Human Stem Cells for Ophthalmology: Recent Advances in Diagnostic Image Analysis and Computational Modelling.

Purpose of Review: To explore the advances and future research directions in image analysis and computational modelling of human stem cells (hSCs) for ophthalmological applications. Recent Findings: hSCs hold great potential in ocular regenerative medicine due to their application in cell-based therapies and in disease modelling and drug discovery using state-of-the-art 2D and 3D organoid models. However, a deeper characterisation of their complex, multi-scale properties is required to optimise their translation to clinical practice. Image analysis combined with computational modelling is a powerful tool to explore mechanisms of hSC behaviour and aid clinical diagnosis and therapy. Summary: Many computational models draw on a variety of techniques, often blending continuum and discrete approaches, and have been used to describe cell differentiation and self-organisation. Machine learning tools are having a significant impact in model development and improving image classification processes for clinical diagnosis and treatment and will be the focus of much future research.

Salt stress and organic fertilization on the growth and biochemical metabolism of Hylocereus costaricensis (red pitaya) seedlings.

Red pitaya (Hylocereus costaricensis) is a promising species, with high cultivation potential due to the organoleptic and functional qualities of its fruits. However, irrigation water salinity can affect the crop yield. Therefore, materials rich in organic substances can minimize the damage caused by excess salts in soil and/or water. Thus, the objective of this study was to evaluate the influence of organic matter sources as attenuators of salt stress on the production and biochemical responses of red pitaya seedlings. A completely randomized design in 4 × 5 factorial scheme, with five sources of organic matter (humus, sheep manure, biofertilizer, organic compost and sand + soil) and four salinities (0.6, 2.6, 4.6 and 6.6 dS m-1) with four replicates and two plants per plot was used. The shoot length, root length, cladode diameter, number of cladodes, number of sprotus, root volume, shoot dry mass, root dry mass and total dry mass, root and shoot dry mass ratio, chlorophyll a, b and total, amino acids and soluble sugars were evaluated at 120 days after the treatments began to be applied. Red pitaya is moderately tolerant to salinity (ECw from 4.0 to 6.0 dS m-1). Organic compost and sheep manure attenuate the harmful effects of salinity on red pitaya seedlings. Under salt stress conditions, red pitaya plants increase their levels of proline, amino acids and total sugars.

Patient-reported outcome measures for a large cohort of serum eye drops recipients in the UK.

INTRODUCTION: Serum eye drops (SED) are an important treatment for patients with chronic and severe ocular surface disease (OSD). Despite a long history of use, there is a paucity of information on patient-reported outcomes, particularly comparing autologous SED (Auto-SED) and allogeneic SED (Allo-SED). National Health Service Blood and Transplant is the national provider of SED service for patients in the UK. PURPOSE: To evaluate and compare patient-reported outcome measures (PROMs) in patients receiving Auto-SED and Allo-SED for severe OSD. MATERIALS AND METHODS: PROMs were retrospectively collected from all new patients commencing treatment with Auto-SED and Allo-SED between January 2017 and September 2018, using the Ocular Surface Disease Index (OSDI) 12-item questionnaire. A linear mixed model was used to evaluate the change in OSDI scores between baseline and follow-up. RESULTS: During the study period, 279 patients who received either Auto-SED (n = 71) or Allo-SED (n = 208) were included in the analysis. Baseline and follow-up OSDI scores were available for 161 of these (49 Auto-SED and 112 Allo-SED). There was a significant reduction in mean OSDI score for both Auto-SED (59.06-24.63, p < 0.001) and Allo-SED (64.21-34.37, p < 0.001). There was no significant difference between Auto-SED and Allo-SED patients in terms of the reduction in the OSDI score (p = 0.27). CONCLUSION: Both Auto-SED and Allo-SED were associated with improvements in the quality of life of patients with chronic and severe OSD. Auto-SED and Allo-SED were equally effective in relieving the symptoms of OSD.

Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions.

Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease.

A systematic review of cellular therapies for the treatment of limbal stem cell deficiency affecting one or both eyes.

