Transplante de células de medula óssea (BMCs) de camundongos em modelo experimental para o desenvolvimento de aterosclerose: aspectos estruturais, ultraestrutuais e moleculares da aorta

As células tronco são caracterizadas pela sua capacidade de se diferenciar em várias linhagens de células e exibir um pontente efeito parácrino. O objetivo deste trabalho foi avaliar o efeito da terapia com células da medula óssea (BMCs) na glicose sanguínea, no metabolismo lipídico e remodelamento da parede da aorta em um modelo experimental para aterosclerose. Camundongos C57BL/6 foram alimentados com uma dieta controle (grupo CO) ou uma dieta aterogênica (grupo AT - 60% gordura). Após 16 semanas, o grupo AT foi dividido em quatro sub grupos: grupo AT 14 dias e o grupo AT 21 dias receberam uma injeção de PBS na veia caudal e mortos 14 e 21 dias após respectivamente; grupo AT-BMC 14 dias e AT-BMC 21 dias que receberam uma injeção com BMCs na veia caudal e mortos 14 e 21 dias após, respectivamente. O grupo CO foi sacrificado juntamente com outros grupos. O transplante BMCs reduziu os niveis de glicose, triglicerídeos e colesterol total no sangue. Não houve diferença significativa em relação à massa corporal entre os grupos transplantados e não transplantados, sendo todos diferentes do grupo CO. Não houve diferença significativa na curva glicemica entre os grupos AT 14 dias, AT-BMC 14 dias e AT 21 dias e estes diferentes do grupo CO e do grupo AT-BMC 21 dias. O Qa (1/mm2) foi quantitativamente reduzido no grupo AT 14 dias e AT 21 dias quando comparado ao grupo CO. Este Qa se mostrou elevado no grupo AT-BMC 21 dias quando comparado a todos os grupos. O aumento da expessura da parede da aorta foi observado em todos os grupos aterogênicos, entretanto o aumento da espessura foi significativamente menor no grupo AT-BMC 21 dias em relação ao grupo AT 14 dias e AT 21 dias. A percentagem de fibras elásticas se apresentou significativamente maior no grupo AT 21 dias quando comparado ao CO e AT-BMC 21 dias. Não houve diferença significativa entre o grupo CO e AT-BMC 21 dias. Vacúolos na túnica média, delaminação e o adelgaçamento das lamelas elásticas foram observados nos grupos AT-14 dias e AT-21 dias. O menor número destes foi visualizado no grupo AT-BMC 14 dias e AT-BMC 21 dias. A imunomarcação para alfa actina de músculo liso (α-SMA) e fator de crescimento vascular e endotelial (VEGF) mostrou menor marcação em grupos transplantados com BMCs. A marcação para antígeno nuclear de proliferação celular (PCNA) mostrou-se mais expressiva no grupo AT-BMC 21 dias grupo. Marcação para CD105, CD133 e CD68 foi observada nos grupos AT 14 dias e AT 21 dias. Estas marcações não foram observadas nos grupos AT-BMC 14 dias e AT-BMC 21 dias. Nas eletromicrografias observamos o remodelamento benéfico no grupo AT-BMC14 dias e AT-BMC 21 dias, com a organização estrutural similar ao grupo CO. Vesículas de pinocitose, projeção da célula muscular lisa e a delaminação da lamina elástica interna são observados nos grupos AT 14 dias e AT 21 dias. Célula endotelial preservada, com lamina elástica interna de contorno regular e contínua é observada no grupo CO e nos grupos AT-BMC 14 dias e AT-BMC 21 dias. Como conclusão, os nossos resultados reforçam o conceito de que, em um modelo aterosclerótico utilizando camundongos e dieta aterogênica, a injeção de BMCs melhora os níveis de glicose, metabolismo lipídico e ocasiona um remodelamento benéfico na parede da aorta.

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

The antioxidative effect of icariin in human erythrocytes against free-radical-induced haemolysis

Icariin (2-(4'-methoxyl phenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3'-methyl-2-butyleny"chromanone) is the major component in Herba Epimedii used in traditional Chinese medicine for the treatment of atherosclerosis. This work focuses on the antioxidative effect of icariin on freeradical-induced haemolysis of human erythrocytes, in which the initial free radical derives from the decomposition of 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) at physiological temperature. To reveal the structure-activity relationship of icariin, the antioxidant effects of two structural analogues of icariin, acacetin (2-(4'-methoxylphenyl)-5,7-dihydroxylchromone) and norwogonin (2-phenyl-5,7,8-trihydroxylchromone), on the same experimental system were examined as well. It was found that all these chromone derivatives (Chm-OHs) dose-dependently protected human erythrocytes against free-radical-induced haemolysis. The order of antioxidative activity was nonvogoni-n > acacetin > icariin by the analysis of the relationship between the concentration of Chm-OHs and the prolongation percentage of the lag time of haemolysis (PP%). It was also proved that the phenyl hydroxyl group attached to the chromone ring at 7-position cannot trap the free radical- On the contrary, phenyl hydroxyl groups at the 5- and 8-position in nonvogonin made it a significant antioxidant in AAPH-induced haemolysis.

