Louise Morgan

Inflammation and dephosphorylation of the tight junction protein occludin in an experimental model of multiple sclerosis.

Multiple sclerosis (MS) is a disease of the CNS in which inflammation, demyelination and neurodegeneration contribute to its initiation and progression. A frequently employed model of MS is experimental autoimmune encephalomyelitis (EAE). Here, to gain new insights into the disease process, an analysis of proteins in extracts of lumbar spinal cord from naïve and EAE rats was undertaken. The data mainly confirm that inflammation and blood-brain barrier (BBB) breakdown are the major hallmarks of disease in this model. Given their importance in the BBB, junctional proteins were further investigated. Occludin, a protein localizing to tight junctions in brain endothelial cells, showed strikingly increased migration in EAE when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increased migration was mimicked by in vitro phosphatase treatment, implying its dephosphorylation in EAE. Occludin dephosphorylation coincided with the onset of inflammation, slightly preceding visible signs of disease, and was just prior to apparent changes in BBB permeability. These findings suggest occludin is a target for signaling processes in EAE, perhaps regulating the response of the BBB to the inflammatory environment as seen in MS.

Protection against blood-brain barrier disruption in focal cerebral ischemia by the type IV phosphodiesterase inhibitor BBB022: a quantitative study.

We examined the effect of BBB022, a type IV phosphodiesterase inhibitor, on blood-brain barrier (BBB) integrity after transient middle cerebral artery occlusion (MCAo) in rats. Male Sprague-Dawley rats were anesthetized with halothane and subjected to 120 min of temporary MCAo by retrograde intraluminal insertion of a nylon suture coated with poly-L-lysine. The drug (BBB022 in saline, 1 mg kg-1 h-1, i.v.) or vehicle (0.9% saline, 1-2 ml kg-1 h-1) was administered by infusion after the onset of MCAo. Four animal groups were studied: Groups A and B were treated by infusion of vehicle or drug over 5 h, and groups C and D over 48 h. Damage to the BBB was judged by extravasation of Evans blue (EB) dye, which was administered i.v. at 3 h after the onset of MCAo in groups A and B; and at 46 h in groups C and D. Fluorometric quantitation of EB was performed 1 or 2 h later in six brain regions. In the 5-h infusion series (group B), BBB022 decreased dye extravasation in the ipsilateral cortex, striatum and hemisphere (hemisphere mean+/-S.E.M. : 41.2+/-5.4 vs. 82.4+/-9.2 microg/g, p=0.005) compared to the vehicle-treated group (A). The 48-h infusion of BBB022 (group D) also decreased dye extravasation in the ipsilateral cortex (7.4+/-2. 5 vs. 29.0+/-8.3 microg/g, p=0.05), striatum (17.2+/-2.2 vs. 50. 8+/-12.1 microg/g, p=0.03) and hemisphere (30.7+/-4.0 vs. 93.2+/-18 microg/g, p=0.01) compared to the vehicle-treated group (C). BBB022 also significantly improved the neurological score at 3 and 5 h after MCAo (in the 5-h infusion group) and at 60 min, 24 h and 48 h (in the 48-h infusion group) compared to the vehicle groups. These data indicate that BBB022 prevents ischemic damage to the BBB after focal cerebral ischemia in rats.

Tissue Engineering the Cornea: The Evolution of RAFT.

Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro.

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system

c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.

Human-derived feeder fibroblasts for the culture of epithelial cells for clinical use

AIM To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. METHODS HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. RESULTS HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. CONCLUSION HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model

A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143-148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate-dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of approximately 125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the approximately 125 kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I-specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release.