PURPOSE: This systematic review (SR) assessed the efficacy, safety and cost-effectiveness of cell-based therapy to manage limbal stem cell deficiency (LSCD), a sight-threatening orphan condition most frequently associated with severe chemical or thermal burns. LSCD has historically been treated by transplanting limbal tissue. In 1997, a new treatment, cultured limbal epithelial autografts, was described for unilateral LSCD. In cases of bilateral disease cultured autologous oral mucosa stem cells have been used. The relative efficacy of different cultured tissue procedures is unknown. METHODS: A protocol was registered with PROSPERO (CRD42017081117). Searches were conducted in 14 databases and 6 conference websites. Two reviewers independently selected studies, conducted data extraction and assessed risk of bias. One reviewer extracted individual patient data (IPD); a second checked extracted data. Data were assessed to determine the feasibility of statistical analysis, with Bayesian synthesis used to estimate improvement achieved by different treatments. RESULTS: Fifty-two studies were eligible for inclusion (1113 eyes); 41 studies (716 eyes) reported IPD. No evidence was identified on cost-effectiveness. This SR was unable to confirm that any of the types of ex vivo cultured stem cell transplants identified for LSCD treatment were statistically superior when assessed against the outcomes of interest. CONCLUSIONS: We believe this SR is the first to include IPD analysis of LSCD data. There is no evidence for the superiority of any method of limbal stem cell transplant. Confirmation of the safety and efficacy of this treatment modality is challenging due to heterogeneity within and between the studies identified. Therefore, recommendations for future research are proposed.

A systematic review of instruments measuring grief after perinatal loss and factors associated with grief reactions.

OBJECTIVE: Expectant parents who live through perinatal loss experience intense grief, which is not always acknowledged or accepted. A screening tool to detect bereaved parents' grief reactions can guide professionals, including perinatal palliative care teams, to provide follow-up for those in need. This review's goal is to identify and synthesize the international published literature on existent instruments specifically measuring the grieving process after any perinatal loss and to identify factors that could moderate grief reactions. METHOD: Systematic review (PROSPERO # CRD42018092555) with critical synthesis. PUBMED, Cochrane, and PsycINFO databases were searched in English language articles using the keywords "perinatal" AND ("grief" OR "bereavement" OR mourning) AND ("scale" OR "questionnaire" OR "measure" OR "assessment") up to May 2018. Eligibility criteria included every study using a measure to assess perinatal grief after all kinds of perinatal losses, including validations and translations to other languages and interventions designed to alleviate grief symptoms. RESULTS: A total of 67 papers met inclusion criteria. Seven instruments measuring perinatal grief published between 1984 and 2002 are described. The Perinatal Grief Scale (PGS) was used in 53 of the selected studies. Of those, 39 analyzed factors associated with grief reactions. Six articles used PGS scores to evaluate pre- and post-bereavement interventions. Studies in English language only might have limited the number of articles. SIGNIFICANCE OF RESULTS: The PGS is the most used standardized measures to assess grief after perinatal loss. All parents living through any kind of perinatal loss should be screened.

Microbiota oral e retal de calitriquídeos (Callithrix sp) em área antropizada de Mata Atlântica, Rio de Janeiro, Brasil / [Oral and rectal microbiota of callitrichids (Callithrix sp) in an anthropized area of the Atlantic Forest in Rio de Janeiro, Brazil]

A proximidade dos primatas não humanos (PNH) com o ser humano pode ser considerada um fator de risco para transmissão de bactérias entre essas duas populações. Neste estudo, foi investigada a microbiota anfibiôntica aeróbica oral e retal de calitriquídeos em um fragmento de Mata Atlântica localizado no Rio de Janeiro, Brasil, e foram realizados testes fenotípicos para detecção de bactérias multirresistentes nos isolados encontrados. Foram capturados 14 calitriquídeos e coletadas 21 amostras (14 de cavidade oral e sete de cavidade retal) em dois pontos da mata próximos às habitações humanas. As espécies mais frequentes, na cavidade oral, foram Klebsiella oxytoca (50,0%), K. pneumoniae (28,6%), Kluyvera ascorbata (21,4%) e Stenotrophomonas maltophilia (21,4%) e, na cavidade retal, K. pneumoniae (85,7%), Escherichia coli (28,6%) e Enterobacter spp. (42,9%). Todos os 48 isolados da família Enterobacteriaceae foram negativos para ESBL (betalactamase de espectro ampliado), mostrando-se não produtores da enzima nos dois métodos utilizados: disco-aproximação e método de detecção automatizado. Na pesquisa de ERC (enterobactérias resistentes a carbapenêmicos), esses mesmos isolados não apresentaram resistência aos antibióticos imipenem, meropenem e ertapenem. Todas as bactérias isoladas apresentam um potencial zoonótico, o que representa um risco à saúde pública e à conservação das espécies.(AU)

