Combined Osteopontin Blockade and Type 2 Classical Dendritic Cell Vaccination as Effective Synergetic Therapy for Conjunctival Melanoma.

Angiogenesis and immune protection are essential at the onset of tumorigenesis. Angiogenesis serves to nourish the tumor, and prevention of immune defenses, for example, by dendritic cells (DCs), allows tumor growth. In this study, we investigated whether there are factors with dual functions that are both angiogenic and immunomodulatory and represent a therapeutic target. We analyzed 1) innate immune responses intratumorally and in draining lymph nodes and 2) angiogenic factors in conjunctival melanoma (CM), a potentially lethal malignant tumor at the ocular surface whose immune and vascular responses are largely unknown. For this purpose, an HGF-Cdk4R24C model in immunocompetent C57BL/6 mice was used and revealed that CD103- type 2 classical DC (cDC2s) were the most abundant DC subtype in healthy conjunctiva, whereas in CM, CD103- cDC2s, CD103+ type 1 cDCs, monocyte-derived DCs, and plasmacytoid DCs were significantly increased. In our analysis of angiogenic factors in CM, the examination of 53 angiogenesis-related factors that might interact with DCs identified osteopontin (OPN) as a major tumor-derived protein that interacts with DCs. Consistent with these findings, 3) a dual therapeutic strategy that inhibited tumor cell function by an OPN blocking Ab while enhancing the immune response by cDC2 vaccination resulted in 35% failure of tumor development. Moreover, tumor progression, monocyte-derived DC infiltration, and intratumoral angiogenesis were significantly reduced, whereas survival and CD8+ T cell infiltration were increased in treated mice compared with the control group. Therefore, we identified OPN blockade in combination with cDC2 vaccination as a potential future therapeutic intervention for early stages of CM by combining antiangiogenic and host immune stimulating effects.

Pseudomonas aeruginosa-induced nociceptor activation increases susceptibility to infection.

We report a rapid reduction in blink reflexes during in vivo ocular Pseudomonas aeruginosa infection, which is commonly attributed and indicative of functional neuronal damage. Sensory neurons derived in vitro from trigeminal ganglia (TG) were able to directly respond to P. aeruginosa but reacted significantly less to strains of P. aeruginosa that lacked virulence factors such as pili, flagella, or a type III secretion system. These observations led us to explore the impact of neurons on the host's susceptibility to P. aeruginosa keratitis. Mice were treated with Resiniferatoxin (RTX), a potent activator of Transient Receptor Potential Vanilloid 1 (TRPV1) channels, which significantly ablated corneal sensory neurons, exhibited delayed disease progression that was exemplified with decreased bacterial corneal burdens and altered neutrophil trafficking. Sensitization to disease was due to the increased frequencies of CGRP-induced ICAM-1+ neutrophils in the infected corneas and reduced neutrophil bactericidal activities. These data showed that sensory neurons regulate corneal neutrophil responses in a tissue-specific matter affecting disease progression during P. aeruginosa keratitis. Hence, therapeutic modalities that control nociception could beneficially impact anti-infective therapy.

Radiosensitizing Effects of Irinotecan versus Oxaliplatin Alone and in Combination with 5-Fluorouracil on Human Colorectal Cancer Cells.

To date, oxaliplatin and irinotecan are used in combination with 5-flourouracil (5-FU) for metastatic colorectal cancer. In this study it was tested whether oxaliplatin and irinotecan and their combinations with 5-FU have an enhanced effect when treated simultaneously with ionizing radiation. In addition, it should be compared whether one combination therapy is more effective than the other. Colorectal cancer cells (HT-29) were treated with irinotecan or oxaliplatin, both alone and in combination with 5-FU, and subsequently irradiated. The cell growth, metabolic activity and proliferation of cells were investigated, and the clonogenic survival was determined. Furthermore, the assessment of radiation-induced DNA damage and the influence of the drugs and their combinations on DNA damage repair was investigated. Treatment with irinotecan or oxaliplatin in combination with 5-FU inhibited proliferation and metabolic activity as well as clonogenic survival and the DNA damage repair capacity of the tumor cells. The comparison of oxaliplatin and irinotecan with simultaneous irradiation showed the same effect of both drugs. When oxaliplatin or irinotecan was combined with 5-FU, tumor cell survival was significantly lower than with monotherapy; however, there was no superiority of either combination regimen. Our results have shown that the combination of 5-FU and irinotecan is as effective as the combination of 5-FU with oxaliplatin. Therefore, our data support the use of FOLFIRI as a radiosensitizer.

