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1.  Defective sphingosine-1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation 
Nature immunology  2013;14(11):1166-1172.
Sphingosine-1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. Sphingosine phosphate receptor 1 (S1P1) agonist, FTY-720 (Gilenya™) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring a S1pr1 gene encoding phosphorylation-deficient receptors [S1P1(S5A)] developed severe experimental autoimmune encephalomyelitis (EAE) due to T helper (TH) 17-mediated autoimmunity in the peripheral immune and nervous system. S1P1 directly activated Janus-like kinase–signal transducer and activator of transcription 3 (JAK-STAT3) pathway via interleukin 6 (IL-6). Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.
doi:10.1038/ni.2730
PMCID: PMC4014310  PMID: 24076635
2.  Intraarterial transplantation of human umbilical cord blood mononuclear cells is more efficacious and safer compared with umbilical cord mesenchymal stromal cells in a rodent stroke model 
Introduction
Stroke is the second leading cause of death worldwide, claims six lives every 60 seconds, and is a leading cause of adult disability across the globe. Tissue plasminogen activator, the only United States Food and Drug Administration (FDA)-approved drug currently available, has a narrow therapeutic time window of less than 5 hours. In the past decade, cells derived from the human umbilical cord (HUC) have emerged as a potential therapeutic alternative for stroke; however, the most effective HUC-derived cell population remains unknown.
Methods
We compared three cell populations derived from the human umbilical cord: cord blood mononuclear cells (cbMNCs); cord blood mesenchymal stromal cells (cbMSCs), a subpopulation of cbMNCs; and cord matrix MSCs (cmMSCs). We characterized these cells in vitro with flow cytometry and assessed the cells’ in vivo efficacy in a 2-hour transient middle cerebral artery occlusion (MCAo) rat model of stroke. cbMNCs, cbMSCs, and cmMSCs were each transplanted intraarterially at 24 hours after stroke.
Results
A reduction in neurologic deficit and infarct area was observed in all three cell groups; however, this reduction was significantly enhanced in the cbMNC group compared with the cmMSC group. At 2 weeks after stroke, human nuclei-positive cells were present in the ischemic hemispheres of immunocompetent stroke rats in all three cell groups. Significantly decreased expression of rat brain-derived neurotrophic factor mRNA was observed in the ischemic hemispheres of all three cell-treated and phosphate-buffered saline (PBS) group animals compared with sham animals, although the decrease was least in cbMNC-treated animals. Significantly decreased expression of rat interleukin (IL)-2 mRNA and IL-6 mRNA was seen only in the cbMSC group. Notably, more severe complications (death, eye inflammation) were observed in the cmMSC group compared with the cbMNC and cbMSC groups.
Conclusions
All three tested cell types promoted recovery after stroke, but cbMNCs showed enhanced recovery and fewer complications compared with cmMSCs.
doi:10.1186/scrt434
PMCID: PMC4055161  PMID: 24690461
3.  Who’s in Favor of Translational Cell Therapy for Stroke: STEPS Forward Please? 
Cell transplantation  2009;18(7):691-693.
A consortium of translational stem cell and stroke experts from multiple academic institutes and biotechnology companies, under the guidance of the government (FDA/NIH), is missing. Here, we build a case for the establishment of this consortium if cell therapy for stroke is to advance from the laboratory to the clinic.
doi:10.3727/096368909X470883
PMCID: PMC3962837  PMID: 19796499
Stem cell transplantation; Tissue regeneration; Cellular therapy; Clinical translation
4.  A Concerted Appeal for International Cooperation in Preclinical Stroke Research 
doi:10.1161/STROKEAHA.113.000734
PMCID: PMC3933930  PMID: 23598526
cerebral ischemia; roadblock; translational medicine
5.  Practical Use of Perioperative Chemotherapy for Muscle-Invasive Bladder Cancer: Summary of Session at the Society of Urologic Oncology Annual Meeting 
Urologic oncology  2012;30(6):772-780.
