Martina C. Herwig

Morphological and immunohistochemical changes after corneal cross-linking.

Purpose: To present light and electron microscopic as well as immunohistochemical findings after corneal cross-linking (CXL). Methods: Six keratoconus corneas after CXL, 12 keratoconus corneas without CXL, and 7 normal corneas were examined by light microscopy, indirect immunohistochemistry using antibodies against proapoptotic BAX, antiapoptotic survivin, and BCL-2, and anti–smooth muscle actin and, in part, by transmission electron microscopy. Direct immunofluorescence with 4′6-diamidino-2-phenylindole was performed to analyze keratocytes/area in the anterior, middle, posterior, peripheral, and central corneal stroma. Results: The period between CXL and keratoplasty ranged from 5 to 30 months. All keratoconus corneas showed the typical histological changes. Increased proapoptotic BAX expression and/or antiapoptotic survivin expression were noticed in keratocytes and endothelium in 2 keratoconus specimens after CXL. Smooth muscle actin was only observed in subepithelial scar tissue of 2 keratoconus corneas without CXL. Keratoconus corneas after CXL revealed a significant reduction in keratocyte counts in the entire cornea (P = 0.003) compared with keratoconus corneas without CXL and normal corneas. This difference was because of a loss of keratocytes in the anterior (P = 0.014) and middle (P = 0.024) corneal stroma. Keratocytes in CXL corneas were reduced in the center (P = 0.028) and the periphery (P = 0.047). Conclusions: CXL in human keratoconus can cause considerable morphologic corneal changes up to 30 months postoperatively. Especially noteworthy is a long-lasting, maybe permanent, keratocyte loss in the anterior and middle corneal stroma involving the central and peripheral cornea. As long-term corneal damage after CXL is of genuine concern, particular care should be taken to perform this procedure only in accordance with investigational protocols.

Endothelial VE-cadherin expression in human lungs

Cell adhesion molecule vascular endothelial cadherin (VE-cadherin) is the major component of endothelial adherence junctions, maintaining endothelial cell integrity. Studies dealing with constitutive VE-cadherin expression patterns in different pulmonary vessel types (arteries, arterioles, capillaries, venules, veins) or with the influence of physiological factors such as age or sex on VE-cadherin expression have not been published yet. Knowledge of constitutive resp. varying expression patterns not only fundamentally contribute to understanding the role of VE-cadherin in the pathogenesis of pulmonary diseases but also help to develop therapies based on immunotargeting. Hence, endothelial VE-cadherin expression was studied in regular lung tissue. Fifty-eight specimens of regular lung tissue (30 females, 28 males between 1 month and 75 years old) were immunohistochemically stained with an antibody against VE-cadherin. There was strong endothelial expression of VE-cadherin in arteries, arterioles, and capillaries but almost no expression in veins and venules. Neither age nor sex had any influence on the expression pattern or staining intensity. There is a vessel type-specific expression pattern for VE-cadherin in regular human lung tissue, which is not influenced by age or sex. Further studies will have to prove whether this is influenced by pathological conditions, e.g., ARDS.

Sebaceous gland carcinoma of the eyelid masquerading as a cutaneous horn in Li–Fraumeni syndrome

A Caucasian man in his 50s was referred to our department with a diagnosis of cutaneous horn. He presented with an exophytic lesion of the right upper eyelid progressively enlarging over the last 6 months with bleeding on some occasions. His past medical and ocular history were unremarkable. His family history (figure 1), however, revealed cancer in at least three generations. One daughter had been diagnosed with early onset colon cancer, and the other with a malignant parotid neoplasm and breast cancer as a teenager. One brother had an unknown cancer of the face, and the other died shortly after birth of unknown cause. His mother was diagnosed with either a sarcoma or a tracheal carcinoma in her 20s. His aunt had a genitourinary malignancy. Slit lamp examination disclosed a conical horn of the right upper eyelid margin. The surface of the lesion appeared dark with a crusty, hyperkeratotic and ulcerative facade. On everting the lid, a yellowish nodular base was observed (figure 2A). Further clinical examination of both eyes was normal and visual acuity was 20/20 OU. A wedge resection was performed, and the lesion was submitted for histopathologic evaluation (figure 2B). The patient died 5 years later due to colon cancer. Figure 1 Family history of the Li–Fraumeni patient. Shaded symbols indicate cancer; cross-hatch indicates deceased members; tumour type is indicated. The Li–Fraumeni syndrome patient (III: 2) is described in this report. Figure 2 (A) Inspection on everting the eyelid showing a …

VE-cadherin and ACE: Markers for sepsis in post mortem examination?

