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Background Image segmentation is often imperfect, particularly in complex image sets such z-stack micrographs of slice cultures and there is a need for sufficient details of parameters used in quantitative image analysis to allow independent repeatability and appraisal. New method For the first time, we have critically evaluated, quantified and validated the performance of different segmentation methodologies using z-stack images of ex vivo glial cells. The BioVoxxel toolbox plugin, available in FIJI, was used to measure the relative quality, accuracy, specificity and sensitivity of 16 global and 9 local threshold automatic thresholding algorithms. Results Automatic thresholding yields improved binary representation of glial cells compared with the conventional user-chosen single threshold approach for confocal z-stacks acquired from ex vivo slice cultures. The performance of threshold algorithms varies considerably in quality, specificity, accuracy and sensitivity with entropy-based thresholds scoring highest for fluorescent staining. Comparison with existing methods We have used the BioVoxxel toolbox to correctly and consistently select the best automated threshold algorithm to segment z-projected images of ex vivo glial cells for downstream digital image analysis and to define segmentation quality. The automated OLIG2 cell count was validated using stereology. Conclusions As image segmentation and feature extraction can quite critically affect the performance of successive steps in the image analysis workflow, it is becoming increasingly necessary to consider the quality of digital segmenting methodologies. Here, we have applied, validated and extended an existing performance-check methodology in the BioVoxxel toolbox to z-projected images of ex vivo glia cells.

Rugby is one of the few contact sports that do not mandate protective headgear, possibly because studies have shown poor efficacy for protection related to concussion pathology with existing headguards.Following innovative material technology utilization to produce headgear believed to have protective capabilities, this study examined the effects of a soft-shell headgear constructed from a novel viscoelastic material, on both behaviour and serum biomarkers after high and average impact force mild traumatic brain injuries (mTBI).Seventy-five male Sprague Dawley rats were divided into five groups: control, average - 37G impact, with and without headgear, and high - 106G impact, with and without headgear. Rats were sacrificed at 3 or 48 hours and serum samples were analyzed for levels of TNF-α, NEF-L, and GFAP. Animals sacrificed at 48 hours also underwent testing for balance and motor coordination, and exploratory/locomotor behaviour.The novel headgear offered significant protection against mTBI symptomology and biomarkers in the group that experienced an average impact force, but only moderated protection for the animals in the high impact group.This innovative headgear may prevent some of the negative sequel associated with concussion pathology.

Conference Paper

Conference Proceeding

ObjectivesIn the aim to develop a usable and wearable head guard for rugby that could reduce impact energy and lessen the likelihood of concussive and subconcussive injury during play, a combination of viscoelastic materials was employed to develop a guard with similar dimensions to those currently used in international rugby.MethodsThe head guard was tested for impact energy reduction following linear acceleration, using drop tests, as required by World Rugby. The head guard was also subjected to pendulum tests, allowing acceleration to be simultaneously measured on two headforms, as well as repeated impacts to mimic ageing and repeated use. Impact following rotational acceleration was determined at two impact locations and at three impact velocities.ResultsThe viscoelastic head guard (N-Pro) was shown to reduce linear impacts by up to 75% in comparison to the use of a commercially available rugby head guard and repeated impacts did not impair the attenuation of impact energy. Rotational impact energy was also reduced by an average of 34% across three speeds and two sites of impact test sites, in comparison to tested bare headforms.ConclusionsThis heralds a new generation of soft-shelled headgear that could help reduce two primary risk factors in sports-induced mild traumatic brain injury: linear and rotational impacts to the head.

