Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage

Producción CientíficaLimbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble the different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improves corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses.This work was supported by Instituto de Salud Carlos III, CIBER‐BBN, Spain (CB06/01/003 MINECO/FEDER, EU); Regional Center for Regenerative Medicine and Cell Therapy, Castilla y León, Spain; Ministry of Science and Innovation, Spain (SAF2010–14900); Ministry of Economy and Competitiveness and European Regional Development Fund, Spain (SAF2015–63594‐R MINECO/FEDER, EU

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Risk factors for infections caused by carbapenem-resistant Enterobacterales: an international matched case-control-control study (EURECA)

Cases were patients with complicated urinary tract infection (cUTI), complicated intraabdominal (cIAI), pneumonia or bacteraemia from other sources (BSI-OS) due to CRE; control groups were patients with infection caused by carbapenem-susceptible Enterobacterales (CSE), and by non-infected patients, respectively. Matching criteria included type of infection for CSE group, ward and duration of hospital admission. Conditional logistic regression was used to identify risk factors. Findings Overall, 235 CRE case patients, 235 CSE controls and 705 non-infected controls were included. The CRE infections were cUTI (133, 56.7%), pneumonia (44, 18.7%), cIAI and BSI-OS (29, 12.3% each). Carbapenemase genes were found in 228 isolates: OXA-48/like, 112 (47.6%), KPC, 84 (35.7%), and metallo-beta-lactamases, 44 (18.7%); 13 produced two. The risk factors for CRE infection in both type of controls were (adjusted OR for CSE controls; 95% CI; p value) previous colonisation/infection by CRE (6.94; 2.74-15.53; <0.001), urinary catheter (1.78; 1.03-3.07; 0.038) and exposure to broad spectrum antibiotics, as categorical (2.20; 1.25-3.88; 0.006) and time-dependent (1.04 per day; 1.00-1.07; 0.014); chronic renal failure (2.81; 1.40-5.64; 0.004) and admission from home (0.44; 0.23-0.85; 0.014) were significant only for CSE controls. Subgroup analyses provided similar results. Interpretation The main risk factors for CRE infections in hospitals with high incidence included previous coloni-zation, urinary catheter and exposure to broad spectrum antibiotics

A non-invasive method for an in vivo assessment of corneal epithelium permeability through tetrapolar impedance measurements

The permeability of the cornea epithelial layer has an important role in optimal function of the cornea. To assess this property quantitatively, methods must be based on the passive electrical properties of living tissues, as they can take advantage of the fundamental role that ionic permeability plays in such properties. For such techniques, measurement of the translayer electrical resistance (TER) has been consistently used to examine the ion transport mechanisms in the corneal epithelial cells; however, this technique has been only possible in vitro. To enhance the applications of this method, in this work we present a novel sensor to perform non-invasive in vivo TER measurements. Herein, the epithelial permeability was assessed using non-invasive tetrapolar impedance measurements that were performed with four electrodes placed on the corneal surface. The geometry of these electrodes was previously optimized to maximize the sensitivity of the corneal epithelium. To evaluate the feasibility of this sensor, the permeability of a rabbit corneal epithelium was monitored by applying a solution of benzalkonium chloride (0.05% BAC). The results validate the capability of the sensor to evaluate the cornea epithelial permeability in vivo.Peer reviewe

Flexible probe for in vivo quantification of corneal epithelium permeability through non-invasive tetrapolar impedance measurements

Studies concerning the functional status of the corneal epithelium are of special interest due to its key role in preventing ocular surface disease and corneal infections. In particular, quantitative measurements of the epithelium permeability translayer electrical resistance (TER) have been proven as a sensitive in vitro test for evaluation of the corneal barrier function. In a recent work from the authors(Guimera et al. 2012), a novel method to non-invasively assess the corneal epithelial permeability by using tetrapolar impedance measurements, based on the same TER theoretical principles, was presented and validated using a rigid sensing device. In this work, the usability of this method has been dramatically improved by using SU-8 photoresist as a substrate material. The flexibility of this novel sensing device makes no need to apply pressure on the cornea to ensure the electrical contact between the electrodes and the corneal surface. The feasibility of this flexible sensor has been evaluated in vivo by increasing the permeability of rabbit corneal epithelium. For that, different concentrations of benzalkonium chloride (BAC) solution were instilled on different rabbit corneas. The obtained results have been compared with measurements of the permeability to sodium fluorescein of different excised corneas, a well-known method used to evaluate the corneal barrier function, to demonstrate the feasibility of this novel flexible sensor for quantifying the corneal epithelium permeability in vivo in a non-invasive way.We thank to Antoni Ivorra for his contribution in the FEM modeling and sensor conception. This work has been funded by the projects SAF2009-14724-C02-02 from the Spanish Ministry of Science and Innovation, IPT-2012-0438-010000 and SAF2012-40227-C02-02 from Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund.Peer reviewe

