Serial step sections at narrow intervals with immunohistochemistry are required for accurate histological assessment of sentinel lymph node biopsy in oral squamous cell carcinoma.

BACKGROUND: Sentinel lymph node (SLN) biopsy is an accurate staging modality in early oral squamous cell carcinoma (OSCC), but its accuracy relies on labor-intensive histopathology protocols. We sought to determine whether serial step sections with immunohistochemistry (SSSIHC) at narrow intervals of the entire SLN are required to accurately exclude metastasis. METHODS: Consecutive SLN biopsies over a 13-year period were retrospectively evaluated. If the index section was negative for carcinoma, the entire SLN was subjected to SSSIHC at 150 µm intervals. The first section level and total number of section levels to contain carcinoma were recorded. RESULTS: One hundred and eighteen SLN+ from 90 patients were included. SSSIHC upstaged the nodal status in 19.5% of patients. Metastasis was identified in 16.7% and 10.2% beyond section levels 4 and 6, respectively. Among SLNs requiring SSSIHC, 47.5% contained carcinoma in a single section level. CONCLUSION: SSSIHC of the entire SLN at 150 µm intervals are required to identify occult metastasis in OSCC.

Common and new acyclovir resistant herpes simplex virus-1 mutants causing bilateral recurrent herpetic keratitis in an immunocompetent patient.

We investigated thymidine kinase (tk) mutants isolated during multiple episodes of recurrent bilateral acyclovir resistant herpes simplex keratitis in an immunocompetent patient. From one eye, we found a single guanine insertion, previously shown to greatly reduce TK expression, and from the other, a previously unidentified substitution, which genetic experiments confirmed confers drug resistance. The substitution, although distant from substrate binding sites, reduced thymidine phosphorylation 10-20-fold, and acyclovir phosphorylation >100-fold. This phenotype should permit reactivation from latency to cause recurrent disease. The results may have implications for the prevalence and prevention of acyclovir resistance in patients with herpes simplex keratitis.

Quantifying changes in corneal neovascularization using fluorescein and indocyanine green angiography.

PURPOSE: To quantify changes in corneal neovascularization in patients with active keratitis after treatment using color imaging, fluorescein angiography (FA), and indocyanine green angiography (ICGA). DESIGN: Prospective, interventional case series. METHODS: Twelve consecutive patients were studied. A comparison of corneal neovascularization parameters was undertaken before and after resolution of the keratitis. A slit-lamp digital camera acquired images of the neovascularization using color imaging, FA, and ICGA. The best-quality images were selected using a grading system, and the neovascular regions of interest were analyzed using automated in-house software. The parameters of analysis were vessel area, diameter, tortuosity, and FA dye leakage. RESULTS: There was a significant reduction in the area of neovascularization after treatment on color imaging (0.78 mm(2); P < .05), FA (2.33 mm(2); P < .01), and ICGA (2.07 mm(2); P < .01). There was also a significant reduction in mean vessel diameter across the region of interest for each patient, more marked on FA (42.74 to 32.52 µm; P < .01) and ICGA (44.77 to 33.29 µm; P < .01) than on color imaging (29.10 to 25.17 µm; P < .01). A significant change in vessel tortuosity was not observed. There was a significant increase in FA dye leakage time (12.41 seconds; P < .05) after treatment. CONCLUSIONS: We demonstrate application of an objective method for analyzing changes in corneal neovascularization. The excellent vessel delineation with ICGA even in the presence of stromal scars makes it an ideal agent for measurement of vessel parameters. FA is useful at detecting vessel leakage, and the time to leakage provides a possible measure of vessel staging.

Hypoxia regulated gene transcription in human optic nerve lamina cribrosa cells in culture.

