"Window of Opportunity" in Ocular Graft-Versus-Host Disease Treatment: Results of a Longitudinal Study and Case Reports.

OBJECTIVE: To perform a longitudinal study for determining the development of ocular graft-versus-host disease (oGVHD) after allogeneic hematopoietic stem cell transplant (HSCT) and report cases that illustrate the "window of opportunity" concept in oGVHD treatment. METHODS: Patients (n=61) were examined at prescheduled clinic visits before HSCT and three-month intervals after HSCT for 2 years. The presence or absence of oGVHD was determined using the international chronic oGVHD consensus group diagnostic criteria. Ocular surface washings (OSW) were obtained at each visit and analyzed for cytokine levels. RESULTS: In the longitudinal study, 26.2% (n=16; progressed group) developed either probable (11.5%, n=7) or definite oGVHD (14.8%, n=9). In the progressed group, clinically significant changes in signs (corneal staining and Schirmer I test) and symptoms at the post-HSCT visit as compared with the pre-HSCT visit occurred at 9 months. Significant differences in clinical signs and symptoms (whether average post-HSCT values or changes in values over pre-HSCT levels) between the progressed and nonprogressed groups occurred at a 9-month visit or later. In the progressed group, 55.6% of eyes that had negative matrix metalloproteinase 9 (MMP-9) test at pre-HSCT turned MMP-9 positive at 3 to 6 months post-HSCT. In the progressed group, interleukin 8 levels in OSW were significantly increased at 6 months post-HSCT. In the case reports, the "window of opportunity" was detected by MMP-9 turning positive, early corneal staining, interleukin 8 increase in OSW, and peripheral corneal epithelial thinning, which resolved with treatment initiation. CONCLUSIONS: A "window of opportunity" exists before patients developing symptomatic tear-deficient dry eye after HSCT for initiating treatment that may preempt oGVHD development; however, larger-scale longitudinal studies are needed for definitive recommendations.

Recombinant Deoxyribonuclease I Eye Drops for Ocular Graft Versus Host Disease: Results of a Randomized Clinical Trial.

OBJECTIVE: We have previously shown that neutrophil extracellular traps (NETs) are present on the ocular surface of patients with ocular graft versus host disease (oGVHD), contributing to inflammation and surface disease. Therefore, we performed a clinical trial using deoxyribonuclease I (DNAase) eye drops to test the hypothesis that reducing the abundance of NETs from the ocular surface will reduce signs and symptoms of oGVHD. METHODS: A prospective, phase I or II, randomized, placebo-controlled, double-masked clinical trial was performed to determine the safety and preliminary efficacy of DNAase (0.1%) eye drops four times daily for 8 weeks in patients with oGVHD (n=58). Intent-to-treat analysis was performed to determine the change in safety outcome measures (drug tolerability and proportion of adverse events) and efficacy outcome measures (ocular surface disease index [OSDI] score and corneal staining) between baseline and week 8. RESULTS: Tolerability and adverse events were similar in the vehicle and DNAase groups. Within the DNAase group (but not the vehicle group), corneal staining showed a statistically significant and clinically meaningful reduction at week 8 (3.50 [2.75; 5.00]) compared with baseline (5.00 [3.00; 7.00]). The OSDI score also showed a statistically significant clinically meaningful reduction of 18.4 (9.16; 33.1) ( P <0.001) at week 8 compared with baseline (45.5 [31.8; 50.0]) within the DNAase group. The proportion of eyes that had improvement in subjective global assessment (SGA) and mucous discharge was significantly greater in the DNAase group (55.6% and 57.7% at weeks 4 and 8, respectively; P <0.0001 at both time points) as compared with the vehicle group (35.7% and 34.0% at weeks 4 and 8, respectively). CONCLUSIONS: Treatment of patients with oGVHD using DNAase eye drops is safe and demonstrates preliminary efficacy. Deoxyribonuclease I eye drops can potentially reduce the severity of signs and symptoms of ocular surface disease in patients with oGVHD.

Combinatorial therapy with immunosuppressive, immunomodulatory and tear substitute eyedrops ("Triple Play") in Recalcitrant Immunological Ocular Surface Diseases.

