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Circulating Tumor Cells (CTCs) may serve as a non-invasive source of tumor material to investigate an individual's disease in real-time. The Parsortix ® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope-independent capture of CTCs with diverse phenotypes based on cell size and deformability. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had CTCs identified using immunofluorescence (IF) or Wright-Giemsa (WG) staining. Peripheral blood from 76 HVs and 76 MBC patients was processed on Parsortix ® PC1 Systems. Harvested cells were cytospun onto a charged slide and immunofluorescently stained for identification of CTCs expressing epithelial markers. The IF slides were subsequently WG-stained and analyzed for CTC identification based on morphological features of malignant cells. All testing was performed by operators blinded to the clinical status of each subject. CTCs were identified on the IF slides in 45.3% (≥ 1) / 24.0% (≥ 5) of the MBC patients (range = 0 - 125, mean = 7) and in 6.9% (≥ 1) / 2.8% (≥ 5) of the HVs (range = 0 - 28, mean = 1). Among the MBC patients with ≥ 1 CTC, 70.6% had only CK + /EpCAM- CTCs, with none having EpCAM + /CK- CTCs. CTC clusters were identified in 56.0% of the CTC-positive patients. On the WG-stained slides, CTCs were identified in 42.9% (≥ 1) / 21.4% (≥ 5) of the MBC patients (range = 0 - 41, mean = 4) and 4.3% (≥ 1) / 2.9% (≥ 5) of the HVs (range = 0 - 14, mean = 0). This study demonstrated the ability of the Parsortix ® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HVs when coupled with both IF and WG cytomorphological assessment. The presence of epithelial cells in subjects without diagnosed disease has been previously described, with their significance being unclear. Interestingly, a high proportion of the identified CTCs did not express EpCAM, highlighting the limitations of using EpCAM-based approaches., (© 2024. The Author(s).)

Introduction: The Parsortix ® PC1 system, Food and Drug Administration (FDA) cleared for use in metastatic breast cancer (MBC) patients, is an epitope-independent microfluidic device for the capture and harvest of circulating tumor cells from whole blood based on cell size and deformability. This report details the analytical characterization of linearity, detection limit, precision, and reproducibility for this device., Methods: System performance was determined using K 2 -EDTA blood samples collected from self-declared healthy female volunteers (HVs) and MBC patients spiked with prelabeled cultured breast cancer cell lines (SKBR3, MCF7, or Hs578T). Samples were processed on Parsortix ® PC1 systems and captured cells were harvested and enumerated., Results: The system captured and harvested live SKBR3, MCF7, and Hs578T cells and fixed SKBR3 cells linearly between 2 and ~100 cells, with average harvest rates of 69%, 73%, 79%, and 90%, respectively. To harvest ≥1 cell ≥95% of the time, the system required 3, 5 or 4 live SKBR3, MCF7 or Hs578T cells, respectively. Average harvest rates from precision studies using 5, 10, and ~50 live cells spiked into blood for each cell line ranged from 63.5% to 76.2%, with repeatability and reproducibility percent coefficient of variation (%CV) estimates ranging from 12.3% to 32.4% and 13.3% to 34.1%, respectively. Average harvest rates using ~20 fixed SKBR3 cells spiked into HV and MBC patient blood samples were 75.0% ± 16.1% (%CV = 22.3%) and 68.4% ± 14.3% (%CV = 21.1%), respectively., Conclusions: These evaluations demonstrate the Parsortix ® PC1 system linearly and reproducibly harvests tumor cells from blood over a range of 1 to ~100 cells., (Copyright © 2023, The Authors.)

Reduced anticonvulsant efficacy of benzodiazepines is a problem in the treatment of status epilepticus, with up to 50% of patients failing to respond to their first dose. KCC2 is a neuronal K + -Cl - co-transporter that helps set and maintain intracellular Cl - concentrations. KCC2 functional downregulation is a potential contributor to benzodiazepine resistance. We tested this idea using male and female doxycycline-inducible, conditional transgenic mice to increase the functional expression of KCC2 in pyramidal neurons. We administered mice with two doses of the chemoconvulsant kainic acid (5 mg/kg, i.p.) 60 min apart and quantified the resultant seizures with electroencephalography (EEG) recordings. Overexpression of KCC2 prior to the chemoconvulsant challenge did not affect seizure latency or other measures of seizure severity, but it did increase diazepam's efficacy in stopping EEG seizures. Spike rate, time in seizure, and EEG spectral power following diazepam (5 mg/kg, i.p) were all significantly lower in KCC2 overexpression mice as compared to control mice. Our results indicate that, in the context of benzodiazepine resistance during sustained seizures, addressing impaired Cl - homeostasis alone appreciably improves the efficacy of γ-aminobutyric acid (GABA)ergic inhibition. We therefore suggest the simultaneous targeting of KCC2 and GABA A receptors as a pathway for improving current anticonvulsant therapeutic strategies., (© 2021 International League Against Epilepsy.)

