome assessment included the Unified PD Rating Scale (UPDRS) motor score at 1 year, standardized questionnaires, and routine follow-up visits. The mean difference between electrode tip location and intended target for all 52 electrodes was less than 2 mm in all axes. Only one electrode was farther than 3 mm from the intended target, and this was the only electrode that had to be replaced due to lack of clinical efficacy (lack of tremor suppression); its reimplantation 4 mm more medially provided excellent tremor control. High correlation coefficients indicate that the MR imaging analysis accurately determined the anatomical location of the electrode tips. Blinded videotape reviews of UPDRS motor scores comparing effects of stimulation in patients who were "on" and "off" medication identified subgroups in whom there was minimal and maximal stimulation response. Patients in these subgroups had no differences between the MR imaging-determined actual electrode tip location and its intended location. Similarly, improvements of dyskinesias and severity of symptoms encountered during the wearing-off period for the drug did not correlate with variations of electrode tip location. CONCLUSIONS: The findings in this study lead the authors to suggest that a DBS electrode placed anywhere within a 6-mm-diameter cylinder centered at the presumed middle of the STN (based on stereotactic atlas coordinates) provides similar clinical efficacy. Future studies may be warranted to evaluate prospectively the degree to which MER modification of the anatomically and/or image-determined target improves clinical efficacy of DBS electrodes.

Implantation of bilateral deep brain stimulators in patients with Parkinson disease and preexisting cardiac pacemakers. Report of two cases.

Deep brain stimulation (DBS) has become an important modality in the treatment of refractory Parkinson disease (PD). In patients with comorbid arrhythmias requiring cardiac pacemakers, DBS therapy is complicated by concerns over a possible electrical interaction between the devices (or with device programming) and the inability to use magnetic resonance imaging guidance for implantation. The authors report two cases of PD in which patients with preexisting cardiac pacemakers underwent successful implantation of bilateral DBS electrodes in the subthalamic nucleus (STN). Each patient underwent computerized tomography-guided stereotactic frame-based placement of DBS electrodes with microelectrode recording. Both extension wires were passed from the right side of the head and neck (contralateral to the pacemaker) to place the cranial pulse generators subcutaneously in the left and right abdomen. The cranial pulse generators were placed farther than 6 in from the cardiac pacemaker and from each other to decrease the chance of interference between the devices during telemetry reprogramming. Postoperative management involved brain stimulator programming sessions with simultaneous cardiological monitoring of pacemaker function and cardiac rhythm. No interference was noted at any time, and proper pacemaker function was maintained throughout the follow-up period. With bilateral STN stimulation, both patients experienced a dramatic improvement in their PD symptoms, including elimination of dyskinesias, reduction of "off" severity, and increase of "on" duration. With some modifications of implantation strategy, two patients with cardiac pacemakers were successfully treated with bilateral DBS STN therapy for refractory PD. To our knowledge, this is the first report on patients with cardiac pacemakers undergoing brain stimulator implantation.

Changes in the immunologic phenotype of human malignant glioma cells after passaging in vitro.

Although immunotherapeutic strategies against glioblastomas have been promising both in vitro and in animal models, similar successes have not been realized in human clinical trials. One reason may be that immunotherapeutic strategies are based on prior studies that primarily have used human glioblastoma cell lines passaged in vitro, which may not accurately reflect the in vivo properties of glioblastoma cells. In this report, we used flow cytometry to quantify the expression of immunological cell surface molecules on human glioblastomas directly ex vivo (prior to any in vitro culturing) and after varying passages in vitro. Furthermore, we used ELISA to quantitate cytokine secretion after various passages in vitro. We demonstrate that in vitro culturing of established cell lines led to increases in the cell surface expression of MHC class I and ICAM-1 and secretion of IL-6 and TGF-beta(2). Furthermore, there were significant changes in the expression of MHC class I, MHC class II, B7-2, ICAM-1, and FasL when comparing ex vivo tumor cells to those after a single passage in vitro. After passaging once in vitro, there were also significant changes in the secretion of TGF-beta(2) and IL-10. This report indicates that in vitro culturing leads to significant changes in both cell surface molecules and secreted cytokines, which are known to affect the ability of immune cells to initiate an anti-tumor immune response. These changes in the immunological phenotype of glioblastomas after in vitro culturing may in part explain the limited success of immunotherapeutic strategies against glioblastomas in human clinical trials.