Adam J. Bentley

Combining immunolabeling and surface-enhanced Raman spectroscopy on cell membranes

We applied surface-enhanced Raman spectroscopy (SERS) to immunolabeled endothelial cells to derive enhanced spectra of the biomolecular makeup of the cellular surface. A two-step immunolabeling protocol with gold-conjugated antibodies coupled with silver enhancement to attach silver nanoparticles to the cell surface was employed. This approach generated ∼50-fold SERS enhancement of spectral signals. The SERS spectra exhibited several SERS-enhanced peaks associated with cell membrane components. The SERS detection of silver nanoparticles proved more far more sensitive than conventional light microscopy techniques. The SERS enhancement allowed us to carry out spectral mapping using wavenumbers associated with membrane components that correlated directly with the distribution of silver nanoparticles. SERS has the potential to detect immunolabeling at lower levels than is possible using conventional immunolabeling methods while simultaneously providing unique, spatially defined, biochemical information.

The use of trehalose-treated freeze-dried amniotic membrane for ocular surface reconstruction

The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM. Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction.

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

Sutureless amniotic membrane transplantation for ocular surface reconstruction with a chemically defined bioadhesive

The purpose of this study was to evaluate the efficiency and safety of a sutureless amniotic membrane transplantation (AMT) for ocular surface reconstruction with a chemically defined bioadhesive (CDB). The CDB was synthesized from aldehyded polysaccharides and epsilon-poly(L-lysine), two kinds of medical and food additives, as starting materials. Biocompatibility assay indicated that the CDB showed excellent biocompatibility with in vitro and in vivo ocular surface tissues and most of the CDB was histologically degraded within 4 weeks. Sutureless AMT using the CDB was safely and successfully performed onto a rabbit scleral surface. Transplanted amniotic membrane (AM) evaluated by histological, electron microscopic- and immunohistochemical examination indicated that the CDB did not affect normal differentiation of the cells or the integrity of the surrounding tissue. Thus, this newly developed CDB was found to be very useful for sutureless AMT for ocular surface reconstruction, without considering the risk of infection. It has the ability to fix AM to the ocular surface for a long time-period without additional inflammation, scarring, or damage to the surrounding tissues.

Effects of Aberrant Pax6 Gene Dosage on Mouse Corneal Pathophysiology and Corneal Epithelial Homeostasis

Background Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6 +/− heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6+/Sey-Neu (Pax6+/−) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77Tg/− transgenics, which over-express Pax6 and model human PAX6 duplication. Methodology/Principal Findings We used electron microscopy to investigate ocular defects in Pax6+/− heterozygotes (low Pax6 levels) and PAX77Tg/− transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZTg/−) mice to investigate corneal epithelial maintenance by LESC clones in Pax6+/− and PAX77Tg/− mosaic mice. PAX77Tg/− mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6+/− mosaics were corrected by introducing the PAX77 transgene (in Pax6+/−, PAX77Tg/− mosaics). Pax6Leca4/+, XLacZTg/− mosaic mice (heterozygous for the Pax6Leca4 missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZTg/− mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6+/− and PAX77Tg/− mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones. Conclusions/Significance Pax6+/− and PAX77Tg/− genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.

Targeted cornea limbal stem/progenitor cell transfection in an organ culture model

PURPOSE To optimize a nonviral gene transfection system targeting the corneal limbal stem/progenitor cells. METHODS A plasmid containing LacZ gene coding for beta-galactosidase (beta-gal) was transfected into human corneal epithelial cells (HCECs) and multilineage progenitor cells (MLPCs) with different transfection reagents, to determine the optimal transfection reagent. In an ex vivo study, the bovine corneal epithelium and limbal stem/progenitor cells were transfected with a microinjection system with a 36-gauge needle that delivered plasmid/transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA) complexes. The transfected corneoscleral discs were cultured in an air-interface culture system. The expression of beta-gal was determined with an X-gal staining assay, and images were acquired with light microscopy and transmission electron microscopy. The expression of cytokeratin K5/14 and K3/K12 in corneal and limbal epithelium was determined by immunohistochemistry. RESULTS The highest percentages of beta-gal expression in HCECs and MLPCs were achieved when the transfection reagent Lipofectamine 2000 was used. Corneal epithelial and limbal basal cells were successfully transfected with the reporter gene by targeted microinjection of plasmid/liposomal complexes. The location of the bovine limbal stem/progenitor cells was confirmed by positive K5/K14 labeling and negative K3/12 labeling. CONCLUSIONS Targeted microinjection of plasmid/liposomal complexes resulted in limbal stem/progenitor cell transfection. This technique has potential for the short-term treatment of corneal diseases.

