Johnathan A. Engh

In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.

The use of bone morphogenetic protein gene therapy in craniofacial bone repair.

&NA; Bone morphogenetic proteins (BMPs) are capable of inducing endochondral bone formation when applied on biologic carriers in numerous mammalian in vivo assay systems. Bone morphogenetic protein gene therapy is also currently being developed to promote osteogenesis for clinical indications such as spinal fusions, craniofacial bone loss, and osteoporosis. In this study, critical‐sized mandibular defects were treated with a control adenoviral vector (Ad‐&bgr;‐gal), a BMP‐2 adenoviral vector (Ad‐BMP‐2), or a BMP‐9 adenoviral vector (Ad‐BMP‐9). Gross tissue examination, radiographic analysis, and histologic analysis demonstrated significant bony healing in the BMP treated groups compared to controls. Osteogenesis was limited to the bony defect, without extension into the surrounding soft tissues. The study suggests that with further development, BMP gene therapy may be potentially useful for repair of bony defects in the craniofacial region.

Notch1 Identified as a Prognostic Factor for Glioma Patients

B ecause of the limited efficacy of surgery, radiation therapy, and chemotherapy in the treatment of malignant gliomas, development of molecular-targeted therapies to treat the disease is an appealing strategy to improve outcome. A critical step toward the development of such therapies is the identification of tumor-specific markers that could potentially be the targets of biologic anti-tumor agents. For example, identification of vascular endothelial growth factor and characterization of its role in glioblastoma (GBM) growth has facilitated the widespread application and popularization of bevacizumab therapy in the setting of relapsed GBM. A recent study performed at the Tangdu Hospital in Xi’an, China, assessed the preponderance of Notch1 expression within fresh glioma specimens of multiple grades. Notch1 is one of a family of transmembrane proteins that have been shown to regulate cell differentiation and proliferation. Paradoxically, Notch1 has been shown to have both proliferative and suppressive effects on the growth of various extracranial solid tumors. However, Notch1 signaling has been recently shown to contribute to glioma proliferation in vitro. Over a 2-year period, the authors acquired tissue from 274 consecutive glioma specimens at their institution, with representation of all 4 World Health Organization (WHO) grades. In addition, 48 samples of ‘‘normal’’ brain tissue from trauma craniotomies and other non-tumoral procedures were also acquired. Paraffinembedded specimens were then processed and stained for anti-Notch1 antibody. Two separate pathologists evaluated each stain to determine SCIENCE TIMES

Anti-convulsants and gene expression in malignant gliomas.

U nderstanding the molecular pathways which mediate tumor cell proliferation and therapeutic resistance in malignant gliomas is fundamental to the development of better treatments. At the present time, the most clinically relevant molecular feature in the setting of glioblastoma (GBM) is the status of the O6-methylguanine-DNA methyltransferase (MGMT) gene. The presence of MGMT promoter methylation in malignant glioma has been demonstrated to correlate both with improvements in overall survival as well as improved therapeutic response to alkylating agents, specifically temozolomide. Although the effects of chemotherapeutic agents on specific molecular pathways have been studied to a great degree, the effects that other drugs may have on such pathways are less recognized. Brain tumor patients are commonly prescribed anti-epileptic drugs (AEDs), steroids, and other therapeutic agents to treat neurologic symptoms related to the intracranial mass lesion. In addition, many of these patients are on other drugs for various medical conditions, including hypertension, diabetes, heart disease, etc. The possible effects of these agents on glioma proliferation may be negligible in most cases, but are largely unknown. The choice of AED agent, in particular, is usually not implicated as a source of bias in randomized trials of anti-tumoral therapies. Levetiracetam (LEV) has been increasingly used as an AED for brain tumor patients over the past decade. This drug may be attractive for these patients because it does not appear to induce cytochrome p450 enzymes, and SCIENCE TIMES

Application of the RELY study to lifetime risks of atrial fibrillation: implications for spontaneous intracranial hemorrhage.

