Interfollicular epidermal stem cells: boosting and rescuing from adult skin

Publicado na série "Methods in Molecular Biology", vol. 989 (2013), ISSN 1064-3745Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the β 1-integrin ligand and Rho- Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f bri /CD71 dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f bri /CD71 dim subpopulations. The stepby- step methodology herein described indulges the boosting and consecutive puri fi cation and separation of interfollicular epidermal stem cells from human keratinocytes cultures.(undefined

An in vivo permeability test protocol using iohexol to reduce and refine the use of laboratory rats in intestinal damage assessment

Assessment of intestinal damage in laboratory rats with experimentally-induced enteropathies is usually carried out by collecting and morphological interpreting tissue samples obtained surgically, endoscopically or at necropsy. Alternatively, changes in the gut mucosa may be less invasively evaluated with intestinal permeability (IP) tests. In contrast to human and veterinary patients, IP test protocols in laboratory rats have been highly variable, which may account for the limited use of this approach by investigators when evaluating intestinal damage. The objective of this study was to establish a refined IP test protocol using iohexol in rats that is able to differentiate between healthy rats and individuals with enteropathies. Iohexol was administered by oral gavage to twenty-eight Sprague-Dawley rats, before and after the induction of inflammatory bowel disease (IBD) with dextran sulphate sodium (DSS). Urine was cumulatively recovered during 24 h, and the presence of iohexol was measured by high-performance liquid chromatography with ultraviolet detection. The median percentage (and interquartile range) of administered iohexol in urine of healthy rats was 0.54% (0.3

Numerical modelling of concrete curing, regarding hydration and temperature phenomena

A numerical model that accounts for the hydration and aging phenomena during the early ages of concrete curing is presented in a format suitable for a finite element implementation. Assuming the percolation of water through the hydrates already formed as the dominant mechanism of cement hydration, the model adopts an internal variable called hydration degree, whose evolution law is easily calibrated and allows an accurate prediction of the hydration heat production. Compressive strength evolution is related to the aging degree, a concept that accounts for the influences of the hydration and curing temperature on the final mechanical properties of concrete. The model capabilities are illustrated by means of a wide set of experimental tests involving ordinary and high performance concretes, and through the simulation of the concrete curing on a viaduct deck of the Öresund Link

Adipose stem cell-derived osteoblasts sustain the functionality of endothelial progenitors from the mononuclear fraction of umbilical cord blood

Vascularization is the most pressing issue in tissue engineering (TE) since ensuring that engineered constructs are adequately perfused after in vivo transplantation is essential for the construct’s survival. The combination of endothelial cells with current TE strategies seems the most promising approach but doubts persist as to which type of endothelial cells to use. Umbilical cord blood (UCB) cells have been suggested as a possible source of endothelial progenitors. Osteoblasts obtained from human adiposederived stem cells (hASCs) were co-cultured with the mononuclear fraction of human UCB for 7 and 21 days on carrageenan membranes. The expression of vWF and CD31, and the DiI-AcLDL uptake ability allowed detection of the presence of endothelial and monocytic lineages cells in the co-culture for all culture times. In addition, the molecular expression of CD31 and VE-cadherin increased after 21 days of coculture. The functionality of the system was assessed after transplantation in nude mice. Although an inflammatory response developed, blood vessels with cells positive for human CD31 were detected around the membranes. Furthermore, the number of blood vessels in the vicinity of the implants increased when cells from the mononuclear fraction of UCB were present in the transplants compared to transplants with only hASC-derived osteoblasts. These results show how endothelial progenitors present in the mononuclear fraction of UCB can be sustained by hASC-derived osteoblast co-culture and contribute to angiogenesis even in an in vivo setting of inflammatory response.Financial support through the Ph.D. Grant SFRH/BD/44893/2008 to R.P. P. by the Portuguese Foundation for Science and Technology (FCT) and through the European Union NoE EXPERTISSUES (NMP3-CT-2004-500283) is acknowledged

Discriminatory ability of next-generation tau PET tracers for Alzheimer's disease

A next generation of tau PET tracers for imaging of Alzheimer’s disease and other dementias has recently been developed. Whilst the new compounds have now entered clinical studies, there is limited information available to assess their suitability for clinical applications. Head-to-head comparisons are urgently needed to understand differences in the radiotracer binding profiles. We characterised the binding of the tau tracers PI2620, RO948, MK6240 and JNJ067 in human post-mortem brain tissue from a cohort of 25 dementia cases and age-matched controls, using quantitative phosphorimaging with tritium labelled radiotracers in conjunction with phospho-tau specific immunohistochemistry. The four tau radiotracers depicted tau inclusions composed of paired helical filaments with high specificity, both in cases with Alzheimer’s disease and in primary tauopathy cases with concomitant Alzheimer’s disease pathology. In contrast, cortical binding to primary tauopathy cases without paired helical filament tau was found to be within the range of age-matched controls. Off-target binding to monoamine oxidase B has been overcome, as demonstrated by heterologous blocking studies in basal ganglia tissue. The high variability of cortical tracer binding within the Alzheimer’s disease group followed the same pattern with each tracer, suggesting that all compounds are suited to differentiate Alzheimer’s disease from other dementias

Fetal stem cells obtained from amniotic fluid and wharton's jelly expanded using platelet lysate for tissue engineering applications