Background: Compared with those with a higher socio-economic position (SEP), individuals with a lower SEP have higher cancer morbidity and mortality. However, the contribution of modifiable risk factors to these inequities is not known. This study aimed to quantify the mediating effects of modifiable risk factors to associations between SEP and cancer morbidity and mortality., Methods: This study used a prospective observational cohort design. We combined eight cycles of the Canadian Community Health Survey (2000/2001-2011) as baseline data to identify a cohort of adults (≥35 years) without cancer at the time of survey administration (n = 309 800). The cohort was linked to the Discharge Abstract Database and the Canadian Mortality Database for cancer morbidity and mortality ascertainment. Individuals were followed from the date they completed the Canadian Community Health Survey until 31 March 2013. Dates of individual first hospitalizations for cancer and deaths due to cancer were captured during this time period. SEP was operationalized using a latent variable combining measures of education and household income. Self-reported modifiable risk factors, including smoking, excess alcohol consumption, low fruit-and-vegetable intake, physical inactivity and obesity, were considered as potential mediators. Generalized structural equation modelling was used to estimate the mediating effects of modifiable risk factors in associations between low SEP and cancer morbidity and mortality in the total population and stratified by sex., Results: Modifiable risk factors together explained 45.6% of associations between low SEP and overall cancer morbidity and mortality. Smoking was the most important mediator in the total population and for males, accounting for 15.5% and 40.2% of the total effect, respectively. For females, obesity was the most important mediator., Conclusions: Modifiable risk factors are important mediators of socio-economic inequities in cancer morbidity and mortality. Nevertheless, more than half of the variance in these associations remained unexplained. Midstream interventions that target modifiable risk factors may help to alleviate inequities in cancer risk in the short term. However, ultimately, upstream interventions that target structural determinants of health are needed to reduce overall socio-economic inequities in cancer morbidity and mortality., (© The Author(s) 2021; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Vitreous substitutes are clinically used to maintain retinal apposition and preserve retinal function; yet the most used substitutes are gases and oils which have disadvantages including strict face-down positioning post-surgery and the need for subsequent surgical removal, respectively. We have engineered a vitreous substitute comprised of a novel hyaluronan-oxime crosslinked hydrogel. Hyaluronan, which is naturally abundant in the vitreous of the eye, is chemically modified to crosslink with poly(ethylene glycol)-tetraoxyamine via oxime chemistry to produce a vitreous substitute that has similar physical properties to the native vitreous including refractive index, density and transparency. The oxime hydrogel is cytocompatible in vitro with photoreceptors from mouse retinal explants and biocompatible in rabbit eyes as determined by histology of the inner nuclear layer and photoreceptors in the outer nuclear layer. The ocular pressure in the rabbit eyes was consistent over 56 d, demonstrating limited to no swelling. Our vitreous substitute was stable in vivo over 28 d after which it began to degrade, with approximately 50% loss by day 56. We confirmed that the implanted hydrogel did not impact retina function using electroretinography over 90 days versus eyes injected with balanced saline solution. This new oxime hydrogel provides a significant improvement over the status quo as a vitreous substitute., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 10 4 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) ( n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Indigenous children are twice as likely to be classified as obese and three times as likely to experience household food insecurity when compared with non- Indigenous Canadian children. The purpose of this study was to explore the relationship between food insecurity and weight status among Métis and off-reserve First Nations children and youth across Canada., Methods: We obtained data on children and youth aged 6 to 17 years (n = 6900) from the 2012 Aboriginal Peoples Survey. We tested bivariate relationships using Pearson chi-square tests and used nested binary logistic regressions to examine the food insecurity-weight status relationship, after controlling for geography, household and school characteristics and cultural factors., Results: Approximately 22% of Métis and First Nations children and youth were overweight, and 15% were classified as obese. Over 80% of the sample was reported as food secure, 9% experienced low food security and 7% were severely food insecure. Off-reserve Indigenous children and youth from households with very low food security were at higher risk of overweight or obese status; however, this excess risk was not independent of household socioeconomic status, and was reduced by controlling for household income, adjusted for household size. Negative school environment was also a significant predictor of obesity risk, independent of demographic, household and geographic factors., Conclusion: Both food insecurity and obesity were prevalent among the Indigenous groups studied, and our results suggest that a large proportion of children and youth who are food insecure are also overweight or obese. This study reinforces the importance of including social determinants of health, such as income, school environment and geography, in programs or policies targeting child obesity.