At the 11th annual meeting of the Society of Urologic Oncology, an expert panel was convened to discuss the practical use of perioperative chemotherapy for muscle-invasive bladder cancer. The discussion was structured as a case-based debate among the panelist. The topics included: neoadjuvant chemotherapy with a focus on T2 disease, pros and cons, survival data, tolerability of cisplatin-based therapy, can we avoid radical cystectomy in complete responders, limitations and alternatives to cisplatin-based therapy, management of ‘suboptimal’ chemotherapy, residual disease after neoadjuvant chemotherapy, adjuvant chemotherapy and key aspects of radical cystectomy and lymph-node dissection in multi-modal therapy. The presentations were derived from published literature. The panelists agreed that patients with muscle-invasive bladder cancer should be managed with a multidisciplinary team including urologist and medical oncologist. Cisplatin-based neoadjuvant chemotherapy has demonstrated improved survival and should be incorporated into the management of all eligible patients with muscle-invasive bladder cancer. However, in some centers neoadjuvant chemotherapy is reserved for patients with >T2 disease or high-risk features. There are no data for the administration of non-cisplatin based neoadjuvant chemotherapy such as carboplatin-combinations. Cisplatin-ineligible patients should proceed directly to surgical extirpation with adjuvant cisplatin-based chemotherapy considered based on pathologic findings. However, the data for adjuvant chemotherapy is less compelling. As our refinement of the selection process continues we may be able to better identify subsets of patients who may be spared chemotherapy, but much work remains to be done in this arena. The current standard for muscle-invasive bladder cancer patients is cisplatin-based neoadjuvant chemotherapy followed by radical cystectomy and pelvic lymph-node dissection.
doi:10.1016/j.urolonc.2012.01.012
PMCID: PMC3524835  PMID: 23218068
Bladder Cancer; neoadjuvant chemotherapy; adjuvant chemotherapy; perioperative chemotherapy; muscle invasive bladder cancer
6.  δPKC mediates microcerebrovascular dysfunction in acute ischemia and in chronic hypertensive stress in vivo 
Brain research  2007;1144:146-155.
Maintaining cerebrovascular function is a priority for reducing damage following acute ischemic events such as stroke, and under chronic stress in diseases such as hypertension. Ischemic episodes lead to endothelial cell damage, deleterious inflammatory responses, and altered neuronal and astrocyte regulation of vascular function. These, in turn, can lead to impaired cerebral blood flow and compromised blood–brain barrier function, promoting microvascular collapse, edema, hemorrhagic transformation, and worsened neurological recovery. Multiple studies demonstrate that protein kinase C (PKC), a widely expressed serine/threonine kinase, is involved in mediating arterial tone and microvascular function. However, there is no clear understanding about the role of individual PKC isozymes. We show that intraperitoneal injection of δV1-1–TAT47–57 (0.2 mg/kg in 1 mL), an isozymespecific peptide inhibitor of δPKC, improved microvascular pathology, increased the number of patent microvessels by 92% compared to control-treated animals, and increased cerebral blood flow by 26% following acute focal ischemia induced by middle cerebral artery occlusion in normotensive rats. In addition, acute delivery of δV1-1–TAT47–57 in hypertensive Dahl rats increased cerebral blood flow by 12%, and sustained delivery δV1-1–TAT47–57 (5 uL/h, 1 mM), reduced infarct size by 25% following an acute stroke induced by MCA occlusion for 90 min. Together, these findings demonstrate that δPKC is an important therapeutic target for protection of microvascular structure and function under both acute and chronic conditions of cerebrovascular stress.
doi:10.1016/j.brainres.2007.01.113
PMCID: PMC3742377  PMID: 17350602
Cerebral blood flow; Hypertension; Microvasculature; Protein kinase C; Stroke; Vasculature
7.  Combined Endovascular and Microsurgical Management of Complex Cerebral Aneurysms 
Cerebral aneurysms are associated with a 50% mortality rate after rupture and patients can suffer significant morbidity during subsequent treatment. Neurosurgical management of both ruptured and unruptured aneurysms has evolved over the years. The historical practice of using microsurgical clipping to treat aneurysms has benefited in the last two decades from tremendous improvement in endovascular technology. Microsurgery and endovascular therapies are often viewed as competing treatments but it is important to recognize their individual limitations. Some aneurysms are considered complex, due to several factors such as aneurysm anatomy and a patient’s clinical condition. A complex aneurysm often cannot be completely excluded with a single approach and its successful treatment requires a combination of microsurgical and endovascular techniques. Planning such an approach relies on understanding aneurysm anatomy and thus should routinely include 3D angiographic imaging. In patients with ruptured aneurysms, endovascular coiling is a well-tolerated early treatment and residual aneurysms can be treated with intervals of definitive clipping. Microsurgical clipping also can be used to reconstruct the neck of a complex aneurysm, allowing successful placement of coils across a narrow neck. Endovascular techniques are assisted by balloons, which can be used in coiling and testing parent vessel occlusion before sacrifice. In some cases microsurgical bypasses can provide alternate flow for planned vessel sacrifice. We present current paradigms for combining endovascular and microsurgical approaches to treat complex aneurysms and share our experience in 67 such cases. A dual microsurgical–endovascular approach addresses the challenge of intracranial aneurysms. This combination can be performed safely and produces excellent rates of aneurysm obliteration. Hybrid angiographic operating-room suites can foster seamless and efficient complementary application of these two modalities.
