Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

Inhibitors of alpha-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson's disease and related disorders.

An abundance of genetic, histopathological, and biochemical evidence has implicated the neuronal protein, alpha-synuclein (alpha-syn) as a key player in the development of several neurodegenerative diseases, the so-called synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. Development of disease appears to be linked to events that increase the intracellular concentration of alpha-syn or cause its chemical modification, either of which can accelerate the rate at which it forms aggregates. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or altered forms of alpha-syn, such as truncations, missense mutations, or chemical modifications by oxidative reactions. Aggregated forms of the protein, especially newly formed soluble aggregates, are toxic to cells, so that one therapeutic strategy would be to reduce the rate at which such oligomerization occurs. We have therefore designed several peptides and also identified small molecules that can inhibit alpha-syn oligomerization and toxicity in vitro. These compounds could serve as lead compounds for the design of new drugs for the treatment of PD and related disorders in the future.

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated.

Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined.

Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination.

Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05).

We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

Objective: To investigate effects of cryopreservation on sperm motility and DNA integrity. Design: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. Setting: A hospital andrology laboratory. Patient(s): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. Intervention(s): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. Main Outcome Measure(s): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. Result(s): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. Conclusion(s): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

A strategy for designing inhibitors of alpha-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders.

Convergent biochemical and genetic evidence suggests that the formation of alpha-synuclein (alpha-syn) protein deposits is an important and, probably, seminal step in the development of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human alpha-syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing alpha-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7-mer peptides spanning the entire alpha-syn sequence, and identified amino acid residues 64-100 of alpha-syn as the binding region responsible for its self-association. Modified short peptides containing alpha-syn amino acid sequences from part of this binding region (residues 69-72), named alpha-syn inhibitors (ASI), were found to interact with full-length alpha-syn and block its assembly into both early oligomers and mature amyloid-like fibrils. We also developed a cell-permeable inhibitor of alpha-syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-syn(A53T), a familial PD-associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)-induced DNA damage. Furthermore, the ASID peptide increased (P

SEPT9_v4 expression induces morphological change, increased motility and disturbed polarity

Several lines of evidence indicate that altered expression of SEPT9 is seen in human neoplasia. In particular there is evidence of altered expression of the SEPT9_v4 isoform. The functional consequences of this remain unclear. We have studied the expression of wild-type- and GTP-binding mutants (G144V and S148N) of the SEPT9_v4 isoform in the MCF7 cell line as a model for its deregulation in neoplasia. We find that SEPT9_v4 expression induces dramatic actin cytoskeletal reorganization with the formation of processes around the cell periphery. Expression of the SEPT9_v4 isoform and a G144V mutant cause delocalization of endogenous SEPT9 from filamentous structures but the S148N mutant does not have this effect. In addition SEPT9_v4 isoform expression enhances cell motility and is associated with perturbation of directional movement. Expression of SEPT9_v4 GTP binding mutants also has potent effects on morphology and motility and causes loss of normal polarity, as judged by Golgi reorientation assays. The phenotypes induced by expression of the SEPT9_v4 isoform and the GTP mutants provide an insight into possible mechanisms of SEPT9_v4 function and suggest that the GTPase functions have both ras- and rab-like features. We propose a model in which overexpression of the SEPT9_v4 isoform in neoplasia is associated with perturbation of SEPT9 complexes, leading to phenotypes associated with neoplasia. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.