Extra-embryonic tissues, such as amniotic fluid (AF) and Wharton´s Jelly (WJ) of umbilical cord, offer many advantages over both embryonic and adult stem cell sources. These tissues are routinely discarded at parturition and the extracorporeal nature of these cell sources facilitates isolation, as well as the comparatively large volume and ease of physical manipulation theoretically increases the number of stem cells that can be isolated. Autologous approaches to use MSCs, namely from bone marrow, have difficulties regarding the limited availability of large amounts of cells from the patient. Fetal stem cells appear to have even more pronounced immunomodulatory properties than adult MSCs (1, 2). This allogeneic escape mechanism may be of therapeutic value, because transplantation of allogeneic human MSCs in stock would be readily available, as opposed to the culture of autologous cells for subsequent transplantation. Cell expansion protocols are based on the use of media supplemented with fetal bovine serum (FBS) as a source of nutrientes and growth factors. The animal serum is not completely safe, once there is a possibility of contamination by animal viroses, prions or others contaminants and it is described that FBS used systematically in MSCs subcultivation induces more humoral immune response (3). Additionally anti-FBS antibodies could be detected in patients after receiving MSCs expanded in FBS (4). Platelet lysate (PL) has enormous possibilities in cell therapy, namely because of the high concentration of growth factors that promotes higher cell expansion, such as tissue regeneration (5). A recent study showed that proliferation of MSCs was much higher on PL gel compared to tissue culture plastic (6). The immunomodulatory properties of MSCs are maintained when expanded in culture medium supplemented with PL (7) Based on these premises we isolated fetal stem cells from AF obtained from amniocentesis and WJ from umbilical cords. These cells were plated and expanded in low density numbers in basal culture medium with FBS or either supplemented with PL. In each passage cells were counted for proliferation kinetics and prepared for flow cytometry analysis. Expanded populations were analysed both population size and complexity and for the MSCs well-known surface markers (CD34, CD45, CD73, CD44, CD106, CD105, CD29, CD90, CD31) and markers related with immune response (HLADR, 80, 83, 86) and embryonic markers SSEA-4 and TRA-1-60

Investigation Into the Humaneness of Slaughter Methods for Guinea Pigs (Cavia porcelus) in the Andean Region

Guinea pigs (Cavia porcelus) are an important source of nonhuman animal protein in the Andean region of South America. Specific guidelines regarding the welfare of guinea pigs before and during slaughter have yet to be developed. This study critically assessed the humaneness of 4 different stunning/slaughter methods for guinea pigs: cervical neck dislocation (n = 60), electrical head-only stunning (n = 83), carbon dioxide (CO(2)) stunning (n = 21), and penetrating captive bolt (n = 10). Following cervical neck dislocation, 97% of guinea pigs had at least 1 behavioral or cranial/spinal response. Six percent of guinea pigs were classified as mis-stunned after electrical stunning, and 1% were classified as mis-stunned after captive bolt. Increased respiratory effort was observed during CO(2) stunning. Apart from this finding, there were no other obvious behavioral responses that could be associated with suffering. Of the methods assessed, captive bolt was deemed the most humane, effective, and practical method of stunning guinea pigs. Cervical neck dislocation should not be recommended as a slaughter method for guinea pigs

Pt(IV)-based nanoscale coordination polymers : antitumor activity, cellular uptake and interactions with nuclear DNA

Cisplatin has been for many years the gold standard chemotherapeutic drug for the treatment of a wide range of solid tumors, even though its use is commonly associated with serious side effects including non-selective toxicity, myelosuppression or development of cisplatin resistance, among others complications. Over the last decade, a number of nanoparticle formulations were developed to reduce its side effects and improve the selectivity and efficacy of this drug. In this study, we have developed a novel nanoparticle platform based on nanoscale coordination polymer named (Zn-Pt(IV)-NCPs) which contains a Pt(IV) prodrug, Zn and the linker ligand 1,4-Bis(imidazol-1-ylmethyl)benzene (bix). The main objective has been to gain insights into the mechanism of action of this nanostructured material in comparison with cisplatin and the free Pt(IV) prodrug in order to establish a correlation between nanostructuration and therapeutic activity. Zn-Pt(IV)-NCPs nanoparticles displayed an average size close to 200 nm as determined by DLS, a good stability in physiologic environments, and a controlled drug release of Pt. In vitro studies demonstrated that Pt(IV)-NCPs showed an enhanced cytotoxic effect against cell culture of cervical cancer, neuroblastoma and human adenocarcinoma cells in comparison with free Pt(IV) prodrug. Although no difference in cell uptake of Pt was observed for any of the three cell lines assayed, a higher amount of Pt bound to the DNA was found in the cells treated with the nanostructured Pt(IV) prodrug. These studies suggest that the nanostructuration of the prodrug facilitate its activation and induce a change in the mechanism of action related to an increased interaction with the DNA as corroborated by the studies of direct interaction of the Pt(IV) prodrug, nanostructured or not, with DNA

Gender and sexual orientation differences in cognition across adulthood : age is kinder to women than to men regardless of sexual orientation

Despite some evidence of greater age-related deterioration of the brain in males than in females, gender differences in rates of cognitive aging have proved inconsistent. The present study employed web-based methodology to collect data from people aged 20-65 years (109,612 men; 88,509 women). As expected, men outperformed women on tests of mental rotation and line angle judgment, whereas women outperformed men on tests of category fluency and object location memory. Performance on all tests declined with age but significantly more so for men than for women. Heterosexuals of each gender generally outperformed bisexuals and homosexuals on tests where that gender was superior; however, there were no clear interactions between age and sexual orientation for either gender. At least for these particular tests from young adulthood to retirement, age is kinder to women than to men, but treats heterosexuals, bisexuals, and homosexuals just the same