The accumulation of iron within the brain occurs in many chronic disorders including Alzheimer's and Parkinson's disease and multiple sclerosis. Outside the CNS, a link between levels of iron and the unfolded protein response has already been established. To determine if such a relationship operates in within the brain, we used our ex vivo hippocampal slice-based model of iron accumulation. Ferrocene addition caused accumulation of iron within slices and loss of oligodendrocytes, an effect that was partially inhibited when ferrocene and ER stressor tunicamycin (Tm) were added together. An upward trend (not found to be statistically significant) in the expression of UPR transcripts in response to ferrocene was demonstrated using real-time PCR, while a significant upregulation of mRNA for B cell immunoglobulin-binding protein (BiP) and C/EBP homologous binding protein (CHOP) occurred following exposure to Tm. In silico analysis revealed consensus DNA-binding sequences for UPR-associated transcription factors within the promoter regions of eight iron-regulatory genes. In addition, dual-staining for CHOP and oligodendrocyte transcription factor 2 (OLIG2) or Ionized calcium binding adaptor molecule 1 (Iba1) showed nuclear expression of CHOP in some oligodendrocyte-lineage cells in response to Tm or Tm+ferrocene, but CHOP was rarely found in microglia. Co-expression of UPR-associated activated transcription factor 6 (ATF6) was detected in the nuclei of some oligodendrocyte-lineage cells exposed to Tm alone, or to Tm and ferrocene, but rarely in microglia. These data highlight the therapeutic potential of targeting UPR-associated proteins when developing novel treatments for chronic brain disorders that are affected by dysregulated iron.

The unfolded protein response (UPR) has been reported during normal development of cortical neurons and cerebellar white matter and may also contribute to the pathogenesis of neurological conditions, such as Marinesco-Sjogren syndrome and Borna virus infection, which result in cerebellar defects. The UPR is initiated when the processing capacity of the endoplasmic reticulum (ER) is overwhelmed. Misfolded proteins accumulate and can activate ER stress sensors; PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activated transcription factor 6 (ATF6) and their downstream targets glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and protein disulfide isomerase (PDI). In order to provide a fuller appreciation of the possible importance of ER stress-associated proteins in the context of cerebellar disease, we have profiled the expression of ER stress sensors and their downstream targets in the developing cerebellar cortex in postnatal rat. Activation of PERK and IRE1 stress sensors was observed for the first time in normally developing granule cell precursors. A second proliferative pPERK-positive population was also detected in the internal granular layer (IGL). In general, the density of UPR protein-positive cells was found to decrease significantly when profiles in early and late postnatal ages were compared. These data may be relevant to studies of medulloblastoma and warrant further investigation.

Although aberrant metabolism and deposition of iron has been associated with aging and neurodegeneration, the contribution of iron to neuropathology is unclear. Well-designed model systems that are suited to studying the putative pathological effect of iron are likely to be essential if such unresolved details are to be clarified. In this review, we have evaluated the utility and effectiveness of the reductionist in vitro platform to study the molecular mechanisms putatively underlying iron perturbations of neurodegenerative disease. The expression and function of iron metabolism proteins in glia and neurons and the extent to which this iron regulatory system is replicated in in vitro models has been comprehensively described, followed by an appraisal of the inherent suitability of different in vitro and ex vivo models that have been, or might be, used for iron loading. Next, we have identified and critiqued the relevant experimental parameters that have been used in in vitro iron loading experiments, including the choice of iron reagent, relevant iron loading concentrations and supplementation with serum or ascorbate, and propose optimal iron loading conditions. Finally, we have provided a synthesis of the differential iron accumulation and toxicity in glia and neurons from reported iron loading paradigms. In summary, this review has amalgamated the findings and paradigms of the published reports modelling iron loading in monocultures, discussed the limitations and discrepancies of such work to critically propose a robust, relevant and reliable model of iron loading to be used for future investigations.