doi:10.3389/fneur.2013.00108
PMCID: PMC3737456  PMID: 23964263
coil embolization; combined therapy; complex aneurysms; microsurgical clipping; revascularization bypass; vessel sacrifice
8.  Radical Cystectomy after BCG Immunotherapy for High-Risk Nonmuscle-Invasive Bladder Cancer in Patients with Previous Prostate Radiotherapy 
ISRN Urology  2013;2013:405064.
Purpose. Intravesical Bacillus Calmette-Guerin (BCG) immunotherapy is indicated for high-grade nonmuscle-invasive bladder cancer (NMIBC). The efficacy of BCG in patients with a history of previous pelvic radiotherapy (RT) may be diminished. We evaluated the outcomes of radical cystectomy for BCG-treated recurrent bladder cancer in patients with a history of RT for prostate cancer (PC). Methods. A retrospective chart review was performed to identify patients with primary NMIBC. We compared the outcomes of three groups of patients who underwent radical cystectomy for BCG-refractory NMIBC: those with a history of RT for PC, those who previously underwent radical prostatectomy (RP), and a cohort without PC or RT exposure. Results. From 1996 to 2008, 53 patients underwent radical cystectomy for recurrent NMIBC despite BCG. Those with previous pelvic RT were more likely to have a higher pathologic stage and decreased recurrence-free survival compared to the groups without prior RT exposure. Conclusion. Response rates for intravesical BCG therapy may be impaired in those with prior prostate radiotherapy. Patients with a history of RT who undergo radical cystectomy after failed BCG are more likely to be pathologically upstaged and have decreased recurrence-free survival. Earlier consideration of radical cystectomy may be warranted for those with NMIBC who previously received RT for PC.
doi:10.1155/2013/405064
PMCID: PMC3730135  PMID: 23956880
9.  Distinctive effects of T cell subsets in neuronal injury induced by co-cultured splenocytes in vitro and by in vivo stroke in mice 
Background and purpose
T cells and their subsets modulate ischemic brain injury. We studied the effects of the absence of T cell subsets on brain infarction after in vivo stroke and then used an in vitro co-culture system of splenocytes and neurons to further identify the roles of T cell subsets in neuronal death.
Methods
Stroke was induced by MCA suture occlusion in mice and infarct sizes were measured 2 days post-stroke.
Splenocytes were co-cultured with neurons, and neuronal survival was measured 3 days later.
Results
A deficiency of both T and B cells (SCID) and the paucity of CD4 or CD8 T cells equally resulted in smaller infarct sizes as measured 2 days post-stroke. Although a functional deficiency of regulatory T cells had no effect, impaired Th1 immunity reduced infarction and impaired Th2 immunity aggravated brain injury, which may be due to an inhibited and enhanced inflammatory response in mice deficient in Th1 and Th2 immunity, respectively. In the in vitro co-culture system, WT splenocytes resulted in dose-dependent neuronal death. The neurotoxicity of splenocytes from the above immunodeficient mice was consistent with their effects on stroke in vivo , except for the mice with the paucity of CD4 or CD8 T cells, which did not alter the ratio of neuronal death.
Conclusion
T cell subsets play critical roles in brain injury induced by stroke. The detrimental versus beneficial effects of Th1 cells and Th2 cells both in vivo and in vitro reveal differential therapeutic target strategies for stroke treatment.
doi:10.1161/STROKEAHA.112.656611
PMCID: PMC3506376  PMID: 22678086
cerebral ischemia; stroke; T cells; Th1; Th2
10.  CGS 19755 (Selfotel): A Novel Neuroprotective Agent Against CNS Injury 
CNS drug reviews  2006;2(3):257-268.