Atrial fibrillation (AF) is a common arrhythmia that can be found in normal individuals or reflect an underlying structural, metabolic, or infectious abnormality. Medical comorbidities associated with an increased risk for stroke in AF include age . 65 years, hypertension, rheumatic heart disease, prior stroke or transient ischemic attack, diabetes mellitus, congestive heart failure, or an abnormal transesophageal echocardiogram. In addition to beta blockade and cardioversion, formal anticoagulation with vitamin K antagonists, such as warfarin, remains the mainstay of therapy for AF in order to reduce the risk of systemic embolization. Drawbacks of warfarin therapy include a substantially increased risk of spontaneous intracranial hemorrhage, an entity all too familiar to most neurosurgeons. Results of the Randomized Evaluation of Long-Term Anticoagulation Therapy study (RELY), published in the New England Journal of Medicine in 2009, demonstrated the effectiveness of a direct thrombin inhibitor, dabigatran, for the treatment of AF.2 Dabigatran has several potential advantages over warfarin, most notably that it does not require direct monitoring of efficacy with routine blood tests. The RELY study was designed to compare 2 standard doses of dabigatran, administered in a blinded manner, compared to an openlabel use of warfarin in patients with atrial fibrillation at an increased risk for stroke. More than 18 000 patients were randomized with a median follow up period of 2 years, and the primary outcome measure was stroke or systemic embolism. The trial demonstrated that low-dose (110 mg) dabigatran vs warfarin had similar rates of stroke (1.44%/y vs 1.57%/y; relative risk [RR], 0.92; P value, .41) with lower rates of major hemorrhage (2.71%/y vs 3.36%/y; RR, 0.80; P value, .03). In contrast, high-dose (150 mg) dabigatran vs warfarin had lower rates of stroke (1.01%/y vs 1.51%/y; RR, 0.64; P value, .001) and similar rates of major hemorrhage, (3.11%/y vs 3.36%/y; RR, 0.93; P value, .31).2 These findings are encouraging, but they do not address potential issues of cost and long-term benefit for patients anticoagulated with dabigatran instead of warfarin. A recent study performed at Bangor University and the University of Liverpool extrapolated the findings of the RELY study in order to simulate the cost effectiveness of the drug relative to warfarin. In the 2011 British Medical Journal, Pink et al demonstrated positive incremental net benefit of 0.094 (95% central range) and 0.146 quality adjusted life years (QALY) in a simulated cohort of 50 000 patients with moderate to high risk of stroke treated with dabigatran. Additionally, their analysis showed that high dose dabigatran is cost effective in those patients with poorly controlled international normalized ratios on warfarin.3 However, no benefit was demonstrated for high dose dabigatran (150 mg bid) over low dose dabigatran (110 mg bid), and no advantage of cost effectiveness was demonstrated for dabigatran in clinics which can achieve good control of international normalized ratio (INR) using warfarin. Together, these data lend further support to the use of dabigatran as an alternative agent to warfarin for the treatment of non-valvular atrial fibrillation.Undoubtedly, such findings will lead to an increase in dabigatran usage as a first line treatment for AF. However, an increase in the popularity of this new anticoagulant poses a worrisome risk to the neurosurgical patient with an intracranial hemorrhage, as the drug has no known antidote. Additionally, there is no widely available laboratory test to objectify the therapeutic effect of dabigatran, although the Ecarin clotting test may be used as a rough measure of therapeutic levels.4 Figure. Economic comparison of dabigatran 150 mg twice daily and dose adjusted warfarin as demonstrated via tornado plot. L 1⁄4 lower end of 95% confidence interval (CI) for parameter set; H 1⁄4 higher end of 95% CI for parameter set. ICER, incremental cost effectiveness ratio; QALY, quality adjusted life year. Dabigatran 150 mg twice daily was within the QALY cost effectiveness threshold for all subgroups of patients except centers with a mean time within therapeutic range of INR of at least 65.5%. Modified, from BMJ 2011;343:d6333. Permission to reprint granted by BMJ Publishing Group Ltd.

Predicting the first site of relapse for cancerous tumors using protein expression profiles.