Aberrant iron deposition in the brain is associated with neurodegenerative disorders including Multiple Sclerosis, Alzheimer’s disease and Parkinson’s disease. To study the collective response to iron loading, we have used hippocampal organotypic slices as a platform to develop a novel ex vivo model of iron accumulation. We demonstrated differential uptake and toxicity of iron after 12 h exposure to 10 μM ferrous ammonium sulphate, ferric citrate or ferrocene. Having established the supremacy of ferrocene in this model, the cultures were then loaded with 0.1–100 μM ferrocene for 12 h. One μM ferrocene exposure produced the maximal 1.6-fold increase in iron compared with vehicle. This was accompanied by a 1.4-fold increase in ferritin transcripts and mild toxicity. Using dual-immunohistochemistry, we detected ferritin in oligodendrocytes, microglia, but rarely in astrocytes and never in neurons in iron-loaded slice cultures. Moreover, iron loading led to a 15% loss of olig2-positive cells and a 16% increase in number and greater activation of microglia compared with vehicle. However, there was no appreciable effect of iron loading on astrocytes. In what we believe is a significant advance on traditional mono- or dual-cultures, our novel ex vivo slice-culture model allows characterization of the collective response of brain cells to iron-loading.

Despite the widespread use of drug eluting stents (DES), in-stent restenosis (ISR), delayed arterial healing and thrombosis remain important clinical complications. Gene-eluting stents (GES) represent a potential strategy for the prevention of ISR by delivering a therapeutic gene via a vector from the stent surface to the vessel wall. To this end, a model in vitro system was established to examine whether cationic liposomes could be used for gene delivery to human artery cells. Three different formulations were compared (DOTMA/DOPE, DDAB/DOPE or DDAB/POPC/Chol) to examine the effects of different cationic and neutral lipids on the transfection efficiency of lipoplex-coatings of metal surfaces. Upon completion of the characterization and optimization of the materials for gene delivery in vitro, these coatings were examined on a range of stents and deployed in a rabbit iliac artery injury model in vivo. Maximal transfection efficiencies for all coatings were observed on day 28, followed by declining, but persisting gene expression 42 days after stent placement, thereby, presenting liposomal coatings for gene eluting stents as treatment options for clinical complications associated with stenting procedures.

The production, transport and integration of myelin components into the membrane during development is a highly coordinated and regulated process that relies heavily on the endoplasmic reticulum (ER), a sub-cellular organelle that is the principal site of membrane assembly. Ribosomes on the rough ER allow translation of proteins such as proteolipid protein (PLP) prior to correct folding, post-translational modification and eventual complexing with nascent smooth ER-synthesised lipids.

Background: The endoplasmic reticulum (ER) stress pathway may play a role in the pathogenesis multiple sclerosis (MS), and while ER stress-associated molecules have been demonstrated in white matter (WM) lesions, these have not been analysed in grey matter (GM) demyelination. Objective: The objective was to characterise the type and frequency of GM lesions and establish expression profiles of ER stress- and hypoxia-associated markers. Methods: Sections from 16 MS cases and 12 non-MS controls were stained for ER stress molecules (BiP and CHOP) and hypoxia-associated D110 antigen. Results: Of the GM lesions analysed, 24% were type 1 (continuous between GM and WM), 22% were type 2 (entirely within GM) and the majority (54%) were type 3 (extending from pia mater). Comparison of GM lesions, MS normal-appearing grey matter (NAGM) and non-MS control tissue showed that NAGM, type 1 and type 3 lesions all had significantly increased levels of CHOP compared to controls. According to morphological and dual-labelling criteria, the majority of CHOP-positive cells were microglia. Approximately 50% of GM lesions contained D110-positive cells. Conclusion: These data suggest that ER stress plays an important role in GM lesion development and may be critical in activation of microglia in pre-lesional NAGM. The high number of lesions containing D110-positive cells suggests a role for hypoxic-like insult in GM lesion development.