Proximity of nonhuman primates (NHP) to humans can be considered a risk factor for transmission of pathogens between these two populations. This study investigated the oral and rectal aerobic bacterial microbiota of marmosets in an anthropized area of the Atlantic Forest located in Rio de Janeiro, Brazil, and performed phenotypic tests for detection of multidrug-resistant bacteria. Twenty-one samples (14 from the oral cavity and seven from the rectum) were collected from 14 Callithrix sp. captured in two sites of the forest near human dwellings. The most frequent species identified from the oral cavity swabs were Klebsiella oxytoca (50.0%), K. pneumoniae (28.6%), Kluyvera ascorbata (21.4%) and Stenotrophomonas maltophilia (21.4%), whereas the species most commonly identified from the rectum swabs were K. pneumoniae (85.7%), Enterobacter spp. (42.9%) and Escherichia coli (28.6%). All isolates of family Enterobacteriaceae showed no extended spectrum ß-lactamase production by disk-diffusion and automated detection tests. In the search for carbapenem-resistant enterobacteriaceae these isolates presented no resistance to the imipenem, meropenem and ertapenem antibiotics. The isolate of Staphylococcus aureus was susceptible to oxacillin and the isolate of Enterococcus was susceptible to vancomycin. All isolated bacteria showed zoonotic potential, thus posing a risk to species conservation and public health.(AU)

Recombinant human MMP-2 associated with monoolein improves bone repair.

OBJECTIVE: The main objective of the present study was to investigate the possible osteostimulatory action of recombinant human matrix metalloproteinase-2 (rhMMP-2) implanted in a bone defect made in calvaria of rats, bounded to the monoolein as carrier. METHODS: Forty-four adult male Wistar rats (about 600 g body weight) underwent surgery in order to create a spherical defect in parietal bone on the right side of the median sagittal suture by using 4 mm diameter of a trephine drill. Animals were divided into three groups: no treatment (control, C), treatment with rhMMP-2 diluted in monoolein liquid crystal (rhMMP-2) and negative control with monoolein (M). The groups were divided into two experimental times, 2- and 4-weeks of experimental time. RESULTS: The rate of new-formed bone, estimated by the number of points on new-formed cancellous bone, was in enhanced rhMMP-2 group in both periods in comparison to C or M groups. CONCLUSION: There was no difference in bone neoformation between second to fourth week within groups. In the present study, monoolein alone had a negative role in the post-operative surgery, but monoolein associated with +rhMMP-2 had a positive role on releasing rhMMP-2 and enhance the rate of new-formed bone (Tab. 1, Fig. 5, Ref. 71).

The impact of two root canal treatment protocols on the oral health-related quality of life: a randomized controlled pragmatic clinical trial.

