Maria Sarris

Mutations in the gene encoding the PML nuclear body protein Sp110 are associated with immunodeficiency and hepatic veno-occlusive disease

We describe mutations in the PML nuclear body protein Sp110 in the syndrome veno-occlusive disease with immunodeficiency, an autosomal recessive disorder of severe hypogammaglobulinemia, combined T and B cell immunodeficiency, absent lymph node germinal centers, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusive disease.

Sutureless nerve repair with laser-activated chitosan adhesive : a pilot in vivo study

OBJECTIVE The anastomosis of peripheral nerves is a demanding procedure that has potential complications due to foreign body reactions elicited by sutures. In this study, the sutureless in vivo anastomosis of rat tibial nerves was successfully performed, using for the first time a chitosan-based laser-activated adhesive. The nerve thermal damage caused by the laser irradiation was quantitatively assessed. MATERIALS AND METHODS A novel adhesive composed of chitosan, indocyanine green, acetic acid, and water, was fabricated in thin sheets. Its adhesive strength was tested in vitro by bonding strips (surface area approximately 20 mm2, thickness approximately 20 microm) onto rat sciatic nerves and sheep intestine by laser activation with low fluence ( approximately 50 J/cm2), using a fiber-coupled diode laser (n = 13). The tensile strength of the adhesive/tissue bonds was measured after tissue repair. The chitosan adhesive was then used to perform sutureless anastomosis of tibial nerves in vivo (n = 6). Adhesive strips were also bonded in vivo onto intact rat sciatic nerves (n = 6) in order to quantitatively assess, by counting myelinated axons, the thermal damage induced by the laser. RESULTS The adhesive bonded well to tissue with a tensile strength of 12.5 +/- 2.6 KPa (mean +/- SD; n = 13). The in vivo anastomosed nerves were in continuity 3 d after surgery. Axon counting showed the number and morphology of myelinated axons were normal proximally ( approximately 96%) compared with intact nerves (100%). Axon demyelination was observed at the operation site ( approximately 49%) and distally ( approximately 27%), and was attributed to laser-induced thermal damage. CONCLUSIONS Nerve anastomosis, performed by the laser-adhesive procedure, was successful 3 d postoperatively. Proximal myelinated axons were not significantly damaged by the low laser fluence.

Localization of the low-affinity nerve growth factor receptor p75 in human limbal epithelial cells

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up‐regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n= 5) and foetal (n= 3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n= 10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin‐embedded archival human skin specimens (n= 4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium‐derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full‐thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium‐derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.

The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities

Aims To investigate the effects of conditioned media from mesenchymal stem cells (MSC) on the wound healing activities of corneal stromal fibroblasts. Methods Cell cycle analysis and early stage activation of apoptosis, chemotactic chambers and fibroblast-populated type I collagen gels were used to assess corneal stromal fibroblast proliferation, migration and contraction, respectively. Fibroblasts were obtained from human donor corneas and MSC from fresh rat bone marrow. MSC conditioned media and fibroblast culture medium (FCM), with and without calf serum supplementation, were compared. Results MSC conditioned media and serum-free FCM had an inhibitory effect on the progression of corneal fibroblasts through the cell cycle. There was a significant increase in the number of cells in the G0–G1 phase for MSC conditioned media and serum-free FCM (p=0.001, p=0.97 respectively). Fibroblast migration and relaxed and stressed gel contraction were significantly inhibited by MSC conditioned media and serum-free FCM compared with FCM with serum (all p=0.001). Glucose and lactate analysis confirmed that these factors were not contributing to this effect. Conclusion MSC conditioned media was found to inhibit the wound healing activities of corneal stromal fibroblasts in vitro. Putative factors secreted by MSC could be developed for therapeutic use in corneal repair.

Sutureless sealing of penetrating corneal wounds using a laser-activated thin film adhesive.

To demonstrate the feasibility of a novel, thin film, laser‐activated adhesive in sealing penetrative corneal wounds with a view to replacing sutures in ophthalmic operations.

Expression of classical components of the renin-angiotensin system in the human eye

Purpose: The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. Methods: We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Results: Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes’ uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. Conclusion: This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.

Opposing Roles for CXCR3 Signaling in Central Nervous System Versus Ocular Inflammation Mediated by the Astrocyte-Targeted Production of IL-12

CXCR3 and its ligands are important for the trafficking of activated CD4(+) T(H)1 T cells, CD8(+) T cells, and natural killer cells during inflammation. Recent functional studies demonstrate a more diverse role of CXCR3 in inflammatory diseases of the central nervous system (CNS). We examined the impact of CXCR3 on a less complex interferon-γ-dependent, type 1 cell-mediated immune response in the CNS, induced in mice by the transgenic production of glial fibrillary acidic protein IL-12 (GF-IL12) by astrocytes and retinal Müller cells. GF-IL12 mice develop ataxia because of severe cerebellar inflammation but have little overt ocular disease. Surprisingly, CXCR3-deficient GF-IL12 mice (GF-IL12/CXCR3KO) have drastically reduced ataxia but developed cataracts, severe ocular inflammation, and eye atrophy. Most GF-IL12/CXCR3KO mice had minimal cerebellar inflammation but severe retinal disorganization, loss of photoreceptors, and lens destruction in the eye. The number of CD3(+), CD11b(+), and natural killer 1.1(+) cells were reduced in the CNS but highly increased in the eyes of GF-IL12/CXCR3KO compared with GF-IL12 mice. High levels of interferon-γ, IL-1, tumor necrosis factor α, CXCL9, CXCL10, and CCL5 were found in GF-IL12 cerebelli and GF-IL12/CXCR3KO eyes. Our findings demonstrate key but paradoxical functions for CXCR3 in IL-12-induced immune disease in the CNS, promoting inflammation in the brain yet restricting it in the eye. We conclude that the function of CXCR3 in cellular immune disease is driven by a common trigger and is controlled by tissue-specific factors.

