RESUMEN
The European Union (EU) initiative on the Digital Transformation of Health and Care (Digicare) aims to provide the conditions necessary for building a secure, flexible, and decentralized digital health infrastructure. Creating a European Health Research and Innovation Cloud (HRIC) within this environment should enable data sharing and analysis for health research across the EU, in compliance with data protection legislation while preserving the full trust of the participants. Such a HRIC should learn from and build on existing data infrastructures, integrate best practices, and focus on the concrete needs of the community in terms of technologies, governance, management, regulation, and ethics requirements. Here, we describe the vision and expected benefits of digital data sharing in health research activities and present a roadmap that fosters the opportunities while answering the challenges of implementing a HRIC. For this, we put forward five specific recommendations and action points to ensure that a European HRIC: i) is built on established standards and guidelines, providing cloud technologies through an open and decentralized infrastructure; ii) is developed and certified to the highest standards of interoperability and data security that can be trusted by all stakeholders; iii) is supported by a robust ethical and legal framework that is compliant with the EU General Data Protection Regulation (GDPR); iv) establishes a proper environment for the training of new generations of data and medical scientists; and v) stimulates research and innovation in transnational collaborations through public and private initiatives and partnerships funded by the EU through Horizon 2020 and Horizon Europe.
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Investigación Biomédica/organización & administración , Nube Computacional , Difusión de Innovaciones , Guías de Práctica Clínica como Asunto , Investigación Biomédica/métodos , Unión Europea , Difusión de la Información/legislación & jurisprudencia , Difusión de la Información/métodosRESUMEN
AIMS: To examine the impact of telephone consent introduced in 2007 on the eye donation rate and to report the changing trend and potential for improvement in eye donation in Newcastle upon Tyne, UK. METHODS: Relevant data were retrospectively collected from the local eye retrieval database for two separate years, namely, 2006 (before the introduction of telephone consent) and 2010. All the hospitals within Newcastle were included in the study. RESULTS: From 2006 to 2010, there was a 3.5-fold increase in eye donation from 32 (of 2479 deaths) to 111 donors per year (of 2213 deaths) in Newcastle (P<0.001). Consent was obtained via face-to-face interview in all 32 (100%) and 59 (53.2%) donors in 2006 and 2010, respectively. Introduction of telephone consent increased the donation rate by an additional 88.1% (from 59 to 111 donors) in 2010 (P<0.001). In addition, there was a significant increase in medical notes of the deceased being reviewed from 27.1% (671/2479 cases) in 2006 to 62.4% (1382/2213 cases) in 2010 (P<0.001). Acceptance rate of eye donation was 45.7% (32/70) in 2006 and 49.6% (111/224) in 2010 (P=0.575). Acceptance rate was positively associated with registration on organ donor register (P<0.001) and telephone consent (P<0.001), but not with age (P=0.883), gender (P=0.234), or location of death (P=0.984) of the potential donors. CONCLUSION: There has been a substantial improvement in eye donation rate in Newcastle over the recent years. Introduction of telephone consent and high-quality eye donation service serve as effective measures for increasing eye donation.
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Ojo , Consentimiento Informado , Teléfono , Donantes de Tejidos/estadística & datos numéricos , Obtención de Tejidos y Órganos/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Bancos de Ojos/estadística & datos numéricos , Femenino , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Donantes de Tejidos/provisión & distribución , Obtención de Tejidos y Órganos/organización & administración , Reino UnidoRESUMEN
PurposeTo report the changing trend in the utilisation rate of donated corneas for keratoplasty and to examine the reasons for unutilised corneas in the North East of England.MethodsRelevant data were retrospectively collected from a local eye retrieval database and the UK Transplant Registry for two separate years; namely, 2006 and 2010.ResultsThe utilisation rate of donated corneas for keratoplasty improved from 57% (52/92) in 2006 to 71% (220/312) in 2010 (P=0.012). Over the same period, there was a marked reduction of failed serological test results from 24% (22/92) to 5% (14/312) (P<0.001). The leading reasons for unutilised corneas were failed serological test results (22/92, 24%) in 2006 and inadequate tissue quality (23/312, 7%) in 2010. The rate of tissue contamination remained similar between 2006 (4%) and 2010 (6%) (P=0.80). Eleven (4%) corneas were not transplanted due to recipient-related factors in 2010. Donor corneas of inadequate tissue quality were associated with older age (P=0.04) but not with gender, donation site, consent method, death-to-enucleation interval, death-to-processing interval, and storage time in the eye bank.ConclusionThere was a substantial improvement in the utilisation rate of corneas donated in the North East of England between 2006 and 2010. The principal reason was a reduction in failed serological test results. High donor age was associated with increased chance of corneas not being used. Utilisation rate of corneas can be further improved if potential modifiable factors are addressed, such as recipient-related factors and microbial contamination.