The hypothesis that excitoxicity is a mechanism of damage following different types of cerebral injury including global and focal ischemia (34), and head and spinal cord trauma (6,7,9,25) has been supported by numerous findings. During ischemia for example, glutamate neurotoxicity is mediated in part through N-methyl-D-aspartate (NMDA) receptors, since selective antagonists to this receptor protect against hypoxic-ischemic injury (10,35,41). In the last few years, different NMDA antagonists have been developed and tested; they can be divided into competitive and noncompetitive antagonists. Noncompetitive NMDA antagonists are extremely lipophilic and reach high levels in the brain after systemic administration. Various studies have demonstrated that these agents provide neuroprotection against hypoxic-ischemic injury (for review see ref. 29).
Many competitive NMDA antagonists are hydrophilic and require direct cerebral administration to obtain high brain levels. Newer competitive NMDA blockers, such as cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755, selfotel), provide neuroprotection against global ischemia, focal ischemia, and trauma when given systemically (2,3,32,33). Selfotel is currently being studied in multicenter safety and efficacy trials for stroke (17) and head trauma (6).
doi:10.1111/j.1527-3458.1996.tb00301.x
PMCID: PMC3678965  PMID: 23766625
Cerebral ischemia; Excitotoxicity; Glutamate; NMDA; Trauma; Anoxia; Neurotoxicity; Neuroprotection; CGS 19755; Selfotel
11.  Phosphorylated MAPK/ERK1/2 may not always represent its kinase activity in a rat model of focal cerebral ischemia with or without ischemic preconditioning 
Neuroscience  2012;209:155-160.
The ERK 1/2 protein require a dual phosphorylation at conserved threonine and tyrosine residues to be fully activated under normal physiological conditions. Thus, ERK1/2 kinase activity is often defined by the quantity of phosphorylated kinase. However, this may not accurately represent its true activity under certain pathological conditions. We investigated whether ERK1/2 kinase activity is proportional to its phosphorylation state in a rat focal ischemia model with and without rapid ischemic preconditioning. We showed that phosphorylated-ERK1/2 protein levels were increased 2.6±0.07 fold, and ERK1/2 kinase activity was increased 10.6±1.9 fold in animals receiving ischemic preconditioning alone without test ischemia compared with sham group (P<0.05, n=6/group), suggesting that phosphorylated-ERK1/2 protein levels represent its kinase activity under these conditions. However, preconditioning plus test ischemia robustly blocked ERK1/2 kinase activity, while it increased phosphorylated-ERK1/2 protein levels beyond those receiving test ischemia alone, suggesting that phosphorylated-ERK1/2 protein levels were not representative of actual kinase activity in this pathological condition. In conclusion, protein phosphorylation levels of ERK1/2 do not always correspond to kinase activity, thus, measuring the true kinase activity is essential.
doi:10.1016/j.neuroscience.2012.02.005
PMCID: PMC3322316  PMID: 22366512
ischemic preconditioning; kinase activity; MAPK; ERK1/2; focal ischemia; stroke
12.  Derivation of Injury-Responsive Dendritic Cells for Acute Brain Targeting and Therapeutic Protein Delivery in the Stroke-Injured Rat 
PLoS ONE  2013;8(4):e61789.
Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs), we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.
doi:10.1371/journal.pone.0061789
PMCID: PMC3627911  PMID: 23613937
13.  Long-term behavioral assessment of function in an experimental model for ischemic stroke 
Journal of neuroscience methods  2011;196(2):247-257.
