Victor L. Perez

Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement

Studies of T cell anergy in vitro have led to the widely accepted view that anergy is induced by T cell antigen recognition without costimulation. We show that the induction of T cell anergy in vivo is due to an abortive T cell response that requires recognition of B7 molecules, since blocking B7 maintains T cells in an unactivated but functionally competent state. Furthermore, the induction of anergy is prevented by blocking CTLA-4, the inhibitory T cell receptor for B7 molecules. Thus, in vivo T cell anergy may be induced not because of a lack of costimulation, but as a result of specific recognition of B7 molecules by CTLA-4. In contrast, blocking CD28 on T cells prevents priming but not the induction of tolerance. Therefore, the outcome of antigen recognition by T cells is determined by the interaction of CD28 or CTLA-4 on the T cells with B7 molecules.

Oxidative damage–induced inflammation initiates age-related macular degeneration

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruchs membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.

ABCB5 is a limbal stem cell gene required for corneal development and repair

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

Prevalence and Risk Factors of Dry Eye Syndrome in a United States Veterans Affairs Population

PURPOSE To evaluate the prevalence of dry eye syndrome (DES) and its associated risk factors in a US Veterans Affairs population receiving ocular care services. DESIGN Retrospective study. METHODS settings: Patients were seen in the Miami and Broward Veterans Affairs eye clinics between 2005 and 2010. patients population: Patients were divided into cases and controls with regard to their dry eye status (cases = ICD9 code for DES plus dry eye therapy; controls = patients without ICD9 code plus no therapy). main outcome measures: The prevalence of DES and its associated risk factors. RESULTS A total of 16 862 patients were identified as either a dry eye case (n = 2056) or control (n = 14 806). Overall, 12% of male and 22% of female patients had a diagnosis of DES, with female gender imparting a 2.40 increased risk (95% confidence interval [CI] 2.04-2.81) over male gender. Several medical conditions were found to increase DES risk including post-traumatic stress disorder (odds ratio [OR] 1.97, 95% CI 1.75-2.23), depression (OR 1.91, 95% CI 1.73-2.10), thyroid disease (OR 1.81, 95% CI 1.46-2.26), and sleep apnea (OR 2.20, 95% CI 1.97-2.46) (all analyses adjusted for gender and age). The use of several systemic medications, including anti-depressant medications (OR 1.97, 95% CI 1.79-2.17), anti-anxiety medication (OR 1.74, 95% CI 1.58-1.91), and anti-benign prostatic hyperplasia medications (OR 1.68, 95% CI 1.51-1.86), was likewise associated with an increased risk of DES. CONCLUSIONS The prevalence of DES was found to be high in both men and women in our eye care population. This is the first study to demonstrate that in a veteran population, several diagnoses were significantly associated with DES, including post-traumatic stress disorder and depression.

Etanercept (Enbrel)-Associated Inflammatory Eye Disease: Case Report and Review of the Literature

Purpose: To report a case of severe anterior uveitis flare following the administration of etanercept (Enbrel) for ankylosing spondylitis and to review the literature pertaining to inflammatory eye disease associated with the use of etanercept. Methods: Clinical data were collected from a 52-year-old female with chronic symptomatic ankylosing spondylitis. The relationship between etanercept administration and the patients systemic and ocular inflammation was assessed. A review of the literature was conducted to identify additional reports. Results: A patient with a history of ankylosing spondylitis exhibited acute exacerbations of uveitis that were temporally related to etanercept injections. Re-challenge was associated temporally with a worsening of symptoms, and drug withdrawal, in concert with aggressive systemic steroid treatment, resulted in rapid resolution. Seventeen cases of inflammatory eye disease (uveitis, scleritis, orbital myositis) believed to be associated with etanercept therapy were found in the recent literature. Conclusions: Ocular inflammation is paradoxically a potential adverse event following the use of etanercept in both previously uninvolved and inflamed eyes. Careful surveillance of patients on etanercept is warranted to assure that flares in ocular inflammation are not secondary to etanercept therapy.

A Hapten Generated from an Oxidation Fragment of Docosahexaenoic Acid Is Sufficient to Initiate Age-Related Macular Degeneration