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Córnea , Bancos de Ojos/estadística & datos numéricos , Queratoplastia Penetrante/tendencias , Obtención de Tejidos y Órganos/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Enfermedades de la Córnea/cirugía , Bases de Datos Factuales , Selección de Donante , Enucleación del Ojo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistema de Registros , Estudios Retrospectivos , Donantes de Tejidos/estadística & datos numéricos , Recolección de Tejidos y Órganos , Reino UnidoRESUMEN
PURPOSE: To determine if donor age and preoperative endothelial cell density (ECD) affect corneal endothelial failure following penetrating keratoplasty (PK). METHODS: Preoperative and postoperative data for PKs performed in the UK between April 1999 and March 2012 were analysed. Donor age was split into three groups (0-60, 61-75 and >75â years) and donor ECD was split into three groups (≤2400, 2401-2600 and >2600 cells/mm2). Cox proportional hazards regression was used to determine whether the selected subgroups of donor age and donor ECD have an impact on endothelial failure and a systematic analysis of the interaction between donor ECD and donor age was conducted. The analysis was stratified for primary corneal diagnosis (Fuchs endothelial dystrophy (FED), pseudophakic bullous keratopathy (PBK) and other) and corrected for potentially confounding factors (human leukocyte antigen matching, donor trephine diameter, deep vascularisation, the occurrence of reversible rejection episodes and receipt of systemic antiviral medication, long-term steroids or other immunosuppressive agents). RESULTS: A total of 9415 patients, from the National Health Service Blood and Transplant UK Transplant Registry, who received their first PK for visual reasons were included in this study. The overall 5-year graft survival rate due to endothelial failure was 89%. Survival rates in recipients with FED, PBK and 'all other indications' were 95%, 83% and 89%, respectively. Our analysis shows that donor ECD did not affect outcome following corneal graft within the preselected categories, irrespective of diagnosis and after allowing for any potential confounding factors. Furthermore, HRs for each level of donor ECD, relative to >2600 cells/mm2, for each combination of age group and indication, were not statistically significant. CONCLUSIONS: We were unable to detect a significant effect of donor age, up to 90â years, and preoperative donor ECD, above the lower limit of 2200 cells/mm2, on endothelial failure at 5â years following PK.
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Pérdida de Celulas Endoteliales de la Córnea/diagnóstico , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/cirugía , Supervivencia de Injerto , Queratoplastia Penetrante/efectos adversos , Donantes de Tejidos , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Recuento de Células , Niño , Preescolar , Pérdida de Celulas Endoteliales de la Córnea/epidemiología , Pérdida de Celulas Endoteliales de la Córnea/etiología , Femenino , Distrofia Endotelial de Fuchs/diagnóstico , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Pronóstico , Reino Unido/epidemiología , Adulto JovenRESUMEN
The use of Nagington and Greaves plug for freezing lymphocytes is very straight-forward but the recovery of cells can be low and variable. The survival of lymphocytes was found to be dependent upon the position of the sample within the plug and the plug position in the lipid nitrogen container. Both of these variables affected the rate of cooling of the samples. It is stressed that the range of cooling rates produced by a plug should be determined to ensure that lymphocytes are cooled at suitable rates.
