A new manual retinal thickness measurement protocol to evaluate high myopia patients.

PURPOSE: To validate a manual measurement protocol for quantifying retinal thickness (RT) using an optical coherence tomography (OCT) device in pathologic myopia patients. METHODS: The macular Cross Hair protocol of Stratus OCT3 (Carl Zeiss Meditec, Inc., Dublin, Calif., USA) was applied and manual RT gauging was performed using the caliper tool. Foveal and paramacular RT, located at 1 and 2 mm distances from the fovea in both vertical and horizontal scans, were measured. Three consecutive RT measurements were taken to assess measurement reliability. The within-subject coefficient of variation (CVw) and intraclass correlation coefficients (ICC) were calculated to validate the manual method. RESULTS: The mean axial length of the 29 eyes assessed was 28.28 ± 2.72 mm and the mean spherical refraction was -13.61 ± 6.68 diopters. CVw ranged from 0.86 to 8.73% and ICC varied from 0.81 to 0.98. CONCLUSION: A manual RT measurement protocol could reliably be used in the daily clinic for assessing pathologic myopic patients when OCT software segmentation fails.

Safety, biocompatibility, and potential functionality of a new accommodative intraocular lens: An experimental study in rabbits.

This study aims to evaluate the safety, biocompatibility, and functionality of a new accommodative intraocular lens (IOL) (LUZ, patent PCT/ES2016/070,813) after implantation in rabbit eyes. LUZ (Study) and EyeCee® plus a capsular ring (Control) were implanted in rabbits (n = 8 each) after phacoemulsification. Intraoperative follow-up, long-term clinical follow-up, and functional IOL studies were carried out periodically for up to 180 days. A macroscopic examination of the eyeballs to reveal abnormalities and determine the implant centering and a microscopic examination to semi-quantify cell and tissue response were performed. Statistical analysis of the collected data was finally achieved. During follow-up, no significant changes in the general condition nor the clinical evaluation were observed between both groups. However, Study IOL remained centered throughout the study and did not present severe complications as observed in the Control group. Functional studies did not reveal significant differences between both materials. Study showed better centering, fewer adhesions, and maintenance of an opening capsular bag compared to the Control. Local biological effects caused by Study implantation are minimal and comparable to the Control. Therefore, LUZ showed no clinical signs or histological response of adverse reaction to the implanted material, according to UNE-EN ISO 11979-5 and 10993-6. Functionality must be confirmed in another animal species with greater lens accommodation capacity than the rabbit. LUZ keeps the capsular bag open, favoring its centering and avoiding fibrosis and adherence to the bag; this allows potential accommodation of this IOL and theoretically enables the patient to focus dynamically.

Live donor kidney transplantation. Situation analysis and roadmap.

Living donor kidney transplantation (LDKT) is the best treatment option for end stage renal disease in terms of both patient and graft survival. However, figures on LDKT in Spain that had been continuously growing from 2005 to 2014, have experienced a continuous decrease in the last five years. One possible explanation for this decrease is that the significant increase in the number of deceased donors in Spain during the last years, both brain death and controlled circulatory death donors, might have generated the false idea that we have coped with the transplant needs. Moreover, a greater number of deceased donor kidney transplants have caused a heavy workload for the transplant teams. Furthermore, the transplant teams could have moved on to a more conservative approach to the information and assessment of patients and families considering the potential long-term risks for donors in recent papers. However, there is a significant variability in the LDKT rate among transplant centers and regions in Spain independent of their deceased donor rates. This fact and the fact that LDKT is usually a preemptive option for patients with advanced chronic renal failure, as time on dialysis is a negative independent factor for transplant outcomes, lead us to conclude that the decrease in LDKT depends on other factors. Thus, in the kidney transplant annual meeting held at ONT site in 2018, a working group was created to identify other causes for the decrease of LDKT in Spain and its relationship with the different steps of the process. The group was formed by transplant teams, a representative of the transplant group of the Spanish Society of Nephrology (SENTRA), a representative of the Spanish Society of Transplants (SET) and representatives of the Spanish National Transplant Organization (ONT). A self-evaluation survey that contains requests about the phases of the LDKT processes (information, donor work out, informed consent, surgeries, follow-up and human resources) were developed and sent to 33 LDKT teams. All the centers answered the questionnaire. The analysis of the answers has resulted in the creation of a national analysis of strengths, weaknesses, opportunities, threats (SWOT) of the LDKT program in Spain and the development of recommendations targeted to improve every step of the donation process. The work performed, the conclusions and recommendations provided, have been reflected in the following report: Spanish living donor kidney transplant program assessment: recommendations for optimization. This document has also been reviewed by a panel of experts, representatives of the scientific societies (Spanish Society of Urology (AEU), Spanish Society of Nephrology Nursery (SEDEN), Spanish Society of Immunology (SEI/GETH)) and the patient association ALCER. Finally, the report has been submitted to public consultation, reaching ample consensus. In addition, the transplant competent authorities of the different regions in Spainhave adopted the report at institutional level. The work done and the recommendations to optimize LDKT are summarized in the present manuscript, organized by the different phases of the donation process.

Live donor kidney transplantation. Situation analysis and roadmap. / Trasplante renal de donante vivo. Análisis de situación y hoja de ruta.