Cadaveric Porcine Model for Teaching and Practicing Conjunctival Autograft Creation.

Purpose: Conjunctival autografting is a technically difficult step of pterygium surgery, and novice surgeons have limited opportunities to develop and practice their surgical skills. The porcine eye model closely approximates the human eye in tissue consistency when preparing conjunctival and limbal–conjunctival grafts. This study assessed the efficacy of a cadaveric porcine model in teaching and improving a novices skills of conjunctival autograft creation. Methods: A novice was taught to prepare 5 × 5 mm conjunctival grafts and created 58 grafts on fresh porcine eyes. The conjunctival graft thickness was measured using standard histological techniques. Results: Between grafts 1–10 and grafts 49–58, there was a statistically significant difference in both thickness (P < 0.0001; mean thickness, 133 ± 27 and 87 ± 23 &mgr;m, respectively) and time of creation (P = 0.037; median time, 191 and 126 seconds, respectively). Conclusions: The cadaveric porcine model may be a useful method for teaching this important technique to novice surgeons.

Limbal Dermoid Epithelium Shares Phenotypic Characteristics Common to Both Hair Epidermal and Limbal Epithelial Stem Cells

Abstract Purpose: To determine putative limbal epithelial stem cell marker expression in human limbal dermoids compared to stem cell niches in normal limbus and hair follicles of normal human dermis. Methods: Human limbal dermoids (n = 7), normal skin (n = 2) and normal limbal (n = 7) tissue were examined. Immunohistochemistry was performed on paraffin embedded specimens using automated and manual immunostaining with primary antibodies to CK15, CK14, Cadherin-P (CDH3), Wnt-3, Wnt-4, Wnt-5a, Dickkopf (DKK)-3, Sox-2, Sox-10, Sox-13, PEDF, NGFR p75 and β-catenin. Results: Positive immunostaining was found for CK15, CK14, CDH3, NGFR p75, PEDF, Sox-2, Sox-10 and Wnt 4 in the basal dermoid epithelium, limbus and hair follicles. Suprabasal epithelium was immunostained with PEDF, Sox-2 and Wnt-4 in these tissues. The sebaceous and sweat glands, vascular endothelium and nerves of the limbal dermoid immunostained with PEDF and Sox-2. Sebaceous and sweat glands stained for Sox-10. DKK-3 immunostaining occurred in the dermoids’ suprabasal epithelium and vascular endothelium but not in the limbus or hair follicle. Conclusion: Human limbal dermoids share a similar antigenic expression profile similar to the basal limbal epithelium and to the stem cell niche of hair follicles. This supports the notion that limbal dermoids could have properties in common with limbal and/or dermal epithelial stem cells.

Differential expression of the nm23 protein in the progression of oesophageal adenocarcinoma.

Aim: Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma. Methods: Paraffin‐embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n=46), some of which were accompanied by tissue from areas with high‐grade dysplasia (n=24) and from metastasis in regional lymph nodes (n=16) were studied. Histologically normal oesophageal glandular tissue (of cardiac‐type) (n=32) obtained from areas of the resections located away from the primary tumour masses and archival tissues of Barretts metaplasia obtained from endoscopic biopsies (n=77) were used as non‐neoplastic controls. Sections were immunohistochemically stained by the labelled streptavidin‐biotin method using NCL‐nm23 antibody. Results: The total or overall amount of nm23 protein expression paralleled that of cytoplasmic expression and was increased in oesophageal adenocarcinomas (36/46 cases, 78%) when compared with normal oesophageal glandular epithelium (2/32, 6%), Barretts metaplasia (8/77, 10%) and dysplasia (14/24, 58%). In metastatic carcinoma in regional lymph nodes, overall nm23 expression was similar in proportion (13/16, 81%) to that seen in primary carcinoma. In the analysis of the sequential development of oesophageal adenocarcinoma based on non‐neoplastic, preneoplastic and neoplastic archival material, it was found that a high level of overall nm23 expression occurred firstly at the transition from Barretts metaplasia (8/77, 10%) to dysplasia (14/24, 58%). Nuclear nm23 expression was low in dysplastic tissue (with none of the cases having a high level of nuclear nm23 expression) followed by increased levels as the lesion progressed to invasive adenocarcinoma (13/46, 28%) and metastatic carcinoma in regional lymph nodes (10/16, 63%). However, nm23 expression did not appear to correlate with sex, age, tumour size, extent of tumour infiltration into the oesophageal wall, presence of lymph node metastasis or overall patient survival. Conclusion: An overall increase in nm23 expression or increase in nm23 expression in the cytoplasm of cells may be important in the early development of oesophageal adenocarcinoma but increased levels of nuclear nm23 occur in its progression to metastatic disease.