Endothelial dysfunction is an important factor in cardiovascular pathology. It has been suggested that pluripotent mesenchymal stem cells (MSCs) may contribute to repair of the endothelium through paracrine pathways. Enhanced re-endothelialization may be associated with a better outcome following angioplasty procedures. We examined the effect of the delivery of MSCs to a denuded vessel in vivo. The right carotid arteries of New Zealand white rabbits were denuded using an uninflated 3-French Fogarty balloon catheter. 1 x 10(5) MSCs in a bolus of 150 microL were then delivered intraluminally and allowed to dwell for 20 min. MSC engraftment was assessed using PKH-26 labeling and transduction with adenoviral reporter genes. Vessels were examined at 2 weeks for levels of endothelialization, as well as for neointimal hyperplasia and vasomotor function. Engraftment of MSCs was noted in the vessel wall following local arterial delivery. Endothelialization was improved following bolus MSC delivery at 2 weeks post-intervention. However, this endothelium is manifestly dysfunctional as indicated by a significant impairment in vasomotor activity and a significant increase in neointimal formation post-bolus delivery. Consistent with the formation of a dysfunctional endothelium, there was a higher rate of vessel occlusions in bolus-treated vessels due to not only predominately thrombosis but also neointimal hyperplasia. Our results suggest that naive MSCs delivered as a bolus to the occluded injured vascular segment generate dysfunctional endothelium presenting a risk of vessel occlusion. Such risks are important and need to be further assessed.

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue. It follows from our examination of the utility of Fiji ImageJ thresholding and binarization algorithms. Describing how we identified the maximum intensity projection as the best of six tested for two dimensional (2D)-rendering of three-dimensional (3D) images derived from a series of z-stacked micrographs, the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein (GFAP), microglial ionized calcium binding adapter molecule 1 (IBA1) and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices. The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections, is also described. Fiji’s BioVoxxel plugin allowed categorisation of algorithms according to their sensitivity, specificity accuracy and relative quality. The Percentile algorithm was deemed best for quantifying levels of GFAP, the Li algorithm was best when quantifying IBA expression, while the Otsu algorithm was optimum for Olig2 staining, albeit with over-quantification of oligodendrocyte number when compared to a stereological approach. Also, GFAP and IBA expression in 3,3′-diaminobenzidine (DAB)/haematoxylin-stained cerebellar tissue was best quantified with Default, Isodata and Moments algorithms. The workflow presented in [Figure 1] could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.

The blood brain barrier (BBB) is formed by capillary endothelial cells with inter-endothelial cell tight junctions and other cells such as pericytes and astrocytes present. Previous studies have shown a role for tight junction abnormalities in BBB leakage in multiple sclerosis (MS) brain. This marks a key stage in the development of inflammatory demyelination in MS. The aim of this study was to identify aberrantly expressed genes involved in BBB changes in MS lesions. A focused endothelial cell biology microarray, capable of detecting changes in expression of 113 endothelial cell-specific genes, was employed to analyse endothelial cell mRNA extracted from post-mortem control white matter, MS normal appearing white matter (NAWM), chronic active or inactive lesions by laser capture microdissection. Microarray analysis found 52 genes out of 113 analysed, predominantly in the activation functional group, to be differentially expressed in lesions compared to control or NAWM (p < 0.01). The majority of the differentially expressed genes were validated by quantitative real time PCR. In addition, the protein expression profiles of ICAM2, MMP2, and VEGFR1 were examined by immunofluorescent staining of selected tissue blocks. ICAM-2 was expressed at a higher level in chronic inactive lesions than control or NAWM, corresponding with the increased mRNA measured by microarray and real time PCR. The data shown, presenting a number of differentially expressed genes in the microvascular compartment of MS lesions, may shed light on the molecular mechanisms that are involved in the breakdown of the BBB. This moves us a step closer to the identification of potential therapeutic targets for repair of the compromised BBB.