Introduction: The purpose of this study was to examine the correlates of current smoking among off-reserve First Nations and Métis adults, two Aboriginal Canadian groups that are at higher risk to smoke and more likely to suffer from chronic health conditions relative to their non-Aboriginal counterparts. A particular focus was on culturally specific factors and their associations with current smoking., Methods: We used data from Statistics Canada's, 2012 Aboriginal Peoples Survey to investigate the correlates of smoking among 12,720 First Nations and Métis adults. Sequential binary logistic regression models were estimated to examine associations between smoking and culturally specific, demographic, geographic, socioeconomic and health-related variables., Results: Overall, 39.4% were current smokers. Multivariate results found that those who had hunted, fished or trapped within the last year were more likely to be smokers. In addition, respondents who were exposed to an Aboriginal language at home or outside the home were more likely to be smokers. Current smoking was significantly associated with being aged 35 to 49 years, living in a small population center, low income, low education, unemployment, being unmarried, low ratings of self-perceived health, heavy drinking and low body mass index. Respondents aged 65 years and older and those living in British Columbia were less likely to smoke., Discussion: The results of this study suggest that it may be useful to consider cultural characteristics, particularly language in efforts to reduce the prevalence of manufactured tobacco use among First Nations and Métis adults. Interventions should also consider demographic, geographic and socioeconomic variables, in addition to co-occurring health-risk behaviors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Aboriginal children in Canada are at a higher risk for overweight and obesity than other Canadian children. In Northern and remote areas, this has been linked to a lack of affordable nutritious food. However, the majority of Aboriginal children live in urban areas where food choices are more plentiful. This study aimed to explore the experiences of food insecurity among Métis and First Nations parents living in urban areas, including the predictors and perceived connections between food insecurity and obesity among Aboriginal children., Methods: Factors influencing children's diets, families' experiences with food insecurity, and coping strategies were explored using focus group discussions with 32 parents and caregivers of Métis and off-reserve First Nations children from Midland-Penetanguishene and London, Ontario. Four focus groups were conducted and transcribed verbatim between July 2011 and March 2013. A thematic analysis was conducted using NVivo software, and second coders ensured reliability of the results., Results: Caregivers identified low income as an underlying cause of food insecurity within their communities and as contributing to poor nutrition among their children. Families reported a reliance on energy-dense, nutrient-poor foods, as these tended to be more affordable and lasted longer than more nutritious, fresh food options. A lack of transportation also compromised families' ability to purchase healthful food. Aboriginal caregivers also mentioned a lack of access to traditional foods. Coping strategies such as food banks and community programming were not always seen as effective. In fact, some were reported as potentially exacerbating the problem of overweight and obesity among First Nations and Métis children., Conclusion: Food insecurity manifested itself in different ways, and coping strategies were often insufficient for addressing the lack of fruit and vegetable consumption in Aboriginal children's diets. Results suggest that obesity prevention strategies should take a family-targeted approach that considers the unique barriers facing urban Aboriginal populations. This study also reinforces the importance of low income as an important risk factor for obesity among Aboriginal peoples.

Objective: To examine the correlates of current smoking among Métis aged 18 years and older, with a particular focus on culturally-specific factors. Cultural factors included spirituality, knowledge of an Aboriginal language, membership in a Métis organization and attendance at Métis cultural events. Demographic, geographic, socio-economic and health-related variables were also considered., Methods: Data from 6,610 adult Métis aged 18 years and older who responded to the 2006 Aboriginal Peoples Survey and Métis supplement were used to examine the correlates of current smoking using sequential binary logistic regression modelling., Results: Overall, 39.9% of adult Métis respondents in the sample were current smokers. Adult Métis who reported a high level of spirituality were less likely to be current smokers. Those who spoke an Aboriginal language, or lived in a house where an Aboriginal language was spoken, were more likely to be current smokers. Being a member of a Métis organization and attending cultural events were not independently associated with current smoking. Métis with higher household income, greater education, higher self-perceived health, and greater physical activity participation were less likely to be current smokers, whereas those who reported heavy alcohol consumption were more likely to be current smokers., Conclusions: The results of this study suggest that interventions aimed at reducing smoking among adult Métis might be more successful if they include some connection to spirituality. It is also evident that co-occurring risk behaviours, in addition to demographic and socio-economic factors, are important considerations when developing interventions to reduce smoking among this population.

