An effective method for culturing functional human corneal endothelial cells using a xenogeneic free culture medium.

Endothelial dysfunction is a leading cause of corneal blindness in developed countries and the only available treatment is the endothelial transplantation. However, the limited availability of suitable donors remains a significant challenge, driving the exploration of alternative regenerative therapies. Advanced Therapy Medicinal Products show promise but must adhere to strict regulations that prohibit the use of animal-derived substances. This study investigates a novel culture methodology using Plasma Rich in Growth Factors (PRGF) as the only source of growth factors for primary cultures of human corneal endothelial cells (CECs). CECs were obtained from discarded corneas or endothelial rings and cultured in two different media: one supplemented with xenogeneic factors and other xenogeneic-free, using PRGF. Comprehensive characterization through immunofluorescence, morphological analyses, trans-endothelial electrical resistance measurements, RNA-seq, and qPCR was conducted on the two groups. Results demonstrate that CECs cultured in the xenogeneic-free medium exhibit comparable gene expression, morphology, and functionality to those cultured in the xenogeneic medium. Notably, PRGF-expanded CECs share 46.9% of the gene expression profile with native endothelium and express all studied endothelial markers. In conclusion, PRGF provides an effective source of xenogeneic-free growth factors for the culture of CECs from discarded corneal tissue. Further studies will be necessary to demonstrate the applicability of these cultures to cell therapies that make clinical translation possible.

Light effects on mitochondrial photosensitizers in relation to retinal degeneration.

The retina captures and converts light between 400-760 nm into electrical signals that are sent to the brain by way of the optic nerve and in the process helps to translate these electrical signals into what is known as vision. The same light that allows vision to occur is nevertheless also potentially toxic to retinal cells in certain situations. The shorter wavelengths of light are known to interact with chromophores in photoreceptors and pigment epithelial cells to cause oxidative stress and severe damage. Indeed it is generally accepted that short wavelength light effects is one cause for loss of photoreceptor function in age-related macular degeneration. Recent studies have demonstrated that light may be a contributing factor for the death of retinal ganglion cells in certain situations. Light as impinging on the retina, especially the short wavelength form, affect mitochondrial chromophores and can result in neurone death. Importantly ganglion cell axons within the eye are laden with mitochondria and unlike the outer retina are not protected from short wavelength light by macular pigments. It has therefore been proposed that when ganglion cell function is already compromised, as in glaucoma, then light impinging on their mitochondria might be a contributor to their eventual demise.

Hepatitis E Virus Infection in Lung Transplant Recipients: A Case Series.

BACKGROUND: Hepatitis E virus (HEV) is one of the common causes of acute and chronic viral hepatitis with a global distribution. Genotypes 1 and 2 only affect humans and produce acute hepatitis epidemics in endemic regions (Asia, Africa). In nonendemic areas (America, Europe), genotypes 3 and 4 are considered a zoonosis and cause sporadic acute hepatitis. HEV has been described in solid organ transplant recipients; however, data on lung transplant patients are limited. OBJECTIVE: To present the first 3 cases of HEV infection in lung transplant recipients in our unit. CASE PRESENTATION: We report 3 cases of HEV infection in post-transplant patients presenting with symptoms and alterations in liver enzymes. All patients have no history of travel outside Spain prior to observing abnormalities in the liver function. Diagnoses were made with in-home polymerase chain reaction and enzyme-linked immunosorbent assay (IgG/IgM). The first patient was not treated and died of progressive hepatic disease, with postmortem diagnosis of HEV infection complications. The other 2 patients were treated with ribavirin after the diagnosis of HEV infection. Ribavirin was discontinued in 1 patient because of anemia necessitating red blood cell transfusions. CONCLUSIONS: HEV should be considered in the differential diagnosis of patients with abnormal liver enzymes after transplant. Early detection and treatment have implications in the prevention of liver failure and mortality. Large prospective seroprevalence studies of HEV in lung transplant patients are warranted to recognize the epidemiology of this infection in lung transplant recipients.

Monitoring multi-class pesticide residues in fresh fruits and vegetables by liquid chromatography with tandem mass spectrometry.

A new analytical method was developed using liquid chromatography with tandem mass spectrometry for the routine analysis of 31 multi-class pesticide residues and applied to approximately 50 fresh fruit and vegetable samples (green bean, cucumber, pepper, tomato, eggplant, watermelon, melon and zucchini). Extraction of the pesticides with ethyl acetate was carried out. The optimal ionisation conditions were selected for each pesticide in the same run. The procedure was validated and the values of some merit figures, such as recovery, precision, linear range, detection limit and quantification limit for each pesticide were calculated together with its calculated expanded uncertainty (U). The average recoveries in cucumber obtained for each pesticide ranged between 74 and 105% at two different fortification levels (n = 10 each) that ranged between 9 and 250 ng g(-1) (depending on the pesticide). The uncertainty associated to the analytical method was lower than 23% for all compounds tested. The calculated limits of detection and quantitation were typically <1 ng g(-1) that were much lower than the maximum residue levels established by European legislation.

