T. Dietrich

Cutting Edge: Lymphatic Vessels, Not Blood Vessels, Primarily Mediate Immune Rejections After Transplantation

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin α5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

Safety profile of topical VEGF neutralization at the cornea.

PURPOSE Bevacizumab eyedrops inhibit corneal neovascularization. The purpose of this study was to analyze the safety profile of VEGF-A neutralization at the ocular surface. METHODS Bevacizumab eyedrops (5 mg/mL) and an antimurine VEGF-A antibody (250 microg/mL) were applied to normal murine corneas five times a day for 7 and 14 days. Subsequently, corneas were analyzed for morphologic changes by light and electron microscopy. In a mouse model of corneal epithelial abrasion, the effects of topically applied anti-VEGF antibodies on epithelial wound healing were analyzed: the treatment group received bevacizumab (5 mg/mL) or the antimurine VEGF-A antibody (250 microg/mL) as eyedrops, and the control group received an equal volume of saline solution. After 12, 18, and 24 hours, corneas were photographed in vivo with and without fluorescein staining for morphometry. Afterwards the mice were killed, and eyes were removed for histology, immunohistochemistry with Ki67/DAPI, and electron microscopy. The effect of midterm anti-VEGF therapy on corneal nerve density was assessed by staining corneas treated with an FITC-conjugated anti-neurofilament antibody and morphometric analysis. RESULTS Murine corneas treated with two different types of anti-VEGF antibody eyedrops did not show obvious corneal morphologic changes at the light and electron microscopic levels. Furthermore, anti-VEGF antibody eyedrops had no significant impact on the wound healing process after corneal epithelial injury or on normal murine corneal nerve fiber density. CONCLUSIONS Topical neutralization of VEGF-A at the corneal surface does not have significant side effects on normal corneal epithelial wound healing, normal corneal integrity, or normal nerve fiber density. Therefore, anti-VEGF eyedrops seem to be a relatively safe option to treat corneal neovascularization.

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation.

PURPOSE To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.

Das trockene Auge

ZusammenfassungDie Pathogenese des trockenen Auges ist multifaktoriell und komplex. Neue Forschungsergebnisse zeigen, dass sich ein Entzündungsprozess der Augenoberfläche als „gemeinsame Endstrecke“ aller chronischen Formen des trockenen Auges herauskristallisiert. Daneben sind lokale Hormonimbalanzen, vor allem ein Androgenmangel, entscheidend für dessen Entstehung. Aktuelle Konzepte zur Pathogenese des trockenen Auges und die diagnostischen Basis- und Zusatzverfahren werden erläutert. Die In-vivo-Konfokalmikroskopie erlaubt erstmals die Quantifizierung von Entzündungszellen der Augenoberfläche zur Indikationsstellung und Verlaufskontrolle antientzündlicher Therapieoptionen des trockenen Auges.AbstractThe etiology of dysfunctional tear syndrome (“dry eye”) is multifactorial and complex. Recent evidence suggests an important role of androgens in regulating tear film secretion onto the ocular surface. In addition, inflammatory processes of the ocular surface seem to be the common final pathway of all chronic forms of dry eye. Novel concepts of pathogenesis and state-of-the-art diagnostic tools are discussed. In vivo confocal microscopy allows quantification of ocular surface inflammatory cells. This is of increasing importance for evaluation of anti-inflammatory treatments in dry eye patients.

HIV and hepatitis B/C infections in patients donating blood for use as autologous serum eye drops.

During recent years eye drops from autologous serum have become increasingly popular for treating ocular surface disorders such as persistent corneal epithelial defects and severe forms of dry eye.1–4 Although very successful, one potential disadvantage of this approach—especially because the drops are used in a domestic setting—is the possibility of transmission of viral infections by the erroneous application of the eye drops to the wrong recipient. In a diagnostic laboratory, one single droplet of serum has been reported to have transmitted HIV to a laboratory technician.5 However, serological testing of patients who donate autologous serum is not generally established and there has been no report published so far analysing the rate of unknown viral infections in potential donors of autologous serum eye drops.1–4 We report the …

[The dry eye. Current concepts on classification, diagnostics, and pathogenesis].