A bioaugmentation failure caused by phage infection and weak biofilm formation ability

Two biological aerated filters (BAF) were setup for ammonia removal treatment of the circulation water in a marine aquaculture. One of the BAFs was bioaugmented with a heterotrophic nitrifying bacterium, Lutimonas sp. H10, where the ammonia removal was not improved and the massive inoculation was even followed by a nitrification breakdown from day 9 to 18. The nitrification was remained stable in control BAF operated under the same conditions. Fluorescent in situ hybridization (FISH) with rRNA-targeted probes and cultivable method revealed that Lutimonas sp. H10 almost disappeared from the bioaugomented BAF within 3 d, and this was mainly due to the infection of a specific phage as revealed by flask experiment, plaque assay and transmission electron observation. Analyses of 16S rRNA gene libraries showed that bacterial groups from two reactors evolved differently and an overgrowth of protozoa was observed in the bioaugmented BAR Therefore, phage infection and poor biofilm forming ability of the inoculated strain are the main reasons for bioaugmentation failure. In addition, gazing by protozoa of the bacteria might be the reason for the nitrification breakdown in bioaugmented BAF during day 9-18.

Workplace sedentary health risks and exercise interventions

Sedentary lifestyle and physical inactivity are causes of major health risks including cardiovascular disease (CVD), diabetes and cancer. Workplace is an ideal setting to understand both the prevalence of these risks and for devising and implementing effective intervention strategies. It is now possible to perform direct workplace assessments to identify the sedentary prevalence and assess the sedentary related health risks, which can include assessing the risks of atherosclerosis, hypertension, hypercholesterolemia, hyperglyaemia and reduced cardiorespiratory capacity. Based on evaluating the workplace health risks, it is possible to identify individuals who may be at higher CVD risk so they can be targeted with a risk-reduction intervention that can also be tailored towards improving healthy behaviours, especially towards physical activity activity and exercise. This chapter explains workplace sedentary risks, and provides examples of CVD risk prevalence, particularly within the university campus workplace, and presents examples of an exercise based targeted interventions aimed at reducing CVD risks amongst high risk sedentary employees.

Charakterystyka ekspresji receptorów scavenger w błonach komórkowych monocytów ludzkich u pacjentów z chorobą niedokrwienną serca

Wydział Biologii: Instytut Biologii Eksperymentalnej

Integracja szlaków sygnalizacyjnych interferonu gamma i receptora TLR4 w procesie zapalnym naczyń krwionośnych, warunkowana czynnikami transkrypcyjnymi STAT1 i IRF

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii Zakład Genetyki Molekularnej Człowieka

Periodontite e Aterosclerose

Dissertação apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Doença de Alzheimer e Periodontite

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

Inibidores PCSK9

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

Isolation, characterization, and functional assessment of novel smooth muscle like stem progenitors

Vascular smooth muscle cells (VSMC) are one of the key players in the pathogenesis of cardiovascular diseases. The origin of neointimal VSMC has thus become a prime focus of research. VSMC originate from multiple progenitors cell types. In embryo the well-defined sources of VSMC include; neural crest cells, proepicardial cells and EPC. In adults, though progenitor cells from bone marrow (BM), circulation and tissues giving rise to SMC have been identified, no progress has been made in terms of isolating highly proliferative clonal population of adult stem cells with potential to differentiate into SMC. Smooth muscle like stem progenitor cells (SMSPC) were isolated from cardiopulmonary bypass filters of adult patients undergoing CABG. Rat SMSPC have previously been isolated by our group from the bone marrow of Fischer rats and also from the peripheral blood of monocrotaline induced pulmonary hypertension (MCT-PHTN) animal model. Characterization of novel SMSPC exhibited stem cell characteristics and machinery for differentiation into SMC. The expression of Isl-1 on SMSPC provided unique molecular identity to these circulating stem progenitor cells. The functional potential of SMSPC was determined by monitoring adoptive transfer of GFP+ SMSPC in rodent models of vascular injury; carotid injury and MCT-PHTN. The participation of SMSPC in vascular pathology was confirmed by quantifying the peripheral blood, and engrafted levels of SMSPC using RT-PCR. In terms of translating into clinical practice, SMSPC could be a good tool for detecting the atherosclerotic plaque burden. The current study demonstrates the existence of novel adult stem progenitor cells in circulation, with the potential role in vascular pathology.

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia.

BACKGROUND: West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease. METHODS: Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis. RESULTS: Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease. CONCLUSIONS: Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.

The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.

Acoustic radiation force impulse imaging (ARFI) on an IVUS circular array.

Our long-term goal is the detection and characterization of vulnerable plaque in the coronary arteries of the heart using intravascular ultrasound (IVUS) catheters. Vulnerable plaque, characterized by a thin fibrous cap and a soft, lipid-rich necrotic core is a precursor to heart attack and stroke. Early detection of such plaques may potentially alter the course of treatment of the patient to prevent ischemic events. We have previously described the characterization of carotid plaques using external linear arrays operating at 9 MHz. In addition, we previously modified circular array IVUS catheters by short-circuiting several neighboring elements to produce fixed beamwidths for intravascular hyperthermia applications. In this paper, we modified Volcano Visions 8.2 French, 9 MHz catheters and Volcano Platinum 3.5 French, 20 MHz catheters by short-circuiting portions of the array for acoustic radiation force impulse imaging (ARFI) applications. The catheters had an effective transmit aperture size of 2 mm and 1.5 mm, respectively. The catheters were connected to a Verasonics scanner and driven with pushing pulses of 180 V p-p to acquire ARFI data from a soft gel phantom with a Young's modulus of 2.9 kPa. The dynamic response of the tissue-mimicking material demonstrates a typical ARFI motion of 1 to 2 microns as the gel phantom displaces away and recovers back to its normal position. The hardware modifications applied to our IVUS catheters mimic potential beamforming modifications that could be implemented on IVUS scanners. Our results demonstrate that the generation of radiation force from IVUS catheters and the development of intravascular ARFI may be feasible.