AIM: To assess the impact of two root canal treatment protocols on the oral health-related quality of life (OHRQoL) of patients in need of root canal treatment on their anterior teeth. METHODOLOGY: The sample consisted of 120 participants (mean age: 34 years) enrolled in a pragmatic randomized clinical trial evaluating two root canal treatment protocols. Anterior teeth with nonvital pulps were allocated for root canal preparation with either hand files and filled with lateral compaction of gutta-percha (manual protocol) or canal preparation with a single file in a reciprocating movement and filled with a single cone technique (Reciproc protocol). OHRQoL data were assessed using the Oral Health Impact Profile instrument (OHIP-14), which was administered before the root canal intervention (baseline), and 6 and 12 months after treatment. Demographic and clinical characteristics of participants were collected at baseline. Data were analysed using bivariate analyses, Poisson univariate and multiple regression (α = 0.05). RESULTS: The drop-out rate from baseline was 27% and 28% at 6 and 12 months after treatment, respectively. Both root canal protocols significantly enhanced patients' OHRQoL, regardless of the follow-up time (P < 0.001). After 6 months, patients treated with the Reciproc protocol had significantly lower OHIP-14 overall scores (P = 0.030), as well as significantly lower scores for psychological discomfort (P = 0.031) and social disability (P = 0.013). After 12 months, no significant difference was observed between the two root canal protocols for OHIP-14 overall scores (P = 0.174). Either large or moderate effect sizes were observed for all domains and overall scores at both evaluation times, irrespective of the protocol. Low-income persons (RR = 2.03) and the Reciproc protocol (RR = 1.52) had a higher likelihood of a positive impact on OHRQoL 12 months after root canal treatment. CONCLUSIONS: The two root canal protocols improved the OHRQoL and differences in scores were observed only after 6 months with poorer OHRQoL for the manual protocol. After 12 months, patients with low-income status and treated with Reciproc were associated with a greater improvement in OHRQoL scores.

Geographical Distribution of Schizotetranychus hindustanicus and Associated Mites in Roraima, Brazil.

The Hindustan citrus mite, Schizotetranychus hindustanicus Hirst (Acari: Tetranychidae), is a quarantine pest present in the state of Roraima, Brazil. This mite, which was described in India in 1924, was reported in 2002 in Venezuela and spread to Roraima, where it was reported in 2008, and to Colombia, where it was reported in 2010. Its possible spread to other regions of Brazil is a threat to Brazilian citriculture. This study reports the current distribution of S. hindustanicus and potential predators of this pest and other mites associated with citrus in Roraima. A survey was conducted in August and September 2015 in the 15 municipalities of the state. In each municipality, orchards and citrus plants in backyards and public areas along highways and in urban areas were sampled. Samples of leaves and fruits were collected to identify the mite and its natural enemies. Schizotetranychus hindustanicus was found in all 15 municipalities in the state of Roraima. In total, 308 associated mites were found, with S. hindustanicus being the most abundant phytophagous mite, followed by Brevipalpus yothersi Baker. Amblyseius aerialis (Muma) was the most abundant predator, followed by Iphiseiodes zuluagai Denmark and Muma and Euseius concordis (Chant). The broad dispersal of S. hindustanicus in Roraima increases the risks of this pest reaching the main citrus-producing regions in Brazil.

The use of Z-scan technique for determination of biochemical parameters in children with solid tumors or leukemias supplemented or not with selenium.

Cancer is a disease that effects cell metabolism causing an imbalance in the health of the patient. On the other hand, malnutrition, presented by oncological patients, is caused by both the disease and its treatment. Some serum biochemical parameters cannot be determined by the traditional method of laboratory blood analysis (spectrophotometry). Among the various techniques that could be used for blood biochemical analysis, we opted for the Z-scan technique, due to its sensitivity to the reading of blood components. Our objective in this work was to compare the data obtained by the Z-scan technique and the spectrophotometry of the serological samples of children with solid tumors and leukemia under treatment, receiving or not selenium supplementation in a randomized, double-blind clinical trial. The biochemical parameters were read based on blood. These blood sampling made at different stages of chemotherapy and selenium supplementation. At each of these stages, the cholesterol, glucose and triglycerides parameters were read using the Z-scan and spectrophotometry techniques. We observed that selenium helps in balancing the health of these patients, and corroborates with our hypothesis that the Z-scan technique may be an alternative for the determination of biochemical parameters.

Ferrule Design Does Not Affect the Biomechanical Behavior of Anterior Teeth Under Mechanical Fatigue: An In Vitro Evaluation.

