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

yesThe 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 variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-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 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, 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 c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker

Introduction: Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. Materials and methods: UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. Results: Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. Conclusions: Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM

The Pediatric and Young Adult Choroidal and Ciliary Body Melanoma Genetic Study, A Survey by the European Ophthalmic Oncology Group

PURPOSE:To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group. METHODS:Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates. RESULTS:The mean age at diagnosis was 17 years (range 5.0–24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018). CONCLUSIONS:We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).</p

The Pediatric and Young Adult Choroidal and Ciliary Body Melanoma Genetic Study, A Survey by the European Ophthalmic Oncology Group

PURPOSE:To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group. METHODS:Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates. RESULTS:The mean age at diagnosis was 17 years (range 5.0–24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018). CONCLUSIONS:We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).</p

Multiple receptor tyrosine kinases are expressed in adult rat retinal ganglion cells as revealed by single-cell degenerate primer polymerase chain reaction

BACKGROUND: To achieve a better understanding of the repertoire of receptor tyrosine kinases (RTKs) in adult retinal ganglion cells (RGCs) we performed polymerase chain reaction (PCR), using degenerate primers directed towards conserved sequences in the tyrosine kinase domain, on cDNA from isolated single RGCs univocally identified by retrograde tracing from the superior colliculi.RESULTS: All the PCR-amplified fragments of the expected sizes were sequenced, and 25% of them contained a tyrosine kinase domain. These were: Axl, Csf-1R, Eph A4, Pdgfrbeta, Ptk7, Ret, Ros, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3. Non-RTK sequences were Jak1 and 2. Retinal expression of Axl, Csf-1R, Pdgfrbeta, Ret, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3, as well as Jak1 and 2, was confirmed by PCR on total retina cDNA. Immunodetection of Csf-1R, Pdgfralpha/beta, Ret, Sky, TrkB, and Vegfr-2 on retrogradely traced retinas demonstrated that they were expressed by RGCs. Co-localization of Vegfr-2 and Csf-1R, of Vegfr-2 and TrkB, and of Csf-1R and Ret in retrogradely labelled RGCs was shown. The effect of optic nerve transection on the mRNA level of Pdgfrbeta, Csf-1R, Vegfr-2, Sky, and Axl, and of the Axl ligands Gas6 and ProteinS, was analysed. These analyses show transection-induced changes in Axl and ProteinS mRNA levels.CONCLUSIONS: The repertoire of RTKs expressed by RGCs is more extensive than previously anticipated. Several of the receptors found in this study, including Pdgfrbeta, Csf-1R, Vegfr-2, Sky, and Axl, and their ligands, have not previously been primarily associated with retinal ganglion cells

Global, regional, and national burden of traumatic brain injury and spinal cord injury, 1990–2016: A systematic analysis for the Global Burden of Disease Study 2016

© 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license Background: Traumatic brain injury (TBI) and spinal cord injury (SCI) are increasingly recognised as global health priorities in view of the preventability of most injuries and the complex and expensive medical care they necessitate. We aimed to measure the incidence, prevalence, and years of life lived with disability (YLDs) for TBI and SCI from all causes of injury in every country, to describe how these measures have changed between 1990 and 2016, and to estimate the proportion of TBI and SCI cases caused by different types of injury. Methods: We used results from the Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study 2016 to measure the global, regional, and national burden of TBI and SCI by age and sex. We measured the incidence and prevalence of all causes of injury requiring medical care in inpatient and outpatient records, literature studies, and survey data. By use of clinical record data, we estimated the proportion of each cause of injury that required medical care that would result in TBI or SCI being considered as the nature of injury. We used literature studies to establish standardised mortality ratios and applied differential equations to convert incidence to prevalence of long-term disability. Finally, we applied GBD disability weights to calculate YLDs. We used a Bayesian meta-regression tool for epidemiological modelling, used cause-specific mortality rates for non-fatal estimation, and adjusted our results for disability experienced with comorbid conditions. We also analysed results on the basis of the Socio-demographic Index, a compound measure of income per capita, education, and fertility. Findings: In 2016, there were 27·08 million (95% uncertainty interval [UI] 24·30–30·30 million) new cases of TBI and 0·93 million (0·78–1·16 million) new cases of SCI, with age-standardised incidence rates of 369 (331–412) per 100 000 population for TBI and 13 (11–16) per 100 000 for SCI. In 2016, the number of prevalent cases of TBI was 55·50 million (53·40–57·62 million) and of SCI was 27·04 million (24·98–30·15 million). From 1990 to 2016, the age-standardised prevalence of TBI increased by 8·4% (95% UI 7·7 to 9·2), whereas that of SCI did not change significantly (−0·2% [–2·1 to 2·7]). Age-standardised incidence rates increased by 3·6% (1·8 to 5·5) for TBI, but did not change significantly for SCI (−3·6% [–7·4 to 4·0]). TBI caused 8·1 million (95% UI 6·0–10·4 million) YLDs and SCI caused 9·5 million (6·7–12·4 million) YLDs in 2016, corresponding to age-standardised rates of 111 (82–141) per 100 000 for TBI and 130 (90–170) per 100 000 for SCI. Falls and road injuries were the leading causes of new cases of TBI and SCI in most regions. Interpretation: TBI and SCI constitute a considerable portion of the global injury burden and are caused primarily by falls and road injuries. The increase in incidence of TBI over time might continue in view of increases in population density, population ageing, and increasing use of motor vehicles, motorcycles, and bicycles. The number of individuals living with SCI is expected to increase in view of population growth, which is concerning because of the specialised care that people with SCI can require. Our study was limited by data sparsity in some regions, and it will be important to invest greater resources in collection of data for TBI and SCI to improve the accuracy of future assessments. Funding: Bill & Melinda Gates Foundation