Pre-incubation of corneal donor tissue with sCD83 improves graft survival via the induction of alternatively activated macrophages and tolerogenic dendritic cells.

Immune responses reflect a complex interplay of cellular and extracellular components which define the microenvironment of a tissue. Therefore, factors that locally influence the microenvironment and re-establish tolerance might be beneficial to mitigate immune-mediated reactions, including the rejection of a transplant. In this study, we demonstrate that pre-incubation of donor tissue with the immune modulator soluble CD83 (sCD83) significantly improves graft survival using a high-risk corneal transplantation model. The induction of tolerogenic mechanisms in graft recipients was achieved by a significant upregulation of Tgfb, Foxp3, Il27, and Il10 in the transplant and an increase of regulatory dendritic cells (DCs), macrophages (Mφ), and T cells (Tregs) in eye-draining lymph nodes. The presence of sCD83 during in vitro DC and Mφ generation directed these cells toward a tolerogenic phenotype leading to reduced proliferation-stimulating activity in MLRs. Mechanistically, sCD83 induced a tolerogenic Mφ and DC phenotype, which favors Treg induction and significantly increased transplant survival after adoptive cell transfer. Conclusively, pre-incubation of corneal grafts with sCD83 significantly prolongs graft survival by modulating recipient Mφ and DCs toward tolerance and thereby establishing a tolerogenic microenvironment. This functional strategy of donor graft pre-treatment paves the way for new therapeutic options in the field of transplantation.

New Therapeutic Approaches for Conjunctival Melanoma-What We Know So Far and Where Therapy Is Potentially Heading: Focus on Lymphatic Vessels and Dendritic Cells.

Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma.

New Technologies in Clinical Trials in Corneal Diseases and Limbal Stem Cell Deficiency: Review from the European Vision Institute Special Interest Focus Group Meeting.

To discuss and evaluate new technologies for a better diagnosis of corneal diseases and limbal stem cell deficiency, the outcomes of a consensus process within the European Vision Institute (and of a workshop at the University of Cologne) are outlined. Various technologies are presented and analyzed for their potential clinical use also in defining new end points in clinical trials. The disease areas which are discussed comprise dry eye and ocular surface inflammation, imaging, and corneal neovascularization and corneal grafting/stem cell and cell transplantation. The unmet needs in the abovementioned disease areas are discussed, and realistically achievable new technologies for better diagnosis and use in clinical trials are outlined. To sum up, it can be said that there are several new technologies that can improve current diagnostics in the field of ophthalmology in the near future and will have impact on clinical trial end point design.

Tyrosinase Is a Novel Endogenous Regulator of Developmental and Inflammatory Lymphangiogenesis.

Lymphangiogenesis is critically involved in tissue fluid balance, graft rejection, and tumor metastasis. Endogenous regulation of lymphangiogenesis is poorly understood. Herein, we use the lymphatic vessel architecture at the limbal border of the normally avascular cornea, a quantitative trait under strong genetic influence, as a model system to identify new candidate genes regulating lymphangiogenesis. Comparing low-lymphangiogenic BALB/cN with high-lymphangiogenic C57BL/6N mice, we performed quantitative trait loci analysis of five phenotypes in a large BALB/cN × C57BL/6N intercross (n = 795) and identified three to eight genome-wide significant loci, the strongest on chromosome 7 containing tyrosinase (Tyr). Tyrosinase-negative mice showed significantly increased limbal lymph vascularized areas, a higher number of lymphatic vessel end points, and branching points and increased inflammation-induced lymphangiogenesis. These findings confirm that tyrosinase is a novel lymphangiogenesis regulator in developmental and inflammatory lymphangiogenesis. Our findings link melanin synthesis with lymphangiogenesis and open new treatment options in lymphangiogenesis-related diseases.

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.

UV light crosslinking regresses mature corneal blood and lymphatic vessels and promotes subsequent high-risk corneal transplant survival.