Myelination is one of the important stages of the nervous system (NS) development and regeneration. Understanding myelination is key to understanding certain diseases of the NS including healing after injuries. In a recent article by Huang et al (Nanotechnology. 2011;22(27):275101), the authors studied myelin structure at various stages of formation using a co-culture of Schwann cells and PC12 cells from a rat pheochromocytoma cell line. These were kept in a myelination medium and studied at 7, 12, 17, and 22 days. Indentation was used to assess the mechanical characteristics of the developing myelin. This was performed using a Berkovich diamond tip of 20 nm of radius. They observed that the optimal cell density PC12/ Schwann cells was 3:2. The myelination process began at day 12 in vitro (Figure 1), when Schwann cells express protein zero (P0). There are 3 stages in myelin structural development (Figure 2). At the latter stage (M-III), myelin becomes denser. Nano-indentation was performed every 5 nm on the axon. Displacements were measured and were a reflection of the myelin thickness. Measured displacements were 784 for M-I, 536 for M-II, and 833 nm for M-III. The work required to penetrate the myelin was -267 for M-I, 63 for M-II, and 301 mN nm for M-III. Resistance 1⁄4 work/ displacement and was—0.34, 0.12, and 0.36 mN respectively. The curves indicate the change in the mechanical properties of the myelin sheaths at various stages of development. Huang et al brought a better understanding of the myelination process using nano-technology to study the stiffness profile of myelinated axons. Development of various factors that influence the myelination process could be key in treating some demyelinating diseases such as GuillainBarré syndrome or multiple sclerosis. On the other hand, it may help develop strategies to promote healing of the central or peripheral NS.

Clinical Characteristics of Ependymoma Patients Based on Patient-Reported Outcomes

1. Di Maio A, Skuba A, Himes BT, et al. In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border. J Neurosci. 2011;31(12):4569-4582. 2. Kerschensteiner M, Schwab ME, Lichtman JW, Misgeld T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med. 2005;11(5):572-577. 3. Misgeld T, Nikic I, Kerschensteiner M. In vivo imaging of single axons in the mouse spinal cord. Nat Protoc. 2007;2(2):263-268. 4. Ramer MS, McMahon SB, Priestley JV. Axon regeneration across the dorsal root entry zone. Prog Brain Res. 2001;132:621-639. 5. Feng G, Mellor RH, Bernstein M, et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000;28(1):41-51. 6. Carlstedt T. Regenerating axons form nerve terminals at astrocytes. Brain Res. 1985;347(1):188-191. 7. Liuzzi FJ, Lasek RJ. Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science. 1987;237(4815):642-645.

Identification of a novel marker of brain tumor stem cells.

T he stem cell theory of cancer has facilitated a paradigm shift in our modern understanding of cancer origins, cancer relapses, and future cancer treatments. Since the definitive identification of leukemic stem cells in 1997, so-called cancer stem cells (CSCs) have been discovered in a number of different solid tumors. In 2003, the identification of CD133 as a marker for cancer stem cells within glioblastomas and medulloblastomas cultivated multiple studies further characterizing these important cells. Since these cells are tumorigenic and multipotent, CSCs have been hypothesized to be the major component of relapse and regrowth of treated tumors. Conventional anti-cancer therapies may not target CSCs as well as the other cells within the solid tumor, partially explaining their noncurative nature. Relevant to the treatment of glioblastoma, cancer stem cells from gliomas have been demonstrated to be resistant to standard therapies, including radiation and alkylating agents. As a result, this cell population has been hypothesized as a promising target for glioma therapy. Appropriate targeting of this cell population requires both an accurate delineation of the cells to be targeted as well as an appropriate therapeutic agent directed at this target. Therefore, discovery of other markers of cancer stem cells within gliomas could potentially enhance our ability to target appropriate therapies against them. In a forthcoming article in Neuro-oncology describing research performed at the Technische Universitat in Munchen, Germany, Rasper et al report the discovery of a novel cancer stem cell marker within glioblastomas. The marker, aldehyde dehydrogenase-1 (ALDH1), is a known stem cell marker of other malignant solid tumors. Their report demonstrates evidence that ALDH1 positive cells from established glioma cell lines will form neurospheres in an appropriate culture medium. In addition, in vitro inhibition of ALDH1 decreased neurosphere formation in their study. Increased levels of ALDH1 in vitro maintained cultured cells in an undifferentiated state. Finally, in sections of the human subventricular zone, ALDH1 positivity was noted within the subependymal ribbon, but not elsewhere (Figure 1). This zone is a known niche for multipotent stem cells within the human brain. Collectively, this data suggests that the authors have identified a novel cancer stem cell marker for human glioblastoma. Further studies will be needed to corroborate this discovery. Identification of this marker, along with other specific markers for CSC, is a significant step toward improving the existing therapies for this devastating disease.