The utility of stem cells and their progeny in adult transplantation models has been limited by poor survival and integration. We designed an injectable and bioresorbable hydrogel blend of hyaluronan and methylcellulose (HAMC) and tested it with two cell types in two animal models, thereby gaining an understanding of its general applicability for enhanced cell distribution, survival, integration, and functional repair relative to conventional cell delivery in saline. HAMC improves cell survival and integration of retinal stem cell (RSC)-derived rods in the retina. The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA. Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function. HAMC also improves the distribution, viability, and functional repair of neural stem and progenitor cells (NSCs). The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aims to explore practice, use, and risk of electroconvulsive therapy (ECT) in pregnancy. A systematic search was undertaken in the databases Medline, Embase, PsycINFO, SveMed and CINAHL (EBSCO). Only primary data-based studies reporting ECT undertaken during pregnancy were included. Two reviewers independently checked study titles and abstracts according to inclusion criteria and extracted detailed use, practice, and adverse effects data from full text retrieved articles. Studies and extracted data were sorted according to before and after year 1970, due to changes in ECT administration over time. A total of 67 case reports were included and studies from all continents represented. Altogether, 169 pregnant women were identified, treated during pregnancy with a mean number of 9.4 ECTs, at mean age of 29 years. Most women received ECT during the 2nd trimester and many were Para I. Main diagnostic indication in years 1970 to 2013 was Depression/Bipolar disorder (including psychotic depression). Missing data on fetus/child was 12 %. ECT parameter report was often sparse. Both bilateral and unilateral electrode placement was used and thiopental was the main anesthetic agent. Adverse events such as fetal heart rate reduction, uterine contractions, and premature labor (born between 29 and 37 gestation weeks) were reported for nearly one third (29 %). The overall child mortality rate was 7.1 %. Lethal outcomes for the fetus and/or baby had diverse associations. ECT during pregnancy is advised considered only as last resort treatment under very stringent diagnostic and clinical indications. Updated international guidelines are urgently needed.

Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neural precursor cells; NPCs) in the adult central nervous system. Administration of CsA in vitro or in vivo promotes the survival of NPCs and expands the pools of NPCs in mice. Moreover, CsA administration is effective in promoting NPC activation, tissue repair and functional recovery in a mouse model of cortical stroke. The mechanism(s) by which CsA mediates this cell survival effect remains unknown. Herein, we examined both calcineurin-dependent and calcineurin-independent pathways through which CsA might mediate NPC survival. To examine calcineurin-dependent pathways, we utilized FK506 (Tacrolimus), an immunosuppressive molecule that inhibits calcineurin, as well as drugs that inhibit cyclophilin A-mediated activation of calcineurin. To evaluate the calcineurin-independent pathway, we utilized NIM811, a non-immunosuppressive CsA analog that functions independently of calcineurin by blocking mitochondrial permeability transition pore formation. We found that only NIM811 can entirely account for the pro-survival effects of CsA on NPCs. Indeed, blocking signaling pathways downstream of calcineurin activation using nNOS mice did not inhibit CsA-mediated cell survival, which supports the proposal that the effects are calcinuerin-independent. In vivo studies revealed that NIM811 administration mimics the pro-survival effects of CsA on NPCs and promotes functional recovery in a model of cortical stroke, identical to the effects seen with CsA administration. We conclude that CsA mediates its effect on NPC survival through calcineurin-independent inhibition of mitochondrial permeability transition pore formation and suggest that this pathway has potential therapeutic benefits for developing NPC-mediated cell replacement strategies., (© 2014. Published by The Company of Biologists Ltd.)

Stroke is a leading cause of disability with no effective regenerative treatment. One promising strategy for achieving tissue repair involves the stimulation of endogenous neural stem/progenitor cells through sequential delivery of epidermal growth factor (EGF) followed by erythropoietin (EPO). Yet currently available delivery strategies such as intracerebroventricular (ICV) infusion cause significant tissue damage. We designed a novel delivery system that circumvents the blood brain barrier and directly releases growth factors to the brain. Sequential release of the two growth factors is a key in eliciting tissue repair. To control release, we encapsulate pegylated EGF (EGF-PEG) in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and EPO in biphasic microparticles comprised of a PLGA core and a poly(sebacic acid) coating. EGF-PEG and EPO polymeric particles are dispersed in a hyaluronan methylcellulose (HAMC) hydrogel which spatially confines the particles and attenuates the inflammatory response of brain tissue. Our composite-mediated, sequential delivery of EGF-PEG and EPO leads to tissue repair in a mouse stroke model and minimizes damage compared to ICV infusion., (© 2013.)

Objectives: To examine the socio-economic, behavioural and Métis-specific factors that predict obesity among Métis children aged 6 to 14 years. Socio-economic factors included household structure and income, parental education and food insecurity. Cultural factors included knowledge of an Aboriginal language, participation in cultural activities, time spent with Elders and parental residential school attendance., Methods: The 2006 Aboriginal Peoples Survey, Children and Youth component collected data about Métis children, including child height and weight, reported by the person most knowledgeable about the child (PMK). Multivariate binary logistic regression was used to predict obesity, defined using IOTF BMI cut-offs. After testing for interactions, models were stratified by age (6-10, 11-14) and gender., Results: An estimated 18.5% of Métis boys and 14.4% of girls were obese. The effects of socio-economic factors and region varied across age and gender groups, although living in a lone-parent family and rural residence had consistent effects. Many effects of cultural variables were unexpected. Although PMK residential schooling was positively associated with obesity generally, the effects were negative among older girls. As expected, children participating in frequent physical activity generally had lower risk, independent of other factors., Conclusions: Although socio-economic factors are related to risk of obesity among Métis children, the effects may not be the same across age groups and for boys and girls. There is some evidence of independent effects of Métis-specific cultural factors, including parental residential schooling, on the risk of child obesity, but further investigation and better data are needed to understand these relationships.

