Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

Multiple oligomeric structures of a bacterial small heat shock protein

Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions.

Aplicación de la Lógica Dominante del servicio (LDS) en el sector turístico: el marketing interno como antecedente de la cultura de co-creación de innovaciones con clientes y empleados

[ES] Este trabajo trata de profundizar en la comprensión del concepto de marketing interno (MI), considerado como un recurso operante desde la óptica de la Lógica Dominante del Servicio (LDS), así como en su influencia en la obtención de resultados empresariales superiores a los de la competencia. Para ello, se examina el efecto del MI en la predisposición de las empresas analizadas a que sus clientes y empleados de primera línea participen en el desarrollo de innovaciones de servicio, ampliando de este modo, de acuerdo con la LDS, las oportunidades de co-creación de valor disponibles para las organizaciones. Para contrastar las hipótesis planteadas se aplica un análisis de ecuaciones estructurales a la información facilitada por los gerentes de 240 hoteles de una muestra de ámbito nacional.

Embryogenesis of Heterobranchus longifilis (Curvier and Valenciennes, 1840)

Studies on development of H. longifilis (Curvier and Valenciennes, 1840) were conducted at a temperature of 25EC ( 1Ec) in aquaria tanks continuous development were monitored with the use of wild Heerbrugy photomacroscope and length of yolk and larva were monitored using Stereo Olympus microscope with ocular micrometer. The division into animal and vegetal poles was observed 22 minutes after activation. The first cleavage occurred 65 minutes after activation while the second division which was perpendicular to the first line of division occurred 74 minutes after activation. This was quickly followed by the third and fourth cleavage at 80th and 82nd minutes after activation respectively. Morular stage was reached at 4 hours 20 minutes with formation of optic bud at 14 hours 35 minutes. (DBO) Developing embryo hatched after 27 hours of activation at a mean length of 6.63 and mean yolk length of 2.17. Yolk size decrease at an average rate of 38.5 % till the 5th day of total absorption. Growth of larvae proceeded faster in tail-anus region than in anus-snout portion of the body. The rate of yolk absorption and larva development (survival) as monitored in this work gives important information in Research and development programme for H. longifilis larva - an important aspect of Research development and implementation of appropriate technologies in small scale fisheries

Influência da superexpressão da claudina-3 na radiorresposta de células de câncer de cólon

O câncer colorretal (CCR) é o terceiro tipo de câncer mais incidente no mundo para o sexo masculino, o segundo para o sexo feminino e a radioterapia é um dos tratamentos de primeira linha no combate a este tipo de câncer. Durante a progressão do CCR as células sofrem alterações morfogenéticas, sendo a desorganização do complexo juncional apical (CJA) um dos eventos iniciais desse processo. As junções oclusivas (JTs) são um dos principais componentes da CJA e desempenham papel importante no controle do fluxo paracelular, na determinação da polaridade celular e na transdução de sinais relacionados com a progressão tumoral. As claudinas são proteínas transmembrana, constituintes das JTs e cumprem um importante papel no controle desses eventos. Alterações na expressão das claudinas são observadas em tumores de diferentes órgãos e têm sido relacionadas com a progressão tumoral. No entanto os mecanismos que regulam essas alterações e sua consequência na progressão do CCR são poucos conhecidos. Desta forma, o presente estudo teve como objetivo avaliar a influência da superexpressão da claudina-3 na radiorresposta de células CCR. Nossos resultados mostraram que a superexpressão de claudina-3 minimiza alterações morfológicas causadas pela radiação, causa diminuição da resistência elétrica transepitelial e não tem efeito na permeabilidade a macromoléculas após a irradiação. Além disso, observamos que a superexpressão de claudina-3 aumenta o potencial proliferativo das células e que esta característica torna as células mais sensíveis a radiação. Porém quando avaliamos eventos celulares relacionados a progressão tumoral observamos que apesar da radiação diminuir a capacidade migratória das progênies, as células que superexpressam claudina-3 apresentam migração mais elevada. Além disso, verificamos que a superexpressão de claudina-3 diminui a invasão e a capacidade de formação de colônias frente ao tratamento com a radiação. Em seguida fomos avaliar o efeito da inibição das vias de proliferação (MEK/ERK) e sobrevivência (PI3K-Akt) na resposta das células que superexpressam claudina-3 frente a radiação. Observamos que a inibição de MEK é capaz de sensibilizar as células que superexpressam claudina-3 à radiação no ensaio de proliferação celular, no entanto a inibição de MEK e PI3K antes da exposição à radiação é capaz aumentar a migração e a capacidade de formação de colônias de células que superexpressam claudina-3 contribuindo para o aumento do potencial maligno. Em conjunto nossos resultados mostram que a superexpressão de claudina-3 contribui para um fenótipo mais maligno, no entanto frente ao tratamento com a radiação é capaz de sensibilizar as células.