Altered expression of endothelial markers - especially adhesion molecules - is diagnostically helpful for diagnosis of ante mortal undiagnosed sepsis. Up to now it is unclear whether (1) expression of Angiotensin converting enzyme (ACE) and/or VE-cadherin (VEC) plays a comparable role, (2) whether expression intensity correlates with post mortem interval. Fifty-nine lung specimens (20 lung specimens with regular morphology from tumour lobectomies, 39 from patients who died of septic ARDS due to microbiologically proven Gram-negative sepsis) were stained with an antibody against ACE (1:80) resp. VEC (1:100). All specimens showed vessel type specific expression patterns for ACE and VEC which was dramatically reduced in sepsis. ACE staining intensity did not correlate with time between death and autopsy. VEC staining was slightly but statistically not significantly reduced with increasing time interval. Pulmonary VEC and ACE expression are reduced in septic ARDS. However, as neither ACE nor VEC expression correlates with time interval between death and post mortem, expression intensities of VEC or ACE are no reliable indices for time elapsed since death.

Intravitreally Injected HCmel12 Melanoma Cells Serve as a Mouse Model of Tumor Biology of Intraocular Melanoma

Abstract Purpose: To establish a mouse model with histologic characteristics of uveal melanoma for investigation of intraocular tumor biology of melanoma. Methods: After injection of 1 × 105 of HCmel12 melanoma cells, a cutaneous melanoma cell line, into the vitreous of CX3CR1+/GFP or C57Bl/6 mice (n = 12), tumor growth patterns, clinicopathological features, angiogenesis and metastatic behavior were analyzed by histology (hematoxylin and eosin, periodic acid-Schiff without hematoxylin) and immunohistochemistry (HMB45/MART-1-Ab, F4/80-Ab, green fluorescent protein (GFP)-Ab and VE-cadherin-Ab). Results: HCmel12 cells formed intraocularly growing tumor masses, which showed histologic features of intraocular melanoma such as angiotropism, intratumoral endothelial-lined vasculature, vasculogenic mimicry including prognostic significant extravascular matrix patterns, and invasion by inflammatory cells, in particular macrophages. There was no difference in tumor growth characteristics between CX3CR1+/GFP and C57Bl/6 mice. Five of 10 mice proceeded to extrascleral tumor growth and three of these developed metastases. Conclusions: Intraocularly injected HCmel12 cells developed tumor masses with histologic characteristics of aggressive melanoma similar to human uveal melanoma. Since hematogenous dissemination to the liver was not observed, intravitreally injected HCmel12 cells do not qualify as a model for metastasizing intraocular melanoma. However, since the eye represents a semi-closed compartment with access to constant blood supply, these intraocular tumors represent a model for studies of isolated parameters in general tumor biology of intraocular melanoma.

Distribution and presumed proliferation of macrophages in inflammatory diseases of the ocular adnexae.

ABSTRACT Purpose: The aim of this article is to investigate whether macrophages show a proliferative activity (as indicated by Ki67 expression) and their distribution at the site of inflammation. Materials and Methods: Six different macrophage-containing lesions from six different patients (four females, two males; age range: 16–58 years) were stained for macrophage markers (CD68, CD163) and Ki67 by immunohistochemistry. Immunofluorescence techniques were used to investigate dual-labeling of the specimens for CD68, CD163 and Ki67, respectively. Results: With immunofluorescence staining, scattered cells in all specimens were dual-labeled for CD68–Ki67 and CD163–Ki67. All lesions were composed of mixed infiltrates of M1 (CD68+CD163−) and M2 (CD68+CD163+) macrophages. The center of epithelioid-cell granulomas and foreign body giant cells was exclusively composed of M1 macrophages. Conclusions: This study shows that CD68+ and CD163+ cells express Ki67, a marker for proliferative activity at the site of inflammation. Until recently, macrophages were regarded as end-differentiated cells without mitotic activity. Since self-renewal of M1 and M2 macrophages has been described in the literature, staining of macrophages with Ki67 may indicate proliferative activity or at least an activation state. The distribution of macrophages in classic granulomatous lesions with only M1 macrophages in the avascular center represents an immune response to foreign body material, whereas the proangiogenic M2 macrophages are located mostly in the surrounding inflammatory tissue and seem to be mandatory for the vascularization of the inflammatory tissue.