Immunologic graft rejection is the main complication after corneal transplant into pathologically prevascularized so-called high-risk eyes. The aim of this study was to evaluate whether ultraviolet (UV) light crosslinking can regress pathologic corneal blood and lymphatic vessels and thereby improve subsequent graft survival. Using the murine model of suture-induced corneal neovascularization, we found that corneal crosslinking with UVA light and riboflavin regressed both preexisting blood and lymphatic vessels significantly via induction of apoptosis in vascular endothelial cells. In addition, macrophages and CD45+ cell counts were significantly reduced. Consistently, corneal crosslinking reduced keratocyte density and corneal thickness without affecting corneal nonvascular endothelial cells, iris, and lens depending on the crosslinking duration. Furthermore, using the murine model of corneal transplant, long-term graft survival was significantly promoted (P < .05) and CD4+ CD25+ FoxP3+ T regulatory cells were upregulated (P < .01) in high-risk eyes preoperatively treated with crosslinking. Our results suggest UV light crosslinking as a novel method to regress both pathologic corneal blood and lymphatic vessels and to reduce CD45+ inflammatory cells. Furthermore, this study demonstrates for the first time that preoperative corneal crosslinking in prevascularized high-risk eyes can significantly improve subsequent graft survival and may become a promising novel therapy in the clinic.

Antilymphangiogenic therapy to promote transplant survival and to reduce cancer metastasis: what can we learn from the eye?

The lymphatic vasculature is - amongst other tasks - essentially involved in inflammation, (auto)immunity, graft rejection and cancer metastasis. The eye is mainly devoid of lymphatic vessels except for its adnexa, the conjunctiva and the limbus. However, several pathologic conditions can result in the secondary ingrowth of lymphatic vessels into physiologically alymphatic parts of the eye such as the cornea or the inner eye. Therefore, the cornea has served as an excellent in vivo model system to study lymphangiogenesis, and findings from such studies have substantially contributed to the understanding of central principles of lymphangiogenesis also with relevance outside the eye. Grafting experiments at the cornea have been extensively used to analyze the role of lymphangiogenesis in transplant immunology. In this regard, we recently demonstrated the crucial role of lymphatic vessels in mediating corneal allograft rejection and could show that antilymphangiogenic therapy increases graft survival. In the field of cancer research, we recently detected tumor-associated lymphangiogenesis in the most common malignant tumors of the eye, such as conjunctival carcinoma and melanoma, and ciliochoroidal melanoma with extraocular extension. These neolymphatics correlate with an increased risk of local recurrence, metastasis and tumor related death, and may offer potential therapeutic targets for the treatment of these tumors. This review will focus on corneal and tumor-associated ocular lymphangiogenesis. First, we will describe common experimentally used corneal lymphangiogenesis models and will recapitulate recent findings regarding the involvement of lymphatic vessels in corneal diseases and transplant immunology. The second part of this article will summarize findings about the participation of tumor-associated lymphangiogenesis in ocular malignancies and their implications for the development of future therapeutic strategies.

IL-10 Indirectly Regulates Corneal Lymphangiogenesis and Resolution of Inflammation via Macrophages.

The role of IL-10, a primarily anti-inflammatory cytokine, in the regulation of inflammatory lymphangiogenesis is undetermined. Herein, we show that IL-10 modulates corneal lymphangiogenesis and resolution of inflammation. IL-10 was not expressed in healthy corneas but was up-regulated in inflamed corneas by infiltrating macrophages. Macrophages up-regulated the expression of prolymphangiogenic vascular endothelial growth factor-C upon stimulation with IL-10. Consistently, corneal inflammation resulted in reduced expression of vascular endothelial growth factor-C and decreased corneal lymphangiogenesis in IL-10-deficient mice (IL-10(-/-)). The effect of IL-10 on lymphangiogenesis was indirect via macrophages, because IL-10 did not directly affect lymphatic endothelial cells. The expression of proinflammatory cytokines and the numbers of infiltrating macrophages increased and remained elevated in inflamed corneas of IL-10(-/-) mice, indicating that IL-10 deficiency led to more severe and prolonged inflammation. The corneal phenotype of IL-10 deficient mice was mimicked in mice with conditional deletion of Stat3 in myeloid cells (lysozyme M Cre mice Stat3(fl/fl) mice), corroborating the critical role of macrophages in the regulation of lymphangiogenesis. Furthermore, local treatment with IL-10 promoted lymphangiogenesis and faster egress of macrophages from inflamed corneas. Taken together, we demonstrate that IL-10 indirectly regulates inflammatory corneal lymphangiogenesis via macrophages. Reduced lymphangiogenesis in IL-10(-/-) and lysozyme M Cre Stat3(fl/fl) mice is associated with more severe inflammatory responses, whereas IL-10 treatment results in faster resolution of inflammation. IL-10 might be used therapeutically to terminate pathological inflammation.

[Anti(lymph)angiogenic Strategies to Improve Corneal Graft Survival]. / Antihämangiogene/antilymphangiogene Strategien zur Verbesserung des Transplantatüberlebens nach Keratoplastik.