Middle cerebral artery occlusion (MCAO) in rats is a well-studied experimental model for ischemic stroke leading to brain infarction and functional deficits. Many preclinical studies have focused on a small time window after the ischemic episode to evaluate functional outcome for screening therapeutic candidates. Short evaluation periods following injury have led to significant setbacks due to lack of information on the delayed effects of treatments, as well as short-lived and reversible neuroprotection, so called false-positive results. In this report, we evaluated long-term functional deficit for 90 days after MCAO in two rat strains with two durations of ischemic insult, in order to identify the best experimental paradigm to assess injury and subsequent recovery. Behavioral outcomes were measured pre-MCAO followed by weekly assessment post-stroke. Behavioral tests included the 18-point composite neurological score, 28-point neuroscore, rearing test, vibrissae-evoked forelimb placing test, foot fault test and the CatWalk. Brain lesions were assessed to correlate injury to behavior outcomes at the end of study. Our results indicate that infarction volume in Sprague-Dawley rats was dependent on occlusion duration. In contrast, the infarction volume in Wistar rats did not correlate with the duration of ischemic episode. Functional outcomes were not dependent on occlusion time in either strain; however, measureable deficits were detectable long-term in limb asymmetry, 18- and 28-point neuroscores, forelimb placing, paw swing speed, and gait coordination. In conclusion, these behavioral assays, in combination with an extended long-term assessment period, can be used for evaluating therapeutic candidates in preclinical models of ischemic stroke.
doi:10.1016/j.jneumeth.2011.01.010
PMCID: PMC3539723  PMID: 21256866
Ischemic stroke; CatWalk; long-term functional recovery; middle cerebral artery occlusion; rat strain
14.  Transplanted stem cell-secreted VEGF effects post-stroke recovery, inflammation, and vascular repair 
Stem cells (Dayton, Ohio)  2011;29(2):10.1002/stem.584.
Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after sub-acute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human VEGF (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair, and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier (BBB) integrity and suppression of inflammation was followed by a delayed spatio-temporal regulated increase in neovascularization. These events coincided with a bi-modal pattern of functional recovery: an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multi-modal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.
doi:10.1002/stem.584
PMCID: PMC3524414  PMID: 21732485
angiogenesis; blood brain barrier; dystroglycan; inflammation; Avastin
15.  Gene expression profiling of blood in brain arteriovenous malformation patients 
Translational stroke research  2011;2(4):575-587.
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
doi:10.1007/s12975-011-0103-3
PMCID: PMC3241209  PMID: 22184505
arteriovenous malformation; blood; gene expression; intracranial hemorrhage; microarray analysis
16.  Arterial Spin-Labeling MRI Can Identify the Presence and Intensity of Collateral Perfusion in Patients With Moyamoya Disease 
Background and Purpose
Determining the presence and adequacy of collateral blood flow is important in cerebrovascular disease. Therefore, we explored whether a noninvasive imaging modality, arterial spin labeling (ASL) MRI, could be used to detect the presence and intensity of collateral flow using digital subtraction angiography (DSA) and stable xenon CT cerebral blood flow as gold standards for collaterals and cerebral blood flow, respectively.
Methods
ASL and DSA were obtained within 4 days of each other in 18 patients with Moyamoya disease. Two neurointerventionalists scored DSA images using a collateral grading scale in regions of interest corresponding to ASPECTS methodology. Two neuroradiologists similarly scored ASL images based on the presence of arterial transit artifact. Agreement of ASL and DSA consensus scores was determined, including kappa statistics. In 15 patients, additional quantitative xenon CT cerebral blood flow measurements were performed and compared with collateral grades.
Results
The agreement between ASL and DSA consensus readings was moderate to strong, with a weighted kappa value of 0.58 (95% confidence interval, 0.52–0.64), but there was better agreement between readers for ASL compared with DSA. Sensitivity and specificity for identifying collaterals with ASL were 0.83 (95% confidence interval, 0.77–0.88) and 0.82 (95% confidence interval, 0.76–0.87), respectively. Xenon CT cerebral blood flow increased with increasing DSA and ASL collateral grade (P<0.05).
Conclusions
ASL can noninvasively predict the presence and intensity of collateral flow in patients with Moyamoya disease using DSA as a gold standard. Further study of other cerebrovascular diseases, including acute ischemic stroke, is warranted.
doi:10.1161/STROKEAHA.111.616466
PMCID: PMC3164217  PMID: 21799169
angiography; arterial spin labeling; cerebral blood flow; cerebral hemodynamics; cerebrovascular disease; collateral flow; neuroradiology; perfusion
17.  Phase III Study of Molecularly Targeted Adjuvant Therapy in Locally Advanced Urothelial Cancer of the Bladder Based on p53 Status  
Journal of Clinical Oncology  2011;29(25):3443-3449.
Introduction
Retrospective studies suggest that p53 alteration is prognostic for recurrence in patients with urothelial bladder cancer and predictive for benefit from combination methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) adjuvant chemotherapy.
