Trevor Gray

Depletion of 26S Proteasomes in Mouse Brain Neurons Causes Neurodegeneration and Lewy-Like Inclusions Resembling Human Pale Bodies

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinsons disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinsons disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment.

Clostridium difficile is the aetiological agent of pseudomembranous colitis, and animal studies suggest the essential role of secreted toxin A in inducing disease. This study examined the biological responses to toxin A by human intestinal epithelial cells. Confluent monolayers of Caco2, HT29, and T84 cells and primary epithelial cells in organ cultures of human colonic biopsy specimens and after detachment with EDTA were studied. Interleukin 8 was assayed using enzyme linked immunosorbent assay (ELISA). Purified C difficile toxin A induced cell rounding and detachment of monolayers of the epithelial cell lines. Cells in detached monolayers initially remained viable while adherent to each other. Subsequently, an increasing number of apoptotic cells appeared in suspension. Exposure to toxin A for 24 hours induced interleukin 8 production in T84 and HT29 cells. Toxin A also induced epithelial cell rounding, detachment, and apoptosis in organ cultures of human colonic biopsy specimens. During culture (in medium only), EDTA detached colonic epithelial cells produced interleukin 8 and cell death occurred by apoptosis. Colonic disease by C difficile may be initiated by toxin A mediated induction of epithelial cell interleukin 8 production and apoptosis after cell detachment from the basement membrane. Studies on isolated (toxin untreated) colonic epithelial cells suggest that interleukin 8 production and apoptosis occur as a consequence of cell injury and detachment.

Human corneal anatomy redefined: a novel pre-Descemet's layer (Dua's layer).

PURPOSE To define and characterize a novel pre-Descemets layer in the human cornea. DESIGN Clinical and experimental study. PARTICIPANTS We included 31 human donor sclerocorneal discs, including 6 controls (mean age, 77.7 years). METHODS Air was injected into the stroma of donor whole globes (n = 4) and sclerocorneal discs (n = 21) as in the clinical deep anterior lamellar keratoplasty procedure with the big bubble (BB) technique. The following experiments were performed: (1) creation of BB followed by peeling of the Descemets membrane (DM); (2) peeling off of the DM followed by creation of the BB, and (3) creation of the BB and continued inflation until the bubble popped to measure the popping pressure. Tissue obtained from these experiments was subjected to histologic examination. MAIN OUTCOME MEASURES Demonstration of a novel pre-Descemets layer (Duas layer) in the human cornea. RESULTS Three types of BB were obtained. Type-1, is a well-circumscribed, central dome-shaped elevation up to 8.5 mm in diameter (n = 14). Type-2, is a thin-walled, large BB of maximum 10.5 mm diameter, which always started at the periphery, enlarging centrally to form a large BB (n = 5), and a mixed type (n = 3). With type-1 BB, unlike type-2 BB, it was possible to peel off DM completely without deflating the BB, indicating the presence of an additional layer of tissue. A type-1 BB could be created after first peeling off the DM (n = 5), confirming that DM was not essential to create a type-1 BB. The popping pressure was 1.45 bar and 0.6 bar for type-1 BB and type-2 BB, respectively. Histology confirmed that the cleavage occurred beyond the last row of keratocytes. This layer was acellular, measured 10.15 ± 3.6 microns composed of 5 to 8 lamellae of predominantly type-1 collagen bundles arranged in transverse, longitudinal, and oblique directions. CONCLUSIONS There exists a novel, well-defined, acellular, strong layer in the pre-Descemets cornea. This separates along the last row of keratocytes in most cases performed with the BB technique. Its recognition will have considerable impact on posterior corneal surgery and the understanding of corneal biomechanics and posterior corneal pathology such as acute hydrops, Descematocele and pre-Descemets dystrophies. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any materials discussed in this article.

Morphological characteristics of the limbal epithelial crypt

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells. Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus. Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3−/CK19+/CD 34−/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)−. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes. Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Optimization of Amniotic Membrane (AM) Denuding for Tissue Engineering

INTRODUCTION Amniotic membrane (AM) has gained increasing popularity as a useful carrier for ex vivo-expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable, and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardized alternative. METHODS AMs preserved for transplantation were denuded using published ethylenediaminetetraacetic acid (EDTA)- and dispase-based methodologies and our novel thermolysin-based procedure. Scanning and transmission electron microscopy and immunohistochemistry, for BM components (collagens IV and VII, laminin 5, and integrins alpha6 and beta4), were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM. RESULTS EDTA- and dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity. CONCLUSION Current procedures for preparing AM are variable and often ineffective, resulting in nonstandard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose that this novel thermolysin procedure may potentially improve overall generation of tissue-engineered constructs using AM.

Differential lamina propria cell migration via basement membrane pores of inflammatory bowel disease mucosa

BACKGROUND & AIMS In active inflammatory bowel disease (IBD), the intestinal mucosa is infiltrated by polymorphonuclear cells (PMNs), lymphocytes, and monocytes from the systemic circulation. Using an ex vivo model, we have investigated luminally directed migration of cells out of the lamina propria. METHODS Fresh untreated and deepithelialized mucosal samples were studied by electron microscopy. Cells migrating out of the lamina propria were investigated by immunohistochemistry and fluorescence-activated cell sorter analysis. RESULTS In intact IBD mucosal samples, tunnels containing cells were prominent in the lamina propria matrix, and PMNs, but not other cell types, were prominent in the epithelium. In deepithelialized mucosal samples, the basement membrane was either destroyed or contained numerous large pores. During culture of deepithelialized mucosal samples, many cells (3.3 [+/-0.8] x 10(5) . g tissue-1 . h-1) migrated out of the lamina propria via basement membrane pores. PMNs and eosinophils were prominent during the first 3 hours of culture, but T cells predominated thereafter. Macrophages also migrated, but B cells were the minority population (<2%) at all times. CONCLUSIONS In active IBD mucosa with an intact epithelium, luminally directed migration of lamina propria cells is restricted mainly to PMNs. After loss of the epithelium, other cell types also migrate into the lumen via numerous, large, basement membrane pores.