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Frío , Linfocitos , Supervivencia Celular , Congelación , Humanos , Técnicas InmunológicasRESUMEN
The Corneal Transplant Follow-up Study has followed 2311 penetrating keratoplasties for up to 450 days after transplant. A total of 207 failures were observed, including 65 classical rejections and 35 endothelial decompensations. At 12 months, graft survival was 89%, and survival free from rejection was 87%. For surviving grafts, risk of failure reduced from 4.8% in the first 75 days and stabilized after 5 months at 1.2% in each 75-day interval. Risk of rejection initially followed a similar pattern, but then increased after 12 months. Multifactorial analyses accounted for differences in recipient characteristics and interrelationships of donor factors. Donor age, sex, cause of death, and method of corneal storage were not found to influence significantly either time to graft failure or time to first rejection. Grafts in prospectively tissue-typed donor-recipient pairs were generally considered before surgery to be at increased risk of either graft failure or rejection. With due allowance, increasing risk of rejection was associated with increasing numbers of mismatches at HLA-A and HLA-B broad antigens. The opposite was true at HLA-DR broad antigens, where increased risk of rejection was observed with no mismatches.
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Trasplante de Córnea/inmunología , Donantes de Tejidos , Adulto , Anciano , Trasplante de Córnea/estadística & datos numéricos , Femenino , Estudios de Seguimiento , Rechazo de Injerto/epidemiología , Supervivencia de Injerto/inmunología , Prueba de Histocompatibilidad , Humanos , Masculino , Persona de Mediana Edad , Preservación de Órganos , Reino Unido/epidemiologíaRESUMEN
PURPOSE: To examine components of the junctional complex and the actin cytoskeleton and the incidence of apoptosis in epithelium and endothelium of organ-cultured human corneas. METHODS: Human corneas, either organ-cultured for 1 to >28 days or excised directly from eyes stored in moist chambers, were stained with antibodies to ZO-1, vinculin, and caspase 3 coupled to FITC-conjugated secondary antibody. These markers were combined with rhodamine-phalloidin staining for F-actin and DAPI labeling for DNA. The corneas were examined by confocal microscopy. RESULTS: The depth of the epithelium was reduced during organ culture, but no changes were observed in the distribution of ZO-1 or vinculin, or in the F-actin cytoskeleton. The appearance of apoptotic epithelial cells positive for caspase 3 or with condensed DNA increased with time after 14 days in organ culture, but there was no correlation with donor age. ZO-1 and F-actin staining patterns in endothelium were similarly undisturbed by organ culture, but apoptotic endothelial cells were only rarely seen and then only after >28 days in organ culture. CONCLUSIONS: Organ culture maintained the integrity of tight junctions and the actin cytoskeleton in epithelial and endothelial cell layers. Apoptosis was evident in epithelium but was observed rarely in the endothelium and then only after extended periods in organ culture.
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Citoesqueleto/fisiología , Endotelio Corneal/citología , Epitelio Corneal/citología , Técnicas de Cultivo de Órganos , Uniones Estrechas/fisiología , Actinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Caspasa 3 , Caspasas/metabolismo , Criopreservación , Endotelio Corneal/metabolismo , Epitelio Corneal/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Humanos , Indoles , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Persona de Mediana Edad , Preservación de Órganos , Fosfoproteínas/metabolismo , Rodaminas , Vinculina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
PURPOSE: To assess the influence of donor and storage factors on the suitability of organ-cultured corneas for penetrating keratoplasty (PKP) using multifactorial regression analysis. METHODS: Corneas (mean donor age, 57 years; standard deviation, 21 years) were stored by organ culture at 34 degrees C for up to 5 weeks (mean, 22 days; standard deviation, 6 days). The endothelium was assessed by light microscopy, and corneas with < 2200 cells/mm2 were considered unsuitable for PKP. RESULTS: Of the 9250 corneas stored between 1992 and 1994, 59% were issued for PKP, 5% were discarded because of bacterial or fungal contamination, and 30% were unsuitable for PKP owing to endothelial deficiencies. Donor age had the strongest influence on suitability for PKP: > 80% of corneas from donors younger than 40 years of age were issued for PKP compared with only 45% of corneas from donors 80 years of age and older. There was an overall decline in the percentage of corneas suitable for PKP with increasing storage time, but the rate of this decline was inversely related to donor age. Cause of death and post mortem times to enucleation and to storage had only a small influence on suitability for PKP. CONCLUSIONS: Criteria based on endothelial assessment rather than on donor age allow corneas from donors of all ages, stored by organ culture for extended periods, to be used for PKP. Organ culture also allows corneas with bacterial or fungal contamination to be identified and discarded before they are grafted.