Myelin production during brain development requires an increase in membrane protein and lipid production in oligodendrocytes and this primarily occurs in the endoplasmic reticulum (ER), an organelle which initiates the Unfolded Protein Response (UPR) when under stress. We hypothesise that the UPR is activated in white matter tracts during myelination in order to expand the ER capacity of oligodendrocytes. Using early and late stage markers, critical myelination time points were identified by immunohistochemistry in developing rat cerebellum. These were correlated to peaks in ER stress signalling by staining for activated UPR transducers (pIRE1, ATF6 and pPERK) and associated downstream molecules (peIF2α, PDI, GRP78, GRP94, CHOP and calreticulin) in cerebellar tracts III and IV. Gene expression in developing cerebellum was assessed by qPCR. Actively myelinating tracts were shown to have differential expression of pIRE1, PERK and ATF6 as well as UPR targets GRP94, GRP78 and PDI. Activated pIRE1-positive cells were widespread at P14 and P17 and at significantly higher numbers during myelination than at other stages. Nuclear-localised ATF6 (indicative of the active transcription factor) peaked at P10, concurrent with the initial phase of myelination. The percentage of cells positive for pPERK was less than 1% at postnatal ages but increased significantly in adult tissue. The downstream targets GRP78, GRP94 and PDI were significantly up-regulated at P17 compared to P7 and remained significantly elevated in adults. The majority of cells positive for these markers and ATF6 were oligodendrocytes as confirmed by dual-labelling. Although gene expression in the cerebellum for GRP78, GRP94 and PDI did not change significantly over time, ATF6 and XBP1s both showed significant fold changes between early and late timepoints. This data helps promote understanding of events occurring during developmental myelination and may have implications for the development of reparative treatments in diseases such as multiple sclerosis.

Gene-eluting stents are being evaluated in animals as an alternative approach to inhibiting in-stent restenosis. Adeno-associated virus type 2 (AAV2) and adenovirus are commonly used for gene transfer applications. We tested the hypothesis that these vectors can achieve prolonged and localized gene delivery to the vessel wall, using stents as delivery platforms. AdbetaGal (5 x 10(9) plaque-forming units) and AAV2betaGal (5.3 x 10(9) DNase-resistant particles) were used to coat BiodivYsio stents with matrix HI coating (Abbott Vascular Devices, Galway, Ireland). After balloon injury, external iliac arteries of New Zealand White rabbits were stented. The reverse transcription-polymerase chain reaction was used to assess viral spread. Expression of LacZ was demonstrated with both vectors at five time points (3, 7, 14, 21, and 28 days). In the adenovirus group the median percentage of cells expressing the transgene on day 3 was 2.73%, which increased to a median expression of 7.31% at 28 days (p0.05). Expression was localized to medial cells on day 3, but was observed predominantly in neointimal cells on day 28. In the AAV group, day 3 expression was 5.78%, which decreased to 2.12% on day 28 (p = 0.05). No systemic dissemination of virus was seen in any group. Adenovirus- and AAV2-coated stents can be used to deliver genes to the blood vessel wall for up to 28 days.

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.

This paper contains a systematic review of the literature relating to whether or not a tracheostomy tube impedes swallowing. Nineteen articles were reviewed; the levels of evidence were generally low. A number of methodological issues were identified. Subject numbers were small and there was wide variation in the diagnostic groups studied, making some comparisons invalid, and this did not allow the data to be aggregated. Many studies included subjects with different types of tracheostomy tubes (cuffed, uncuffed, fenestrated, unfenestrated), which also rendered comparisons difficult. In conclusion, scientific evidence, as opposed to clinical opinion, does not currently exist to support the notion that a tracheostomy tube impedes swallowing function.

Mesenchymal stem cells (MSCs) suppress T helper (Th)17 cell differentiation and are being clinically pursued for conditions associated with aberrant Th17 responses. Whether such immunomodulatory effects are enhanced by coadministration of MSCs with other agents is not well known. In the present study, individual and combined effects of MSCs and the vitamin D receptor (VDR) agonist paricalcitol on Th17 induction were investigated in vitro and in a mouse model of sterile kidney inflammation (unilateral ureteral obstruction). In vitro, MSCs and paricalcitol additively suppressed Th17 differentiation, although only MSCs suppressed expression of Th17-associated transcriptions factors. Combined administration of MSCs and paricalcitol resulted in an early ( day 3) reduction of intrarenal CD4+and CD8+T cells, CD11b+/lymphocyte antigen 6G+neutrophils, and inflammatory (lymphocyte antigen 6Chi) monocytes as well as reduced transcript for IL-17 compared with untreated animals. Later ( day 8), obstructed kidneys of MSC/paricalcitol double-treated mice, but not mice treated with either intervention alone, had reduced tubular injury and interstitial fibrosis as well as lower numbers of neutrophils and inflammatory monocytes and an increase in the ratio between M2 (CD206+) and M1 (CD206−) macrophages compared with control mice. Adjunctive therapy with VDR agonists may enhance the immunosuppressive properties of MSCs in the setting of pathogenic Th17-type immune responses and related inflammatory responses.