PURPOSE: Vascular hypoperfusion, extracellular matrix remodeling and axon loss are pathological characteristics of the glaucomatous optic nerve head. We report a novel study demonstrating transcriptional responses in optic nerve lamina cribrosa (LC) cells exposed to in vitro hypoxic stress. METHODS: Primary cultures of human glial fibrillary acid protein (GFAP) negative LC cells were generated from four donors. Cells were exposed to 24 hours of hypoxic stress (1% O2) or normoxia (21% O2). Hypoxia responsive genes were identified using Affymetrix HG-U133A microarrays (n = 3) and validated with real time PCR (n = 3). Secreted protein was measured by ELISA (n = 4) and cellular protein by Western blot (n = 4). Expression data were annotated with NIH DAVID software and putative transcription factor sites in hypoxia-responsive gene promoters were identified using Core_TF software. RESULTS: Hypoxia-sensitive genes included those involved in apoptosis (e.g., BNIP3), neurogenesis (e.g., STC1), extracellular matrix (e.g., MIF, DDR1/TrkE, and IGFR2), mitochondrion (e.g., CYP1B1) and angiogenesis (e.g., VEGF). Real time PCR for selected genes supported the expression changes identified by microarray. ELISA and Western blot validated corresponding changes in protein production. Promoter sequence interrogation revealed putative conserved transcription factor binding sites (e.g., HIF and CREB) in the promoters of the hypoxia responsive genes. CONCLUSIONS: Our data show that LC cell gene expression is sensitive to reduced oxygen levels in vitro and provides bioinformatic evidence of the potential transcriptional regulators of this response.

Differential global and extra-cellular matrix focused gene expression patterns between normal and glaucomatous human lamina cribrosa cells.

PURPOSE: Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. We report the first study of global and ECM-focused gene transcription differentials between GFAP-negative lamina cribrosa (LC) cells from normal and POAG human donors. METHODS: GFAP-negative LC cell lines were generated from the optic nerve tissue of four normal (n=4) and four POAG (n=4) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was performed using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data. RESULTS: 183 genes were upregulated by greater than 1.5 fold and 220 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, transforming growth factor beta 1 (TGFbeta1), secreted acid protein cysteine rich (SPARC), periostin (POSTN), thrombospondin-1 (THBS1), cartilage linking protein-1 (CRTL-1), and collagen type I (COL1A1), collagen type V (COL5A1), and collagen type XI (COL11A1). Downregulated ECM genes in POAG included fibulin 1 (FBLN1), decorin (DCN), and collagen type XVIII (COL18A1). All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells. CONCLUSIONS: This study reports for the first time that POAG LC cells in-vitro demonstrate upregulated ECM and pro-fibrotic gene expression compared with normal LC cells. This may be a pathological characteristic of this cell type in POAG in-vivo. We believe that the LC cell may be a pivotal regulator of optic nerve head ECM remodeling in POAG and an attractive target for molecular therapeutic strategies in the future.

Influence of cyclical mechanical strain on extracellular matrix gene expression in human lamina cribrosa cells in vitro.

PURPOSE: The mechanical effect of raised intraocular pressure is a recognised stimulus for optic neuropathy in primary open angle glaucoma (POAG). Characteristic extracellular matrix (ECM) remodelling accompanies axonal damage in the lamina cribrosa (LC) of the optic nerve head in POAG. Glial cells in the lamina cribrosa may play a role in this process but the precise cellular responses to mechanical forces in this region are unknown. The authors examined global gene expression profiles in lamina cribrosa cells exposed to cyclical mechanical stretch, with an emphasis on ECM genes. METHODS: Glial fibrillary acid protein negative primary LC cells were generated from the optic nerve head tissue of three normal human donors. Confluent cell passages (n=4) were exposed to 15% stretch at 1 Hz or static conditions for 24 h using the Flexercell system. Gene expression was assessed using Affymetrix U133A microarrays with pooled RNA. Expression levels were normalized using robust multi-chip average (RMA). Expression data was annotated using NIH DAVID software. ECM-related gene expression was validated in an independent experiment using quantitative real-time PCR and protein synthesis was measured using ELISA and immunohistochemistry. RESULTS: Compared with static controls, 805 genes were upregulated and 644 were downregulated by +/-1.5 fold in stretched LC cells. Gene ontologies included ECM, cell proliferation, growth factor activity, and signal transduction. Differentially expressed ECM genes included elastin, collagens (IV, VI, VIII, IX), thrombospondin 1, perlecan, and lysl oxidase. Quantitative PCR demonstrated that the expression of TGF-beta2, BMP-7, elastin, collagen VI, biglycan, versican, EMMPRIN, VEGF, and thrombomodulin were reproducible and consistent with the microarray data. VEGF and TGF-beta2 protein levels were also significantly (p<0.05) increased in stretched cell media supernatants. Immunohistochemistry demonstrated increased EMMPRIN (an extracellular matrix metalloproteinase inducer) protein in human POAG optic nerve head tissue compared to nonglaucomatous controls. CONCLUSIONS: These findings demonstrate that LC cells respond to mechanical stimuli in vitro by transcription of several components and modulators of the ECM. Some of the upregulated ECM genes identified are novel in the context of glaucomatous optic neuropathy (biglycan, versican, EMMPRIN, and BMP-7). The LC cell may represent both an important pro-fibrotic cell type in the optic nerve head and an attractive target for novel therapeutic intervention in POAG.