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Córnea/fisiología , Queratoplastia Penetrante , Técnicas de Cultivo de Órganos , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Causas de Muerte , Recuento de Células , Supervivencia Celular , Criopreservación , Endotelio Corneal/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Preservación de Órganos , Factores de Tiempo , Donantes de TejidosRESUMEN
Solutions developed for cardioplegia and myocardial protection during elective ischemic cardiac arrest have also been used for the storage of heart grafts at 0 degrees C. In this study we show that a solution based upon the principles established in renal preservation experiments gives superior preservation of rabbit hearts at 0 degrees C. The solution contained K+ 25 mmol/L, Mg++ 10 mmol/L, sufficient glucose to prevent fluid uptake during storage (30 mmol/L), and a low concentration of calcium (0.1 mmol/L). The complete omission of calcium was detrimental to function after prolonged storage.
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Congelación , Paro Cardíaco Inducido , Corazón , Preservación de Órganos , Animales , Bicarbonatos , Calcio , Cloruro de Calcio , Glucosa , Magnesio , Potasio , Cloruro de Potasio , Conejos , Cloruro de Sodio , Factores de TiempoRESUMEN
Cryopreservation techniques for umbilical cord blood (UCB) have been based on methods established for marrow (BM) and peripheral blood progenitor cells (PBPC) with varying degrees of success. The aim of this study was to optimise cryopreservation of UCB haemopoietic cells based on sound cryopreservation principles. UCB samples were cryopreserved with different combinations of DMSO and hydroxyethyl starch (HES) by a variety of freezing protocols. After cooling at 1 degree C/min in solutions containing 4% HES and various concentrations of DMSO there was a dramatic fall in CD34+ recovery from 85.4% (s.d. 28.4) to 12.2% (s.d. 10.0) as DMSO concentration was reduced from 5 to 2.5%. Varying HES concentration in solutions containing 5% DMSO did not have a significant effect on CD34+ cell recovery. Increasing cooling rate from 1 to 10 degrees C/min significantly reduced CD34+ recovery (P < 0.0001) while increasing DMSO concentration up to 10% had little effect (P = 0.8, two-way ANOVA). Good recovery of UCB CD34+ cells can be achieved with 5-10% DMSO at a controlled cooling rate of 1 degrees C/min. There was a significant difference (P < 0.0001) in the apparent recovery of CD34+ cells between paired aliquots thawed in the presence (recovery = 76.8%, s.d. 26.0) and absence (32.5%, s.d. 18.7) of DNase. In conclusion, conditions for cryopreserving UCB for clinical banking that yield optimal recovery of CD34+ cells have been established.
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Criopreservación/métodos , Sangre Fetal , Antígenos CD34/sangre , Eliminación de Componentes Sanguíneos/métodos , Supervivencia Celular , Crioprotectores , Desoxirribonucleasas , Dimetilsulfóxido , Estudios de Evaluación como Asunto , Femenino , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Derivados de Hidroxietil Almidón , Técnicas In Vitro , Recién Nacido , EmbarazoRESUMEN
AIM: To compare a cohort of corneal graft patients in east Jerusalem with one in Sweden, concerning diagnosis, sex, patient age, preoperative visual acuity in both eyes, and type of operation. METHODS: Standard forms developed for the Swedish Corneal Transplant Register were used for data collection at the time of operation. RESULTS: In east Jerusalem, keratoconus accounted for 51% of the grafts compared with only 27% in Sweden and the male:female ratio was reversed. There were very few patients with endothelial disease. The Palestinian patients had overall worse visual acuity both in the eye to be operated and the fellow eye compared with patients in Sweden. CONCLUSION: Significant differences were found between the Palestinian and Swedish cohorts in the distribution of indications for transplantation and preoperative visual acuity.