OBJECTIVES: To investigate the survival and failure mode of fiber-post resin restorations over preparations with different ferrule designs when submitted to a fatigue load test. METHODS AND MATERIALS: Fifty bovine incisors were selected and divided into five groups (n=10) according to ferrule design: a no-ferrule group, a 2-mm circumferential ferrule group, a 2-mm buccal ferrule group, a 2-mm lingual ferrule group, and a 2-mm buccal and lingual ferrule group. The fiberglass post was cemented and the composite core was built up and prepared, followed by cementation of a full composite crown. The samples were subjected to a cyclic fatigue test with loading applicator at 135°; a staircase approach was used until fracture. Survival (cycles to fracture) and failure modes were recorded. Survival data were analyzed with the log-rank test, while Kruskal-Wallis and Fisher exact tests were used to analyze failure mode data (α=0.05). RESULTS: The median number of cycles to fracture ranged from 215,000 to 236,153. The log-rank test showed no statistically significant difference in survival rates among the groups (p=0.82). Regarding failure mode, three types were observed: I, post and/or core fracture; II, root fracture in the cervical third; and III, root fracture in the middle third. No statistical difference was observed among the groups (Kruskal-Wallis test, p=0.147). CONCLUSION: The ferrule design had no effect on fatigue resistance or failure mode of endodontically treated incisor teeth restored with a fiber post, composite core buildup, and composite crown.

Prognosis of Juvenile myoclonic epilepsy with eye-closure sensitivity.

PURPOSE: Eye closure sensitivity (ECS) has been described as a reflex trait in juvenile myoclonic epilepsy (JME). However, there is no consensus regarding its significance on prognosis. The aim of this study is to clarify the long-term impact of ECS documented by a clinical interview and a video-EEG neuropsychological protocol (VNPP) in a series of 133 JME patients. METHODS: Data from 22 JME patients with ECS confirmed by a VNPP (Group 1) were compared with those of 20 JME patients without any reflex traits (Group 2). They were followed for a mean of 8.21 years (SD=±5.044). The frequency of seizures was assessed using a diary. Except for photosensitivity (PS), any other reflex traits occurrence, drugs/alcohol abuse intake, noncompliance, and Jeavons syndrome, were considered exclusion criteria. RESULTS: Group 1 had a lower age at epilepsy onset (p = 0.028), higher incidence of febrile seizures (13.6%), and familial history of epilepsy (p = 0.023). Only 18.2% had self-perception of eyelid myoclonia (EM) (kappa coefficient = 0.193), which persisted in 77.3% of patients. Limb myoclonia, tonic-clonic seizures (TCS) and/or myoclonic-tonic-clonic seizures (MTCS), as well as absences were more frequent (p = 0.015; p = 0.013; p = 0.011, respectively) in Group 1. PS did not influenced frequency of EM (p = 1.0), absences (p = 0.648), or TCS/MTCS (p = 0.934). Psychiatric comorbidities were not different between groups. CONCLUSIONS: ECS is related to a worse outcome regarding control of all seizure types, persistence of EM, and higher frequency of limb myoclonia, as well as the total number of TCS and/or MTCS.

On the stability in alkaline conditions and electrochemical performance of A2BO4-type cathodes for liquid fuel cells.

Typical direct liquid fuel cells (DLFCs) use a liquid fuel and O2 as the oxidant. However, for applications where O2 is not available (e.g., space and underwater), the gas has been replaced by H2O2 as a liquid oxidant. This work presents a study of various ceramic disc electrodes with K2NiO4 structure and nominal compositions La2NiO4, La2CuO4, La1.9Pr0.1CuO4, La1.9Sr0.1CuO4, La1.8Ce0.2NiO4, La1.9Pr0.1NiO4, La1.8Pr0.2NiO4 and La1.9Sr0.1NiO4 to assess their stability and activity for the hydrogen peroxide reduction reaction (HPRR) in alkaline media. Stability tests conducted in 2 M NaOH show that Ni and Cu are readily dissolved, as occurs for substituting elements such as Sr, in agreement with calculated Pourbaix diagrams. Such degradation affects the surface of the materials, which is depleted of transition metals. This has consequences for the ORR and HPRR activity due to formation of a La-rich passivation layer on the surface. Only La2CuO4 and La1.8Ce0.2NiO4 display HPRR activity at around -0.25 V vs. RHE. An attempt is made to correlate the composition, chemical stability and electrochemical behaviour of these materials based on known molecular-orbital models proposed for the oxygen reduction reaction.