The protein adduct carboxyethylpyrrole (CEP) is present in age-related macular degeneration (AMD) eye tissue and in the blood of AMD patients at higher levels than found in age-matched non-AMD tissues. Autoantibodies to CEP are also higher in AMD blood samples than in controls. To test the hypothesis that this hapten is causally involved in initiating an inflammatory response in AMD, we immunized C57BL/6J mice with mouse serum albumin (MSA) adducted with CEP. Immunized mice develop antibodies to CEP, fix complement component-3 in Bruch’s membrane, accumulate drusen below the retinal pigment epithelium during aging, show decreased a- and b-wave amplitudes in response to light, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. Inflammatory cells are present in the region of lesions and may be actively involved in the pathology observed. We conclude that early immunization of mice with CEP-adducted MSA sensitizes these animals to the ongoing production of CEP adducts in the outer retina where DHA is abundant and the conditions for oxidative damage are permissive. In response to this early sensitization, the immune system mounts a complement-mediated attack on the cells of the outer retina where CEP adducts are formed. This animal model for AMD is the first that was developed from an inflammatory signal discovered in eye tissue and blood from AMD patients. It provides a novel opportunity for dissecting the early pathology of AMD and the immune response contributing to this disorder. The availability of a mouse with a mechanistically based AMD-like disease that progresses rapidly is highly desirable. Such a model will allow for the efficient preclinical testing of the much-needed therapeutics quickly and inexpensively.

Depression, Post-traumatic Stress Disorder, and Dry Eye Syndrome: A Study Utilizing the National United States Veterans Affairs Administrative Database

PURPOSE To study the scope of dry eye syndrome (DES) in veterans on a national level and to evaluate the relationship between psychiatric diagnoses and DES. DESIGN Case-control study. METHODS SETTING Patients were seen in a Veterans Affairs (VA) eye clinic between 2006 and 2011. PATIENT POPULATION Patients were divided into cases and controls with regard to their dry eye status (cases = ICD-9 code for DES plus dry eye therapy; controls = patients without ICD-9 code plus no therapy). MAIN OUTCOME MEASURES The prevalence of DES and the influence of psychiatric diagnoses on the risk of DES. RESULTS A total of 2 454 458 patients were identified as either a dry eye case (n = 462 641) or control (n = 1 991 817). Overall, 19% of male patients and 22% of female patients had a diagnosis of DES, with female sex imparting an increased risk of DES at each decade compared to male sex (odds ratio [OR] 1.22-2.09). Several conditions were found to increase DES risk, including post-traumatic stress disorder (OR 1.92, 95% CI 1.91-1.94) and depression (OR 1.92, 95% CI 1.91-1.94) (analyses adjusted for sex and age). The use of several systemic medications was likewise associated with an increased risk of DES, including antidepressant medications (OR 1.97, 95% CI 1.79-2.17) and antianxiety medication (OR 1.74, 95% CI 1.58-1.91). Multivariate analysis (adjusted for age and sex) revealed that for psychiatric diagnoses, both the use of medication and the diagnosis remained significant risk factors when considered concomitantly, although the magnitude of each association decreased. CONCLUSIONS DES is a disease associated with depression and post-traumatic stress disorder, and is prevalent among male and female veterans receiving eye care services. The association could be driven by underlying disease physiology or medications used to treat psychiatric conditions. Regardless of the causal link, this suggests that individuals with a known psychiatric diagnosis should be questioned about dry eye symptoms and, if applicable, referred to an eye care physician.

Impact of ocular surface symptoms on quality of life in a United States veterans affairs population.

PURPOSE To evaluate the impact of ocular surface symptoms on quality of life in a veteran population receiving eye care services. DESIGN Cross-sectional survey study. METHODS setting: Miami Veterans Affairs Medical Center (VAMC). patient population: Patients seen at the eye clinic between June and August 2010 were asked to fill out the Dry Eye Questionnaire 5 (DEQ5) and the Impact of Dry Eye on Everyday Life (IDEEL) questionnaire. main outcome measures: Correlation between ocular surface symptoms and functionality. RESULTS Four hundred eighty-nine patients elected to fill out the DEQ5 questionnaire (36% response rate). The mean age of respondents was 66 years (standard deviation 12). Ninety-four percent were male; 62% were white and 37% were black. Using the DEQ5 as a surrogate measure of ocular surface symptoms, 65% of respondents reported at least mild ocular surface symptoms (DEQ5 ≥6) and 27% of them reported severe symptoms (DEQ5 ≥12). Black subjects had a 2-fold increased risk of severe symptoms compared to white subjects (odds ratio 2.06, 95% confidence interval 1.33-3.19). Several medications were associated with a significantly increased risk of severe symptoms, including glaucoma medications (1.7-fold increase), antidepressants (2.3-fold increase), and antihistamines (2.1-fold increase). There was an inverse correlation between DEQ5 and IDEEL scores with regard to ability to perform activities of daily living (n = 391, r = -0.54, P < .001), emotional well-being (n = 386, r = -0.63, P < .001), and the ability to work (n = 205, r = -0.57, P < .001). Fifty percent of patients with severe symptoms had documentation that their symptoms were addressed during the visit. CONCLUSION Severe ocular surface symptoms reduce the quality of life of Miami VAMC veterans. Eye care professionals should be vigilant in eliciting ocular surface complaints from their patients.

Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

High-resolution, noninvasive longitudinal live imaging of immune responses

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.