[Diagnosis of dominant renal polycystosis in adults by analysis of DNA polymorphism]. / Diagnóstico de la poliquistosis renal dominante del adulto mediante análisis de los polimorfismos del ADN.

BACKGROUND: Adult renal polycystosis (ARP) is a dominant autosomic disease. The gene responsible for this disease in most families has been located on the short arm of chromosome 16 (16 p) by restriction analysis of the DNA polymorphisms (RFLP). METHODS: The existence of several polymorphic markers flanking this gene permits the diagnosis of any member of an affected family. A series of proximal and distal genetic markers have been used to study the segregation of the disease in a group of families with more than one affected member. RESULTS: The clinical and genetic results obtained from a study of 10 Spanish families with ARP have been reported. A high percentage of the members under 30 years of age (40%) did not present renal cysts. CONCLUSIONS: Restriction analysis of DNA are fundamentally for a disease in which a high percentage of carriers remain asymptomatic within the reproductive age.

[Changes in blood calcium during liver transplant in children]. / Variaciones de la calcemia durante el trasplante hepático pediátrico.

We have evaluated the changes in plasma total and ionic calcium levels in twenty hepatic transplantations in pediatric patients. Direct intraoperative monitoring of ionic calcium is fundamental, because its variability is unrelated with total calcium levels; in addition, normal ionic calcium levels contribute to the hemodynamic stability of the patient. Although at the end of the operation total and ionic calcium levels were similar to the postinduction measurement, their values were dissociated in the perianhepatic period. In the anhepatic phase ionic calcium reached its lowest value (1.00 mmol/l) although total calcium increased above postinduction level from 2.13 to 2.46 mmol/l (p less than 0.05). In hepatic transplantation in pediatric patients calcium administration is indicated during the transfusion of citrated blood, being particularly necessary during the anhepatic phase to prevent ionic hypocalcemia.

A prospective study of a rapid method for diagnosing cytomegalovirus infections in immunosuppressed patients.

A rapid assay is described for detection of cytomegalovirus in peripheral blood lymphocytes. It consists of an indirect immunofluorescence technique for detection of cytomegalovirus antigens by means of a monoclonal antibody directed against early viral coded proteins. This assay was compared with the conventional cell culture system. Patients transplanted between December 1986 and May 1988 were studied, and of 27 patients identified, two were excluded due to early graft failure. A total of 320 blood specimens obtained were studied, and from 12 patients cytomegalovirus was isolated in at least one specimen by conventional cell culture (in total 25 specimens). Results were available with the new technique within 6 h, whereas the cell culture took an average of 13 days to develop the typical cytopathic effects changes. Sensitivity and specificity compared with that of viral isolation in conventional cell culture was 92% and 95%, respectively. This technique provides an accurate and rapid diagnosis of cytomegalovirus infections, and allows specific antiviral therapy to be started earlier.

Genetic and clinical studies in autosomal dominant polycystic kidney disease type 1 (ADPKD1).

Thirteen Spanish families with autosomal dominant polycystic kidney disease were studied. In one family the disease did not segregate with polymorphic markers around the PKD1 locus. All subjects over the age of 30 years carrying a mutation at the PKD1 locus showed renal ultrasonographic cysts, but 40% of carriers of the PKD1 mutation younger than 30 years did not have renal cysts. Hypertension was found to be more frequent in those with renal cysts. Recombinants between 16p polymorphic loci and the PKD1 locus are described.

Cytomegaloviraemia and T cell subpopulations in renal transplant patients.

We studied 54 consecutive recipients of renal transplants to evaluate their immunological responses to cytomegalovirus (CMV) infection. Forty-three (79.6%) patients developed CMV infection, and all of them subsequently recovered. Fourteen of these infected patients (32.6%) developed viraemia during the infectious process, four of whom then manifested the disease. The number of lymphocytes and their main subpopulations was normal before the appearance of CMV infection. During the infection there was a significant growth (P < 0.001) in the CD8+DR+ subset, corresponding to activated T suppressor/cytotoxic lymphocytes, whereas the natural killer measured subsets remained within normal limits during the whole infectious process. As all viraemic patients recovering from the infection developed CD8+DR+ activation, we conclude that this recovery is associated with the immunological activation.

DNA microsatellite analysis of families with autosomal dominant polycystic kidney disease types 1 and 2: evaluation of clinical heterogeneity between both forms of the disease.

We studied 17 large families affected by adult dominant polycystic kidney disease (ADPKD). Ultrasonographic analysis was performed on all the family members. DNA microsatellite markers closely linked to PKD1 on 16p13.3 were analysed, and linkage of the disease to this locus was determined. Families showing a negative linkage value were evaluated for linkage to the PKD2 locus on 4q. Five of the 17 families showed negative linkage for the 16p13.3 markers. In these families significant linkage to 4q was obtained. Renal cysts developed at an earlier age in PKD1 mutation carriers, and end stage renal failure occurred at an older age in people affected with PKD2. Analysis of large families with ADPKD in a Spanish population indicates that this is a genetically heterogeneous disorder, but mutations at only two loci are responsible for the development of the disease in most if not all the families. Clinicopathological differences between both forms of the disease occur, with subjects with ADPKD2 having a better prognosis than those with mutations at PKD1.