Pedram Hamrah

Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity.

There are no studies so far linking molecular regulation of lymphangiogenesis and induction of adaptive immunity. Here, we show that blockade of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling significantly suppresses corneal antigen-presenting (dendritic) cell trafficking to draining lymph nodes, induction of delayed-type hypersensitivity and rejection of corneal transplants. Regulating the function of VEGFR-3 may therefore be a mechanism for modulating adaptive immunity in the periphery.

Corneal immunity is mediated by heterogeneous population of antigen‐presenting cells

Corneal antigen‐presenting cells (APC), including dendritic cells (DC), were thought to reside exclusively in the peripheral cornea. Here, we present recent data from our group demonstrating that the central cornea is indeed endowed with a heterogeneous population of epithelial and stromal DC, which function as APC. Although the corneal periphery contains mature and immature resident bone marrow‐derived CD11c+ DC, the central cornea is endowed exclusively with immature and precursor DC, both in the epithelium and the stroma, wherein Langerhans cells and monocytic DC reside, respectively. During inflammation, a majority of resident DC undergo maturation by overexpressing major histocompatibility complex class II and B7 (CD80/CD86) costimulatory molecules. In addition to the DC, macrophages are present in the posterior corneal stroma. In transplantation, donor‐derived DC are able to migrate to host cervical lymph nodes and activate host T cells via the direct pathway when allografts are placed in inflamed host beds. These data revise the tenet that the cornea is immune‐privileged as a result of lack of resident lymphoreticular cells and suggest that the cornea is capable of diverse cellular mechanisms for antigen presentation.

Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts.

To examine the widely accepted dogmas that corneal grafts lack passenger leukocytes or cells capable of migrating directly to lymph nodes (LNs), we tracked the migration of corneal graft-derived transgenic green fluorescent protein (GFP; Iab) cells into the draining LNs of allogeneic (Iad) recipients. GFP+ cells were identified in cervical LNs several hours after transplantation, and this traffic was significantly enhanced when grafts were placed in inflamed recipient beds. Draining cells were Iab+, CD45+, and CD11c+, and examination of ungrafted corneas revealed numerous similarly CD45+CD11c+CD3−CD8α− cells that uniformly lacked major histocompatibility complex (MHC) class II expression; transmission electron microscopy confirmed the presence of morphologically similar cells. After transplantation, or placement in culture, these CD11c+ cells became class II+ in a time-dependent manner and were capable of allostimulatory function. However, the stimulatory capacity of these cornea-derived dendritic cells (DCs) was suppressed compared with splenic controls. These results demonstrate for the first time that the cornea is endowed with resident DCs that are universally MHC class II− but that are capable of expressing class II antigen after surgery and migrating to draining LNs of allografted hosts. These data refute the tenet that the cornea is immune privileged due to lack of resident lymphoreticular cells or due to antigenic sequestration from systemic immunity.

Twenty-five-year panorama of corneal immunology: emerging concepts in the immunopathogenesis of microbial keratitis, peripheral ulcerative keratitis, and corneal transplant rejection.

Purpose. To describe the most recent advances in our understanding of the cellular and molecular mechanisms involved in the immunopathogenesis of corneal immunoinflammatory disorders including microbial keratitis, peripheral ulcerative keratitis, and allograft rejection. Methods. Review of the published peer-reviewed literature that has contributed significantly to our modern understanding of corneal immunology. In addition, the authors have summarized the information in conceptual diagrams that highlight the critical cellular and molecular pathways that lead to corneal immune responses in the two most thoroughly studied corneal immune disorders, herpes simplex keratitis (HSK) and transplant rejection. Results. In spite of the wide array of molecular and cellular factors that mediate corneal immunity, critical mechanistic facets are shared by the various corneal immunoinflammatory disorders. These include activation and migration of local antigen-presenting cells (APCs), including Langerhans cells (LCs), upregulation in pleiotropic proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alfa (TNF-&agr;) that can mediate a wide array of immune functions in addition to upregulating protease expression, and chemokines that play a critical role on the one hand in attracting nonantigen-specific inflammatory cells such as neutrophils and on the other in attracting CD4+ T helper type 1 (Th1) cells that mediate most of the destruction in the cornea. Conclusions. In the last 25 years, we have seen our field develop from a descriptive stage into a new phase where the fundamental processes that mediate and effect corneal immunity are being accurately deciphered. It is anticipated that this new knowledge will allow development of specific molecular and genetic therapeutic strategies that could target critical steps in the immunopathogenesis of disease without the untoward side-effects of nonspecific generalized immune suppression that still remains the standard of care today.