Transforming growth factor-beta-regulated gene transcription and protein expression in human GFAP-negative lamina cribrosa cells.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head (ONH) include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. Transforming growth factor-beta (TGF-beta) is an important pro-fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non-glaucomatous controls. We hypothesize that in POAG, lamina cribrosa (LC) glial cells respond to elevated TGF-beta, producing a remodeled ONH ECM. Using Affymetrix microarrays, we report the first study examining the effect of TGF-beta1 on global gene expression profiles in glial fibrillary acidic acid (GFAP)-negative LC glial cells in vitro. Prominent among the differentially expressed genes were those with established fibrogenic potential, including CTGF, collagen I, elastin, thrombospondin, decorin, biglycan, and fibromodulin. Independent TaqMan and Sybr Green quantitative PCR analysis significantly validated genes involved in regulation of cell proliferation (platelet-derived growth factor [PDGF-alpha]), angiogenesis (vascular endothelial growth factor [VEGF]), ECM accumulation and degradation (CTGF, IL-11, and ADAMT-S5), and growth factor binding (ESM-1). Bioinformatic analysis of the ESM-1 promoter identified putative Smad and Runx transcription factor binding sites, and luciferase assays confirmed that TGF-beta1 drives transcription of the ESM-1 gene. TGF-beta1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro-fibrotic cell type and an attractive target for novel therapeutic strategies.

Effect of cyclical mechanical stretch and exogenous transforming growth factor-beta1 on matrix metalloproteinase-2 activity in lamina cribrosa cells from the human optic nerve head.

PURPOSE: Extensive remodeling of the lamina cribrosa extracellular matrix occurs in primary open angle glaucoma. The transforming growth factor-beta (TGF-beta) and matrix metalloproteinase (MMP) protein families are implicated in this process. The authors investigated (a). the effect of cyclical mechanical stretch on TGF-beta1 mRNA synthesis, TGF-beta1 protein secretion, MMP-2 protein activity and (b). the effect of exogenous TGF-beta1 on MMP-2 protein activity in human lamina cribrosa cells in vitro. METHODS: Primary human lamina cribrosa cells grown on flexible and rigid plates were exposed to cyclical stretch (1Hz, 15%) or static conditions for 12 and 24 hours. Cells grown on 100-mm plates were exposed to human TGF-beta1 (10 ng/ml) or vehicle (4 mM HCl/1% BSA) for 24 hours. TGF-beta1 mRNA synthesis in stretched and static cells was measured using real-time polymerase chain reaction. TGF-beta1 protein secretion in stretched and static cell media was measured using enzyme linked immunosorbent assay. Gelatin zymography measured MMP-2 activity in stretched, static, TGF-beta1- treated and vehicle-treated cell media. RESULTS: Cyclical stretch induced significant increases in TGF-beta1 mRNA synthesis after 12 hours (**P < 0.01) and TGF-beta1 protein secretion after 24 hours (*P < 0.05). Cyclical stretch significantly (*P < 0.05) increased MMP-2 activity in cell media after 24 hours. Exogenous TGF-beta 1 induced a significant (**P < 0.01) increase in cell media MMP-2 activity after 24 hours. CONCLUSIONS: These results suggest that cyclical stretch and TGF-beta1 modulate MMP-2 activity in human lamina cribrosa cells. TGF-beta 1 and MMP-2 release from lamina cribrosa cells may facilitate matrix remodeling of the optic nerve head in primary open angle glaucoma.