Few quantitative diagnostic and monitoring, tools are available to clinicians treating patients with Alzheimer's disease. Further, many of the promising quantitative imaging tools under development lack clear specificity toward different types of Amyloid-β (Aβ) pathology such as vascular or oligomeric species. Antibodies offer an opportunity to image specific types of Aβ pathology because of their excellent specificity. In this study, we developed a method to translate a panel of anti-Aβ antibodies, which show excellent histological performance, into live animal imaging contrast agents. In the TgCRND8 mouse model of Alzheimer's disease, we tested two antibodies, M64 and M116, that target parenchyma aggregated Aβ plaques and one antibody, M31, that targets vascular Aβ. All three antibodies were administered intravenously after labeling with both poly(ethylene glycol) to enhance circulation and (64)Cu to allow detection via positron emission tomography (PET) imaging. We were clearly able to differentiate TgCRND8 mice from wild type controls by PET imaging using either M116, the anti-Aβ antibody targeting parenchymal Aβ or M31, the antivascular Aβ antibody. To confirm the validity of the noninvasive imaging of specific Aβ pathology, brains were examined after imaging and showed clear evidence of binding to Aβ plaques.

Purpose: Citalopram is a selective serotonin reuptake inhibitor (SSRI) antidepressant that is widely used in clinical practice. Recent data have indicated that high therapeutic citalopram doses may cause electrocardiographic abnormalities, and the regulatory authorities have amended its licenced dosage. The present manuscript reviews the available data concerning citalopram and cardiac toxicity., Methods: Published data concerning the cardiac effects of citalopram were ascertained, and clinical data were considered separately between adverse effects arising from therapeutic use versus toxicity in the setting of intentional overdose., Results: The occurrence of electrocardiographic abnormalities has long been recognised as a complication of acute citalopram overdose; a dose-effect relationship for QT prolongation has been described in a number of large case series, including several cases of torsades de pointes. In contrast, few data indicate the occurrence of QT prolongation and arrhythmia after therapeutic doses, and a dose-effect relationship within the therapeutic range has only recently been established. Citalopram is more likely to cause QT prolongation in patients with metabolic disturbance or pre-existing cardiac disease., Conclusions: A dose-effect relationship for QT prolongation exists across a broad range of citalopram doses, such that caution must be exercised when prescribing high doses or if there are co-existent risk factors for QT effects. The available data illustrate how clinical toxicity data may offer an earlier signal of cardiac effects than ascertained from conventional pharmacovigilance methods.

