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The objective of this study was to analyze the effectiveness of two intravitreal antiangiogenic drugs, ranibizumab and aflibercept, in a Mexican population over a period of 5 years, evaluating the improvement in visual acuity (VA) and central retinal thickness (CRT) in a real-world scenario. This is a retrospective study with subjects diagnosed with diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR), and diabetic macular edema (DME) receiving intravitreal injections of ranibizumab and/or aflibercept. In this study, we analyzed 588 eyes of 294 patients who received intravitreal antiangiogenic injections. The results showed an improvement regardless of antiangiogenic treatment or diagnosis in both VA and CRT. We found that both aflibercept and ranibizumab improved VA, while subjects with DME responded less to antiangiogenic treatment (p < 0.05), and that this difference did not correspond to the CRT measured by OCT. These results support evidence that intravitreal antiangiogenic medications are effective for ophthalmic complications of diabetes in our population; however, damage to visual structures is not reversed in most patients. And that the perception by the patient (VA) and that of the ophthalmologist (CRT) do not completely correlate in our study. [ABSTRACT FROM AUTHOR]

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release. [ABSTRACT FROM AUTHOR]

The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus ( ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4+ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells. [ABSTRACT FROM AUTHOR]

Purpose: Pterygium is an ocular surface disease characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva to the cornea and is associated with abnormal tear function caused by changes in tear composition and osmolarity. In this study, the effect of two different surgical techniques to remove primary pterygium: conjunctival autograft surgery (CAG) and amniotic membrane transplantation (AMT), on changes in MUC2 and MUC5AC tear mucins concentration were evaluated., Methods: Forty-four patients (>18 years old) with primary unilateral pterygium (> 1.0 mm long, measured from the limbus to the apex on the cornea) were randomly enrolled, and assigned to the AMT or CAG group by using the permuted block technique. Patients with systemic inflammatory diseases or other eye comorbidities were excluded from the study. Tear break-up time (TBUT) and best-corrected visual acuity (BCVA) assessments were performed before surgery and at 1, 3, and 6 months after surgery. Tears were collected concurrently with the clinical evaluations, and MUC2 and MUC5AC concentrations were subsequently measured by means of ELISA., Results: At 6 months after CAG or AMT, TBUT and BCVA were significantly lower (P < 0.05) in comparison with the baseline values in the study subjects. The tear mucin concentrations of both MUC2 and MUC5AC were significantly higher (P < 0.0001) in patients with pterygium before any surgical procedure than in healthy individuals. The concentration of MUC2 increased at 1 and 3 months after CAG surgery and decreased at 6 months; however, the MUC2 concentration decreased on the AMT group in all time point measurements. Interestingly, the MUC5AC concentration significantly increased at 1 month after AMT or CAG and then decreased at 3 and 6 months after surgery. Finally, an inverse correlation was found between both MUC2 and MUC5AC tear mucins concentration and the TBUT., Conclusions: These results suggest that pterygium excision via both CAG or AMT changes the concentrations of the tear mucins MUC2 and MUC5AC during the evaluated times, and these changes could affect tear film stability and clinical recovery after pterygium treatment., Translational Relevance: The tear film stability during pterygium excision was evaluated to determine adequate treatments.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C -terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ β -catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ β -catenin pathway. CEMP1-p4 stimulation upregulated the expression of β -catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of β -catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the β -catenin signaling cascade.

Bilateral limbal stem cell deficiency (LSCD) treatment requires the need to obtain allogenic limbal tissue for transplantation. Outcomes of different surgical techniques depend on multiple factors, including the underlying etiology, ocular surface, eyelid status and used surgical intervention. Some of the management options for bilateral LSCD include cadaveric, living related or living non-related conjunctival limbal allograft (CLAL), keratolimbal allograft (KLAL), allogenic cultured limbal epithelial transplantation (CLET) and allogenic simple limbal epithelial transplantation (SLET). Systemic immunosuppressive therapy plays a pivotal role in survival of transplanted tissue. The present review focuses on different systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation, with specific emphasis on different surgical techniques and their outcomes. We included all reports with details of different systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation. Oral cyclosporine A at different doses is the most commonly used immunosuppressive agent in limbal allograft and allogenic limbal epithelial cell transplantation. However, different studies using oral mycophenolate mofetil and tacrolimus also reported good results. In conclusion, systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation are not standardized. Further studies regarding different surgical techniques should assess outcomes and adverse effects of such protocols., Competing Interests: Ethical issues have been completely observed by the authors. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship of this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published. No conflict of interest has been presented. Funding/Support: None. The datasets analyzed during this study are available from the corresponding author on reasonable request., (© 2020, Author(s).)

The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4(+) T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4(+) T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4(+) T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4(+) T cells.