Dose terapêutica de radiação ionizante induz um fenótipo migratório em células de carcinoma de colon humano (caco-2): vias de sinalização envolvidas

O câncer colo-retal é a terceira neoplasia mais frequente em todo o mundo e a recorrência local e neoplasia refratária são desafios no tratamento do câncer colo-retal após a cirurgia convencional. Com o intuito de controlar a recorrência e aumentar a média de sobrevida dos pacientes, uma estratégia multidisciplinar que combina a radioterapia (RT) e a quimioterapia com o processo cirúrgico tem sido protocolo clínico de escolha. Embora esta combinação seja capaz de otimizar o tratamento, nem todos os pacientes são beneficiados com o protocolo quimio-rádio combinado, uma vez que existem os insucessos terapêuticos relacionados com a incidência de neoplasias secundárias tardias em pacientes que foram submetidos à RT para tratamento de neoplasias anteriores. Além da doença refratária, outro agravante da RT são os efeitos colaterais produzidos pela radiação ionizante (IR), em especial àqueles do trato gastrointestinal. Estes efeitos estão relacionados com alterações da homeostase do epitélio intestinal, através da desorganização dos complexos juncionais. Porém, os mecanismos que medeiam estes efeitos ainda não estão elucidados. Este estudo avaliou as vias de sinalização que medeiam os efeitos da IR em células Caco-2. Foi observado que a IR causa uma desorganização da junção aderente via Src, EGFR e MAPK, sendo estas alterações acompanhadas por desorganização do citoesqueleto de actina em todo o volume celular. Src, EGFR e MAPK participam de maneira diferenciada na modulação destes efeitos. Observamos também que a radiação aumenta a motilidade dessas células via Src e MAPK e não induz alteração na proliferação celular até 48 horas após o tratamento. Este é o primeiro trabalho que correlaciona vias de sobrevivência celular como Src, EGFR e MAPK com alterações nas proteínas de junção aderente, alterações do citoesqueleto e migração celular. Estes eventos são relacionados aos efeitos colaterais primários e tardios induzidos pela IR, e podem favorecer à aquisição de um fenótipo maligno herdável durante o fracionamento de doses na RT, favorecendo a progressão tumoral do câncer colo-retal. Logo, além da correlação das vias de sinalização envolvidas nos eventos induzidos pela IR mostrados neste estudo, os resultados também corroboram para um melhor entendimento da atividade farmacológica dos inibidores químicos utilizados, uma vez que muitos deles encontram-se em fase de ensaios pré-clínicos e clínicos.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. (C) 2009 Elsevier Ltd. All rights reserved.

Regulação Farmacológica da Ovulação

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

A randomised phase II study of two different schedules of pegylated liposomal doxorubicin in metastatic breast cancer (EORTC-10993).

One hundred and sixteen women with measurable metastatic breast cancer participated in a randomised phase II study of single agent liposomal pegylated doxorubicin (Caelyx) given either as a 60 mg/m2 every 6 weeks (ARM A) or 50 mg/m2 every 4 weeks (ARM B) schedule. Patients were over 65 years of age or, if younger, had refused or been unsuitable for standard anthracyclines. The aims of the study were to evaluate toxicity and dose delivery with the two schedules and obtain further information on the response rate of liposomal pegylated doxorubicin as a single agent in anthracycline nai ve advanced breast cancer. Twenty-six patients had received prior adjuvant chemotherapy (including an anthracycline in 10). Sixteen had received non-anthracycline-based first-line chemotherapy for advanced disease. One hundred and eleven patients were evaluable for toxicity and 106 for response. The delivered dose intensity (DI) was 9.8 mg/m2 (95% CI, 7.2-10.4) with 37 (69%) achieving a DI of >90% on ARM A and 11.9 mg/m2 (95% CI, 7.5-12.8) with 37 (65%) achieving a DI of >90% on ARM B. The adverse event profiles of the two schedules were distinctly different. Mucositis was more common with the every 6 weeks regimen (35% CTC grade 3/4 in ARM A, 14% in ARM B) but palmar plantar erythrodysesthesia (PPE) was more frequent with the every 4 weeks regimen (2% CTC grade 3/4 in ARM A, 16% in ARM B). Confirmed objective partial responses by RECIST criteria were seen with both schedules; 15/51 (29%) on ARM A and 17/56 (31%) on ARM B. Liposomal pegylated doxorubicin showed significant activity in advanced breast cancer with a generally favourable side-effect profile. The high frequency of stomatitis seen with 6 weekly treatment makes this the less preferred of the two schedules tested.

Clinical outcome of breast cancer patients with liver metastases alone in the anthracycline-taxane era: a retrospective analysis of two prospective, randomised metastatic breast cancer trials.

