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1.  Stem cell therapy for acute cerebral injury: What do we know and what will the future bring? 
Current opinion in neurology  2013;26(6):617-625.
Purpose of review
The central nervous system has limited capacity for regeneration after acute and chronic injury. An attractive approach to stimulate neural plasticity in the brain is to transplant stem cells in order to restore function. Here we discuss potential mechanisms of action, current knowledge and future perspectives of clinical stem cell research for stroke and traumatic brain injury.
Recent findings
Preclinical data using various models suggest stem cell therapy to be a promising therapeutic avenue. Progress has been made in elucidating the mechanism of action of various cell types used, shifting the hypothesis from neural replacement to enhancing endogenous repair processes. Translation of these findings in clinical trials is currently being pursued with emphasis on both safety as well as efficacy.
Summary
Clinical trials are currently recruiting patients in phase I and II trials to gain more insight in the therapeutic potential of stem cells in acute cerebral injury. A close interplay between results of these clinical trials and more extensive basic research is essential for future trial design: Choosing the optimal transplantation strategy and selecting the right patients.
doi:10.1097/WCO.0000000000000023
PMCID: PMC4465754  PMID: 24136128
Neural repair; recovery; stem cell therapy; stroke; traumatic brain injury
2.  Stepwise Recruitment of Transcellular and Paracellular Pathways Underlies Blood-Brain Barrier Breakdown in Stroke 
Neuron  2014;82(3):603-617.
SUMMARY
Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a novel transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 h post-stroke, TJs display profound structural defects only after two days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 h post-stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.
doi:10.1016/j.neuron.2014.03.003
PMCID: PMC4016169  PMID: 24746419
3.  Manufacturing neurons from human embryonic stem cells: biological and regulatory aspects to develop a safe cellular product for stroke cell therapy 
Regenerative medicine  2009;4(2):251-263.
Demographic trends, particularly those related to longer life expectancy, suggest that the demand for tissue and organ transplants will further increase since many disorders result from degeneration, injury or organ failure. The most urgent problem in transplantation medicine is the shortage or lack of suitable donor organs and tissue, leading to ethical and societal problems such as organ trafficking. The discovery of stem cells in the inner cell mass of developing embryos and in adult tissue has revolutionized the medical field by introducing new therapeutic dimensions to consider for previously untreatable diseases and injuries. The unlimited self-renewal ability and pluripotent capacity to become any cell type of the organism make human embryonic stem cells (hESCs) a compelling source of cells to study tissue histogenesis and to apply in a wide array of tissue engineering, cell transplantation therapy and drug discovery applications. In this article, we will focus on hESCs and address the derivation of therapeutic neural stem cell lines from hESCs, as well as the biological and regulatory aspects to developing a safe cellular product for stroke cell therapy.
doi:10.2217/17460751.4.2.251
PMCID: PMC4337782  PMID: 19317644
cell therapy; cGMP; good manufacturing practice; human embryonic stem cells; human neural stem cells; investigational new drug application; manufacturing neurons; master cell bank; neuroplasticity; product development; regulation of stem cell products; stroke
4.  Sex disparities in diagnosis of bladder cancer after initial presentation with hematuria: a nationwide claims-based investigation 
Cancer  2013;120(4):555-561.
Background
Women have disproportionately higher mortality rates relative to incidence for bladder cancer. Multiple etiologies have been proposed, including delayed diagnosis and treatment. Guidelines recommend rule-out of malignancy in men and women presenting with hematuria. We aimed to determine the difference in timing from presentation with hematuria to diagnosis of bladder cancer in women versus men.
Methods
This is a retrospective population-based study examining the timing from presentation with hematuria to diagnosis of bladder cancer, based on data from the MarketScan databases, which include enrollees of more than 100 health insurances plans of approximately 40 large US employers from 2004 through 2010. All study patients presented with hematuria and were subsequently diagnosed with bladder cancer. The primary outcome measure was number of days between initial presentation with hematuria and diagnosis of bladder cancer by gender.
Results
5416 men and 2233 women met inclusion criteria. Mean days from initial hematuria claim to bladder cancer claim was significantly longer in women (85.4 vs. 73.6 days, p<0.001), and the proportion of women with >6 month delays in bladder cancer diagnosis significantly higher (17.3% vs. 14.1%, p<0.001). Women were more likely to be diagnosed with urinary tract infection (OR 2.32 [95% CI 2.07–2.59]) and less likely to undergo abdominal or pelvic imaging (OR 0.80 [95% CI 0.71–0.89]).
Conclusions
Both men and women experience significant delays between presentation with hematuria and diagnosis of bladder cancer, with longer delays for women. This may be partly responsible for the gender-based discrepancy in outcomes associated with bladder cancer.
doi:10.1002/cncr.28416
PMCID: PMC3916781  PMID: 24496869
Urinary Bladder Neoplasms; Hematuria; Diagnosis; Insurance Claim Review; Standards
5.  Moderate Hypothermia Inhibits Brain Inflammation and Attenuates Stroke-induced Immunodepression in Rats 
CNS neuroscience & therapeutics  2013;20(1):10.1111/cns.12160.
