The Effect of Amniotic Membrane Transplantation or Conjunctival Autografts on the Tear Mucins MUC5A and MUC2 After Pterygium Resection: A Six-Month Follow-Up.

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.

Intravitreal Antiangiogenic Treatment for Diabetic Retinopathy: A Mexican Real-Life Scenario Experience.

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.

Risk Factors for Diabetic Retinopathy in Latin America (Mexico) and the World: A Systematic Review and Meta-Analysis.

Diabetic retinopathy (DR) is one of the main complications of diabetes, and the management of the main control parameters explains only an 11% reduction in the risk of progressing to DR, leaving 89% to be explained by other factors or correlations between the usual factors that are currently unknown. The objective of this systematic review and meta-analysis is to evaluate the similarities and differences between the possible risk factors for developing DR when comparing the world to Latin American populations. The search was performed first for Latin American (LA) populations and a second search for non-Latin American (Non-LA) populations. Using the PRISMA guidelines, five articles were found to be relevant for each of the groups. The patients who had elevated systolic blood pressure (SBP) developed DR more frequently than the patients without retinopathy (Z = 2.1, p = 0.03), an effect measured in the population at a global level (GL), behavior that becomes not significant when the LA and non-LA populations are grouped separately; relevant to this is that the diagnosis of hypertension (HBP) grouped globally and stratified does not present a risk factor for DR (Z = 0.79, p = 0.42). This indicates that SBP is a risk factor for the world population and that, by separating it into different regions, the omission could cause it not to be considered a possible risk factor. In conclusion, the relationship between the increase in DR associated with the risk factors present in different populations, the limited research conducted in Latin America, and the cultural, social, economic, and genetic differences makes for a complex condition, which reflects the necessity of researching in a more integrated way.

Production of Reactive Oxygen (ROS) and Nitrogen (RNS) Species in Macrophages J774A.1 Activated by the Interaction between Two Escherichia coli Pathotypes and Two Probiotic Commercial Strains.

Probiotics play an important role against infectious pathogens, such as Escherichia coli (E. coli), mainly through the production of antimicrobial compounds and their immunomodulatory effect. This protection can be detected both on the live probiotic microorganisms and in their inactive forms (paraprobiotics). Probiotics may affect different cells involved in immunity, such as macrophages. Macrophages are activated through contact with microorganisms or their products (lipopolysaccharides, endotoxins or cell walls). The aim of this work was the evaluation of the effect of two probiotic bacteria (Escherichia coli Nissle 1917 and Bifidobacterium animalis subsp. lactis HN019 on macrophage cell line J774A.1 when challenged with two pathogenic strains of E. coli. Macrophage activation was revealed through the detection of reactive oxygen (ROS) and nitrogen (RNS) species by flow cytometry. The effect varied depending on the kind of probiotic preparation (immunobiotic, paraprobiotic or postbiotic) and on the strain of E. coli (enterohemorrhagic or enteropathogenic). A clear immunomodulatory effect was observed in all cases. A higher production of ROS compared with RNS was also observed.

Spontaneous Neutrophil Extracellular Traps Release Are Inflammatory Markers Associated with Hyperglycemia and Renal Failure on Diabetic Retinopathy.

Diabetic retinopathy (DR) is the major microvascular complication of diabetes and causes vitreous traction and intraretinal hemorrhages leading to retinal detachment and total blindness. The evolution of diabetes is related to exacerbating inflammation caused by hyperglycemia and activation of inflammatory cells. Neutrophils are cells able to release structures of extracellular DNA and proteolytic enzymes called extracellular traps (NETs), which are associated with the persistence of inflammation in chronic pathologies. The purpose of the study was to determine the usefulness of neutrophil traps as indicators of DR progression in patients with type 2 diabetes (T2DM). We performed a case-control study of seventy-four cases classified into five groups (non-proliferative DR, mild, moderate, severe, and proliferative) and fifteen healthy controls. We found correlations between NETs and a diagnostic time of T2DM (r = 0.42; p < 0.0001), fasting glucose (r = 0.29; p < 0.01), glycated hemoglobin (HbA1c) (r = 0.31; p < 0.01), estimated glomerular filtration rate (eGFR) (r = -0.29; p < 0.01), and plasma osmolarity (r = 0.25; p < 0.01). These results suggest that due to NETs being associated with clinical indicators, such as HbA1c and eGFR, and that NETs are also associated with DR, clinical indicators might be explained in part through an NET-mediated inflammation process.