XXX Congreso Nacional de Técnico Superior en Laboratorio Clínico y Biomédico y Técnico Superior de Anatomía Patológica y Citodiagnóstico. "Genómica y Terapias Dirigidas".

Producción CientíficaIntroducción: Las células madre responsables de la regeneración continua del epitelio corneal se localizan en el denominado “limbo esclero-corneal”, la zona de transición entre la córnea y la esclera-conjuntiva. Cuando se produce una disfunción o pérdida de estas células madre limbares se desarrolla el Síndrome de Insuficiencia Límbica (SIL), patología que se caracteriza principalmente por la presencia de conjuntivalización, neovascularización y defecto epitelial corneal y por la aparición de dolor crónico y pérdida de visión. Actualmente, la reparación del epitelio de la superficie ocular después de haber sufrido un SIL es un gran desafío. Con el fin de estudiar con precisión la eficacia de nuevas terapias en el tratamiento de esta patología, es necesario desarrollar nuevos modelos animales que reproduzcan los diferentes grados de severidad del SIL en humanos. Objetivo: Desarrollar y clasificar modelos de SIL que engloben diferentes grados de inflamación de la superficie ocular (de leve a severo), y relacionarlos con los diferentes grados de severidad de SIL en humanos. Materiales y método: Se desarrolló un modelo parcial de SIL en 6 conejos y un modelo total de SIL en 10 conejos. Para ello, en el ojo derecho de cada conejo se realizó una desepitelización completa de la córnea con n-heptanol y una peritomía limbar quirúrgica de 180º (modelo parcial) o 360º (modelo total). Se evaluó semanalmente la neovascularización corneal, la opacificación y el defecto epitelial utilizando una escala de 0 a 4 (de menor a mayor severidad) y realizando un seguimiento total de 11 semanas. Posteriormente, los conejos fueron eutanasiados y se recogieron los globos oculares para realizar un estudio histológico. Los tejidos se fijaron y se incluyeron en parafina, después se realizaron cortes en el micrótomo y se tiñeron con la técnica de tinción de PAS. Se evaluó el grado de daño tisular, la presencia de infiltrados inflamatorios y el número de células caliciformes en el limbo y la córnea central. Resultados: Las córneas de conejo de los modelos parcial y total de SIL desarrollaron opacidad, defectos epiteliales y neurovascularización durante las 3-4 semanas posteriores a la lesión. La opacidad corneal, la ulceración y la neovascularización fueron significativamente mayores en el modelo total que en el modelo parcial. En 4 de los 10 conejos del modelo total se observó mayor severidad de los signos clínicos, desarrollándose una conjuntivalización que les cubría la córnea totalmente. En la histología se observó que el estroma corneal y limbar del modelo de SIL total presentaba una mayor desorganización y un número significativamente mayor de células inflamatorias y de células caliciformes con respecto al modelo de SIL parcial. Los resultados mostraron el desarrollo de diferentes grados progresivos de la enfermedad. Se desarrolló un SIL leve en la superficie ocular del 100% de los conejos con SIL parcial. En cambio, en el modelo total de SIL, se observó el desarrollo de un SIL moderado en la superficie ocular del 60% de los conejos y un SIL severo en el 40% de los conejos. Conclusiones: Se ha desarrollado un modelo experimental eficiente de inflamación después de producir un SIL parcial o total. Se han observado grados progresivos de severidad (leve, moderado y severo), que se asemejan mucho a los grados que se producen en esta patología en humanos y por lo tanto, estos modelos serán de utilidad para el estudio preliminar de la eficacia de nuevos tratamientos para el SIL antes de realizar un posible ensayo clínico.Ministerios de Economía y Competitividad (SAF2015-63594-R MINECO/FEDER, UE) y de Ciencia e Innovación, España (SAF2010-14900); Instituto de Salud Carlos III, España (CIBER-BBN, CB06/01/003 MINECO/FEDER). Centro de Medicina Regenerativa y Terapia Celular, de Castilla y León, España