Patients and Methods
Patients with pT1/T2N0M0 disease whose tumors demonstrated ≥ 10% nuclear reactivity on centrally performed immunohistochemistry for p53 were offered random assignment to three cycles of adjuvant MVAC versus observation; p53-negative patients were observed. By using a log-rank test with one-sided α = .05 and β = .10, 190 p53-positive patients were planned to be randomly assigned to detect an absolute improvement in probability of recurring by 3 years from 0.50 to 0.30.
Results
A total of 521 patients were registered, 499 underwent p53 assessment, 272 (55%) were positive, and 114 (42%) were randomly assigned. Accrual was halted on the basis of the data and safety monitoring board review of a futility analysis. Overall 5-year probability of recurring was 0.20 (95% CI, 0.16 to 0.24) with no difference on the basis of p53 status. Only 67% of patients randomly assigned to MVAC received all three cycles with 12 patients receiving no treatment. There was no difference in recurrence in the randomly assigned patients (hazard ratio, 0.78; 95% CI, 0.29 to 2.08; P = .62).
Conclusion
Neither the prognostic value of p53 nor the benefit of MVAC chemotherapy in patients with p53-positive tumors was confirmed, but the high patient refusal rate, lower than expected event rate, and failures to receive assigned therapy severely compromised study power.
doi:10.1200/JCO.2010.34.4028
PMCID: PMC3164246  PMID: 21810677
18.  The Use of Regenerative Medicine in the Management of Invasive Bladder Cancer 
Advances in Urology  2012;2012:653652.
Muscle invasive and recurrent nonmuscle invasive bladder cancers have been traditionally treated with a radical cystectomy and urinary diversion. The urinary diversion is generally accomplished through the creation of an incontinent ileal conduit, continent catheterizable reservoir, or orthotopic neobladder utilizing small or large intestine. While radical extirpation of the bladder is often successful from an oncological perspective, there is a significant morbidity associated with enteric interposition within the genitourinary tract. Therefore, there is a great opportunity to decrease the morbidity of the surgical management of bladder cancer through utilization of novel technologies for creating a urinary diversion without the use of intestine. Clinical trials using neourinary conduits (NUC) seeded with autologous smooth muscle cells are currently in progress and may represent a significant surgical advance, potentially eliminating the complications associated with the use of gastrointestinal segments in the urinary reconstruction, simplifying the surgical procedure, and greatly facilitating recovery from cystectomy.
doi:10.1155/2012/653652
PMCID: PMC3457671  PMID: 23019421
19.  Dopaminergic Neurons from Midbrain-Specified Human Embryonic Stem Cell-Derived Neural Stem Cells Engrafted in a Monkey Model of Parkinson’s Disease 
PLoS ONE  2012;7(7):e41120.
The use of human embryonic stem cells (hESCs) to repair diseased or injured brain is promising technology with significant humanitarian, societal and economic impact. Parkinson’s disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet therapeutic need. We report on the isolation and perpetuation of a midbrain-specified self-renewable human neural stem cell line (hNSCs) from hESCs. These hNSCs grew as a monolayer and uniformly expressed the neural precursor markers nestin, vimentin and a radial glial phenotype. We describe a process to direct the differentiation of these hNSCs towards the DA lineage. Glial conditioned media acted synergistically with fibroblastic growth factor and leukemia inhibitory factor to induce the expression of the DA marker, tyrosine hydroxylase (TH), in the hNSC progeny. The glial-derived neurotrophic factor did not fully mimic the effects of conditioned media. The hNSCs expressed the midbrain-specific transcription factors Nurr1 and Pitx3. The inductive effects did not modify the level of the glutamic acid decarboxylase (GAD) transcript, a marker for GABAergic neurons, while the TH transcript increased 10-fold. Immunocytochemical analysis demonstrated that the TH-expressing cells did not co-localize with GAD. The transplantation of these DA-induced hNSCs into the non-human primate MPTP model of PD demonstrated that the cells maintain their DA-induced phenotype, extend neurite outgrowths and express synaptic markers.
doi:10.1371/journal.pone.0041120
PMCID: PMC3398927  PMID: 22815935
20.  The CCR2/CCL2 Interaction Mediates the Transendothelial Recruitment of Intravascularly Delivered Neural Stem Cells to the Ischemic Brain 
Background and Purpose
The inflammatory response is a critical component of ischemic stroke. In addition to its physiological role, the mechanisms behind transendothelial recruitment of immune cells also offer a unique therapeutic opportunity for translational stem cell therapies. Recent reports have demonstrated homing of neural stem cells (NSC) into the injured brain areas after intravascular delivery. However, the mechanisms underlying the process of transendothelial recruitment remain largely unknown. Here we describe the critical role of the chemokine CCL2 and its receptor CCR2 in targeted homing of NSC after ischemia.
