Experimental and economic evaluation of different culture systems for mesenchymal stromal/stem cell expansion for clinical applications

The translation of cell therapies into clinical practice requires a scalable, efficient and cost-effective manufacturing process. This study presents an integrated experimental and cost analysis of different cell culture technologies for commercial manufacture of a novel umbilical cord-derived cell therapy, currently in early phase clinical trials for the treatment of acute graft-versus-host disease (aGvHD). The experimental analysis assessed the expansion and harvest potential of mesenchymal stromal cells (MSCs), derived from umbilical cord matrix (UCM-MSCs), in different scalable cell culture technologies: a multi-layer vessel (ML), a stirred tank bioreactor with microcarrriers (STR), a hollow fiber bioreactor (HF) and a packed-bed bioreactor (PB). The presentation will highlight differences in cell proliferation rate, expansion fold and harvesting efficiency across the technologies. The cells retained their functional properties post culture in all the technologies evaluated. The experimental results were incorporated into a bioprocess economics tool comprising a stochastic cost of goods (COG) and sizing model to evaluate the commercial economic feasibility and robustness of the technologies. The financial and risk rank orders predicted by the tool will be presented, as well as their sensitivity to the reimbursement scenario selected. The model determined industrially relevant scenarios for which no technology will yield a satisfactory gross margin, indicating that many studies are still needed to establish an optimized manufacturing process

The Effects of Dietary Polyphenols on Circulating Cardiovascular Disease Biomarkers and Iron Status:A Systematic Review

The prevalence of cardiovascular disease (CVD) is rising worldwide, remaining the major cause of death in developed countries. Polyphenols have been shown to have cardioprotective properties; however, their impact on iron bioavailability and potential impact on other aspects of health is unclear. A systematic review was undertaken to evaluate the current status of the relationship between habitual polyphenol consumption, iron status, and circulating biomarkers of CVD. Following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2009 guidelines, searches were performed across 5 electronic databases (PubMed, Cochrane Library, Scopus, Web of Science, and CINAHL) to identify randomized controlled trials which investigated the effects of polyphenol consumption on inflammatory markers, serum lipid profile, and iron absorption and bioavailability. In total, 1174 records were identified, with only 7 studies meeting the inclusion criteria. The selected studies involved 133 participants and used a variety of foods and supplements, including olive oil and cherries, rich in polyphenols including hydroxytyrosol, quercetin, and resveratrol, as well as catechin enriched drinks. The duration of the studies ranged from between 56 and 145 days, with total polyphenolic content of the food items and supplements ranging from 45 to 1015 mg (per 100 g). Polyphenols did not appear to interfere with iron status, and most studies reported improvements in inflammatory markers and lipid profile. While these results are promising, the limited number of studies and considerable heterogeneity across the interventions support the need for more extensive trials assessing the relationship between polyphenol intake, iron bioavailability, and CVD risk

Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2007/56146-2]Comissao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)[1429/08-6

Optimal neural network feature selection for spatial-temporal forecasting

In this paper, we show empirical evidence on how to construct the optimal feature selection or input representation used by the input layer of a feedforward neural network for the propose of forecasting spatial-temporal signals. The approach is based on results from dynamical systems theory, namely the non-linear embedding theorems. We demonstrate it for a variety of spatial-temporal signals, with one spatial and one temporal dimensions, and show that the optimal input layer representation consists of a grid, with spatial/temporal lags determined by the minimum of the mutual information of the spatial/temporal signals and the number of points taken in space/time decided by the embedding dimension of the signal. We present evidence of this proposal by running a Monte Carlo simulation of several combinations of input layer feature designs and show that the one predicted by the non-linear embedding theorems seems to be optimal or close of optimal. In total we show evidence in four unrelated systems: a series of coupled Henon maps; a series of couple Ordinary Differential Equations (Lorenz-96) phenomenologically modelling atmospheric dynamics; the Kuramoto-Sivashinsky equation, a partial differential equation used in studies of instabilities in laminar flame fronts and finally real physical data from sunspot areas in the Sun (in latitude and time) from 1874 to 2015.Comment: 11 page

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. Methods: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. Results: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in na\uefve and primed pluripotent states. Conclusions: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation

Epidemiological factors related to slow progression of the acquired immune deficiency syndrome (AIDS)

Com o objetivo de identificação de fatores envolvidos na progressão lenta para aids, realizou-se estudo transversal para avaliação de dados epidemiológicos de indivíduos infectados pelo Vírus da Imunodeficiência Humana tipo 1 (HIV-1), atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto-USP. Foram selecionados pacientes, conforme critérios definidos, constituindo duas populações: população 1, composta por lentos progressores (P1), que possuía anticorpos anti-HIV há mais de oito anos e com ocorrência de menos de duas doenças oportunistas no último ano, e a população 2 (P2), pacientes rápidos progressores, com diagnóstico de infecção pelo HIV e doença manifesta a menos de dois anos e com mais de duas doenças oportunistas, diagnosticadas no último ano. Todos os indivíduos foram submetidos a questionário, contendo dados demográficos, profissão, ocorrência de outras doenças sexualmente transmissíveis, forma de contágio, data de diagnóstico e hábitos. O período do estudo foi de março de 1998 a outubro de 1999. Obtivemos na P1: doze homens e quatro mulheres, idade média 30,7 anos, forma de contágio predominantemente sangüínea, tempo de progressão da doença 10,5 anos; P2: 12 homens e 4 mulheres; idade média 34,8 anos, forma de contágio predominantemente sexual, tempo de progressão da doença de 1,5 anos. Tabagismo foi o principal vício em ambas as populações. Quando interrogados sobre a causa do bom estado de saúde, os indivíduos da P1 responderam estar ela relacionada à fé e ao uso adequado das medicações. Os pacientes da P2 não foram interrogados sobre a causa de seu estado de saúde. Quanto à prática sexual, nove pacientes da P1 mantinham relações, sendo cinco sem uso regular do preservativo. Na P2, apenas um paciente utilizava preservativo. Dois pacientes da P1 e um da P2 revelaram ter apresentado DST, Sífilis e Papiloma Vírus Humano. Em vista do reduzido número de pacientes, não foi possível estabelecer associação entre as variáveis estudadas e os padrões de progressão da doença. Os dados sobre hábitos não parecem contribuir para o padrão de desenvolvimento da doença. O estudo oferece um perfil epidemiológico dessas populações de pacientes.To determine the factors involved in slow progression to aids, a transverse study was conducted for the evaluation of the epidemiological data of individuals infected with type 1 human immunodeficiency virus seen at the University Hospital of the Faculty of Medicine of Ribeirão Preto-USP. Patients were choosed in conformity some judgment, establish two populations: population 1, consisting of slow progressors (P1), had been carrying HIV antibodies for more than 8 years, with the occurrence of less than 2 opportunistic diseases in the last year, and population 2 (P2), consisting of rapid progressors, had a diagnosis of HIV infection and overt disease detected less than 2 years before and more than 2 opportunistic diseases diagnosed during the last year. All patients responded to a questionnaire concerning demographic data, profession, occurrence of other sexually transmissible diseases, form of contagion, date of diagnosis, and habits. The study was conducted from March 1998 to October 1999. We obtain in the P1: 12 men and 4 women, mean age 30.7 years, predominant form of contagion: blood route, time of disease progression 10.5 years; P2 12 men and 4 women; mean age 34,8 years, predominant form of contagion: sexual, time of disease progression 1.5 years. Tabagisme was the principal vice in about populations. When asked about motivation yours good health, the subjects of P1 answered to be relationed the faith and medications use. The patients of the P2 did not answer about yours health state. Whatever the sexual custom, 9 patients of the P1 had sexual relations, five without regular use of condom. In the P2 only um patient used condom. Two patients of the P1 and one of P2 declared to have STD, syphilis and Human Papiloma Virus (HPV). Because there are reduces number of patients it’s impossible to make asociation between the variables studies and mesures of the disease progression. The dates about habits don’t contribute for the disease development. The study offers an epidemiological profile of these patient populations

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1-kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (+/- 931.7)- and 295,400 (+/- 75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (+/- 493,700)- and 308,000 (+/- 139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/mu g protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.FINEP [01.07.0652.00]FINEPFAPESPFAPESP [1998/14247-6]CNPq [314458/2009-3, 2008/57877-3]CNP