Protocol for the treatment of cystoid macular edema secondary to retinitis pigmentosa and other inherited retinal dystrophies.

Inherited retinal dystrophies (IRD) are the leading cause of legal blindness in the working population. Cystic macular edema (CME) is one of the treatable causes of visual loss, affecting up to 50% of the patients. A bibliographic review has been carried out combining "inherited retinal dystrophy", "retinitis pigmentosa", "macular oedema" and a diagnostic-therapeutic protocol according to the levels of evidence and recommendations of the "US Agency for Healthcare Research and Quality". This protocol has been discussed in the monthly meetings of the XAREA DHR group with the participation of more than 25 ophthalmologists, creating a consensus document. The etiology of CME is multifactorial: dysfunction of the blood-retinal barrier, retinal pigment epithelium, and Müller cells, inflammation, and vitreous traction. OCT is the test of choice for the diagnosis and follow-up of CME associated with IRD. The drugs with the highest degree of scientific evidence are carbonic anhydrase inhibitors (IAC). Intravitreal corticosteroids, anti-VEGF, and vitrectomy with peeling of the internal limiting membrane do not have sufficient evidence. A treatment scheme is proposed for the CME in IRD in adults, another for pediatric patients and another for IRD and cataract surgery. Oral and topical IACs are effective in the treatment of CME secondary to IRD. Treatment with corticosteroids, anti-VEGF, and vitrectomy are second-line options. Randomized clinical trials are required to establish the therapeutic scale in these patients.

Translation and validation of the Visual Function and Corneal Health Status (V-FUCHS) questionnaire into Spanish language.

PURPOSE: To translate and validate the V-FUCHS questionnaire into Spanish in a population of patients with Fuchs endothelial dystrophy (DEF). METHODS: The V-FUCHS consists of 15 short, easily understandable questions that assess visual aspects of quality of life in patients with DEF, which can be gathered into a group of seven that assess the "Visual Difficulty" factor and another group of eight that assess the "Glare Factor". For the translation and cultural adaptation, the standardized norms for this process were followed, among other phases, a translation, a back-translation and an application in patients with DEF. RESULTS: In the first phase, consensus was reached on the Spanish translation of the V-FUCHS. Subsequently, 25 patients were included to carry out the pre-test phase with the aim of assessing the applicability and feasibility of the test. The score obtained a minimum value of -0.88 and a maximum value of +2.44, according to the Rasch probabilistic scale. The mean value obtained from the Visual Difficulty factor was 0.61 (±0.71), while the mean for the Glare Factor was 0.41 (±0.51). CONCLUSION: The validation of the V-FUCHS questionnaire, after its translation and adaptation into Spanish, proved to be a useful tool for assessing the visual quality of patients with DEF. Patients with a more advanced stage of the disease presented a greater severity in the test result. Likewise, the Glare Factor (Glare) correlates better with the pachymetric increase than with the visual acuity of the patient.

Pterygium surgery with lyophilized versus cryopreserved amniotic membrane graft.

PURPOSE: To evaluate surgical outcomes (recurrence rate, aesthetics and symptoms) of pterygium surgery with two different amniotic membrane preservation approaches - lyophilized (LAM) and cryopreserved (CAM). METHODS: Primary pterygium patients were randomized to either LAM or CAM surgery. Demographic data, ocular surface disease index (OSDI), aesthetic grading (1 to 4), recurrences and complications were recorded over a 6-month follow-up period. RESULTS: Twenty-nine patients were recruited. Recurrence at month 6 was detected in 11 cases (37.9%) and was more prevalent with CAM grafts, without reaching statistical significance (P=0.196). Aesthetic outcome grading showed no differences between LAM and CAM at month 6 (P=0.124). Aesthetic results were mostly unsatisfactory (grade 3 and 4) without statistical differences between groups (P=0.514). Baseline OSDI was similar in both groups (P=0.888), and it significantly decreased by the last follow-up visit (P<0.001) for both the LAM and CAM groups. This decrease did not significantly differ between amniotic membrane preservation approach surgery groups (P=0.714). CONCLUSION: LAM might be considered a legitimate alternative to CAM, showing no inferiority in outcomes, since clinical and aesthetic outcomes were similar for both groups.

Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix.

BACKGROUND: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure. METHODS: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue. RESULTS: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue. CONCLUSIONS: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients.

Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Determination of the Culture Time Point to Induce Corneal Epithelial Differentiation in Induced Pluripotent Stem Cells.

BACKGROUND: Limbal stem cells (LSC) are progenitor cells in the ocular surface that renew the corneal epithelium. Limbal stem cell deficiency usually induces blindness through the loss of corneal transparency, and bilateral cases do not an accurate treatment because of the lack of an autologous source of stem cells. METHODS: Induced pluripotent stem cells (iPSC) are promising for use in cell therapy because of their autologous origin and the capability to differentiate into corneal epithelial cells. However, there are not standardized protocols to achieve a complete corneal epithelial differentiation. We examined the expression of several markers in a human episomal iPSC line after an induction period from embryoid bodies. RESULTS: Progenitor LSC and corneal epithelial differentiation markers, some extracellular matrix protein adhesion molecules, and wingless signaling pathway were studied. Overall, LSC progenitor and corneal epithelium differentiation markers increased after maintaining cell culture in specific conditions for 14 days, whereas pluripotency markers decreased. CONCLUSIONS: Our approach indicated that the optimal time point to initiate iPSC differentiation into LSC and corneal phenotypes, with the use of specific medium, is from 14 days after initial embryoid bodies treatment induction.