Living donor kidney transplantation (LDKT) is the best treatment option for end stage renal disease in terms of both patient and graft survival. However, figures on LDKT in Spain that had been continuously growing from 2005 to 2014, have experienced a continuous decrease in the last five years. One possible explanation for this decrease is that the significant increase in the number of deceased donors in Spain during the last years, both brain death and controlled circulatory death donors, might have generated the false idea that we have coped with the transplant needs. Moreover, a greater number of deceased donor kidney transplants have caused a heavy workload for the transplant teams. Furthermore, the transplant teams could have moved on to a more conservative approach to the information and assessment of patients and families considering the potential long-term risks for donors in recent papers. However, there is a significant variability in the LDKT rate among transplant centers and regions in Spain independent of their deceased donor rates. This fact and the fact that LDKT is usually a preemptive option for patients with advanced chronic renal failure, as time on dialysis is a negative independent factor for transplant outcomes, lead us to conclude that the decrease in LDKT depends on other factors. Thus, in the kidney transplant annual meeting held at ONT site in 2018, a working group was created to identify other causes for the decrease of LDKT in Spain and its relationship with the different steps of the process. The group was formed by transplant teams, a representative of the transplant group of the Spanish Society of Nephrology (SENTRA), a representative of the Spanish Society of Transplants (SET) and representatives of the Spanish National Transplant Organization (ONT). A self-evaluation survey that contains requests about the phases of the LDKT processes (information, donor work out, informed consent, surgeries, follow-up and human resources) were developed and sent to 33 LDKT teams. All the centers answered the questionnaire. The analysis of the answers has resulted in the creation of a national analysis of strengths, weaknesses, opportunities, threats (SWOT) of the LDKT program in Spain and the development of recommendations targeted to improve every step of the donation process. The work performed, the conclusions and recommendations provided, have been reflected in the following report: Spanish living donor kidney transplant program assessment: recommendations for optimization. This document has also been reviewed by a panel of experts, representatives of the scientific societies (Spanish Society of Urology (AEU), Spanish Society of Nephrology Nursery (SEDEN), Spanish Society of Immunology (SEI/GETH)) and the patient association ALCER. Finally, the report has been submitted to public consultation, reaching ample consensus. In addition, the transplant competent authorities of the different regions in Spain have adopted the report at institutional level. The work done and the recommendations to optimize LDKT are summarized in the present manuscript, organized by the different phases of the donation process.

Boundary sequences stabilize transgene expression from subtle position effects in retroviral vectors.

Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-beta SAR and/or 5'HS4 beta-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5'HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5'HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects.

Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit model.

BACKGROUND: Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. METHODS: Rabbit fetuses received 8.5 x 10(6) HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post-in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. RESULTS: Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. CONCLUSIONS: At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer.

Lipophilic substances in intraocular silicone oil.

PURPOSE: To examine intravitreal silicone oil samples for lipophilic substances, including cholesterol, fatty acids, and derived methyl esters. DESIGN: Clinical interventional case series study. METHODS: The study included 53 patients (53 eyes; mean age 57.6 years) who underwent removal of intravitreal silicone oil used for intraocular tamponade with pars plana vitrectomy for retinal detachment. The silicone oil was removed 8.3 +/- 5.7 months after pars plana vitrectomy and analyzed using gas chromatography with a flame ionization detector. RESULTS: Cholesterol was present in all samples with a mean concentration of 65 +/- 32.3 microg/ml (95% confidence interval: 56.1 to 73.9 microg/ml). The concentration increased (P < .001) with the time of tamponade, and decreased (P = .003) with the age of the patients. Fatty acids along with derived methyl acids were detected in low concentrations in 49 samples (92.5%). CONCLUSIONS: Cholesterol and lipophilic acids accumulate in intravitreal silicone oil used in intraocular tamponade.

The ß-interferon scaffold attachment region confers high-level transgene expression and avoids extinction by epigenetic modifications of integrated provirus in adipose tissue-derived human mesenchymal stem cells.

Because of their abundance and ease of isolation, multilineage differentiation, and paracrine and immunoregulatory capabilities, genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) might combine cell- and gene therapy-based strategies for efficacious tissue repair/regeneration. In this report, we aimed to analyze and influence the long-term dynamics of transgene expression in ASCs transduced with different gammaretroviral vector configurations incorporating the human ß-interferon scaffold attachment region (IFN-SAR) and/or chicken 5'HS4 ß-globin insulator sequences. In our undifferentiated ASC culture model, naked retroviral vectors experienced EGFP transgene extinction correlating with increases in both H3 histone deacetylation and CpG dinucleotide methylation within the 5' long terminal repeat-primer-binding site proviral region. Retroviral configurations incorporating the referred boundary elements alone or combined were able to prevent the development of the above epigenetic events and to reduce transgene extinction to different degrees. Particularly, the IFN-SAR sustained the highest levels of H3 histone acetylation and transgene expression throughout the study. Analogously, ASCs differentiating to adipocytes or osteocytes experienced a gradual decline of EGFP expression using naked retroviral vectors. In contrast, only retroviral configurations including the IFN-SAR alone were able to overcome the epigenetic pressure, yielding high-level, uniform transgene expression throughout both lineage differentiation processes. Thus, embedding the IFN-SAR in retroviral vectors should have positive implications in gene repair strategies using ASCs.