Corneal transplantation (keratoplasty) is the most frequently performed form of transplantation worldwide. A rejection reaction against the transplant is the main complication occurring after transplantation in an already vascularized, so-called "high-risk" recipient eye. Our group has shown that clinically invisible lymphatic vessels play a crucial role in the induction of a rejection reaction against the corneal graft, and that anti-(lymph)angiogenic therapies in the mouse model of keratoplasty can significantly improve transplant survival. The underlying mechanisms, which improve transplant survival through anti-lymphangiogenic therapies have not been well understood. We assume that the blockade of lymph vessel sprouting leads to a tolerance (and not to a simple ignorance) of the transplant, in which the antigen-presenting cells are held longer in the cornea and, thus, an immunomodulation of these cells occurs. Therefore, an important goal of our project is to find out whether and when transplant tolerance comes from a corneal anti-lymphangiogenic therapy. We assume that the antigen-presenting cells will have a different maturity level and that more tolerogenic effector cells (regulatory T cells, Tregs) develop in the absence of lymphatic vessels. Current anti(lymph)angiogenic therapies have the disadvantage that they are primarily effective on actively growing vessels. Most patients who receive high-risk keratoplasty often present in the clinic with already established, mature corneal blood and lymphatic vessels. At present, there are no lymph vessel regressing strategies, and the mechanisms regulating the maturation of the lymphatics are largely unknown. Therefore, our second goal is to develop new strategies for the regression of existing, pathological lymphatic vessels in the cornea. We are testing both destructive strategies, such as photodynamic therapy and diathermy as well as strategies for the molecular destabilization of the lymph vessel endothelium. Thus, our project identifies the precise mechanisms by which anti-lymphangiogenic therapies improve transplant survival, and we are developing new strategies to push back mature lymphatics in the high-risk setting.

[New Therapeutic Approaches in Inflammatory Diseases of the Eye - Targeting Lymphangiogenesis and Cellular Immunity: Research Unit FOR 2240 Presents Itself]. / Neue Therapieansätze bei entzündlichen Augenerkrankungen durch Modulation von Lymphangiogenese und zellulärer Immunität: Die DFG-Forschergruppe 2240 stellt sich vor.

Background Ophthalmology, principally, is a very successful subdiscipline in medicine. Nonetheless, there are still unmet medical needs which necessitate translational research. Methods The funding instrument of a Research Unit (RU) of the German Research Foundation (DFG) is presented as exemplified by the RU 2240 at the Department of Ophthalmology at the University of Cologne. Results The Research Unit integrates different research groups working on pathologic ocular inflammation, macrophages/microglia and (lymph)angiogenesis to collaborate in a synergistic way. Rotation positions allow young clinicians to rotate into research labs for a defined period of time. A Research Unit is also a powerful strategic tool to strengthen clinical and experimental ophthalmology at individual medical faculties. Conclusions The funding instrument of a Research Unit is highly suitable for fostering translational research in a medical subdiscipline such as ophthalmology, supporting the next generation of (clinician) scientists in ophthalmology and finding new cures for our patients.

Topical application of soluble CD83 induces IDO-mediated immune modulation, increases Foxp3+ T cells, and prolongs allogeneic corneal graft survival.

Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.

High-dose subconjunctival cyclosporine a implants do not affect corneal neovascularization after high-risk keratoplasty.

PURPOSE: To test whether subconjunctival cyclosporine A (CsA) implants affect the incidence and the degree of corneal neovascularization occurring after penetrating keratoplasty. DESIGN: Prospective, randomized, multicenter, controlled phase 2/3 clinical trial. The study comprised 43 trial sites in Germany, India, and the United States. PARTICIPANTS: Enrolled patients (n = 97) were randomized to 1 of 3 groups: treatment group A (n = 36), treatment group B (n = 40), and the control group (n = 21). METHODS: Patients from each group received either of 2 doses of subconjunctival CsA (group A, low-dose CsA; group B, high-dose CsA) or placebo (carrier only) implants at the time of high-risk penetrating keratoplasty. MAIN OUTCOME MEASURES: The incidence and degree of corneal neovascularization occurring after penetrating keratoplasty were evaluated in a substudy (LX201-01 study: NCT00447187). A web-based image upload system was developed. Standardized digital slit-lamp pictures were quantitatively and objectively evaluated using CellˆF morphometry software. RESULTS: No statistically significant difference in incidence and degree of corneal neovascularization developing after penetrating keratoplasty was found between treatment groups and placebo group. Mean corneal neovascularization area at week 52 (visit 12) was 2.32±1.79% in treatment group A versus placebo (2.79±2.11%; P = 0.45) and 2.74±2.22% in treatment group B versus placebo (2.79±2.11%; P = 0.94). CONCLUSIONS: High-dose subconjunctival CsA implants do not significantly affect corneal neovascularization after high-risk penetrating keratoplasty. This suggests that local CsA has negligible antiangiogenic effects in the human cornea, at least in the transplant setting.

