We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to ...assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.
Several molecular subtypes of sporadic Creutzfeldt–Jakob disease have been identified and electroencephalogram and cerebrospinal fluid biomarkers have been reported to support clinical diagnosis but ...with variable utility according to subtype. In recent years, a series of publications have demonstrated a potentially important role for magnetic resonance imaging in the pre-mortem diagnosis of sporadic Creutzfeldt–Jakob disease. Magnetic resonance imaging signal alterations correlate with distinct sporadic Creutzfeldt–Jakob disease molecular subtypes and thus might contribute to the earlier identification of the whole spectrum of sporadic Creutzfeldt–Jakob disease cases. This multi-centre international study aimed to provide a rationale for the amendment of the clinical diagnostic criteria for sporadic Creutzfeldt–Jakob disease. Patients with sporadic Creutzfeldt–Jakob disease and fluid attenuated inversion recovery or diffusion-weight imaging were recruited from 12 countries. Patients referred as ‘suspected sporadic Creutzfeldt–Jakob disease’ but with an alternative diagnosis after thorough follow up, were analysed as controls. All magnetic resonance imaging scans were assessed for signal changes according to a standard protocol encompassing seven cortical regions, basal ganglia, thalamus and cerebellum. Magnetic resonance imaging scans were evaluated in 436 sporadic Creutzfeldt–Jakob disease patients and 141 controls. The pattern of high signal intensity with the best sensitivity and specificity in the differential diagnosis of sporadic Creutzfeldt–Jakob disease was identified. The optimum diagnostic accuracy in the differential diagnosis of rapid progressive dementia was obtained when either at least two cortical regions (temporal, parietal or occipital) or both caudate nucleus and putamen displayed a high signal in fluid attenuated inversion recovery or diffusion-weight imaging magnetic resonance imaging. Based on our analyses, magnetic resonance imaging was positive in 83% of cases. In all definite cases, the amended criteria would cover the vast majority of suspected cases, being positive in 98%. Cerebral cortical signal increase and high signal in caudate nucleus and putamen on fluid attenuated inversion recovery or diffusion-weight imaging magnetic resonance imaging are useful in the diagnosis of sporadic Creutzfeldt–Jakob disease. We propose an amendment to the clinical diagnostic criteria for sporadic Creutzfeldt–Jakob disease to include findings from magnetic resonance imaging scans.
Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe ...the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease.
Summary
Background Equine heaves is a naturally occurring organic dust‐induced asthma characterized by airway neutrophilia, mucus hypersecretion and obstructive lung dysfunction. However, the ...relative role of different dust components in disease severity remains unclear.
Objective This study investigated the relative contribution of inhaled endotoxin and organic dust particulates (mainly mould spores) in inducing heaves in heaves‐susceptible horses.
Methods Control and heaves‐susceptible horses received inhalation challenges with hay dust suspension (HDS) before and after lipopolysaccharide (LPS) depletion. Heaves‐susceptible horses also received inhalation challenge with HDS particulates with and without the addition of LPS and were housed in two separate dusty environments during which mould and endotoxin exposure was measured. The airway inflammatory and functional response to each challenge was measured.
Results Depletion of endotoxin from HDS attenuated the airway neutrophilia and abrogated the airway dysfunction induced in heaves horses by inhaled HDS. The airway response was re‐established by adding back LPS to the depleted HDS, confirming that the attenuation in airway response was due specifically to endotoxin depletion. Interestingly, the magnitude of alteration in airway response following endotoxin depletion and add‐back was greater than that which could be attributed solely to endotoxin per se, indicating that the LPS activity was enhanced by the other dust components. Consistent with this possibility, washed particulates harvested from HDS enhanced the airway response to inhaled LPS in heaves horses. Heaves horses given two different hay/straw challenges had a significantly different severity of airway inflammation and dysfunction, despite airborne dust and endotoxin concentrations in the horses' breathing zones being similar.
Conclusion Although inhaled endotoxin appears not to be the only determinant of disease severity in heaves, it does contribute significantly to the induction of airway inflammation and dysfunction. This contribution is largely via the synergistic action of inhaled endotoxin and organic dust particulates, although other soluble dust components also contribute to a lesser degree.
Highlights ► Aging altered behavioural function, including learning and memory. ► Aging altered NOS activity and protein expression in memory-related structures. ► Agmatine improved spatial working ...memory and object recognition memory in aged rats.
Highlights • Aging affects brain stem concentrations of l -arginine and its metabolites. • Aging alters spinal cord concentrations of l -arginine and its metabolites. • Regional variations in the ...arginine metabolic profiles. • Altered clusters as a function of aging.
Abstract l -arginine, one of the most metabolically versatile amino acids, can be metabolized to form a number of bioactive molecules. The present study systematically investigated age-related ...changes in l -arginine and its metabolites in the hippocampus, parahippocampal region, and prefrontal cortex at the tissue (crude homogenates) and synaptoneurosome (a subcellular preparation enriched for synaptic material) levels. As aging leads to reduced water content in the brain, age-related changes in neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level were compared. There were significant differences in l -arginine, l -citrulline, l -ornithine, agmatine, putrescine, spermidine, spermine, and glutamate, but not GABA, in the CA1, CA2/3, and dentate gyrus sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, and postrhinal cortices in 24 (aged) and 4 (young) months old rats in a region-specific manner. The overall pattern of age-related changes in amino acids ( l -arginine, l -citrulline, l -ornithine, glutamate, and GABA) was largely similar between homogenates and synaptoneurosomes, whereas the pattern for the amines (agmatine, putrescine, spermidine, and spermine) was quite different. Furthermore, the pattern of age-related changes in neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level was very similar for all 9 neurochemicals measured. These findings suggest that there are differential effects of aging on l -arginine metabolism at the tissue and synaptoneurosome levels and that the way of data normalization (tissue weight vs. protein level) has no or very minor effects on 9 neurochemicals measured.
With respect to sporadic Creutzfeldt-Jakob disease (sCJD), six molecular subtypes (MM1, MM2, MV1, MV2, VV1, and VV2) have been described, which vary with respect to age at disease onset, disease ...duration, early symptoms, and neuropathology. MRI signal alterations were reported to correlate with distinct Creutzfeldt-Jakob disease (CJD) subtypes. This multicenter, international study aimed to describe the brain MRI findings associated with each of the sCJD molecular subtypes.
Pathologically confirmed sCJD cases with codon 129 genotype (MM, MV, and VV), PrP(Sc) type, and fluid-attenuated inversion recovery (FLAIR) or diffusion-weighted imaging (DWI) were collected in seven countries. All MRI scans were assessed for signal changes according to a standard protocol encompassing seven cortical regions, basal ganglia, thalamus, and cerebellum.
MRI scans were evaluated in 211 CJD patients (98 MM1, 23 MM2, 19 MV1, 30 MV2, 9 VV1, and 32 VV2). Basal ganglia hyperintensities occurred most frequently in MV2, VV2, and MM1 subtypes (79, 77, and 70%). Wide cerebral cortical signal increase was most common in VV1, MM2, and MV1 subtypes (86, 77, and 77%). Thalamic hyperintensities occurred most often in VV2 (45%) and MV2 (43%). The most consistent finding across most subtypes was high signal in basal ganglia, with these abnormalities found in 63% (FLAIR) and 71% (DWI).
Cortical signal increase and hyperintensities in the basal ganglia and thalamus are detected by MRI across all molecular sporadic Creutzfeldt-Jakob disease subtypes. Our findings argue that characteristic MRI lesion patterns may occur for each molecular subtype.
Abstract Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory ...responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.
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