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Enfermedades de la Córnea/cirugía , Trasplante de Córnea/métodos , Adulto , Distribución por Edad , Anciano , Árabes , Estudios de Cohortes , Enfermedades de la Córnea/fisiopatología , Distrofias Hereditarias de la Córnea/cirugía , Trasplante de Córnea/estadística & datos numéricos , Femenino , Distrofia Endotelial de Fuchs/fisiopatología , Distrofia Endotelial de Fuchs/cirugía , Humanos , Queratocono/fisiopatología , Queratocono/cirugía , Masculino , Persona de Mediana Edad , Sistema de Registros/estadística & datos numéricos , Distribución por Sexo , Suecia , Agudeza Visual/fisiologíaRESUMEN
AIM: To assess visual outcome and the incidence of complications at 2 years postoperatively in corneal grafts reported to the Swedish Corneal Transplant Register. METHODS: Preoperative and 2 year follow up data were submitted to the Swedish Corneal Transplant Register by surgeons in eight corneal transplant clinics in Sweden. Preoperative data on 1957 grafts and 520 grafts with 2 year follow up were included in the analysis. Data were analysed by multiple linear and logistic regression methods, as appropriate. RESULTS: The major diagnostic categories were keratoconus (29%), bullous keratopathy (21%), and "other diagnosis" (32%). Fuchs' endothelial dystrophy and stromal dystrophies accounted for 15% and 3% of grafts, respectively. At 2 years the overall incidence of complications, other than rejection and regrafting, was 26%, with an increasing frequency from keratoconus < Fuchs' dystrophy < bullous keratopathy < "other diagnosis." Rejection was observed in 15% of grafts and was more likely in the bullous keratopathy (OR 3.1, 95% CI 1.1 to 9.0, p=0.04) and "other diagnosis" (OR 2.6, 95% CI 1.1 to 5.9, p=0.03) groups. Regrafting, which occurred in 10% of cases, was not influenced by diagnosis, but it was related to the incidence of rejection (OR 14.8, 95% CI 6.1 to 35.9, p<0.001) and other complications (OR 4.4, 95% CI 1.9 to 10.4, p=0.001), and to the presence of other sight threatening pathology in the eye (OR 3.6, 95% CI 1.3 to 9.9, p=0.01). Visual acuity was improved in a high proportion of the patients, especially those with keratoconus and Fuchs' dystrophy where, respectively, 86% and 54% of grafts achieved a visual acuity of > or =0.5 at 2 years, compared with only 31% with bullous keratopathy and 35% in the "other diagnosis" group. 60% of grafts for keratoconus and Fuchs' dystrophy achieved a visual acuity equal to or better than the other eye. Postoperative astigmatism was higher in the bullous keratopathy (p=0.01) group. Patients with high astigmatism benefited from refractive surgery, showing a reduction from 7.9 (95%CI 6.9, 8.7) to 3.2 (95% CI 2.6, 3.9) dioptres (p<0.001). A centre effect was evident in visual outcome. CONCLUSION: The overall incidence of complications was related to diagnosis. Complications other than rejection and regrafting were most likely in the "other diagnosis" group, and further analysis of this group is therefore planned. The best improvement in visual acuity and the lowest astigmatism were achieved in the keratoconus and Fuchs' dystrophy groups; but the influence of diagnosis on astigmatism was small and, overall, the statistical model accounted for only 8% of the variability in astigmatism. Refractive surgery was, however, effective in reducing astigmatism. It is hoped that a better understanding of the factors that determine the visual outcome of grafts will emerge from future analyses of the Swedish Corneal Transplant Register, helping to refine the criteria for patient selection and to guide clinical practice.