The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer)

Background The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemets layer (Duas layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. Methods The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. Results EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemets membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. Conclusions This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

Histologic Features of Transplanted Amniotic Membrane: Implications for Corneal Wound Healing

PURPOSE To evaluate the histologic changes occurring in the transplanted amniotic membrane in human eyes. DESIGN Observational consecutive case series. PARTICIPANTS Seven consecutive patients who underwent amniotic membrane transplantation (AMT) for bullous keratopathy and subsequently had a penetrating keratoplasty (PK). METHODS Corneal buttons obtained at PK were examined by light and electron microscopy and by immunohistology with antibodies against CD34 (keratocytes), alpha smooth muscle actin and vimentin (myofibroblasts and fibroblasts respectively). Time from AMT to PK ranged from 2 to 32 months. MAIN OUTCOME MEASURES Immunophenotypic characteristics of cells populating transplanted amniotic stroma. RESULTS Amniotic tissue was covered with stratified corneal epithelium with well-defined desmosomes and hemidesmosomes. Transformed corneal stroma-derived cells (CSDCs) could be seen migrating from the anterior stroma, through breaks in the Bowmans zone, into connective tissue of the amniotic membrane. Immunohistology showed that the cells populating amniotic stroma were CD34 negative but positive for vimentin and alpha smooth muscle actin. In 2 samples in which corneal transplants were performed approximately 1 year or more after AMT, some cells in the amniotic stroma showed CD34+ staining. Features of increased metabolic activity and formation of new collagen were seen on electron microscopy. In 2 cases, epithelial cell nests were seen in the amniotic stroma. CONCLUSIONS The amniotic basement membrane facilitates epithelial cell migration and adhesion. The amniotic stroma supports CSDCs and epithelial cells. Repopulation of the amniotic stroma by CSDCs migrating through breaks in Bowmans zone integrates the amnion with corneal tissue and allows for rebuilding of corneal stroma. Over time, some CSDCs may revert to the resting keratocyte immunophenotype.

Phenotypic and functional characterisation of myofibroblasts, macrophages, and lymphocytes migrating out of the human gastric lamina propria following the loss of epithelial cells

BACKGROUND The basement membrane of human colonic mucosa contains numerous discrete pores. We have recently shown that following loss of the surface epithelium, many cells migrate out of the colonic lamina propria via basement membrane pores. AIMS To characterise cells migrating out via basement membrane pores of the human gastric lamina propria, following loss of the surface epithelium. METHODS Fresh human gastric mucosal samples were completely denuded of epithelial cells and placed in culture. Tissue samples were studied by electron microscopy (EM) and cells by EM, FACS analysis, immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR). RESULTS EM showed numerous discrete pores (0.65–8.29 μm in diameter) in the subepithelial basement membrane. During culture of mucosal samples denuded of epithelial cells, lymphocytes, macrophages, and myofibroblasts migrated out of the lamina propria via the basement membrane pores. The lymphocytes were predominantly CD45RO+ and CD69+ T cells. Macrophages were shown to express cyclooxygenase (COX) 1 and 2 enzymes. Myofibroblasts were established in culture and, despite prolonged culture and passage, retained their phenotype. They expressed mRNA and protein for COX 1 and 2 enzymes and their release of prostaglandin E2 was inhibited by selective COX 1 and 2 inhibitors. CONCLUSIONS Lamina propria cells migrating out of cultured denuded gastric mucosal samples have been characterised phenotypically and functionally. Such cells would be suitable for studies of their interactions with epithelial cells and also with Helicobacter pylori and its products.

Kinetics of immune cell migration at the human ocular surface

Aim: The conjunctiva has a resident population of intraepithelial and stromal immune cells. These cells play an active part in ocular surface defence and disease. Our aim was to study the migration of immune cells in the human conjunctiva, across the basement membrane and to characterize their phenotypes. Methods: Organ cultures of human conjunctival samples, denuded of the epithelium, were maintained for varying time periods. Cells migrating on to the surface were harvested and analysed by flow cytometry. Conjunctival samples were also studied by immunohistology and electron microscopy. Results: A preferential unidirectional migration of immune cells from the stroma, through pores in the basement membrane, towards the surface was noted. Cells migrated through intrastromal channels, communicating with the surface basement membrane pores. CD3+ T cells (76.18%) were the predominant migrating phenotype. The ratio of CD4:CD8 T cells was approximately 4:1 as compared with the control conjunctiva where the ratio was approximately 2:1. Various other phenotypes including NK, NKT and B cells were also detected. Only 8.41% of the migrating population expressed the Human mucosal lymphocyte-1marker of intraepithelial lymphocytes. Conclusions: Immune cells migration at the ocular surface is an active process involving the formation of intrastromal channels, and cell egression through intact basement membrane pores. The preferential migration of CD4 T cells indicates that this is a specific response of conjunctival lymphoid tissue and not a passive movement of cells. A wide range of immune cell phenotypes exist at the ocular surface. This model can serve to test in vitro the effects of injurious agents on the ocular surface.