PURPOSE: The current paradigm for therapy of recalcitrant ocular surface diseases (OSD) consists of a sequential, step-up treatment approach. A combinatorial topical therapy (anti-inflammatory/immunosuppressive [steroid] with immunomodulatory [pooled human immune globulin] and tear substitute [serum]) that simultaneously targets several immunological pathways may be more efficacious. This report evaluates if the combinatorial therapy resulted in clinical benefit in patients with recalcitrant OSD. METHODS: We performed a retrospective case study of patients receiving topical, preservative-free, compounded formulations of steroids, pooled human immune globulin, and serum tears. Outcome measures included visual acuity, ocular surface disease index (OSDI), ocular discomfort score, subjective global assessment (SGA), corneal staining, conjunctival redness, and slit lamp photographs. RESULTS: Patients consisted of one male and 11 females ranging in age from 27 to 87 years old. Pathologies included ocular graft-versus-host disease (n = 4), Sjögren's syndrome (n = 3), ocular cicatricial pemphigoid (n = 1), pemphigus vulgaris (n = 1), peripheral ulcerative keratitis (n = 1), Stevens-Johnson syndrome (n = 1), and giant papillary conjunctivitis (n = 1). All patients were "improved" or "much improved" on SGA after combinatorial therapy. There was a clinically meaningful reduction in OSDI, ocular discomfort, corneal staining, and conjunctival injection. Additionally, three patients had improvement in their visual acuity (one from 20/400 to 20/20). Adverse effects included increased intraocular pressure in two patients, presumably due to topical steroid use. CONCLUSIONS: Combinatorial therapy provides clinical benefit by reducing the symptoms and signs in recalcitrant OSD. Our study provides the rationale for performing prospective clinical trials to evaluate the efficacy of combinatorial therapy for treating recalcitrant OSD.

Entry receptor bias in evolutionarily distant HSV-1 clinical strains drives divergent ocular and nervous system pathologies.

PURPOSE: Herpes simplex virus-1 (HSV-1) infection leads to varying pathologies including the development of ocular lesions, stromal keratitis and encephalitis. While the role for host immunity in disease progression is well understood, the contribution of genetic variances in generating preferential viral entry receptor usage and resulting immunopathogenesis in humans are not known. METHODS: Ocular cultures were obtained from patients presenting distinct pathologies of herpes simplex keratitis (HSK). Next-generation sequencing and subsequent analysis characterized genetic variances among the strains and estimated evolutionary divergence. Murine model of ocular infection was used to assess phenotypic contributions of strain variances on damage to the ocular surface and propagation of innate immunity. Flow cytometry of eye tissue identified differential recruitment of immune cell populations, cytokine array probed for programming of local immune response in the draining lymph node and histology was used to assess inflammation of the trigeminal ganglion (TG). Ex-vivo corneal cultures and in-vitro studies elucidated the role of genetic variances in altering host-pathogen interactions, leading to divergent host responses. RESULTS: Phylogenetic analysis of the clinical isolates suggests evolutionary divergence among currently circulating HSV-1 strains. Mutations causing alterations in functional host interactions were identified, particularly in viral entry glycoproteins which generated a receptor bias to herpesvirus entry mediator, an immune modulator involved in immunopathogenic diseases like HSK, leading to exacerbated ocular surface pathologies and heightened viral burden in the TG and brainstem. CONCLUSIONS: Our data suggests receptor bias resulting from genetic variances in clinical strains may dictate disease severity and treatment outcome.

Kainic Acid Activates TRPV1 via a Phospholipase C/PIP2-Dependent Mechanism in Vitro.

Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca2+ influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEKTRPV1 cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca2+ induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca2+-induced neurotoxicity.

Pathological consequences of anti-citrullinated protein antibodies in tear fluid and therapeutic potential of pooled human immune globulin-eye drops in dry eye disease.

PURPOSE: To investigate the role of Anti-Citrullinated Protein autoantibodies (ACPAs) in the pathology of dry eye disease (DED) and the therapeutic potential of pooled human immune globulin-eye drops in these patients. METHODS: We investigated the presence of citrullinated proteins and ACPAs in ocular surface wash (OSW) and conjunctival impressions from patients with DED and determined the pathological consequences of OSW with high ACPA using in vitro experiments and in vivo murine models. We performed a randomized, double-masked, pilot clinical trial to determine the safety, tolerability and preliminary efficacy of using pooled human immune globulin-eye drops to treat DED patients with ACPAs in OSW. RESULTS: We found that neutrophils are a source of citrullinated proteins on the ocular surface of DED patients. We detected significantly higher immunoglobulin amount and presence of several species of ACPAs in OSW from DED patients. We also found that OSW with high ACPA contributes to production of NETs, and that ACPAs cause ocular surface disease in murine eyes, both of which are reduced with addition of Immune globulins. As compared to Vehicle treatment, pooled human immune globulin-eye drops (IVIG 4 mg/mL) twice a day for 8 weeks caused significant reduction in signs and symptoms of DED with no difference in tolerability or adverse events. CONCLUSIONS: This is the first report demonstrating ACPAs in OSW of DED patients and their contribution to ocular surface disease. The first-in-human clinical trial suggests that pooled immune globulin-eye drops are a potential new class of biologic therapies for Dry Eye patients.

Presence of Histatin-1 in Human Tears and Association with Aqueous Deficient Dry Eye Diagnosis: A Preliminary Study.