Topical Bevacizumab in the Treatment of Corneal Neovascularization Results of a Prospective, Open-Label, Noncomparative Study

OBJECTIVE To study the safety and efficacy of topical bevacizumab in the treatment of corneal neovascularization (NV). DESIGN In a prospective, open-label, noncomparative study, 10 eyes from 10 patients with stable corneal NV were treated with topical bevacizumab, 1.0%, for 3 weeks and followed up for up to 24 weeks. MAIN OUTCOME MEASURES The primary safety variables were the occurrence of ocular and systemic adverse events throughout the course of the study. The primary efficacy variables were neovascular area, the area of the corneal vessels themselves; vessel caliber, the mean diameter of the corneal vessels; and invasion area, the fraction of the total corneal area covered by the vessels. RESULTS From baseline visit to the last follow-up visit, mean reductions were 47.1% (standard deviation [SD], 36.7%) for neovascular area, 54.1% (SD, 28.1%) for vessel caliber, and 12.2% (SD, 42.0%) for invasion area. The decreases in neovascular area and vessel caliber were statistically significant (P= .001 and P< .001, respectively). However, changes in invasion area did not achieve statistical significance (P= .19). Visual acuity and central corneal thickness showed no significant changes. Topical bevacizumab was well tolerated with no adverse events. CONCLUSIONS Short-term topical bevacizumab therapy reduces the severity of corneal NV without local or systemic adverse effects. APPLICATION TO CLINICAL PRACTICE Topical bevacizumab provides an alternative therapy in the treatment of stable corneal NV. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00559936.

Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study.

PURPOSE To study and correlate corneal sensation in patients with herpes simplex keratitis (HSK) with density and morphologic features of subbasal corneal nerves by in vivo confocal microscopy (IVCM). DESIGN Prospective, cross-sectional, controlled, single-center study. PARTICIPANTS Thirty-one eyes with the diagnosis of acute (n = 7) or chronic (n = 24) HSK and their contralateral clinically unaffected eyes were studied and compared with normal controls (n = 15). METHODS In vivo confocal microscopy (Confoscan 4; Nidek Technologies, Gamagori, Japan) and corneal esthesiometry (Cochet-Bonnet; Luneau Ophthalmlogie, Chartres, France) of the central cornea were performed bilaterally in all patients and controls. Patients were grouped into normal (> 5.5 cm), mild (> 2.5-5.5 cm), and severe (≤ 2.5 cm) loss of sensation. MAIN OUTCOME MEASURES Changes in corneal nerve density, total nerve number, main nerve trunks, branching, and tortuosity were evaluated after IVCM and were correlated to corneal sensation, disease duration, and number of recurrences. RESULTS Herpes simplex keratitis eyes, as compared with controls, demonstrated significant (P < 0.001) decrease in mean nerve density (448.9 ± 409.3 vs. 2258.4 ± 989.0 μm/frame), total nerve number (5.2 ± 4.5 vs. 13.1 ± 3.8), main nerve trunks (2.3 ± 1.6 vs. 4.7 ± 1.2), and nerve branches (3.2 ± 4.3 vs. 9.8 ± 3.3). In contralateral unaffected eyes, mean nerve density (992.7 ± 465.0 μm/frame), total nerve number (7.8 ± 3.3), and branches (4.5 ± 2.3) were decreased significantly as compared with controls (P < 0.002). Reduced nerve density, total nerve count, and main trunks in HSK eyes were correlated significantly with corneal sensation across all subgroups (P < 0.001). Nerve density decreased within days of infection and was correlated to frequency of episodes in patients with HSK (P < 0.02). CONCLUSIONS In vivo confocal microscopy revealed that the loss of corneal sensation in HSK correlates strongly with profound diminishment of the subbasal nerve plexus after herpes simplex virus infection. Surprisingly, the contralateral, clinically unaffected eyes also demonstrated a diminishment of the subbasal nerve plexus as compared with normal subjects, revealing bilateral nerve alteration in an apparently unilateral disease.

Novel expression of vascular endothelial growth factor receptor (VEGFR)-3 and VEGF-C on corneal dendritic cells.