ZusammenfassungDie Pathogenese des trockenen Auges ist multifaktoriell und komplex. Neue Forschungsergebnisse zeigen, dass sich ein Entzündungsprozess der Augenoberfläche als „gemeinsame Endstrecke“ aller chronischen Formen des trockenen Auges herauskristallisiert. Daneben sind lokale Hormonimbalanzen, vor allem ein Androgenmangel, entscheidend für dessen Entstehung. Aktuelle Konzepte zur Pathogenese des trockenen Auges und die diagnostischen Basis- und Zusatzverfahren werden erläutert. Die In-vivo-Konfokalmikroskopie erlaubt erstmals die Quantifizierung von Entzündungszellen der Augenoberfläche zur Indikationsstellung und Verlaufskontrolle antientzündlicher Therapieoptionen des trockenen Auges.AbstractThe etiology of dysfunctional tear syndrome (“dry eye”) is multifactorial and complex. Recent evidence suggests an important role of androgens in regulating tear film secretion onto the ocular surface. In addition, inflammatory processes of the ocular surface seem to be the common final pathway of all chronic forms of dry eye. Novel concepts of pathogenesis and state-of-the-art diagnostic tools are discussed. In vivo confocal microscopy allows quantification of ocular surface inflammatory cells. This is of increasing importance for evaluation of anti-inflammatory treatments in dry eye patients.

Tumour-associated lymphangiogenesis in conjunctival malignant melanoma

Background: To evaluate whether tumour-associated lymphangiogenesis, that is the formation of new lymphatic vessels (LVs) induced by a tumour, occurs in and around conjunctival malignant melanoma (MM). Methods: Clinical files and conjunctival specimens of 20 patients with histologically diagnosed conjunctival MM were analysed. Sections were stained with LYVE-1 and podoplanin antibodies as specific lymphatic endothelial markers and Ki67 as proliferation marker. The tumour area and the area covered by LV (LVA), LV number (LVN) and LV density (LVD) were measured within the tumour and in the peritumoural area in digital images of the specimen. The LV results were correlated with the histopathological characteristics, tumour location, recurrence rate, mitomycin C therapy and presence of metastases. Results: LVs were detected in all specimens within the tumour and peritumourally. Significantly more Ki67+ proliferating lymphatic endothelial cells were detected in the tumour and in the peritumoural tissue up to 300 μm compared with the surrounding normal conjunctiva (>300 μm distance). There was a slightly positive correlation between the tumour size and the LVN and LVA in the 50 μm zone adjacent to the tumour. We did not find any significant correlations between LVs and histopathological and clinical characteristics (location, shape, relapses, metastases), possibly due to the small sample sizes. Non-limbal tumours with involvement of tarsus or fornix showed a tendency towards a higher LVD compared with limbal tumours. Conclusion: Conjunctival MMs display tumour-associated LV within and around the tumour. The MM seems to induce lymphangiogenesis not only in the tumour, but also in its proximity.

Penetrating keratoplasty for endothelial decompensation in eyes with buphthalmos.

