Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

Differential Distribution of Dynamin Isoforms in Mammalian Cells

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in Myoclonus Epilepsy and Ragged-Red Fibers (MERRF) syndrome by a multiplex Molecular Beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15+ myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.

Estimativa de dose nos pulmões para procedimentos de tomografia computadorizada

Desde o seu desenvolvimento na década de 1970 a tomografia computadorizada (TC) passou por grandes mudanças tecnológicas, tornando-se uma importante ferramenta diagnóstica para a medicina. Consequentemente o papel da TC em diagnóstico por imagem expandiu-se rapidamente, principalmente devido a melhorias na qualidade da imagem e tempo de aquisição. A dose de radiação recebida por pacientes devido a tais procedimentos vem ganhando atenção, levando a comunidade científica e os fabricantes a trabalharem juntos em direção a determinação e otimização de doses. Nas últimas décadas muitas metodologias para dosimetria em pacientes têm sido propostas, baseadas especialmente em cálculos utilizando a técnica Monte Carlo ou medições experimentais com objetos simuladores e dosímetros. A possibilidade de medições in vivo também está sendo investigada. Atualmente as principais técnicas para a otimização da dose incluem redução e/ou modulação da corrente anódica. O presente trabalho propõe uma metodologia experimental para estimativa de doses absorvidas pelos pulmões devido a protocolos clínicos de TC, usando um objeto simulador antropomórfico adulto e dosímetros termoluminescentes de Fluoreto de Lítio (LiF). Sete protocolos clínicos diferentes foram selecionados, com base em sua relevância com respeito à otimização de dose e frequência na rotina clínica de dois hospitais de grande porte: Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (InRad) e Instituto do Câncer do Estado de São Paulo Octávio Frias de Oliveira (ICESP). Quatro protocolos de otimização de dose foram analisados: Auto mA, Auto + Smart mA, Baixa Dose (BD) e Ultra Baixa Dose (UBD). Os dois primeiros protocolos supracitados buscam redução de dose por meio de modulação da corrente anódica, enquanto os protocolos BD e UBD propõem a redução do valor da corrente anódica, mantendo-a constante. Os protocolos BD e UBD proporcionaram redução de dose de 72,7(8) % e 91(1) %, respectivamente; 16,8(1,3) % e 35,0(1,2) % de redução de dose foram obtidas com os protocolos Auto mA e Auto + Smart mA, respectivamente. As estimativas de dose para os protocolos analisados neste estudo são compatíveis com estudos similares publicados na literatura, demonstrando a eficiência da metodologia para o cálculo de doses absorvidas no pulmão. Sua aplicabilidade pode ser estendida a diferentes órgãos, diferentes protocolos de CT e diferentes tipos de objetos simuladores antropomórficos (pediátricos, por exemplo). Por fim, a comparação entre os valores de doses estimadas para os pulmões e valores de estimativas de doses dependentes do tamanho (Size Specific Dose Estimates SSDE) demonstrou dependência linear entre as duas grandezas. Resultados de estudos similares exibiram comportamentos similares para doses no reto, sugerindo que doses absorvidas pelos uma órgãos podem ser linearmente dependente dos valores de SSDE, com coeficientes lineares específicos para cada órgão. Uma investigação mais aprofundada sobre doses em órgãos é necessária para avaliar essa hipótese.

Validation of the Spanish version of mackey childbirth satisfaction rating scale

Background: The “Mackey Childbirth Satisfaction Rating Scale” (MCSRS) is a complete non-validated scale which includes the most important factors associated with maternal satisfaction. Our primary purpose was to describe the internal structure of the scale and validate the reliability and validity of concept of its Spanish version MCSRS-E. Methods: The MCSRS was translated into Spanish, back-translated and adapted to the Spanish population. It was then administered following a pilot test with women who met the study participant requirements. The scale structure was obtained by performing an exploratory factorial analysis using a sample of 304 women. The structures obtained were tested by conducting a confirmatory factorial analysis using a sample of 159 women. To test the validity of concept, the structure factors were correlated with expectations prior to childbirth experiences. McDonald’s omegas were calculated for each model to establish the reliability of each factor. The study was carried out at four University Hospitals; Alicante, Elche, Torrevieja and Vinalopo Salud of Elche. The inclusion criteria were women aged 18–45 years old who had just delivered a singleton live baby at 38–42 weeks through vaginal delivery. Women who had difficulty speaking and understanding Spanish were excluded. Results: The process generated 5 different possible internal structures in a nested model more consistent with the theory than other internal structures of the MCSRS applied hitherto. All of them had good levels of validation and reliability. Conclusions: This nested model to explain internal structure of MCSRS-E can accommodate different clinical practice scenarios better than the other structures applied to date, and it is a flexible tool which can be used to identify the aspects that should be changed to improve maternal satisfaction and hence maternal health.

Moya moya disease : literature review and case report

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Amaurose e alterações neuropsiquiátricas como apresentação de neurossífilis

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Human naïve regulatory T-cells feature high steady-state turnover and are maintained by IL-7

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.

Inflamação na aterosclerose : impacto na doença coronária

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Surdez súbita : a marcha diagnóstica

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Perfil de risco cardiovascular nos pais de crianças dos 3 aos 6 anos

A doença cardiovascular constitui a causa de morte mais relevante em toda a Europa, incluindo Portugal, e é atualmente considerada como uma junção de doença arterial coronária nas suas diversas apresentações clínicas, eventos cerebrovasculares, doença arterial periférica e insuficiência cardíaca. De modo a contribuir para o estudo da importância de uma intervenção baseada numa estratégia populacional integrada na promoção de estilos de vida saudáveis, a principal finalidade deste estudo consistiu em definir o perfil de risco cardiovascular tendo por base os dados de prevalência de alguns fatores de risco, numa amostra de adultos (792 de ambos os sexos), em várias regiões de Portugal Continental. Para tal foram: caraterizados os hábitos alimentares, o contexto sociodemográfico dos adultos; analisadas as correlações entre o peso, perímetro da cintura, índice de massa corporal e a pressão arterial para ambos os sexos. Os resultados revelaram uma prevalência do excesso de peso, de obesidade e das respetivas caraterísticas, como o aumento do perímetro abdominal, e do IMC, sugerindo um contínuo de risco de doença cardiovascular. Verificou-se também uma elevada prevalência de hipertensão nos sujeitos com excesso de peso, sugerindo a existência de um risco cardiovascular acrescido. Os resultados obtidos neste estudo sustentam a necessidade de serem desenvolvidos planos de intervenção que contribuam para a redução do risco cardiovascular nos adultos. Palavras-chave: Estilos de vida; Hipertensão arterial; Índice de Massa Corporal; Obesidade; Risco Cardiovascular.

Approaches and Strategy for Cancer Research and Surveillance Data: Integration, Information Pipeline, Data Models, and Informatics Opportunities

Thesis (Ph.D.)--University of Washington, 2016-04