[Availability of resources for patients with wet age-related macular degeneration. Optimal study]. / Disponibilidad de recursos para pacientes con degeneración macular asociada a la edad de tipo húmedo. Estudio Optimal.

OBJECTIVE: The aim of the study was to assess the availability of resources for patients with wet age-related macular degeneration (wAMD) in current clinical practice. METHODS: Observational, cross-sectional and multicenter study. Eligible subjects were ≥ 18 years old, with primary/secondary active subfoveal AMD-related choroidal neovascularization diagnosed 12-18 months prior to inclusion in the study. RESULTS: A total of 266 patients were included (39 centers). The mean age (SD) was 76.1 (8.1) years, of whom 55.6% were female. According to the investigator assessment a median (Q1-Q3) of 20.0 (10.0-50.0) patients were visited weekly. A mean of 100.0 (45.0-250.0) were currently under treatment mainly performed in operating rooms (61.5%). Centers had 1.0 (1.0-2.0) operating rooms available for treatment 2.0 (2.0-5.0) days/week. In 74.4% they were located on different floors/buildings from ophthalmology services. Waiting time until visit was 40.0 (30.0-60.0) min, and duration of treatment was 20.0 (15.0-50.0) min. The time between request until medical visit was 20.0 (15.0-30.0) days, and from diagnosis to treatment was 7.0 (5.0-10.0) days. Clinicians considered there was insufficient staff for examinations (84.6%), and treatment (46.2%). About 30.8% and 20.5% mentioned lack of diagnostic tools, such as optical coherence tomography and fluorescein angiography. CONCLUSIONS: More resources for diagnosis and treatment of wAMD are required. These results, together with the current policy of reducing the budget in the Spanish Health System, could lead to possible delays in the diagnosis and treatment of wAMD.

Pathological findings in the lens capsules and intraocular lens in chronic pseudophakic endophthalmitis: an electron microscopy study.

PURPOSE: The aim of this study was to describe the pathological findings in lens capsules and intraocular lens (IOL) studied by scanning and/or transmission electron microscopy (SEM and TEM, respectively) in a series of four eyes with chronic pseudophakic endophthalmitis (CPE). PATIENTS AND METHODS: We performed a retrospective study of four patients presenting CPE in whom surgical treatment with pars plana vitrectomy, capsulectomy with extraction of the IOL, and intravitreous antibiotic therapy was thereafter performed. The extracted IOL and the capsular remains were studied by SEM and/or TEM and microbiologic analysis of aqueous humour and vitreous aspirate was also carried out in all the cases. RESULTS: The presence of microorganisms was observed in the material analysed in all the cases studied. The use of TEM identified bacterial contamination by Staphylococcus spp and mixed contamination with microorganisms presenting a bacillar morphology suggestive of infection by Propionibacterium acnes in addition to the presence of cocci in the capsular remains. In another two cases, SEM localized colonies of Staphylococcus spp on the surface of the IOL in one case and mixed bacterial colonization with cocci plus filamentous bacteria in the other. The presence of macrophages associated with bacteria was observed in the capsular remains. CONCLUSIONS: Microorganisms were found in the IOL or the capsular material in the four cases studied, thereby explaining the refractoriness and severity of infection. The possible presence of polymicrobial infections, especially in the cases with filamentous bacteria, also explains the recurrence of infection.

[Acute posterior multifocal placoid pigment epitheliopathy. Study of 16 cases]. / Epiteliopatía pigmentaria placoide posterior multifocal aguda. Estudio de 16 casos.

OBJECTIVE: Acute Posterior Multifocal Placoid Pigment Epitheliopathy (APMPPE) is a rare disease with a probable inflammatory component which mostly affects young patients. The aim of our study was to analyse the demographic and clinical features of this disease in a group of 16 patients. METHODS: Sixteen patients with APMPPE were included in this study. We analyzed their demographic data (age, sex) and the most relevant clinical findings: visual acuity and retinal disease outcome, association with other systemic diseases and response to treatment. We also collected data from fluorescence angiography, autofluorescence and optical coherence tomography (OCT) in some of the patients. RESULTS: Average age at diagnosis was 26.75 years with no sex predilection. Average final visual acuity (Snellen Scale) in our study was 0.73. Four patients presented with a systemic disease related to the APMPPE. Eleven patients were treated with oral steroids (one patient with steroids and cytotoxic agents) while the remaining 5 patients received no treatment. CONCLUSIONS: In our patients, the average age at diagnosis was less than 30 years, with no sex predilection, as previously described by many authors. The visual outcome is usually good regardless of the treatment given, although there are cases with a bad visual outcome, especially those with foveal involvement when initially seen.