Liver metastases have long been known to indicate an unfavourable disease course in breast cancer (BC). However, a small subset of patients with liver metastases alone who were treated with pre-taxane chemotherapy regimens was reported to have longer survival compared with patients with liver and metastases at other sites. In the present study, we examined the clinical outcome of breast cancer patients with liver metastases alone in the context of two phase III European Organisation for Research and Treatment of Cancer (EORTC) trials which compared the efficacy of doxorubicin (A) versus paclitaxel (T) (trial 10923) and of AC (cyclophosphamide) versus AT (trial 10961), given as first-line chemotherapy in metastatic BC patients. The median follow-up for the patients with liver metastases was 90.5 months in trial 10923 and 56.6 months in trial 10961. Patients with liver metastases alone comprised 18% of all patients with liver metastases, in both the 10923 and 10961 trials. The median survival of patients with liver metastases alone and liver plus other sites of metastases were 22.7 and 14.2 months (log rank test, P=0.002) in trial 10923 and 27.1 and 16.8 months (log rank test, P=0.19) in trial 10961. The median TTP (time to progression) for patients with liver metastases alone was also longer compared with the liver plus other sites of metastases group in both trials: 10.2 versus 8.8 months (log rank test, P=0.02) in trial 10923 and 8.3 versus 6.7 months (log rank test, P=0.37) in trial 10961. Most patients with liver metastases alone have progression of their disease in their liver again (96 and 60% of patients in trials 10923 and 10961, respectively). Given the high prevalence of breast cancer, improved detection of liver metastases, encouraging survival achieved with currently available cytotoxic agents and the fact that a significant portion of patients with liver metastases alone have progression of their tumour in the liver again, a more aggressive multimodality treatment approach through prospective clinical trials seems worth exploring in this specific subset of women.

Baseline health-related quality-of-life data as prognostic factors in a phase III multicentre study of women with metastatic breast cancer.

The potential value of baseline health-related quality-of-life (HRQOL) and clinical factors in predicting prognosis was examined using data from an international randomised phase III trial which compared doxorubicin and paclitaxel with doxorubicin and cylophosphamide as first line chemotherapy in 275 women with metastatic breast cancer. The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and the related breast module (QLQ-BR23) were used to assess baseline HRQOL data. The Cox proportional-hazards regression model was used for both univariate and multivariate analyses of survival. In the univariate analyses, performance status (P<0.001) and number of sites involved (P=0.001) were the most important clinical prognostic factors. The HRQOL variables at baseline most strongly associated with longer survival were better appetite, physical and role functioning, as well as less fatigue (P<0.001). The final multivariate model retained performance status (P<0.001) and appetite loss (P=0.005) as the variables best predicting survival. Substantial loss of appetite was the only independent HRQOL factor predicting poor survival and was strongly correlated (/r/>0.5) with fatigue, role and physical functioning. In addition to known clinical factors, appetite loss appears to be a significant prognostic factor for survival in women with metastatic breast cancer. However, the mechanism underlying this association remains to be precisely defined in future studies.

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.

La greffe de moelle dans les maladies héréditaires.

Since 1968, bone marrow transplantation became the first line therapy for selected metabolic and immunological hereditary disorders. Actually, advances in the supportive care in bone marrow transplantation and a better knowledge of the immunology of BMT complications has been associated with a better disease correction and an increase in long term survival. New approaches are under investigation and include: hematopoietic growth factors, enzymatic replacement and gene therapy. However at the present time BMT is still the only curative treatment for selected hereditary disorders.

Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential.

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(3)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

Accessibility, availability and affordability of anti-malarials in a rural district in Kenya after implementation of a national subsidy scheme.

BACKGROUND: Poor access to prompt and effective treatment for malaria contributes to high mortality and severe morbidity. In Kenya, it is estimated that only 12% of children receive anti-malarials for their fever within 24 hours. The first point of care for many fevers is a local medicine retailer, such as a pharmacy or chemist. The role of the medicine retailer as an important distribution point for malaria medicines has been recognized and several different strategies have been used to improve the services that these retailers provide. Despite these efforts, many mothers still purchase ineffective drugs because they are less expensive than effective artemisinin combination therapy (ACT). One strategy that is being piloted in several countries is an international subsidy targeted at anti-malarials supplied through the retail sector. The goal of this strategy is to make ACT as affordable as ineffective alternatives. The programme, called the Affordable Medicines Facility - malaria was rolled out in Kenya in August 2010. METHODS: In December 2010, the affordability and accessibility of malaria medicines in a rural district in Kenya were evaluated using a complete census of all public and private facilities, chemists, pharmacists, and other malaria medicine retailers within the Webuye Demographic Surveillance Area. Availability, types, and prices of anti-malarials were assessed. There are 13 public or mission facilities and 97 medicine retailers (registered and unregistered). RESULTS: The average distance from a home to the nearest public health facility is 2 km, but the average distance to the nearest medicine retailer is half that. Quinine is the most frequently stocked anti-malarial (61% of retailers). More medicine retailers stocked sulphadoxine-pyramethamine (SP; 57%) than ACT (44%). Eleven percent of retailers stocked AMFm subsidized artemether-lumefantrine (AL). No retailers had chloroquine in stock and only five were selling artemisinin monotherapy. The mean price of any brand of AL, the recommended first-line drug in Kenya, was $2.7 USD. Brands purchased under the AMFm programme cost 40% less than non-AMFm brands. Artemisinin monotherapies cost on average more than twice as much as AMFm-brand AL. SP cost only $0.5, a fraction of the price of ACT. CONCLUSIONS: AMFm-subsidized anti-malarials are considerably less expensive than unsubsidized AL, but the price difference between effective and ineffective therapies is still large.