Seroprevalence of SARS-CoV-2 in Mexican Health Care Workers after Two Years of the Pandemic: The Picture of an Ophthalmic Medical Centre.

OBJECTIVE: In this study, we investigated the impact of the SARS-CoV-2 vaccination on seroprevalence in a cohort of healthcare workers (HCW) at an ophthalmic medical center. METHODS: IgG antibodies against the N, S1, and S2 antigens of SARS-CoV-2 as well as their serum neutralizing activity were determined. RESULTS: In the present study, we observed that 98.4% of HCW were seropositive for S1/S2 proteins of SARS-CoV-2 due to the national vaccination program. Interestingly, 78.4% of the participants had anti-N protein antibodies, suggesting previous COVID-19 infection. We also evaluated the neutralizing antibodies and found that the mean value was high (90.7%). CONCLUSION: These results indicate that our HCWs cohort presented a robust hybrid humoral response owing to the massive national vaccination program and natural infections.

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature.

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 °C and 4 °C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.

Human Amniotic Membrane Mesenchymal Stem Cell-Synthesized PGE2 Exerts an Immunomodulatory Effect on Neutrophil Extracellular Trap in a PAD-4-Dependent Pathway through EP2 and EP4.

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.

SARS-CoV-2 Seroprevalence among the Health Care Staff of an Ophthalmological Reference Centre, a Cross Sectional Study.

PURPOSE: During the COVID-19 pandemic, healthcare workers (HCWs) are at a considerable risk of being infected with SARS-CoV-2; among them, HCWs from ophthalmology departments are more prone to develop severe symptoms. In Mexico City, the prevalence of SARS-CoV-2 infection among HCWs is 30%. The present work aims to describe the seroprevalence among HCWs at an Ophthalmological Reference Centre in Mexico City. METHODS: A self-report questionnaire, RT-PCR test and detection of serum IgG/IgM antibodies against SARS-CoV-2 were performed among HCWs at the Institute of Ophthalmology "Conde de Valenciana". RESULTS: A total of 169 HCWs participated in the study. None of the participants declared severe symptoms, and only 15% showed three or more symptoms. The results showed that 32% of the participants were RT-PCR+ (54/169), and 20% (35/169) presented IgG antibodies against SARS-CoV-2. Thirteen percent of the RT-PCR+ subjects were IgG positive, and 7.6% of the RT-PCR- participants were IgG positive. The presence of three or more symptoms correlated with the presence of IgG antibodies, as well as Ct values of < 32 (p < 0,05). CONCLUSION: Most of the HCW cohort showed mild symptoms, and 69% of the RT-PCR+ participants did not show IgG antibodies against SARS-CoV-2. Seroprevalence was significantly associated with the presentation of COVID-19-associated symptoms.

AS1411 Nucleolin-Specific Binding Aptamers Reduce Pathological Angiogenesis through Inhibition of Nucleolin Phosphorylation.

Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.

Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts.

Background: Coinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection. Objective: To determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers. Results: The biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited. Conclusion: This is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

Amniotic membrane conditioned medium (AMCM) reduces inflammatory response on human limbal myofibroblast, and the potential role of lumican.