The extent of apoptotic cell death was examined in central nervous system (CNS) tissues from three cases of subacute sclerosing panencephalitis (SSPE). Apoptosis was demonstrated by in situ end-labelling of DNA in formalin-fixed, paraffin-embedded tissue sections. Measles virus and cell types were labelled by immunohistochemistry and/or in situ hybridization. Furthermore, bcl-2 expression in SSPE was examined by immunohistochemistry. All three cases exhibited varying degrees of apoptosis in all CNS areas studied. Brain tissue from a non-neurological control case did not show any significant apoptosis. Characterization of cell types demonstrated neurons, oligodendrocytes, lymphocytes and microglia undergoing apoptosis. A linear relationship could not be established between virus burden and the extent of apoptosis in any particular area. Virus-negative cells were observed which were undergoing apoptosis. Bcl-2 immunoreactivity in SSPE was confined to the infiltrating cell population. These results suggest that apoptosis of various cell types may contribute to the neuropathogenesis of measles virus infection in the human CNS, either as a direct effect of viral infection or by cytokine-mediated responses.

Long-term high-level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to `switch off' some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of β-galactosidase reporter plasmids, promoter `switch off' is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle-directed gene therapy.

Mesenchymal stem cell (MSC) therapy offers the potential to promote recovery after myocardial infarction (MI). However, therapeutic efficacy may be limited by poor survival and retention of transplanted cells. A combination of gene and cell therapy has the capacity to prevent donor cell death and augment the reparative and regenerative effects of cell transfer. The present study investigates the effect of exogenous heat shock protein 27 (Hsp27) expression in MSCs in an in vitro model of ischemia and in an in vivo rat MI model and aims to determine if this could enhance the therapeutic benefit associated with cell delivery. Hsp27 overexpression by lentivirus vector modification resulted in increased MSC survival in vitro and in vivo. Furthermore, decreased apoptosis in the infarcted tissue and improved cardiac function was observed in the Hsp27 group, enhancing the therapeutic effect of MSCs. Together, these data demonstrate that ex vivo genetic modification-specifically Hsp27 overexpression-offers the possibility of enhancing the efficacy of MSC therapy in MI.

Background Calreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This ‘ecto-CRT’ may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Demyelination was induced in the spinal cord by intradermal injection of recombinant mouse MOG mixed with incomplete Freund’s adjuvant (IFA) at the base of the tail. Tissue samples were analysed by semi-quantitative scoring of immunohistochemically stained frozen tissue sections. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. CRT present in rat serum and in a cohort of human serum derived from 14 multiple sclerosis patients and 11 healthy controls was measured by ELISA. Results Stained tissue scores revealed significantly (p Conclusion This data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.

In this study, the antigenic expression of CD34, a 110 kDa glycoprotein which is expressed on human haemopoietic progenitor cells and vascular endothelium, has been assessed in a variety of neuropathological conditions, including infectious and demyelinating disease. Using immunoperoxidase staining on paraffin sections, the immunohistochemical results show that CD34 antigen is expressed widely on human CNS endothelium in grey and white matter, in the eye including retina, and in the anterior and posterior lobes of the pituitary. In demyelinating disease CD34 antigen expression was not detected in acute lesions, whereas strong expression was observed in old lesions. CD34 endothelial positivity was observed in areas of gliosis, vasogenic oedema, vascular disease and in Alzheimer's and Parkinson's disease pathology. A general pattern emerged, with CD34 antigen reactivity predominantly negative in areas of inflammation with demyelination but positive in adjacent non-inflamed tissue, irrespective of myelin pathology. We conclude that perivascular inflammation is a key factor in the absence of immunoreactivity of CD34 in the CNS in demyelinating disease.