Methods
Twenty-four hours after induction of stroke using the hypoxia-ischemia model in mice CCR2+/+ and CCR2−/− reporter NSC were intra-arterially delivered. Histology and bioluminescence imaging were used to investigate NSC homing to the ischemic brain. Functional outcome was assessed with the horizontal ladder test.
Results
Using NSC isolated from CCR2+/+ and CCR2−/− mice, we show that receptor deficiency significantly impaired transendothelial diapedesis specifically in response to CCL2. Accordingly, wild-type NSC injected into CCL2−/− mice exhibited significantly decreased homing. Bioluminescence imaging showed robust recruitment of CCR2+/+ cells within 6 hours after transplantation in contrast to CCR2−/− cells. Mice receiving CCR2+/+ grafts after ischemic injury showed a significantly improved recovery of neurological deficits as compared to animals with transplantation of CCR2−/− NSC.
Conclusions
The CCL2/CCR2 interaction is critical for transendothelial recruitment of intravascularly delivered NSC in response to ischemic injury. This finding could have significant implications in advancing minimally invasive intravascular therapeutics for regenerative medicine or cell-based drug delivery systems for central nervous system diseases.
doi:10.1161/STROKEAHA.110.606368
PMCID: PMC3371396  PMID: 21836091
chemokines; intravascular transplantation; neural stem cells; regenerative medicine; stroke; transendothelial recruitment
21.  Lithium Treatment Reduces Brain Injury Induced by Focal Ischemia with Partial Reperfusion and the Protective Mechanisms Dispute the Importance of Akt Activity 
Aging and Disease  2012;3(3):226-233.
Lithium is a mood stabilizer shown to have neuroprotective effects against several chronic and acute neuronal injuries, including stroke. However, it is unknown whether lithium treatment protects against brain injury post-stroke in a rat model of permanent distal middle cerebral artery occlusion (MCAo) combined with transient bilateral common carotid artery occlusion (CCAo), a model that mimics human stroke with partial reperfusion. In addition, whether lithium treatment alters Akt activity as measured by the kinase activity assay has not been reported, although it is known to inhibit GSK3β activity. After stroke, Akt activity contributes to neuronal survival while GSK3β activity causes neuronal death. We report that a bolus of lithium injection at stroke onset robustly reduced infarct size measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 48 h post-stroke and inhibited cell death in the ischemic penumbra, but not in the ischemic core, as shown by TUNEL staining performed 24 h post-stroke. However, lithium treatment did not alter the reduction in Akt activity as measured by Akt kinase assay. We further showed that lithium did not alter phosphorylated GSK3β protein levels, or the degradation of β-catenin, a substrate of GSK3β, which is consistent with previous findings that long-term treatment is required for lithium to alter GSK3β phosphorylation. In summary, we show innovative data that lithium protects against stroke in a focal ischemia model with partial reperfusion, however, our results dispute the importance of Akt activity in the protective effects of lithium.
PMCID: PMC3375079  PMID: 22724081
Lithium; Akt; Cerebral focal ischemia; GSK3β; β-catenin
22.  Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation 
PLoS ONE  2012;7(5):e36669.
Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.
doi:10.1371/journal.pone.0036669
PMCID: PMC3349679  PMID: 22590586
23.  Stem Cells and Stroke: Opportunities, Challenges, and Strategies 
INTRODUCTION
Stroke remains the leading cause of disability in the Western world. Despite decades of work, no clinically effective therapies exist to facilitate recovery from stroke. Stem cells may have the potential to minimize injury and promote recovery after stroke.