Purpose: Viral infections such as herpetic keratitis (HSK) activate the innate immune response in the cornea triggering opacity and loss of vision. This condition is performed mainly by myofibroblasts that exacerbate secretion of inflammatory cytokines. Amniotic membrane transplantation (AMT) reduces ocular opacity and scarring inhibiting secretion of inflammatory cytokines and proliferation of myofibroblasts. We previously reported that the amniotic membrane (AM) favors an anti-inflammatory microenvironment inhibiting the secretion of inflammatory cytokines, expression of innate immune receptors, and translocation of nuclear NF-κB on human limbal myofibroblasts (HLMs). The aim of the present study was to determine whether the soluble factors of the AM decrease the immune response of HLMs stimulated with polyinosinic-polycytidylic acid sodium salt (poly I:C). Methods: The AM was incubated in Dulbecco's modified eagle medium (DMEM)/F12, and the supernatant was collected to obtain amniotic membrane conditioned medium (AMCM). HLMs were isolated from cadaveric sclera-corneal rims. HLMs were cultured in DMEM/F12 or AMCM and stimulated or not with poly I:C (10 µg/ml) for 12 h to analyze synthesis of CCL2, CCL5, CXCL10, MDA5, RIG-1, and TLR3 or for 2 h to analyze translocation of nuclear NF-kB, IRF3, and IRF7. The proteins contained on AMCM were analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the acquired peptide ions were analyzed with the Mascot program using both National Center for Biotechnology Information (NCBI) and expressed sequence tag (EST) databases. Results: AMCM downregulated the mRNA levels of CCL2, CCL5, CXCL10, MDA5, RIG-1, and TLR3. In addition, AMCM decreased secretion of CCL2, CCL5, and CXCL10 and translocation of nuclear NF-κB. Interestingly, AMCM increased translocation of nuclear IRF3 and synthesis and secretion of type I IFN-ß. We also identified small leucine-rich proteoglycan lumican in the AMCM. The administration of rh-lumican to poly I:C-stimulated HLMs reduced the mRNA levels of CCL2, CCL5, and CXCL10. Conclusions: These results suggest that the AM can trigger an anti-inflammatory response on HLMs through soluble factors, and that lumican could play an important role in these effects.

Future Perspectives of Therapeutic, Diagnostic and Prognostic Aptamers in Eye Pathological Angiogenesis.

Aptamers are single-stranded DNA or RNA oligonucleotides that are currently used in clinical trials due to their selectivity and specificity to bind small molecules such as proteins, peptides, viral particles, vitamins, metal ions and even whole cells. Aptamers are highly specific to their targets, they are smaller than antibodies and fragment antibodies, they can be easily conjugated to multiple surfaces and ions and controllable post-production modifications can be performed. Aptamers have been therapeutically used for age-related macular degeneration, cancer, thrombosis and inflammatory diseases. The aim of this review is to highlight the therapeutic, diagnostic and prognostic possibilities associated with aptamers, focusing on eye pathological angiogenesis.

Comparison of amniotic membrane transplantation and carpal tunnel syndrome release surgery (CTRS) and CTRS alone: Clinical outcomes at 1-year follow-up.

Carpal tunnel syndrome (CTS) is the most common focal entrapment mononeuropathy, comprising medium nerve chronic inflammation and fibrosis. Although carpal tunnel release surgery (CTRS) has demonstrated to be effective, around 3% to 25% of CTRS show recurrence. Amniotic membrane transplantation (AMT) has been used in different pathologies inhibiting inflammation and fibrosis and promoting nerve repair. The aim of this study was to determine the efficacy of AMT in CTRS. The present study comprised a randomized, single-blind controlled trial to compare the 1-year follow-up outcomes of AMT in CTRS (AMT group) or CTRS alone (control group) in patients with CTS. Thirty-five patients with unilateral or bilateral CTS were enrolled, and 47 wrists were randomized into two groups: the AMT group and the control group. To compare the outcomes, three different questionnaires scores (Boston Carpal Tunnel Syndrome Questionnaire, Disabilities of the Arm, Shoulder, and Hand, and Historical-Objective scale) were used. Evaluations were assessed at baseline and at 15 days, 1, 3, 6, and 12 months after surgery. Compared with the control group, the AMT group showed significant (p < 0.05) reductions in all scores from 6 months after surgery until the end of the study. Both AMT and control groups showed significant intragroup differences in all scores, since the first month after surgery until the end of the study in comparison with the baseline scores. Taken together, these results indicate that CTRS in conjunction with AMT is more effective than CTRS alone in patients with CTS at 1-year follow-up. Clinical Trial: NCT04075357; Amniotic Membrane in Carpal Tunnel Syndrome.