Purpose. To evaluate the prognosis and complications of penetrating keratoplasty (PKP) for corneal decompensation in eyes with buphthalmos and to analyze the risk factors for graft failure. Patients and Methods. Clinical records of 13 adult and three pediatric patients who underwent PKP for endothelial decompensation with a previous diagnosis of congenital glaucoma of a total of 3,663 corneal transplantations performed in our department between January 1987 and December 2001 were reviewed retrospectively. During the study period, a total of 33 PKPs was performed in 20 eyes with buphthalmos. The median age of the patients at the time of PKP was 39 years (range, 3 to 72). All patients had a history of intraocular surgery, including multiple glaucoma surgeries, cataract extraction, and PKP. The impact of pre-, intra-, and postoperative factors on graft failure and duration of graft clarity was analyzed. Results. Fifty-five percent (11/20) of the eyes received only one graft, 25% (5/20) received two, and 20% (4/20) received three grafts. During a mean follow-up of 87.2 months (range, 4.5–72), graft failure occurred in 18 of 33 grafts (54%). Seven (7/18, 39%) had immunologic graft rejection, and 11 (11/18, 61%) had nonimmunologic graft failure. At the end of the follow-up, 75% (15/20) of the eyes had clear grafts. Duration of graft clarity was found to be significantly shorter in regrafts compared with that of primary grafts (27.0 ± 27.7 versus 56.4 ± 41.0 months, p = 0.02). After PKP, intraocular pressure (IOP) was uncontrolled in 12 (12/33, 36%) grafts. Nine of 20 eyes (45%) required an average of 3.2 cyclodestructive procedures per eye for pharmacologically resistant elevated IOP. The final postoperative vision improved in 70% (14/20) of the eyes and the best visual acuity postoperatively (75% ≥20/400) was significantly better than the preoperative visual acuity (25% ≥20/400, p = 0.0001). Conclusions. Endothelial decompensation due to congenital glaucoma is a very rare indication for PKP. The incidence of graft failure is high, and nonimmunologic reasons are the leading causes of graft failure in this high-risk population. Visual acuity can be significantly improved but is usually still very limited by advanced glaucomatous optic nerve damage and amblyopia. Efficient control of IOP before and after PKP is mandatory in eyes with buphthalmos to avoid graft failure and progress of glaucomatous optic nerve atrophy.

Herstellung von Eigenserumaugentropfen zur ambulanten Therapie

BACKGROUND Autologous serum eye drops are an important therapy option in severe ocular surface disorders and the therapeutic effectiveness has been demonstrated in many clinical studies. The production and use of autologous serum eye drops is strictly controlled by legal regulations in Germany: Both the German Medicines Act (AMG) and the Blood Transfusion Act regulate production, distribution and application, unless it is carried out by one person under controlled conditions in a hospital setting. MATERIAL AND METHODS In cooperation with the ophthalmic clinic and the department of transfusion medicine, a standard operating procedure (SOP) was developed and a license for production and delivery of autologous serum eye drops was obtained from the appropriate local authorities. The experiences of the first two years of practice were analyzed. RESULTS By an interfaculty cooperation, the possibility of legal and feasible out-patient treatment with autologous eye drops has been established at the University Hospital Erlangen. From 07/2005 to 07/2007, there ware 240 prescriptions for autologous serum eye drops. Unexpectedly, a relatively high rate (3.3%) of patients with primarily unknown viral or bacterial infectious diseases were found, which were diagnosed during the screening. These patients had to be excluded from autologous serum eye drop therapy. CONCLUSION The treatment with autologous serum eye drops in an out-patient setting is possible, when the infrastructure for manufacture and delivery is provided in accordance with existing regulations.

[Manufacture of autologous serum eye drops for out-patient therapy : cooperation between ophthalmic clinic and transfusion medicine department].

BACKGROUND Autologous serum eye drops are an important therapy option in severe ocular surface disorders and the therapeutic effectiveness has been demonstrated in many clinical studies. The production and use of autologous serum eye drops is strictly controlled by legal regulations in Germany: Both the German Medicines Act (AMG) and the Blood Transfusion Act regulate production, distribution and application, unless it is carried out by one person under controlled conditions in a hospital setting. MATERIAL AND METHODS In cooperation with the ophthalmic clinic and the department of transfusion medicine, a standard operating procedure (SOP) was developed and a license for production and delivery of autologous serum eye drops was obtained from the appropriate local authorities. The experiences of the first two years of practice were analyzed. RESULTS By an interfaculty cooperation, the possibility of legal and feasible out-patient treatment with autologous eye drops has been established at the University Hospital Erlangen. From 07/2005 to 07/2007, there ware 240 prescriptions for autologous serum eye drops. Unexpectedly, a relatively high rate (3.3%) of patients with primarily unknown viral or bacterial infectious diseases were found, which were diagnosed during the screening. These patients had to be excluded from autologous serum eye drop therapy. CONCLUSION The treatment with autologous serum eye drops in an out-patient setting is possible, when the infrastructure for manufacture and delivery is provided in accordance with existing regulations.