The aims of this study were to determine if histatin-1 (H1) is present in normal human tears and whether tear levels of H1 varied between normal patients and those with aqueous deficient dry eye disease (ADDE). Patient samples were obtained from 11 normal patients and 11 severe ADDE patients. Relevant patient characteristics, including age, sex, and dry eye disease (DED) diagnostic parameters were collected. Multiple qualitative and quantitative methods were used to compare the concentration of H1 between patient groups. Mixed linear modeling was used to compare H1 levels between groups, and diagnostic performance was assessed using the receiver-operator-characteristic (ROC). ADDE patients had significantly lower H1 concentrations (85.9 ± 63.7 ng/ml) than the normal group (891.6 ± 196.5 ng/ml) (p < 0.001), while controlling for age and sex. ROC analysis indicated that H1 concentration is potentially a biomarker for ADDE (area under curve = 0.96). Reclassification of patients by DED parameters including, Ocular Surface Disease Index (OSDI) (≤13, >13) and Schirmer I (without anesthesia) (<10 mm, ≥10 mm) showed significant differences in H1 level (OSDI, p = 0.004) and Schirmer I ((p = 0.010). In conclusion, this is the first preliminary report of the presence of H1 in human tears. H1 concentrations are lower in ADDE patients and H1 may have diagnostic potential in evaluation ADDE patients.

A Phase I/II Placebo-Controlled Randomized Pilot Clinical Trial of Recombinant Deoxyribonuclease (DNase) Eye Drops Use in Patients With Dry Eye Disease.

PURPOSE: To determine whether DNase eye drops have the potential to reduce signs and symptoms of dry eye disease (DED). METHODS: A placebo-controlled, randomized clinical trial was performed to compare the safety and efficacy of DNase eye drops 0.1% four times a day for 8 weeks in patients with severe tear deficient DED. The change in safety outcome measures (drug tolerability and proportion of adverse events) and efficacy outcome measures (Ocular Surface Disease Index [OSDI] score, corneal and conjunctival staining) were analyzed between baseline and week 8. RESULTS: Tolerability and adverse events were similar in placebo group and DNase group. Within the DNase group (but not placebo group), corneal staining showed a statistically significant and clinically meaningful reduction at week 8 compared with baseline. The OSDI score also showed a significant median reduction of 27.3 at week 8 compared with baseline within the DNase group. The median reduction in corneal staining and mucoid debris/strands was significantly greater in the DNase group as compared with the placebo group. In the DNase group, the median reduction in OSDI (-20.75) was more than placebo group (-8.43); however, the difference between groups was borderline significant. CONCLUSIONS: In this pilot study, treatment of severe tear deficient DED patients with DNase eye drops appears safe, well tolerated, and has the potential to reduce the severity of signs and symptoms. TRANSLATIONAL RELEVANCE: Data from this pilot clinical trial demonstrate the therapeutic potential of DNase eye drops in dry eye disease, possibly due to degradation neutrophil extracellular traps (NETs) from the ocular surface.

Neutrophil extracellular traps (NETs) contribute to pathological changes of ocular graft-vs.-host disease (oGVHD) dry eye: Implications for novel biomarkers and therapeutic strategies.

PURPOSE: To investigate the role of neutrophil extracellular traps (NETs) and NET-associated proteins in the pathogenesis of oGVHD and whether dismantling of NETs with heparin reduces those changes. METHODS: Ocular surface washings from oGVHD patients and healthy subjects were analyzed. Isolated peripheral blood human neutrophils were stimulated to generate NETs and heparinized NETs. We performed in vitro experiments using cell lines (corneal epithelial, conjunctival fibroblast, meibomian gland (MG) epithelial and T cells), and in vivo experiments using murine models, and compared the effects of NETs, heparinized NETs, NET-associated proteins and neutralizing antibodies to NET-associated proteins. RESULTS: Neutrophils, exfoliated epithelial cells, NETs and NET-associated proteins (extracellular DNA, Neutrophil Elastase, Myeloperoxidase, Oncostatin M (OSM), Neutrophil gelatinase-associated lipocalin (NGAL) and LIGHT/TNFSF14) are present in ocular surface washings (OSW) and mucocellular aggregates (MCA). Eyes with high number of neutrophils in OSW have more severe signs and symptoms of oGVHD. NETs (and OSM) cause epitheliopathy in murine corneas. NETs (and LIGHT/TNFSF14) increase proliferation of T cells. NETs (and NGAL) inhibit proliferation and differentiation of MG epithelial cells. NETs enhance proliferation and myofibroblast transformation of conjunctival fibroblasts. Sub-anticoagulant dose Heparin (100 IU/mL) dismantles NETs and reduces epithelial, fibroblast, T cell and MG cell changes induced by NETs. CONCLUSION: NETs and NET-associated proteins contribute to the pathological changes of oGVHD (corneal epitheliopathy, conjunctival cicatrization, ocular surface inflammation and meibomian gland disease). Our data points to the potential of NET-associated proteins (OSM or LIGHT/TNFSF14) to serve as biomarkers and NET-dismantling biologics (heparin eye drops) as treatment for oGVHD.

Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo.

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1-/- mice. Endothelial cell (EC)-specific Atox1-/- mice and gene transfer of nuclear-target Atox1 in Atox1-/- mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1-/- mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2- production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.

Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function.

Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1(-/-) mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.