Comparative analysis of inflammatory response in the BALB/c and C57BL/6 mouse strains in an endotoxin-induced uveitis model.

Uveitis is an inflammatory disease associated with diverse systemic and autoimmune diseases, defined as the inflammation of any given segment of the uveal tract, uveitis contributes with 5-20% cases of blindness in the USA/Europe and >25% of cases in third-world countries. To understand its pathogenic mechanisms, BALB/c and C57BL/6 mice were induced to develop the condition by a single intraperitoneal injection of E. coli lipopolysaccharide, the aim of the present work is to determine leukocyte infiltration in an endotoxin-induced uveitis (EIU) in two non-related mouse strains. Histopathological findings and clinical analysis were conducted 24 and 48 h postinjection. Both strains presented conventional clinical signs of uveitis 24 h post LPS injection and the highest inflammatory leukocyte infiltration in the uveal tract was found in the BALB/c mouse strain. This article will give an insight on the difference of the inflammatory response in the EIU model in two different mouse strains and the relationship between the pathologic response.

Anti-Inflammatory and Anti-Fibrotic Effects of Human Amniotic Membrane Mesenchymal Stem Cells and Their Potential in Corneal Repair.

Acute ocular chemical burns are ophthalmic emergencies requiring immediate diagnosis and treatment as they may lead to permanent impairment of vision. The clinical manifestations of such burns are produced by exacerbated innate immune response via the infiltration of inflammatory cells and activation of stromal fibroblasts. New therapies are emerging that are dedicated to repair mechanisms that improve the ocular surface after damage; for example, transplantation of stem cells (SC) has been successfully reported for this purpose. The pursuit of easily accessible, noninvasive procedures to obtain SC has led researchers to focus on human tissues such as amniotic membrane. Human amniotic mesenchymal SC (hAM-MSC) inhibits proinflammatory and fibrotic processes in different diseases. hAM-MSC expresses low levels of classical MHC-I and they do not express MHC-II, making them suitable for regenerative medicine. The aim of this study was to evaluate the effect of intracameral injection of hAM-MSC on the clinical manifestations, the infiltration of inflammatory cells, and the activation of stromal fibroblasts in a corneal alkali-burn model. We also determined the in vitro effect of hAM-MSC conditioned medium (CM) on α-SMA+ human limbal myofibroblast (HLM) frequency and on release of neutrophil extracellular traps (NETs). Our results show that intracameral hAM-MSC injection reduces neovascularization, opacity, stromal inflammatory cell infiltrate, and stromal α-SMA+ cells in our model. Moreover, in in vitro assays, CM from hAM-MSC decreased the quantity of α-SMA+ HLM and the release of NETs. These results suggest that intracameral hAM-MSC injection induces an anti-inflammatory and anti-fibrotic environment that promotes corneal wound healing. Stem Cells Translational Medicine 2018;7:906-917.

Human Amniotic Membrane Mesenchymal Stem Cells inhibit Neutrophil Extracellular Traps through TSG-6.

The mesenchymal stem cells obtained from human amniotic membrane (hAMSC) possess immunosuppressive functions through soluble factors such as prostanoids and proteins; thus, they have been proposed to ameliorate inflammatory processes. On the other hand, activated neutrophils are cells of the first line of immune defense that are able to release extracellular traps (NETs). NETs are formed of DNA and granular components; however, the excessive release of NETs is associated with the development of autoimmune and chronic inflammatory diseases. In this study, we identified that conditioned medium (CM) from hAMSC was able to diminish NETs release, as well as the production of reactive oxygen species (ROS) and the mitochondrial membrane potential from LPS-stimulated mouse bone marrow-derived neutrophils (BMN). Interestingly, NETs inhibition, ROS levels decrease and mitochondrial membrane potential loss were reverted when LPS-stimulated murine derived BMN were exposed to the CM from hAMSC transfected with TSG-6-siRNA. Finally, rhTSG6 was able to significantly diminish NETs release in BMN. These data suggest an inhibition mechanism of NETs ROS-dependent in which TSG-6 participates. Consequently, we propose the hAMSC use as a therapeutic candidate in the treatment of inflammatory diseases in which NETs are involved.