The importance for children and young people to be able to communicate openly about the death of a parent is evident from the literature. This small-scale investigation uses a case-study approach to illustrate the impact on siblings of the sudden death of a father. The abundance of comments from the young people in the study such as “talking is the only thing that helps” and “everybody has to get it out” emphasise the important role of communication within the family. Children tend to take their emotional cues from other family members and, paradoxically, restrict communication of their own grief in an attempt to protect others. Even if painful in the short term, certain lines of communication may need to be established if family members are to be able to support each other in dealing with the distressing experience of the death in a healthy manner. The study suggests that those who work with young people in such circumstances should take cognisance of these issues.

Microwave antigen retrieval methods were assessed for a panel of antibodies on formalin-fixed, paraffin-embedded sections from a range of human central nervous system (CNS) tissues taken at post-mortem. The immunoreactivity of a number of antigens (synaptophysin, PGP9.5, GFAP, carbonic anhydrase II, CD68, ferritin, HLA-DR, alpha beta-crystallin, measles and herpes viruses) was markedly enhanced by this procedure compared with other methods of antigen unmasking, such as trypsinization. Enhancement was noted both by an increased number of positive cells and by the intensity of reactions within individual cells and their processes. Furthermore, the microwave method produced uniform immunostaining over large surface area sections with no loss of morphological detail. Large cryostat sections of CNS tissue can be difficult to obtain with good morphology. The ability to retrieve a wide range of antigen in formalin-fixed, paraffin-embedded CNS tissue will greatly increase the range of investigations that can be carried out on this type of stored material.

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.

The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery.In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro.We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.

Dysfunction of the BBB (blood–brain barrier) is a major hallmark of MS (multiple sclerosis). Studies in our laboratories over the last decade have shown that increased BBB permeability is associated with decreased expression of TJ (tight junction) proteins in brain capillary endothelial cells. Results have revealed that TJ abnormalities were most common in active lesions (42% of vessels affected), but were also present in inactive lesions (23%) and in MS normal-appearing white matter (13%). Importantly, TJ abnormality was also positively associated with leakage of the serum protein fibrinogen which has recently been shown to be an activator of microglia. TJ abnormality and the resultant vascular permeability in both lesional and non-lesional white matter may impair tissue homoeostasis, which may have effects on disease progression, repair mechanisms and drug delivery.

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.

Drug-eluting stents for coronary artery disease results in inhibition of smooth muscle cell (SMC) and endothelial cells which may increase the risk of stent thrombosis. In this study, we attempted to enhance re-endothelialization of deployed stents while simultaneously inhibiting intimal hyperplasia by overexpression of endothelial nitric oxide synthase (eNOS) delivery in the vasculature using an adenovirus gene-eluting stent. Re-endothelialization was significantly greater in vessels obtained from normocholesterolemic animals at day 14 (85.34% +/- 7.38 versus 62.66% +/- 10.49; P < 0.05) and day 28 (91.1% +/- 10 versus 63.1% +/- 22; P < 0.05) and hypercholesterolemic animals (96.97% +/- 3.2 versus 28.33% +/- 38.76; P < 0.05) at day 28 with AdeNOS-eluting stents. At day 28, there was a significant increase in the lumen size [AdeNOS 2.73 mm(2) +/- 1.18, Ad beta Gal 0.98 mm(2) +/- 0.98, phosphorylcholine (PC) 1.87 mm(2) +/- 1.18; P < 0.05], and a significant reduction in neointimal formation (AdeNOS 2.32 mm(2) +/- 1.13, Ad beta Gal 3.73 mm(2) +/- 0.95, PC 3.2 mm(2) +/- 0.94; P < 0.05), and percent restenosis (AdeNOS 45.23 +/- 20.81, Ad beta Gal 79.6 +/- 20.31, PC 70.16 +/- 22.2; P < 0.05) in AdeNOS-stented vessels in comparison with controls from hypercholesterolemic animals, assessed by morphometry and quantitative coronary angiography (AdeNOS 15.95% +/- 7.63, Ad beta Gal 56.9% +/- 38.6, PC 58 +/- 34.6; P < 0.05). Stent-based delivery of AdeNOS results in enhanced endothelial regeneration and reduction in neointimal formation as compared with controls. This seems to be a promising treatment strategy for preventing in-stent restenosis (ISR) while simultaneously reducing the risk of stent thrombosis.