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Enfermedades de la Córnea/cirugía , Trasplante de Córnea , Agudeza Visual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Astigmatismo/etiología , Astigmatismo/cirugía , Niño , Preescolar , Enfermedades de la Córnea/fisiopatología , Femenino , Estudios de Seguimiento , Rechazo de Injerto , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Sistema de Registros , Resultado del TratamientoRESUMEN
Rabbit corneas were perfused in vitro with an irrigation solution for 90 minutes. This was followed by 6 hours of perfusion with tissue culture medium TC199 during which endothelial function was assessed by monitoring rates of swelling during a period of perfusion in the absence of bicarbonate ions, and subsequent rates of thinning when bicarbonate ions were restored to the perfusate. Corneal thickness (measured with an ultrasonic pachymeter) immediately following excision was 401 microns (SD 19, n = 23). During the 90 minute perfusion at 35 degrees C, corneas exposed to balanced salt solution (BSS), Hartmann's solution or 0.9% NaCl (all initially at room temperature) swelled, respectively, at 14 (SD 2.3, n = 4), 11 (SD 2.6, n = 4), and 70 (SD 4.3, n = 4) microns/h. Cold Hartmann's solution (initially at 4 degrees C) caused corneas to swell at 9 (SD 2.3, n = 4) microns/h. On the other hand, corneas perfused with BSS Plus thinned at 9 (SD 3.4, n = 4) microns/h and TC199 with Earle's salts had little effect on thickness. Rates of swelling and thinning during the following assessment perfusion showed no apparent effects of prior exposure to any of the irrigation solutions on the barrier properties or pump function of the endothelium. Despite this, the increased thickness of corneas exposed initially to BSS, cold Hartmann's solution, or 0.9% NaCl was not fully reversed, even by the end of the 6 hour assessment perfusion. In contrast, the swelling observed in corneas exposed to Hartmann's solution at room temperature was reversed and these corneas had returned to their normal thickness by the end of the assessment period. All corneas, even those exposed to 0.9% NaCl, had an intact endothelial mosaic with no evidence of damage or cell loss, although morphological differences in cell shape and the appearance of cell borders were evident compared with freshly isolated cornea.
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Endotelio Corneal/fisiología , Soluciones Isotónicas/farmacología , Animales , Bicarbonatos , Córnea/anatomía & histología , Endotelio Corneal/anatomía & histología , Conejos , Lactato de Ringer , Irrigación Terapéutica , Factores de Tiempo , Equilibrio HidroelectrolíticoRESUMEN
AIMS: Organ culture medium for corneas contains labile components, such as L-glutamine, whose loss could be a limiting factor to the length of storage. The medium is also supplemented with fetal bovine serum (FBS), which can vary significantly between different batches. The aim of this study was to establish the need for FBS during corneal organ culture, and to determine whether substitution of L-glutamine by the stable dipeptide L-analyl-L-glutamine was beneficial. METHODS: Porcine corneoscleral discs were suspended in 80 ml of organ culture medium (HEPES buffered Eagle's MEM with Earle's salts, 26 mmol/l NaHCO3, penicillin, streptomycin, and amphotericin B) and kept at 34 degrees C. The medium contained either 2 mmol/l L-glutamine or 2 mmol/l L-analyl-L-glutamine, and was either serum free or contained 2% FBS. At weekly intervals, five corneas from each group were stained with trypan blue and alizarin red S, and the surface area and shape of 100 endothelial cells were determined for each cornea. RESULTS: No differences were observed between corneas in organ culture medium with L-glutamine or L-analyl-L-glutamine. In serum free medium, endothelial cell density remained constant for the first week, but then declined rapidly over the next 2 weeks. With 2% FBS, there was no loss of endothelial cells for the first 2 weeks, but cell density had halved by the fourth week of organ culture. CONCLUSION: The presence of 2% FBS extended the period of endothelial stability, but no advantage was gained from the stabilised form of L-glutamine. The overall loss of endothelial cells was much greater than would be expected for human corneas.