Stimulation of endogenous neural stem/progenitor cells (NSPCs) with therapeutic factors holds potential for the treatment of stroke. Cyclosporin A (CsA) is a particularly promising candidate molecule because it has been shown to act as a survival factor for these cells over a period of weeks both in vitro and in vivo; however, systemically-delivered CsA compromises the entire immune system, necessitating sustained localized delivery. Herein we describe a local delivery strategy for CsA using an epi-cortical hydrogel of hyaluronan-methylcellulose (HAMC) as the drug reservoir. Three methods of incorporating the drug into the hydrogel (solubilized, particulate, and poly(lactic-co-glycolic) acid (PLGA) microsphere-encapsulated) resulted in tunable release, spanning a period of hours to weeks. Importantly, PLGA-encapsulated CsA released from the hydrogel had equivalent bioactivity to fresh drug as measured by the neurosphere assay. Moreover, when CsA was released from the PLGA/HAMC composite that was injected on the cortex of adult mice, CsA was detected in the NSPC niche at a constant concentration for at least 24days post-implant. Thus this hydrogel composite system may be promising for the treatment of stroke., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Alzheimer's disease is a neurodegenerative disease characterized pathologically by amyloid-β (Aβ) aggregates in the brain. Notwithstanding many promising therapeutics that are under development, early diagnosis of Alzheimer's disease is limited. By targeting the Aβ aggregates, diagnosis can be improved and disease progression reduced. Molecular imaging using monoclonal antibodies to target specific isoforms of Aβ aggregates offer increased specificity in comparison to conventional imaging tracers; however, antibodies that are widely used in histology do not necessarily show similar binding in a dynamic in vivo environment. In this study, the diffusion and binding were studied of a classical monoclonal antibody, 6E10, in the brain of the TgCRND8 mouse model of AD. After intracranial injection of fluorescent 6E10, we observed broad and rapid labelling of Aβ deposits in the cortex and corpus callosum within 4h. Aβ plaques were detected up to 2.5mm away from the injection site in TgCRND8 mice and not in wild type mice at all, demonstrating specificity of binding. The apparent diffusivity and elimination constant of the anti-Aβ antibody were found to be independent of both the age of the animal and the accumulation of Aβ in the extracellular space, suggesting broad applicability of this targeting molecule. Mathematical modelling of the diffusion profiles of the anti-Aβ antibody in the brain parenchyma provides insights into the utility of antibodies as molecular imaging tools and targeted therapeutics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Drug delivery to the brain is challenging because systemic delivery requires high doses to achieve diffusion across the blood-brain barrier and often results in systemic toxicity. Intracerebroventricular implantation of a minipump/catheter system provides local delivery, yet results in brain tissue damage and can be prone to infection. An alternate local delivery strategy, epi-cortical delivery, releases the biomolecule directly to the brain while causing minimal tissue disruption. We pursued this strategy with a hyaluronan/methyl cellulose (HAMC) hydrogel for the local release of erythropoietin to induce endogenous neural stem and progenitor cells of the subventricular zone to promote repair after stroke injury in the mouse brain. Erythropoeitin promotes neurogenesis when delivered intraventricularly, thereby making it an ideal biomolecule with which to test this new epi-cortical delivery strategy. We investigated HAMC in terms of the host tissue response and the diffusion of erythropoeitin therefrom in the stroke-injured brain for neural repair. Erythropoietin delivered from HAMC at 4 and 11 days post-stroke resulted in attenuated inflammatory response, reduced stroke cavity size, increased number of both neurons in the peri-infarct region and migratory neuroblasts in the subventricular zone, and decreased apoptosis in both the subventricular zone and the injured cortex. We demonstrate that HAMC-mediated epi-cortical administration is promising for minimally invasive delivery of erythropoeitin to the brain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Traumatic injury to the central nervous system (CNS) is highly debilitating, with the clinical need for regenerative therapies apparent. Neural stem/progenitor cells (NSPCs) are promising because they can repopulate lost or damaged cells and tissues. However, the adult CNS does not provide an optimal milieu for exogenous NSPCs to survive, engraft, differentiate, and integrate with host tissues. This review provides an overview of tissue engineering strategies to improve stem cell therapies by providing a defined microenvironment during transplantation. The use of biomaterials for physical support, growth factor delivery, and cellular co-transplantation are discussed. Providing the proper environment for stem cell survival and host tissue integration is crucial in realizing the full potential of these cells in CNS repair strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Antibody-mediated imaging of amyloid β (Aβ) in Alzheimer's disease (AD) offers a promising strategy to detect and monitor specific Aβ species, such as oligomers, that have important pathological and therapeutic relevance. The major current limitation of antibodies as a diagnostic and imaging device is poor blood-brain-barrier permeability. A classical anti-Aβ antibody, 6E10, is modified with 10 kDa polyethylene glycol (PEG) and a positron emitting isotope, Copper-64 (t(½) = 12.7 h), and intravenously delivered to the TgCRND8 mouse model of Alzheimer's disease. Modification of 6E10 with PEG (6E10-PEG) increases accumulation of 6E10 in brain tissue in both TgCRND8 and wild type control animals. 6E10-PEG differentiates TgCRND8 animals from wild type controls using positron emission tomography (PET) and provides a framework for using antibodies to detect pathology using non-invasive medical imaging techniques.

One of the challenges in treating central nervous system (CNS) disorders with biomolecules is achieving local delivery while minimizing invasiveness. For the treatment of stroke, stimulation of endogenous neural stem/progenitor cells (NSPCs) by growth factors is a promising strategy for tissue regeneration. Epidermal growth factor (EGF) enhances proliferation of endogenous NSPCs in the subventricular zone (SVZ) when delivered directly to the ventricles of the brain; however, this strategy is highly invasive. We designed a biomaterials-based strategy to deliver molecules directly to the brain without tissue damage. EGF or poly(ethylene glycol)-modified EGF (PEG-EGF) was dispersed in a hyaluronan and methylcellulose (HAMC) hydrogel and placed epi-cortically on both uninjured and stroke-injured mouse brains. PEG-modification decreased the rate of EGF degradation by proteases, leading to a significant increase in protein accumulation at greater tissue depths than previously shown. Consequently, EGF and PEG-EGF increased NSPC proliferation in uninjured and stroke-injured brains; and in stroke-injured brains, PEG-EGF significantly increased NSPC stimulation. Our epi-cortical delivery system is a minimally-invasive method for local delivery to the brain, providing a new paradigm for local delivery to the brain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL-002., Materials and Methods: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real-time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers., Results: Undifferentiated KCL-002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox-2, Oct-4 and TRA 1-60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR-2, α-actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives., Conclusions: The data presented suggest that the KCL-002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells., (© 2011 Blackwell Publishing Ltd.)