Electroporation is a powerful method for gene delivery to dystrophic muscle in the mdx mouse model of Duchenne muscular dystrophy. Successful transfer of reporter and therapeutic plasmids and antisense oligonucleotides has been demonstrated. However, the efficiency falls with increasing plasmid size. Although it is unlikely that the electrotransfer approach will be useful clinically, it is an important experimental tool, particularly in testing potential immune responses to gene transfer in the absence of vector proteins.

Electroporation is a powerful method for gene delivery to dystrophic muscle in the mdx mouse model of Duchenne muscular dystrophy. Successful transfer of reporter and therapeutic plasmids and antisense oligonucleotides has been demonstrated. However, the efficiency falls with increasing plasmid size. Although it is unlikely that the electrotransfer approach will be useful clinically, it is an important experimental tool, particularly in testing potential immune responses to gene transfer in the absence of vector proteins.

The change in communicative satisfaction of twenty laryngectomees (ten oesophageal and ten Servox speakers) and their spouses was assessed through two questionnaires conducted in interview form at one week apart. One interview assessed pre-operative satisfaction, the other post-operative satisfaction, in a variety of situations and with a variety of speaking partners. The communicative satisfaction of the laryngectomees as a group and the spouses as a group was compared pre- and post-operatively. The changes in communicative satisfaction post-laryngectomy experienced by the Servox speakers was compared with those experienced by the oesophageal speakers. Similarly, the changes in communicative satisfaction experienced by the laryngectomees were compared with those experienced by their spouses. A paired t test revealed a significant decrease in communicative satisfaction of the laryngectomees and their spouses post-laryngectomy. In two situations (when speaking on the telephone and with strangers) the laryn...

Background Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. Methods Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. Results In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. Conclusions AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.

By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.

Mesenchymal stem cells (MSCs) have been proposed for use in combinatorial gene and cell therapy protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.

Top of pageAbstract Introduction: Gene eluting stents are now being evaluated in animals as an alternative approach to inhibiting in stent restenosis. AAV and particularly adenovirus are commonly used for gene transfer applications. However, it is unknown whether these vectors can be eluted from a stent over a prolonged period. We tested the hypothesis that these vectors can achieve a prolonged and localized gene delivery to vessel wall using stents as delivery platform. Methods: Ad|[beta]|Gal and AAV2|[beta]|Gal at a concentration of 5 |[times]| 109 pfu and 5.3 |[times]| 109 drp respectively were used to coat BiodivYsio Hi Matrix stents. Gene transfer was assessed at five time points (3, 7, 14, 21, 28 days) using X-gal staining. After balloon injury, 3.0/11 mm stents were deployed in the external iliac arteries of NZW rabbits. 21 stents were used in the adenovirus group and 23 stents were used in the AAV group. 8 PC coated stents were used as controls. RT-PCR was used to assess distal spread of virus in multiple organs (n=6). Results: Expression of LacZ was demonstrated with both vectors at all time points. In the adenovirus group mean percentage of cells expressing transgene at 3 days was 5.15% which increased gradually to a mean expression of 9.85% at 28 days (p>0.005). Expression was localized to medial cells at 3 day, but it was predominantly seen in the neointimal cells at 28 days. In the AAV group 3 day expression was 8.45% which gradually decreased to 1.73% at 28day (p

The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.