[Efficacy of sodium carboxymethylcellulose in the treatment of dry eye syndrome]. / Eficacia de la carboximetilcelulosa sódica para el tratamiento del síndrome del ojo seco.

AIM: To assess the efficacy of sodium carboxymethylcellulose in the treatment of dry eye. MATERIAL AND METHODS: We carried out a prospective, randomized, masked-observer, control/problem group, single-center clinical assay during a period of 12 months in 19 patients that presented mild or moderate forms of dry eye. Patients were clinically evaluated each 3 months and treated with a 0.5% isotonic solution of sodium carboxymethylcellulose (CMC) or balanced salt solution. Subjective symptoms, functional tests and conjunctival impression cytology were performed according preexistent schedule study visits. To compare data between groups chi squared (chi2) analysis was applied. RESULTS: We observed a significant (p<0.05) decrease in the frequency of subjective symptoms and a significant (p<0.05) improvement of tearfilm interface stability after CMC treatment. There was a tendency to improve the degree of corneal surface wettability and the tearfilm integrity with higher percentage improvements in the CMC group compared to controls. Improved baseline values in at least one of the objective functional tests carried out (p<0.05) was also observed in an elevated percentage of patients in the CMC group (83.3%) compared with controls (34%). Furthermore, we observed a tendency to diminish the frequency of associated subjective symptoms after treatment. Conjunctival impression cytology did not provide significant differences related with therapeutic response. CONCLUSIONS: The results show a significant beneficial effect of CMC to improve clinical parameters in mild and moderate forms of dry eye.

[Utility of ultrasonography in toxoplasmic uveitis]. / Utilidad de la ecografía en las uveítis por toxoplasmosis.

PURPOSE: To analyze the main indications and the most common ultrasonographic features observed in uveitis due to toxoplasmosis. MATERIAL AND METHODS: We carried out a retrospective, observational and descriptive study performed in 97 exams corresponding to 89 patients with uveitis during 7 consecutive years (1994-2000) using B-ultrasonography evaluation. RESULTS: The main ultrasonographic indication in toxoplasmosis was vitreous opacity. We observed that the most frequent findings were: a) intravitreous punctiform echoes, b) thickening of posterior hyaloid, c) partial or total posterior vitreous detachment and d) focal retinochoroidal thickening. This last finding should be highlighted due to its significant correlation (p<0.01) with toxoplasmosis. CONCLUSIONS: Our results suggest that ultrasonography plays an important role in the diagnosis and clinical follow-up of toxoplasmic uveitis.

Cloning and characterization of human syndecan-3.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.

Syndecan-2 expression in colorectal cancer-derived HT-29 M6 epithelial cells induces a migratory phenotype.

Members of the heparan sulfate proteoglycan family, the syndecans have emerged as integrators of extracellular signals, such as ECM components or growth factors, that activate cytoplasmic signaling cascades and regulate cytoskeletal functions. Specifically, syndecan-2 has been implicated in various cellular processes, from differentiation to migration, including its participation in cell-cell and cell-matrix adhesion. Here, we focused on the involvement of syndecan-2 in epithelial versus mesenchymal differentiation. Colorectal cancer-derived HT-29 M6 epithelial cells were stably transfected with full-length syndecan-2 cDNA, and the effect on cell morphology, adhesion, and mobility was evaluated. Characteristic features of migratory cells such as loss of intercellular contacts, flatter shape and multiple membrane projections were observed in syndecan-2 transfectants. Western blot analysis of the major component of epithelial adherens junctions, E-cadherin, revealed decreased expression levels. Furthermore, syndecan-2 induced stronger adhesion to collagen type I, specifically inhibited by heparin. This was correlated with an increased ability for migration, as demonstrated by wound healing experiments and transwell assays, without affecting their growth rate. These results indicate that syndecan-2 expression in mucus-secreting HT-29 M6 cells induces differentiation toward a migratory mesenchymal-like phenotype.

Integrating signals from T-cell receptor and serum by T cells enhance translation of tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF-alpha in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF-alpha due to serum could be inhibited by the phosphatidylinositol (PI) 3-K inhibitors, wortmannin and LY294002, suggesting that PI 3-K is involved in the translational control of TNF-alpha by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF-alpha secretion and this was up-regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3-Kalpha increased the production of TNF-alpha in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen-activated protein kinase (MAPK), potentially downstream of PI 3-kinase, PD98059 and SB203580. Differently from with PI 3-K inhibitors, the accumulation of TNF-alpha mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3-K is an important step in controlling TNF-alpha protein synthesis in response to growth factors.

Rab27a: A key to melanosome transport in human melanocytes.

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells.

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

[The use of collagen in the cicatrization of wounds]. / El uso del colágeno en la cicatrización de las heridas.

The authors review the use of collagen in the cicatrization of wounds, analyzing what this process consists of and what its regeneration and reparation phases are. The authors also summarize some fundamental biological aspects collagen has, their functions in hemostasia and in cicatrization; they develop the use of heterologous collagen in the cicatrization process. Expressive illustrations and a selection of bibliographical references accompany this article.