Preterm diagnosis of choristoma and choroidal coloboma in Goldenhar's syndrome.

In addition to general pathological findings characteristic of Goldenhars syndrome, we report ocular findings in a 22-week-old fetus with hemifacial microsomia, endorsing this diagnosis. After abortion the fetus was examined via a standard paidopathological autopsy including ophthalmopathologic macroscopic and microscopic examination of both eyes. Postmortem findings included left hemifacial microsomia with ipsilateral microtia, atresia of the acoustic meatus, microphthalmia, a ventricular septal defect, and abnormalities of the ribs. Ophthalmopathological examination of the affected microphthalmic eye revealed a scleral choristoma (cartilage), choroidal/retinal pigment epithelium coloboma, and staphyloma. General pathology findings plus the ocular findings allowed the diagnosis of Goldenhars syndrome. The cartilaginous choristoma present in the patient has previously not been reported in association with this syndrome. A discussion of differential diagnoses is provided, confirming that the ophthalmopathological investigation of fetal eyes can be of great value for classifying syndromes associated with microphthalmia.

Corneal clouding in Alport syndrome.

Purpose: Alport syndrome is a hereditary basement membrane disease that typically involves the kidney, the cochlea, and the eyes. Characteristic ocular problems include posterior polymorphous corneal dystrophy, lenticonus, and dot-and-fleck retinopathy. Methods: A 48-year-old male patient with Alport syndrome presented with corneal and retinal changes. In 2003, he was diagnosed with posterior polymorphous corneal dystrophy and received a corneal transplant in his left eye in 2007 because of progressive deterioration in visual acuity. At this time, a lamellar macular hole was diagnosed in his right eye. The removed corneal button was examined by light and electron microscopy and by immunohistochemistry. Results: Histology revealed not only endothelial changes but also a marked irregular thickening of the epithelial basement membrane and of Bowman layer. Alcian blue staining demonstrated an accumulation of mucopolysaccharides in the Bowman layer. Conclusions: The presented changes underline the great variation of ocular disorders related to Alport syndrome. To our knowledge, this is one of the first reports describing histologic corneal findings in Alport syndrome. Only a few cases with accumulation of mucopolysaccharides in the Bowman layer have been described previously, none of them being associated with Alport syndrome. Besides, anterior corneal alterations and corneal clouding seem to be uncommon in patients suffering from Alport syndrome.

Immunolocalization of Different Collagens in the Cornea of Human Fetal Eyes: A Developmental Approach

Purpose: To study the corneal development in the human fetal eye with particular emphasis on the epithelial basement membrane and Bowman’s layer. Thus, immunohistochemical markers supposed to stain this region were employed. Material and Methods: 19 formalin-fixed fetal eyes and a 16-day-old newborn’s cornea without any obvious irregularities of the anterior segment were investigated. The age of the fetal eyes ranged from 11 to 38 week of gestation (WoG). The eyes (including the corneal thickness) were measured and, in addition to routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, immunohistochemical labeling with antibodies to collagen IV, V, IX, and XVII was performed. Results: Analysis of the H&E stains revealed that measurements of corneal thickness correlated well with corneal development as a basic indicator for maturation. In a more detailed immunohistochemical analysis, collagen IV was expressed in the epithelial basement membrane (BM) of the cornea, conjunctiva, and Descemet’s membrane in fetal eyes up to the age of 23 WoG. In fetal eyes older than 23 WoG, staining was confined to the limbal area only. With the antibody against collagen V, the corneal stroma and the BM were intensely stained. Bowman’s layer (first detected at 17 WoG by light microscopy) was not labeled. Anti-collagen IX labeled predominantly the conjunctival and corneal epithelium. With anti-collagen XVII, the BM of the cornea and conjunctiva was stained in all fetal eyes, whereas intracellular expression in the epithelium increased with age. Conclusion: Our results indicate maturation-associated variations of collagen expression in the human cornea. Measurements of the corneal thickness may serve as an additional parameter to narrow down the developmental age with possible implications for pediatric pathology and forensic issues.

Reactive lymphoid hyperplasia of the ocular surface: clinicopathologic features and search for infectious agents

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