Subconjunctival injection of mesenchymal stromal cells protects the cornea in an experimental model of GVHD.

PURPOSE: To evaluate the therapeutic effect of subconjunctival injection of human mesenchymal stromal cells (hMSCs) in the cornea of mice with graft versus host disease (GVHD). METHODS: GVHD was induced in mice after hematopoietic stem cell transplantation (HSCT) between MHC-mismatched mouse strains. Subconjunctival injection of hMSCs was applied at day 10 post-HSCT. Infiltration of CD3+ cells in the cornea and epithelial alterations were analyzed by immunofluorescence. Tear was assessed using the PRT test and TearLab Osmolarity System. qPCR was used to evaluate changes in cytokines, Pax6 and Sprr1b expression. To evaluate the effect of irradiation, we analyzed the expression of these genes in TBI mice. RESULTS: Immune cell invasion occurs in mice with GVHD, as shown by the presence of CD3+ cells in the cornea. Interestingly, eyes treated with hMSC did not present CD3+ cells. Tear osmolarity was increased in GVHD eyes, but not in treated eyes. TNFa expression was highly increased in all corneas except in Control and treated eyes. Pax6 in corneal epithelium showed a similar pattern in GVHD and Control mice, and its gene expression was enhanced in GVHD corneas. In contrast, Pax6 was reduced in GVHD + MSC corneas. We also found an increase in SPRR1B staining in GVHD eyes that was lower in GVHD + MSC mice, demonstrating that corneal keratinization is less frequent after treatment with hMSC. CONCLUSIONS: The treatment with hMSCs by subconjunctival injection is effective in reducing corneal inflammation and squamous metaplasia in ocular GVHD (oGVHD). Local treatment with hMSCs is a promising strategy for oGVHD.

Assessment of dry eye in a GVHD murine model: Approximation through tear osmolarity measurement.

Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Given the increasing evidence that tear osmolarity is an important player of dry eye disease, our purpose in this study was to validate the use of a reliable method to assess tear osmolarity in mice: the electrical impedance method. Then, we wanted to test its utility with an oGVHD model. Tear volume assessment was also performed, using the phenol red thread test. We found differences in tear osmolarity in mice that received a transplant with cells from bone marrow and spleen (the GVHD group) when compared with mice that only received bone marrow cells (the BM group) at day 7 (362 ± 8 mOsm/l and 345 ± 9 mOsm/l respectively; P < 0.01) and day 21 (348 ± 19 mOsm/l vs. 326 ± 15 mOsm/l; P < 0.05). We found also differences in tear volume at day 14 (2.30 ± 0.61 mm in oGVHD group and 2.89 ± 0.62 mm in BM group; P = 0.06) and at day 21 (2.10 ± 0.30 mm in oGVHD group and 2.89 ± 0.32 mm in BM group; P < 0.01). Besides this, we observed reduction in epithelial thickness between the GVHD and BM groups (37.0 ± 6.2 µm and 43.6 ± 3.3 µm respectively; P < 0.05). These data show the usefulness of the electrical impedance method to measure tear osmolarity in mice. We can also conclude that this oGVHD model mimics the tear film alterations found in human dry eye disease, what contributes to give relevance to this model for the study of GVHD.

Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice.

Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP(+)-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP(-) cells, which confirms their differentiation into corneal tissues. GFP(+)-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.

Predictive value of VEGF A and VEGFR2 polymorphisms in the response to intravitreal ranibizumab treatment for wet AMD.

BACKGROUND: To determine whether gene polymorphisms of the vascular endothelial growth factor A (VEGF A) and its receptor (VEGFR) influence the response to a variable-dosing treatment regimen with ranibizumab for age-related macular degeneration. METHODS: This prospective cohort study included 94 patients (94 eyes) with exudative age-related macular degeneration (AMD) treated with ranibizumab. Patients underwent a 1-year treatment as in the Study of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (SUSTAIN). Injections were administered monthly during 3 months to all the patients diagnosed of neovascular AMD; reinjections were made when a patient lost 5 letters on the Early Treatment Diabetic Retinopathy Study chart or gained 100 µm in central subfield retinal thickness measured by OCT. Genotypes (VEGF A (rs 699947, rs833061) and VEGFR (rs 2071559)) were analyzed using TaqMan probes. Best-corrected visual acuity (BCVA), subjective improvement, and macular thickness measured with OCT values were compared with VEGF A and VEGFR genotypes. Multiple regression analysis was used to assess the statistical significance. RESULTS: We found statistically significant differences in allelic distribution of VEGF A rs833061 polymorphism in relation with the response to intravitreal ranibizumab regarding to visual acuity improvement [p = 0,.34; OR: 1.619 (1.098-2.386)]. Patients carrying "protector" genotype CC had higher probability of best corrected visual acuity improvement. When we analyzed VEGF A rs699947 polymorphism we found that patients expressing AA genotype had a higher chance of increasing their best corrected visual acuity [p:0,022; OR 1,532 (1,015-2,313)]. We did not find statistically significant differences reagarding VEGFR rs2071559 polymorphism and treatment response. CONCLUSIONS: Polymorphisms of VEGF A seem to influence the different response to antiangiogenic treatment in patients with AMD in our population, although further investigation is needed to know the mechanisms of this relationship.

Cistoid macular edema as first manifestation of sarcoidosis.

The purpose of this study is to report a case of cystoid macular edema (CME) as a rare first manifestation of ocular sarcoidosis after cataract surgery. A 60-year-old male developed a CME following uneventful phacoemulsification cataract extraction on his left eye. It resolved with conventional medical therapy. One year later the patient was diagnosed with bilateral CME. Oral corticosteroid therapy produced a significant regression. His medical and ocular histories were unremarkable and all tests for etiological diagnosis were negative. There were inflammation recurrences in his left eye, which were also treated with steroids. Optical coherence tomography showed complete resolution of foveal thickening without improvement in vision. Four years later, the patient presented with CME in both eyes. The laboratory tests included high angiotensin-converting enzyme levels and a gallium scan which were also consistent with sarcoidosis. Azathioprine was needed for management of ocular involvement, but it was withheld due to side-effects. At the present time, the CME is controlled with low-dose corticoids. Ocular involvement in sarcoidosis occurs in 20-50 % of patients. CME is not often the initial manifestation of the disease, but ocular sarcoidosis may present with a wide variety of ocular symptoms in all parts of the eye. Therefore, sarcoidosis should be kept in mind when evaluating a patient with ocular inflammation.

Limbus damage in ocular graft-versus-host disease.

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality among patients undergoing allogeneic hematopoietic stem cell transplantation. Although animal models have been clearly established for the study of skin, liver, and gut, currently there is no equivalent experiemental model for analyzing ocular involvement, which is rather common, especially among patients diagnosed with chronic GVHD. In the current study we have developed a murine model of ocular GVHD and, for the first time, we describe the histopathologic features involving cornea and limbus, which could play a role in the physiopathology of the disease at the ocular level. Our results represent a major finding that allows us to define a model for evaluating new therapeutic strategies for treating ocular GVHD prior to their use in clinical setting.