Novel Sutureless Keratoplasty with a Chemically Defined Bioadhesive

PURPOSE The purpose of this study was to evaluate sutureless keratoplasty using a chemically-defined bioadhesive (CDB) made from food or medical additives. METHODS Sutureless automated lamellar therapeutic keratoplasty (ALTK) using a CDB was performed on three rabbit eyes. Allogenic lamellar graft was transplanted onto the recipient bed using either suture fixation or a sutureless technique using the CDB. Slit-lamp examination was performed at selected intervals to evaluate the grade of epithelialization and the corneal clarity. The rabbits were killed at 90 days after operation and the eyes processed for histology, electron microscopic examination, and immunohistochemistry for cytokeratins and cell junction-related proteins. RESULTS Sutureless keratoplasty was successfully performed with appropriate handling time before the CDB gelatinized. All the glued grafts were rapidly epithelialized within 7 days, and thereafter remained clear and attached for 90 days. Histologic and ultrastructural findings on the sutureless group showed the normal feature of stromal and epithelial cells and the grafts to be closely adhered with no inflammatory or scarring changes on the interface. Immunohistochemistry of the epithelial cells on the sutureless group revealed a similar expression pattern to the control group. CONCLUSIONS These results demonstrate that sutureless keratoplasty using the CDB is easy to perform, with reliable attachment and no fear of toxic effects or disease transmissions. The authors expect the CDB to become a major choice for corneal treatment, especially in lamellar keratoplasty, posterior keratoplasty, and amniotic membrane transplantation on corneas.

Near-field photothermal microspectroscopy for adult stem-cell identification and characterization

The identification of stem cells in adult tissue is a challenging problem in biomedicine. Currently, stem cells are identified by individual epitopes, which are generally tissue specific. The discovery of a stem‐cell marker common to other adult tissue types could open avenues in the development of therapeutic stem‐cell strategies.

Imaging sclera with hard X-ray microscopy

In this study, the organization of collagen fibrils within the sclera of the eye was investigated using the 7 keV hard X-ray microscope of the Pohang light source and compared to images from electron and atomic force microscopy. From the captured X-ray images, individual collagen fibrils were observed clearly in a spatial resolution much better than 100 nm, both in longitudinal sections and in transverse sections. Some of the collagen fibrils showed evidence of axial periodicity. In some regions of the samples, we could see cross-bridge like structures between adjacent collagen fibrils. The X-ray microscope also allowed the observation of keratocytes and the lamella structure of the scleral stroma. The X-ray microscope has some unique advantages in the nano-scale imaging of bio-samples relative to other established imaging techniques.

Expression of ERα, its ERαΔ3 splice variant and γ-SYNUCLEIN in ovarian cancer: a pilot study.

AIMS: Ovarian cancer has the highest mortality of any gynaecological malignancy; this is due to rapid peritoneal spread of tumour cells and neovascularization. Understanding the mechanisms underlying this is critical to developing early diagnostic or treatment strategies. We devised a pilot study to examine the role of γ-SYNUCLEIN (γ-SYN), oestrogen receptor (ER)α, and the splice variant ERαΔ3. METHODOLOGY: With ethical approval, ovarian tissue was collected from patients (n=24) undergoing oopherectomy for non-ovarian pathology or primary surgery for suspected ovarian cancer. Quantitative gene expression analysis was employed for γ-SYN, ERα, and ERαΔ3. To identify the in situ localization, immunofluorescence for γ-syn was carried out. RESULTS: Ovarian tumour tissue exhibited an elevated expression of γ-SYN and high-grade tumours had an elevated ERαΔ3:ERα ratio compared with benign tissue. The majority of previous studies point to the γ-syn protein being present in epithelial cells of high-grade disease. Our study supports this, but additionally we conclusively identify its presence in the endothelial cells of vasculature surrounding low-grade disease; immunofluorescence was strongest in the apical cells surrounding the lumen. CONCLUSION: Our results demonstrate for the first time that there are readily-expressed levels of γ-SYN and ERαΔ3 in normal ovarian tissue and ovarian tumours. In high-grade disease, γ-syn and an elevated ERαΔ3:ERα ratio might confer metastatic potential to the tumourigenic cells and promote neoangiogenesis. Future in vitro studies might be necessary to delineate such a mechanism, which could potentially be the basis of early intervention.