Summary
Aims
Stroke causes both brain inflammation and immunodepression. Mild to moderate hypothermia is known to attenuate brain inflammation but its role in stroke-induced immunodepression (SIID) of the peripheral immune system remains unknown. This study investigated the effects in rats of moderate intra-ischemic hypothermia on SIID and brain inflammation.
Methods
Stroke was induced in rats by permanent distal MCA occlusion combined with transient bilateral CCA occlusion while body temperature was reduced to 30°C. Real-time PCR, flow cytometry, in vitro T cell proliferation assays and confocal microscopy were used to study SIID and brain inflammation.
Results
Brief Intra-Ischemic hypothermia helped maintain certain leukocytes in the peripheral blood and spleen, and enhanced T cell proliferation in vitro and delayed-type hypersensitivity in vivo, suggesting that hypothermia reduces SIID. In contrast, in the brain, brief intra-Ischemic hypothermia inhibited mRNA expression of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines INF-γ, TNF-α, IL-2, IL-1β and MIP-2. Brief intra-Ischemic hypothermia also attenuated the infiltration of lymphocytes, neutrophils (MPO+ cells) and macrophages (CD68+ cells) into the ischemic brain, suggesting that hypothermia inhibited brain inflammation.
Conclusions
Brief intra-ischemic hypothermia attenuated SIID and protected against acute brain inflammation.
doi:10.1111/cns.12160
PMCID: PMC3867545  PMID: 23981596
focal cerebral ischemia; hypothermia; inflammation; immunodepression; leukocytes
6.  Human brain arteriovenous malformations express lymphatic-associated genes 
Objective
Brain arteriovenous malformations (AVMs) are devastating, hemorrhage-prone, cerebrovascular lesions characterized by well-defined feeding arteries, draining vein(s) and the absence of a capillary bed. The endothelial cells (ECs) that comprise AVMs exhibit a loss of arterial and venous specification. Given the role of the transcription factor COUP-TFII in vascular development, EC specification, and pathological angiogenesis, we examined human AVM tissue to determine if COUP-FTII may have a role in AVM disease biology.
Methods
We examined 40 human brain AVMs by immunohistochemistry (IHC) and qRT-PCR for the expression of COUP-TFII as well as other genes involved in venous and lymphatic development, maintenance, and signaling. We also examined proliferation and EC tube formation with human umbilical ECs (HUVEC) following COUP-TFII overexpression.
Results
We report that AVMs expressed COUP-TFII, SOX18, PROX1, NFATC1, FOXC2, TBX1, LYVE1, Podoplanin, and vascular endothelial growth factor (VEGF)-C, contained Ki67-positive cells and heterogeneously expressed genes involved in Hedgehog, Notch, Wnt, and VEGF signaling pathways. Overexpression of COUP-TFII alone in vitro resulted in increased EC proliferation and dilated tubes in an EC tube formation assay in HUVEC.
Interpretation
This suggests AVM ECs are further losing their arterial/venous specificity and acquiring a partial lymphatic molecular phenotype. There was significant correlation of gene expression with presence of clinical edema and acute hemorrhage. While the precise role of these genes in the formation, stabilization, growth and risk of hemorrhage of AVMs remains unclear, these findings have potentially important implications for patient management and treatment choice, and opens new avenues for future work on AVM disease mechanisms.
doi:10.1002/acn3.142
PMCID: PMC4284124  PMID: 25574473
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  Immune response profiling identifies autoantibodies specific to Moyamoya patients 
Background
Moyamoya Disease is a rare, devastating cerebrovascular disorder characterized by stenosis/occlusion of supraclinoid internal carotid arteries and development of fragile collateral vessels. Moyamoya Disease is typically diagnosed by angiography after clinical presentation of cerebral hemorrhage or ischemia. Despite unclear etiology, previous reports suggest there may be an immunological component.
Methods
To explore the role of autoimmunity in moyamoya disease, we used high-density protein arrays to profile IgG autoantibodies from the sera of angiographically-diagnosed Moyamoya Disease patients and compared these to healthy controls. Protein array data analysis followed by bioinformatics analysis yielded a number of auto-antibodies which were further validated by ELISA for an independent group of MMD patients (n = 59) and control patients with other cerebrovascular diseases including carotid occlusion, carotid stenosis and arteriovenous malformation.
Results
We identified 165 significantly (p < 0.05) elevated autoantibodies in Moyamoya Disease, including those against CAMK2A, CD79A and EFNA3. Pathway analysis associated these autoantibodies with post-translational modification, neurological disease, inflammatory response, and DNA damage repair and maintenance. Using the novel functional interpolating single-nucleotide polymorphisms bioinformatics approach, we identified 6 Moyamoya Disease-associated autoantibodies against APP, GPS1, STRA13, CTNNB1, ROR1 and EDIL3. The expression of these 6 autoantibodies was validated by custom-designed reverse ELISAs for an independent group of Moyamoya Disease patients compared to patients with other cerebrovascular diseases.
Conclusions
We report the first high-throughput analysis of autoantibodies in Moyamoya Disease, the results of which may provide valuable insight into the immune-related pathology of Moyamoya Disease and may potentially advance diagnostic clinical tools.
doi:10.1186/1750-1172-8-45
PMCID: PMC3648437  PMID: 23518061
Autoantibodies; Cerebrovascular disease; Moyamoya; Protein microarray
21.  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
22.  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
23.  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
24.  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
25.  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

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