AREAS COVERED
Transplanted stem cells have been shown in animal models to migrate to the injured region, secrete neurotrophic compounds, promote revascularization, enhance plasticity and regulate the inflammatory response, thereby minimizing injury. Endogenous neural stem cells also have a remarkable propensity to respond to injury. Under select conditions, subventricular zone progenitors may be mobilized to replace lost neurons. In response to focal infarcts, neuroblasts play important trophic roles to minimize neural injury. Importantly, these endogenous repair mechanisms may be experimentally augmented, leading to robust improvements in function. Ongoing clinical studies are now assessing the safety and feasibility of cell-based therapies for stroke.
EXPERT OPINION
We outline the unique challenges and potential pitfalls in the clinical translation of stem cell research for stroke. We then detail what we believe to be the specific basic science and clinical strategies needed to overcome these challenges, fill remaining gaps in knowledge and facilitate development of clinically viable stem cell-based therapies for stroke.
doi:10.1517/14712598.2011.552883
PMCID: PMC3087443  PMID: 21323594
stem cell; neurogenesis; neuroregeneration; stroke; ischemic brain injury; neural progenitor cell; neuroblast; neuroprotection; clinical trial; translational research; plasticity; subventricular zone; migration; differentiation
24.  An Insult-Inducible Vector System Activated by Hypoxia and Oxidative Stress for Neuronal Gene Therapy 
Translational stroke research  2011;2(1):92-100.
Gene therapy has demonstrated the protective potential of a variety of genes against stroke. However, conventional gene therapy vectors are limited due to the inability to temporally control their expression, which can sometimes lead to deleterious side effects. Thus, an inducible vector that can be temporally controlled and activated by the insult itself would be advantageous. Using hypoxia responsive elements (HRE) and antioxidant responsive elements (ARE), we have constructed an insult-inducible vector activated by hypoxia and reactive oxygen species (ROS). In COS7 cells, the inducible ARE−HRE-luciferase vectors are highly activated by oxygen deprivation, hydrogen peroxide treatment, and the ROS-induced transcription factor NF-E2-related factor 2 (Nrf2). Using a defective herpes virus, the neuroprotective potential of this inducible vector was tested by over-expressing the transcription factor Nrf2. In primary cortical cultures, expression of the inducible ARE−HRE–Nrf2 protects against oxygen glucose deprivation, similar to that afforded by the constitutively expressed Nrf2. This ARE+HRE vector system is advantageous in that it allows the expression of a transgene to be activated not only during hypoxia but also maintained after reperfusion, thus prolonging the transgene expression during an ischemic insult. This insult-inducible vector system will be a valuable gene therapy tool for activating therapeutic/protective genes in cerebrovascular diseases.
doi:10.1007/s12975-010-0060-2
PMCID: PMC3097421  PMID: 21603078
Insult-inducible; Gene therapy; Hypoxia; HIF1; Nrf2; Reactive oxygen species
25.  Intraoperative blood flow analysis of direct revascularization procedures in patients with moyamoya disease 
Moyamoya disease is characterized by the progressive stenosis and often occlusion of the terminal internal carotid arteries, which leads to ischemic and hemorrhagic injuries. The etiology is unknown and surgical revascularization remains the mainstay treatment. We analyzed various hemodynamic factors in 292 patients with moyamoya disease, representing 496 revascularization procedures, including vessel dimension and intraoperative blood flow, using a perivascular ultrasonic flowprobe. Mean middle cerebral artery (MCA) flow rate was 4.4±0.26 mL/min. After superficial temporal artery (STA)–MCA bypass surgery, flows at the microanastomosis were increased fivefold to a mean of 22.2±0.8 mL/min. The MCA flows were significantly lower in the pediatric (16.2±1.3 mL/min) compared with the adult (23.9±1.0 mL/min; P<0.0001) population. Increased local flow rates were associated with clinical improvement. Permanent postoperative complications were low (<5%), but very high postanastomosis MCA flow was associated with postoperative stroke (31.2±6.8 mL/min; P=0.045), hemorrhage (32.1±10.2 mL/min; P=0.045), and transient neurologic deficits (28.6±5.6 mL/min; P=0.047) compared with controls. Other flow and vessel dimension data are presented to elucidate the hemodynamic changes related to the vasculopathy and subsequent to surgical intervention.
doi:10.1038/jcbfm.2010.85
PMCID: PMC3049490  PMID: 20588321
cerebral blood flow; extracranial to intracrania bypass; hemodynamic; moyamoya; STA–MCA; vessel diameter

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