Vascular endothelial growth factor-3 (VEGFR-3) plays a critical role in embryonic cardiovascular development and is thought to be expressed exclusively on the lymphatic endothelium, high endothelial venules, and rarely on adult vascular endothelium. Recent evidence also suggests expression of VEGFR-3 on some tumor-associated macrophages. We have studied the expression of VEGFR-3, its ligand VEGF-C and the co-receptor neuropilin-2, in normal and inflamed corneas and characterized the phenotype and distribution of VEGFR-3(+) cells. Our data demonstrate, for the first time, the expression of VEGFR-3 on corneal dendritic cells (DC) and its up-regulation in inflammation. VEGFR-3(+) DC are CD11c(+)CD45(+)CD11b(+), and are mostly major histocompatibility (MHC) class II(-)CD80(-)CD86(-), indicating immature DC of a monocytic lineage. During inflammation, there is rapid increase in the number of VEGFR-3(+) DC in the cornea associated with heightened membranous expression as compared to a mostly intracellular expression in uninflamed tissue. VEGFR-3(+) DC in normal corneas are VEGF-C(-)neuropilin-2(-), but express VEGF-C in inflammation. Interestingly, similar cells are absent both in the normal and inflamed skin. These data demonstrate, for the first time, the expression of VEGFR-3 and VEGF-C on tissue DC, which implicate a novel potential relationship between lymphangiogenesis and leukocyte trafficking in the eye.

In Vivo Confocal Microscopy of Corneal Nerves: Analysis and Clinical Correlation

Corneal confocal microscopy is a growing technique for the study of the cornea at the cellular level, providing images comparable to ex vivo histochemical methods. In vivo confocal microscopy (IVCM) has an enormous potential, being a noninvasive procedure that images the living cornea, to study both its physiological and pathological states. Corneal nerves are of great interest to clinicians and scientists due to their important roles in regulating corneal sensation, epithelial integrity, proliferation, wound healing, and for their protective functions. IVCM enables the noninvasive examination of corneal nerves, allowing the study of nerve alterations in different ocular diseases, after corneal surgery, and in systemic diseases. To date, the correlation of sub-basal corneal nerves and their function has been studied in normal eyes, keratoconus, dry eye, contact lens wearers, and in neurotrophic keratopathy, among others. Further, the effect of corneal surgery on nerves has been studied, demonstrating the regenerative capacity of corneal nerves and the recovery of sensation. Moreover, IVCM has been applied in the diagnosis of peripheral diabetic neuropathy and the assessment of progression in this systemic disease. The purpose of this review is to describe the principles, applications, and clinical correlation of IVCM in the study of corneal nerves in different ocular and systemic diseases.

In vivo confocal microscopy of the ocular surface: from bench to bedside.

Abstract In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

Unilateral Herpes Zoster Ophthalmicus Results in Bilateral Corneal Nerve Alteration: An In Vivo Confocal Microscopy Study

PURPOSE Herpes zoster ophthalmicus (HZO), thought to be a unilateral disease, results in loss of corneal sensation, leading to neurotrophic keratopathy. This study aimed to analyze bilateral corneal nerve changes in patients with HZO by in vivo confocal microscopy (IVCM) and their correlation with corneal sensation as a measure of nerve function. DESIGN Prospective, cross-sectional, controlled, single-center study. PARTICIPANTS Twenty-seven eyes with the diagnosis of HZO and their contralateral clinically unaffected eyes were studied and compared with normal controls (n = 15). METHODS In vivo confocal microscopy (Confoscan 4; Nidek Technologies, Gamagori, Japan) and corneal esthesiometry (Cochet-Bonnet; Luneau Ophthalmologie, Chartres, France) of the central cornea were performed bilaterally in all patients and controls. Patients were grouped into normal (>5.5 cm), mild (>2.5-5.5 cm), and severe (<2.5 cm) loss of sensation. MAIN OUTCOME MEASURES Changes in corneal nerve density, total nerve number, main nerve trunks, branching, and tortuosity were evaluated after IVCM and were correlated to corneal sensation, disease duration, and number of recurrences. RESULTS Eyes with herpes zoster ophthalmicus had a significant (P<0.001) decrease in total nerve length (595.8±358.1 vs. 2258.4±989.0 μm/frame), total number of nerves (5.4±2.8 vs. 13.1±3.8), number of main nerve trunks (2.3±1.1 vs. 4.7±1.2), and number of nerve branches (3.2±2.3 vs. 8.4±3.7) as compared with controls. In the contralateral clinically unaffected eyes, total nerve length (1053.1±441.4 μm/frame), total number of nerves (8.3±2.9), and main nerve trunks (3.1±1.0) also were decreased significantly as compared with controls (P<0.01). Reduced nerve density, total nerve count, main trunks, and tortuosity was correlated significantly with corneal sensation across all subgroups (P<0.001). CONCLUSIONS Patients with unilateral HZO demonstrated a profound and significant bilateral loss of the corneal nerve plexus as compared with controls, demonstrating bilateral changes in a clinically unilateral disease. Loss of corneal sensation strongly correlated with subbasal nerve plexus alterations as shown by IVCM. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.