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Endotelio Corneal/citología , Sangre Fetal/fisiología , Glutamina/farmacología , Animales , Bovinos , Células Cultivadas , Medios de Cultivo , Endotelio Corneal/efectos de los fármacos , Glutamina/análogos & derivados , Técnicas de Cultivo de Órganos , PorcinosRESUMEN
Current methods for the production of lenticules for epikeratophakia involve rapid freezing, cryolathing, and slow warming of the donor cornea. We have found that this procedure causes structural damage to the epithelial basement membrane in the donor cornea which may subsequently contribute to poor postoperative re-epithelialisation of the implant, leading to graft failure. Endeavouring to overcome these problems, the effects of cryoprotection of donor cornea were investigated, using dimethyl sulphoxide, in conjunction with different cooling and warming rates as part of the protocol for cryolathing. The structural integrity of the epithelial basement membrane zone (BMZ) was then assessed by electron microscopy and by immunofluorescence microscopy using antibodies to types IV and VII collagen, components of the basal lamina and anchoring fibrils respectively, and an antibody to a component of the anchoring filaments. No differences in the pattern of immunostaining for these components were detected, indicating that the composition of the BMZ was unaltered by the different treatment regimens applied. However, electron microscopy showed that preservation of basement membrane ultrastructure was markedly improved when cornea was warmed rapidly rather than slowly, both in cryoprotected and non-cryoprotected tissue. Epithelial cell retention and preservation of stromal architecture appeared superior in cryoprotected samples, while keratocyte structure was heterogeneous throughout the experimental groups. Further work is in progress to assess the efficacy of these protocols in the preservation of keratocyte viability in association with improved basement membrane structure in donor tissue for epikeratophakia.
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Córnea , Criopreservación , Epiqueratofaquia , Conservación de Tejido/métodos , Membrana Basal/ultraestructura , Córnea/ultraestructura , Humanos , Microscopía Electrónica , Microscopía FluorescenteRESUMEN
This study reports corneal graft survival, endothelial cell changes, and visual outcome in 20 patients who received some of the first corneas stored by organ culture in the Corneal Transplant Service Eye Bank in Bristol. Mean donor age was 48 years (SD 15, n = 20) and corneas were stored for an average of 21 days (SD 7, n = 20). Preoperative endothelial cell density was 2334 cells/mm2 (SD 235, n = 18) and this fell by 8% (SD 12) to 2158 cells/mm2 (SD 372) within the first 2 months following transplantation. In 13 patients, endothelial cell density thereafter declined exponentially with a half-life of 41 months (SD 17, n = 12; one patient excluded as an outlier). Corneas that suffered rejection episodes showed the highest rates of loss of endothelial cells. Endothelial cell loss 4 years after transplantation was 46% (SD 16, n = 12), which was similar to the postoperative decline in cell density reported for corneas stored for far shorter periods in McCarey-Kaufman medium at 4 degrees C.
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Trasplante de Córnea/fisiología , Endotelio Corneal/fisiología , Supervivencia de Injerto , Adulto , Trasplante de Córnea/patología , Endotelio Corneal/patología , Femenino , Rechazo de Injerto , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Factores de Tiempo , Resultado del TratamientoRESUMEN
AIM: To study the influence of cryoprotectant, cooling rate, and warming rate on recovery and viability of keratocytes from corneas for cryolathing. METHODS: Corneas were frozen at -50 degrees C for 2 minutes either after exposure to 10% dimethyl sulphoxide in Eagle's MEM for 15 minutes at room temperature (about 22 degrees C), or without earlier exposure to the cryoprotectant. Corneas were cooled either rapidly (20 degrees C/min) or slowly (1 degree C/min), and they were warmed either rapidly (> 50 degrees C/min) by direct transfer into medium at 22 degrees C or slowly (< 20 degrees C/min) in air at 22 degrees C. The cryoprotectant was removed by dilution in medium containing 0.5 mol/l sucrose. Recovery of keratocytes was determined by using collagenase digestion to release the cells from the stroma and trypan blue staining. Viability was assessed by the outgrowth of cells from stromal explants in primary tissue culture. RESULTS: The use of a cryoprotectant before freezing was beneficial, irrespective of the different cooling and warming regimens. Both collagenase digestion and tissue culture revealed that keratocyte survival was improved when corneas were warmed rapidly rather than slowly. The collagenase digestion assay showed an apparently higher recovery of keratocytes after slow cooling (54.3%) than after rapid cooling (34.1%), but no differences in cell viability could be demonstrated by primary tissue culture. CONCLUSION: Although in these experiments slow cooling apparently provided the best recovery of keratocyte numbers (though not viability), previous work had revealed some disruption of the epithelial basement membrane after slow cooling. If viable keratocytes and good preservation of epithelial basement membrane are considered to be prerequisites for epikeratophakia lenticules then it is suggested that corneas should be prepared for cryolathing by freezing rapidly after exposure to 10% dimethyl sulphoxide and, following cryolathing, they should be warmed rapidly.