Background: Vernal keratoconjunctivitis (VKC) is a severe form of allergic conjunctivitis, in which inflammatory infiltrates of the conjunctiva are characterized by CD3+ and CD30+ cells. Until today, the functional involvement of CD30+ T cells in VKC was unclear. Our aim was to evaluate the functional characteristics of CD30+ T cells after allergen stimulation in peripheral blood mononuclear cells obtained from patients with VKC., Methods: Seventeen consecutive patients at the Institute of Ophthalmology with active forms of VKC were included., Results: After allergen stimulation, we observed the frequency of CD30+ T cells increased compared with non-stimulated cells (p<0.0001). The CD30+ T cells responded to the specific allergen-inducing expression of intracellular interleukin-4 (IL-4), IL-5, and interferon-gamma (IFN-γ) compared with the CD30- T cells (p<0.0001). Increased early secretion of soluble CD30 was observed in the supernatant of the cultured cells from patients with keratoconjunctivitis, compared with healthy controls (p=0.03). Blockage with IL-4 significantly diminished CD30 frequency in the allergen-stimulated cells., Conclusions: Our results suggest that after allergenic stimulation, CD4+CD30+ cells are the most important source of IL-4, IL-5, and IFN-γ. IL-4 acts as an activation loop that increases CD30 expression on T cells after specific stimulation. These findings suggest that CD4+CD30+ T cells are effector cells and play a significant role in the immune pathogenic response in patients with vernal keratoconjunctivitis.

Purpose: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue., Methods: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-β1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA)., Results: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFβ1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFβ1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFβRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFβ1 secreted by L-MSC was able to suppress T cell proliferation., Conclusions: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.

Purpose: The pterygium is characterized by a fibrovascular neoformation from the bulbar conjunctiva into the cornea. The recent discovery that abnormal markers associated with tumor diseases are identified in the pterygium strengthens the theory that the pterygium is a tumor-like disease rather than a degenerative disease. The CD30 molecule has been identified in neoplastic and normal epithelial proliferating cells. The aim of this study was to determine the expression of the CD30 molecule in the pterygium., Methods: Immunohistochemical staining using an antibody to CD30 and to the Ki-67 nuclear antigen was performed on 25 pterygial specimens and 10 healthy conjunctivas., Results: Strong immunostaining against CD30 was observed in all pterygium specimens, in contrast to the healthy conjunctivas that showed weak immono-positivity in only three cases. The staining was diffused, predominantly to the basal epithelium. The Ki-67 antigen was observed in the nucleus of the basal epithelium of the pterygial specimens, and no staining was observed in the healthy conjunctiva. When serial sections were stained with CD30 and Ki-67, the cells that expressed CD30 also expressed Ki-67., Conclusions: The present study identified for the first time the existence of CD30 molecules in the basal epithelium of a pterygium. The fact that the positivity to Ki-67 in the basal epithelium of the pterygium correlated with the CD30 reactivity suggested that this protein could be associated with the uncontrolled cell proliferation of the epithelium in this pathology. The CD30 molecule could therefore be a suitable target to be inhibited using chimerical antibodies in pterygium diseases.

Purpose: Keratoconus (KC) is a common progressive corneal disease characterized by excessive stromal thinning, central or paracentral conical protrusion, and disruptions in Bowman's layer. The etiology of KC is largely unknown, and a combination of genetic and environmental factors is believed to play a role in the origin of the disease. Recently, the absence of transcripts of the water channel, aquaporin-5 (AQP5), was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR) in KC tissues and was proposed as a possible marker for KC. In this study, we sought to evaluate AQP5 mRNA and protein expression in KC and non-KC corneal tissues using a combination of techniques., Methods: A total of 69 samples of corneal tissue were analyzed including 39 corneal buttons from patients with advanced KC, 16 samples of non-KC corneal epithelium belonging to patients who underwent surface refractive surgery, 12 sclerocorneal rims obtained from healthy donor subjects, and two healthy corneal buttons. Determination of AQP5 transcript and protein expression patterns was performed by means of real time RT-PCR, immunohistochemistry, immunocytochemistry, and flow cytometry methods. Cell culture was performed to identify AQP5 protein expression in KC epithelial cells., Results: AQP5 mRNA was expressed with no significant differences between KC and non-KC tissues. Moreover, AQP5 protein expression analysis did not reveal differences in protein levels and/or cell location among KC and non-KC tissues. Interestingly, AQP5 expression continues for up to 21 days in the isolated KC corneal epithelial cells., Conclusions: Our results do not support a role for AQP5 in KC etiopathogeny or as a disease marker. Genetic background differences or a distinct pathogenetic KC cascade specific to the analyzed population could account for the dissimilarities observed in KC-related AQP5 expression.

Introduction: Human limbal epithelial cells (huLEC) have been used for clinical purposes in ocular surface diseases to promote rapid re-epithelisation and restore corneal epithelium integrity. However, in Mexico this technique has not been fully developed. This study was conducted to characterize the huLEC phenotype expanded in vitro using a cell culture technique., Material and Methods: Cells were obtained from limbal tissue, cultured in KSFM medium and analyzed for the expression of vimentin, K, K19, p63, K12, by flow cytometry and immuno-fluorescence., Results: The phenotype of cultured cells was vimentin+K+K19+ p63+K12-., Conclusions: Our results suggest that under these culture conditions huLEC maintained their stem cell phenotype. This culture technique could be used for clinical purposes in Mexico.