Stroke is a neurological disorder that currently has no cure. Intrathecal delivery of growth factors, specifically recombinant human epidermal growth factor (rhEGF), stimulates endogenous neural precursor cells in the subventricular zone (SVZ) and promotes tissue regeneration in animal models of stroke. In this model, rhEGF is delivered with an invasive minipump/catheter system, which causes trauma to the brain. A less invasive strategy is to deliver rhEGF from the brain cortex; however, this requires the protein to diffuse through the brain, from the site of injection to the SVZ. Although this method of delivery has great potential, diffusion is limited by rapid removal from the extracellular space and hence for successful translation into the clinic strategies are needed to increase the diffusion distance. Using integrative optical imaging we investigate diffusion of rhEGF vs. poly(ethylene glycol)-modified rhEGF (PEG-rhEGF) in brain slices of both uninjured and stroke-injured animals. For the first time, we quantitatively show that PEG modification reduces the rate of growth factor elimination by over an order of magnitude. For rhEGF this corresponds to a two to threefold increase in predicted brain penetration distance, which we confirm with in vivo data., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination., (Copyright 2009 Wiley Periodicals, Inc.)

Regenerative strategies for retinal degenerative diseases are limited by poor cellular survival, distribution and integration after transplantation to the sub-retinal space. To overcome this limitations a stem cell delivery system was developed, taking advantage of the minimally-invasive, injectable and biodegradable properties of a blend of hyaluronan and methylcellulose (HAMC). The physical and biological properties of this unique HAMC formulation were studied. HAMC supported retinal stem-progenitor cell (RSPC) survival and proliferation in vitro. The blend was a viscous solution, exhibiting properties ideal for delivery to the sub-retinal space. In vivo transplantation studies in mice were carried out to investigate both the biodegradability of HAMC in the sub-retinal space over 7 days and the potential of HAMC as a cell delivery vehicle. RSPCs delivered in HAMC were more evenly distributed in the sub-retinal space than those delivered in traditional saline solutions, suggesting that HAMC is a promising vehicle for cellular delivery to the degenerating retina overcoming previously reported barriers to tissue integration in the retina such as cellular aggregation and non-contiguous distribution., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Embryonic stem cells (ESC) are a unique cell population with the ability to self-renew and differentiate into all three germ layers. Human ESC express the telomerase reverse transcriptase (TERT) gene and the telomerase RNA (TR) and show telomerase activity, but TERT, TR, and telomerase are all downregulated during the differentiation process. To examine the role of telomerase in human ESC self-renewal and differentiation, we modulated the expression of TERT. Upregulation of TERT and increased telomerase activity enhanced the proliferation and colony-forming ability of human ESC, as well as increasing the S phase of the cell cycle at the expense of a reduced G1 phase. Upregulation of TERT expression was associated with increases in CYCLIN D1 and CDC6 expression, as well as hyperphosphorylation of RB. The differentiated progeny of control ESC showed shortening of telomeric DNA as a result of loss of telomerase activity. In contrast, the differentiated cells from TERT-overexpressing ESC maintained high telomerase activity and accumulated lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Although the TERT-overexpressing human ESC are able to form teratoma composed of three germ layers in vivo, their in vitro differentiation to all primitive and embryonic lineages was suppressed. In contrast, downregulation of TERT resulted in reduced ESC proliferation, increased G1, and reduced S phase. Most importantly, downregulation of TERT caused loss of pluripotency and human ESC differentiation to extraembryonic and embryonic lineages. Our results indicate for the first time an important role for TERT in the maintenance of human ESC pluripotency, cell cycle regulation, and in vitro differentiation capacity.

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.

Pluripotent stem cells transplanted into immune-deficient mice form complex teratomas. Although such tumors are generally haphazard in their organization, they do contain some structures that resemble tissues normally seen in the embryo. As a consequence, the teratoma model is useful for exploring the developmental potential of stem cells and studying certain aspects of tissue development. To further our understanding of this process, we examined whether the anatomical location into which human pluripotent stem cells were grafted influenced their growth in situ. Here we report that cells grafted into the liver rapidly produced large tumors containing predominantly immature cells. In contrast, subcutaneous implants were significantly slower growing and eventually formed tumors composed of differentiated tissues. The alternative growth patterns recorded between these two graft sites indicates how environmental cues affect stem cell behavior. This approach may lead to the identification of new ways to control stem cell growth and differentiation.