Quality of life on hemodialysis and inflammation: a descriptive analysis.

Chronic kidney disease (CKD) is highly prevalent worldwide. Patients with CKD on hemodialysis are more likely to present behavioral changes and worse quality of life as a result of their routine and complications. They also have higher levels of cytokines. The aim of this study is to assess the relationship between the inflammatory profile and quality of life measured by KDOQL-SF36 in hemodialysis outpatients. Patients older than 21 years of age and on routine hemodialysis for at least 6 months with treatment on a regular weekly basis were included and their anthropometric parameters and serum inflammatory markers were evaluated. Thirty patients consented to participate. Homocysteine (Hcy) levels were correlated with worse glomerular filtration rate (GFR; P=0.003) and creatinine (P=0.002). IL-6 was not correlated with worse nutritional status taking into account body mass index (BMI; kg/m2; P=0.83). On the other hand, TNF-alpha was positively correlated with albumin (P=0.008), nutritional status by BMI (P=0.04), and nutritional status by arm circumference area (P=0.04). IL-6 was correlated with activity limitation (P=0.02) and Hcy with work status (P=0.04). Hcy was correlated with nutritional status and inflammatory markers. In this population, the majority of the sections in KDOQL-SF36 were not correlated with cytokines levels.

Microbial keratitis in corneal grafts: predisposing factors and outcomes.

PurposeTo identify the nature of microbial keratitis in corneal grafts and the clinical outcomes at a tertiary hospital in the United Kingdom.Patients and methodsA retrospective case series of microbial keratitis in corneal grafts at the Royal Victoria Infirmary, Newcastle upon Tyne over a 17-year period (1997-2014).ResultsA total of 759 consecutive corneal grafts were identified from the Cornea Transplantation database. Of these, 59 episodes of microbial keratitis occurred in 41 eyes of 41 patients (5.4%; 19 male, 46.3%). Median patient age was 73 years (SD=19.4 years). The most common indication for corneal transplantation was bullous keratopathy (11/41, 26.8%). There were 34/59 (57.6%) episodes of culture-positive graft keratitis; Streptococcus pneumoniae and Staphylococcus aureus were each isolated in 5/34 (14.7%) culture-positive episodes. In all, 35/59 (59.3%) episodes of microbial keratitis occurred in 22 previously failed grafts and 3 de novo graft failures. Gram-negative keratitis was more likely to cause reduced BCVA after (χ2-test, P=0.02). Median graft duration was 49.5 months (SD=43.7 months). Failed grafts were significantly older (median 69 vs 27 months, P=0.009).ConclusionThis represents the longest published follow-up data on microbial keratitis and is the only of its kind in the United Kingdom. The incidence of 5.4% is comparable to that within the developed world. Graft age was significantly associated with graft failure in microbial keratitis; the ongoing risk of microbial keratitis warrants providing patients with long-term open access to hospital eye services.

Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis / Acurácia da reação em cadeia da polimerase quantitativa em amostras de pele íntegra congelada e parafinada no diagnóstico da leishmaniose visceral canina

The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery.(AU)

O objetivo deste trabalho foi avaliar a acurácia da PCR quantitativa (qPCR) realizada em amostras de pele íntegra congelada (FT) e parafinada (FFPE). Trata-se de um estudo de validação, com amostras provenientes de 46 cães de uma área endêmica no Brasil. Após as coletas de amostras, as extrações de DNA foram realizadas utilizando-se kits comerciais, e a qPCR foi orientada para alvos do kDNA da espécie Leishmania infantum. Os resultados obtidos para as amostras FFPE foram 63,6% de sensibilidade e 77,1% de especificidade; para as amostras FT, 100% e 48,6%, respectivamente. A concordância dos resultados da técnica de qPCR com amostras FT e FFPE foi pobre. Os resultados sugerem que o congelamento é o método mais adequado de conservação para banco de amostras para recuperação de DNA.(AU)

Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.