Asunto(s)
Córnea , Criopreservación/métodos , Crioprotectores/uso terapéutico , Epiqueratofaquia/métodos , Animales , Frío , Técnicas de Cultivo , Conejos , Tasa de SupervivenciaRESUMEN
AIM: On the basis of finalised data from the Corneal Transplant Follow up Study to identify and quantify factors influencing corneal graft outcome in terms of graft survival, rejection, visual acuity, and astigmatism. METHODS: Multifactorial analysis of 2777 grafts registered by the UK Transplant Support Service from July 1987 to June 1991. RESULTS: Several recipient factors influencing graft survival, rejection, and visual acuity were identified, but no donor factors. Of the operative factors amenable to change, mixed suturing was associated with reduced graft survival, and larger grafts with increased risk of rejection but better visual acuity when surviving. There was increased risk of rejection with poor matching at HLA class I antigens, but mismatched HLA-DR grafts suffered less rejection than those with zero HLA-DR mismatches. Recipient age below 10 years was associated with increased risk of both rejection and graft failure. However, whereas increasing age above 10 years was not associated with differential graft survival, it was significantly associated with decreasing risk of rejection. CONCLUSIONS: While confirming possible benefits of HLA-A and B matching, the expense and delay involved in awaiting matched HLA-DR tissue is unlikely to be justified. Other donor factors are unrelated to graft outcome following screening of tissue by eye banks. The highest rates of graft failure and rejection happen in the early postoperative period, and factors influencing visual outcome are also apparent at this stage.
Asunto(s)
Trasplante de Córnea , Rechazo de Injerto/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Seguimiento , Prueba de Histocompatibilidad , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Análisis Multivariante , Modelos de Riesgos Proporcionales , Factores de Riesgo , Agudeza VisualRESUMEN
OBJECTIVE: The aim of this work was to investigate single-nucleotide polymorphisms (SNPs) in multiple genes on chromosome 6p in corneal transplant recipients known to be at increased risk of failure through immunologic rejection (ie, "high-risk" corneal transplants). Tumor necrosis factor alpha (TNF-α) is a key immunoregulatory cytokine in the ocular environment, interacting with a variety of factors in a synergistic way and playing a crucial role in many stages of the inflammatory response. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors, supporting both hemangiogenesis and lymphangiogenesis, both key in transplant tolerance and rejection. Interleukin-17 (IL-17) is a multifunctional cytokine produced by T-helper 17 cells, exerting specific effector functions during an immune response. Association of SNPs in all 3 genes with corneal transplant outcome was therefore investigated. METHODS: Three hundred five corneal transplant recipients were followed for 3 years, and episodes of allograft rejection were recorded. With the use of patient DNA, 6 SNPs of 3 different genes on chromosome 6p were investigated. The TNF-α promoter SNP -308 G/A (rs1800629) was analyzed with the use of induced heteroduplex generation; 2 VEGF-A functional variants were analyzed, -2578 (rs699947) C/A and -1154 (rs1570360) G/A, with the use of Taqman genotyping assays; and 3 nonsynonymous IL-17F SNPs in exon 3 (negative strand), (rs2397084) A/G, (rs11465553) G/A, and (rs763780) A/G, were investigated with the use of direct sequencing. Haplotypes were inferred with the use of PHASE using positive strand alleles, and exact measures of association were determined with the use of Mid-P exact chi-square. RESULTS: Six common haplotypes were inferred, with the haplotype TNF-α (rs1800629), VEGF-A (rs699947), (rs1570360), IL-17F (rs763780), (rs11465553), and (rs2397084) ACGTCT having a significant association with corneal transplant rejection (odds ratio, 1.78; 95% confidence interval, 1.01-3.11; P = .04). CONCLUSIONS: The results suggest that patients carrying a combination of SNPs for TNF-α, VEGF-A, and IL-17F of ACGTCT haplotype may have an increased risk of corneal allograft rejection compared with patients carrying other haplotypes.