It has been claimed that blockade of receptors for N-methyl-D-aspartate (NMDA) can enhance adenosine receptor function on single neurones. Previous work has also indicated that the NMDA channel blocker dizocilpine, and the A1 selective agonist N6-cyclopentyladenosine (CPA) both had anxiolytic profiles in the elevated plus-maze. The anxiolytic effect of dizocilpine was accompanied by an increase in locomotor activity. In the present study, the elevated plus-maze has been used to determine whether dizocilpine's effects on behaviour are mediated through activation of adenosine receptors. When co-administered with dizocilpine (0.05 mg/kg), CPA (0.05 mg/kg) reduced the anxiolytic and locomotor effects of dizocilpine. The A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX, 0.05 mg/kg) had no effect when administered alone. When co-administered with dizocilpine, CPX reversed the anxiolytic and increased locomotor effects induced by dizocilpine. The A2 receptor selective agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)ethyladenosine (DPMA) (1 mg/kg) reversed both the anxiolytic effect and the increased locomotion induced by dizocilpine, while the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (1 mg/kg) did not. It is concluded that at least part of the anxiolytic and locomotor stimulant properties of dizocilpine may be explained by the release of endogenous adenosine acting at A1, but not A2 receptors.

The Y-maze was used to assess spontaneous alternation behaviour in mice to examine possible interactions between the N-methyl-D-aspartate receptor channel blocker dizocilpine and purine receptor agonists and antagonists. Scopolamine reduced spontaneous alternation. Dizocilpine also produced a dose-dependent reduction in alternation scores, which was accompanied by an increase in locomotion. The selective A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) had no effect when administered alone, or in combination with scopolamine. However, when co-administered with dizocilpine, CPX reversed both the deficit in alternation behaviour and also the increase in locomotion induced by dizocilpine. The A1 selective agonist N6-cyclopentyladenosine (CPA) had no effect on either locomotion or alternation scores when administered alone, but in combination with scopolamine, CPA attenuated the scopolamine-induced deficit. CPA had no significant effect on the dizocilpine-induced deficit. The A2 selective agonist N6-[2-(3, 5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA), had no effect on spontaneous alternation when administered alone, but did cause a depression of locomotion. DPMA had no significant effect when co-administered with scopolamine, but reversed the deficit in spontaneous alternation, and the increase in locomotion induced by dizocilpine. The A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) had no effect when given alone or in combination with scopolamine, but when co-administered with dizocilpine, DMPX reversed the reduction in spontaneous alternation caused by dizocilpine. It is concluded that dizocilpine has a detrimental effect on spontaneous alternation which is mediated partly by A1 and A2 adenosine receptors.

The N-methyl-D-aspartate (NMDA) receptor polyamine site antagonist, ifenprodil, had no effect on spontaneous alteration or locomotor activity in the Y-maze when given alone. The NMDA receptor/ion channel blocker, dizocilpine, induced a deficit in spontaneous alteration, but when ifenprodil was co-administered with dizocilpine, it showed a strong tendency to attenuate the dizocilpine-induced deficit. In the plus-maze, ifenprodil had an anxiolytic profile which was accompanied by an increase in locomotion. Dizocilpine had an anxiolytic profile in this model and increased locomotor activity. When co-administered with dizocilpine, ifenprodil reduced both the anxiolytic and locomotor effects of dizocilpine. When co-administered with ifenprodil, cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentylxanthine (CPX) reduced the anxiolytic effect of ifenprodil. CPA and CPX in combination did not reverse the anxiolytic effect of ifenpropil. It is concluded that NMDA antagonists with different sites of action can show distinct behavioural profiles, with dizocilpine but not ifenprodil inducing a deficit in working memory, while both are anxiolytic. Blockade of NMDA receptors by ifenprodil, however, can preclude any response to dizocilpine. The anxiolytic activity of ifenprodil may involve the release of purines acting at adenosine receptors.

A field trial was set up to test the prophylactic properties of the organophosphorous drug metrifonate (Bilarcil Bayer AG). Subjects were rural African children living in an area of Rhodesia where Schistosoma haematobium and S. mansoni are highly endemic. The trial was conducted in three stages, a preliminary period of therapy followed by two six-month periods of prophylaxis. Parasitological and haematological tests were carried out monthly and major assessments (including clinical examinations) were carried out prior to the start of the trial and at the end of each of the three stages. Drug was given to the appropriate groups at a dose rate of 7-5 mg/kg once per fortnight for three doses during the therapy stage and four-weekly during the prophylaxis stage. Results with S. haematobium were very good. A 60% cure-rate was observed six weeks aection was obtained in those children continuing to receive the drug as a prophylactic, even during the season of highest transmission; intensities of infection in those who became infected were very low. Infection rates in the treated but unprotected group rose steadily from 40% at week 11 to 95% at week 70. There was a sigificant effect upon the intensity of S. mansoni infections only when pre- and post-trial data were compared. Apart from the anticipated (and previously reported) depression of plasma cholinesterase values no side effects were recorded. Drug tolerance and acceptibility were very high. It is likely that the costs of a year's protection against S. Haematobium using metrifonate will be significantly lower than protection by molluscicidal techniques or single courses of treatment with established drugs.