Aganirsen antisense oligonucleotide eye drops inhibit keratitis-induced corneal neovascularization and reduce need for transplantation: the I-CAN study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 µg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Blockade of the VEGF isoforms in inflammatory corneal hemangiogenesis and lymphangiogenesis.

BACKGROUND: The VEGF-A family plays a crucial role in the induction of pathological corneal neovascularization. The role of the different VEGF-A isoforms during lymphangiogenesis is only little-known. Current anti-angiogenic therapies in the eye and other organs inhibit all VEGF-A isoforms, and have effects on both blood and lymphatic vessels. Here we investigate whether selective targeting of the isoform VEGF 165 is able to inhibit corneal lymphangiogenesis under inflammatory conditions. METHODS: The mouse model of suture-induced corneal neovascularization was used to assess the antihem- and antilymphangiogenic effect of topically applied pegaptanib. Corneal blood and lymph vascularized areas were analyzed morphometrically. Furthermore, we analyzed the proliferative effects of VEGF A 121, 165, and 189 on blood and lymphatic endothelial cells (BEC/LEC) via a cell-proliferation assay. RESULTS: Pegaptanib significantly inhibited inflammatory corneal hemangiogenesis (p < 0.01), but not lymphangiogenesis in vivo (p > 0.05), both topically as well as systemically, in the inflamed cornea. In vitro, BECs were more susceptible to pegaptanib than LECs. CONCLUSIONS: Targeting VEGF-A 165 significantly inhibits hem- but not lymphangiogenesis, suggesting VEGF-A 165 to be critical for hem-, but dispensable for lymphangiogenesis, at least in the inflamed cornea.

Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis in vivo.

PURPOSE: To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels in vivo. METHODS: Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. RESULTS: All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. CONCLUSIONS: A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

Impact of 68Ga-PSMA PET/CT on radiation treatment planning of prostate cancer patients.

AIM: This study aimed to assess the impact of 68Ga-PSMA PET/CT on radiation treatment (RT) planning in prostate cancer patients with salvage (sRT) or definitive (dRT) radiotherapy. METHODS: 38 patients (27 sRT, median PSA 0.79 ng/ml (range 0.06-12.1); 11 dRT, median PSA 4.35 ng/ml (range 1.55-55.5) underwent 68Ga-PSMA PET/CT before RT. Influence of 68Ga-PSMA PET/CT on the extent of planning target volume (PTV) and addition of PET-based boosts were assessed. Median follow up was 12 months (range 3-24). RESULTS: 68Ga-PSMA PET/CT showed positive findings in 23/38 patients (8/23: local recurrence (LR), 11/23: nodal metastasis, 1/23: LR and nodal, 2/23: solitary bone metastasis, 1/23: oligometastatic nodal/ bone metastases). In sRT primary PTV was changed in 16/27 patients extending the PTV to the lymphatic drainage (10/16), PSMA-positive LR (3/16), bone metastases (2/16) and both nodal/bone metastases (1/16). PET-based increase of primary PTV was 116%. PET-based boosts were administered in 19/27 patients (8/19: local, 10/19: nodal, 1/19: both), median boost volume was 31.3 cm3 (range 17.2-80.2) (local) and 19.7 cm3 (range 3.0-109.3) (nodal). PTV was changed in 1/11 (9%) of dRT patients (extension of primary PTV to the lymphatic drainage (RT volume of 644.5 cm3), additional nodal boost (volume of 2.7 cm3, 23.1 Gy)). All patients showed biochemical response (mean PSA decrease 88.8 +/- 14.0%). Nadir PSA was reached 10 months (range 1-17) after end of RT (median 0.07 ng/ml, range 0.002-3.96). Within a median 12 months follow-up (range 3-22/8-24 in sRT/dRT), median PSA was 0.05 ng/ml (range 0.002-8.5) (sRT) and 0.26 ng/ml (range 0.02-2.68) (dRT). CONCLUSIONS: 68Ga-PSMA PET/CT influenced sRT planning in almost 63% and dRT in 9% of patients by change of PTV and additional boosts.