Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the α1 domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/−1.7 vs. 54.7 +/−3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.

Variabilidade intratípica de HPV 16 em relação à origem étnica e HLA de uma população de alto risco para o câncer do colo do útero

A infecção por papilomavirus é a principal causa de desenvolvimento de neoplasias intraepiteliais cervicais (NIC) e câncer do colo do útero (CCU). Estudos epidemiológicos têm demonstrado que a persistência do genoma viral encontra-se associado a variantes moleculares específicas de papilomavirus humano (HPV) de alto risco. As moléculas HLA de classe II têm um importante papel na resposta imune. Associações entre HLA e CCU ou infecção por HPV tem sido demonstrado em diferentes populações. O nosso objetivo foi verificar se a variabilidade de HLA-DRB1 e DQB1 estavam associada ao CCU e NIC III em mulheres de Belém, uma população formada pelos 3 principais grupos étnicos humanos e uma área de alto risco para o CCU no Norte do Brasil. Foi investigada a existência de diferenças na distribuição de alelos HLA entre mulheres com CCU e NIC III portadoras de diferentes variantes de HPV-16 e mulheres citologicamente normais. Os genes HLA DQB1 e DRB1 foram tipados pelo método de PCR-SSO em 95 casos e 287 controles de mulheres com citologia normal atendidas em um centro de prevenção do colo do útero na mesma cidade. As variantes de HPV-16 foram tipadas por sequenciamento de um fragmento da região controladora do genoma viral (LCR). O polimorfismo na posição 350 do gene E6 foi tipado baseado em um protocolo de hibridização em pontos, para identificar a alteração na posição 350T→G. A magnitude das associações foi estimada por odds ratio (OR) e os respectivos intervalos de confiança (IC), ajustados para potenciais fatores de confusão. Uma associação positiva foi observada entre CCU e os haplótipos DRB1* 150 l-DQB1*0602, DRB1*04-DQB1*0301 e DRB1*1602-DQB1*0301. Ao contrário, DRB1*01-DQB1*0501 mostrou um efeito protetor. Os alelos DRB1*0804, DQB1*0402 apresentaram efeito protetor contra positividade por HPV. O alelo DQB1*0502 e o grupo DRB1*15 foram positivamente associados. Os nossos resultados mostram que as associações positivas de DRB1*1501 e DRB1*1602 podem ser atribuídas a variantes asiático-americanas quando comparado a variantes européias. O risco conferido a DRB1*1501 foi encontrado associado tanto a variantes E6350G quanto a variantes E6350T, entretanto, o maior efeito foi devido às variantes E6250T. A associação positiva de DRB1*1602 foi significativa somente no grupo de mulheres positivas para E6350G. Estes resultados estão de acordo com a composição étnica da população estudada bem como um maior potencial oncogênico de certas variantes. Nossos dados sugerem que a contribuição dos alelos HLA na susceptibilidade genética ao CCU difere de acordo com a distribuição das variantes de HPV em uma dada região geográfica ou grupo étnico.

Doença celíaca num doente com síndrome poliglandular autoimune tipo III : caso clínico

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

Potent T cell response to a class I-binding 13-mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer ((56)LPOGQLTAy(64)), 11-mer ((54)EPLPQGQLTAy(64)), and 13-mer ((52)LPEPLPQGQLTAY(64)) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HILA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156 Leucine; B*3508, 156 Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Predictive vaccinology: Optimisation of predictions using support vector machine classifiers

Promiscuous human leukocyte antigen (HLA) binding peptides are ideal targets for vaccine development. Existing computational models for prediction of promiscuous peptides used hidden Markov models and artificial neural networks as prediction algorithms. We report a system based on support vector machines that outperforms previously published methods. Preliminary testing showed that it can predict peptides binding to HLA-A2 and -A3 super-type molecules with excellent accuracy, even for molecules where no binding data are currently available.

Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis

Background - Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods - The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results - Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion - Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

The classification of HLA supertypes by GRID/CPCA and hierarchical clustering methods

Biological experiments often produce enormous amount of data, which are usually analyzed by data clustering. Cluster analysis refers to statistical methods that are used to assign data with similar properties into several smaller, more meaningful groups. Two commonly used clustering techniques are introduced in the following section: principal component analysis (PCA) and hierarchical clustering. PCA calculates the variance between variables and groups them into a few uncorrelated groups or principal components (PCs) that are orthogonal to each other. Hierarchical clustering is carried out by separating data into many clusters and merging similar clusters together. Here, we use an example of human leukocyte antigen (HLA) supertype classification to demonstrate the usage of the two methods. Two programs, Generating Optimal Linear Partial Least Square Estimations (GOLPE) and Sybyl, are used for PCA and hierarchical clustering, respectively. However, the reader should bear in mind that the methods have been incorporated into other software as well, such as SIMCA, statistiXL, and R.

Overview of risk factors for age-related macular degeneration

Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make life style choices that may reduce the risk of disease. This review discusses the role of genetics, sunlight, diet, cardiovascular factors, smoking, and alcohol as possible risk factors for AMD. Genetics plays a more significant role in AMD than previously thought, especially in younger patients, histocompatibility locus antigen (HLA) and complement system genes being the most significant. Whether the risk of AMD is increased by exposure to sunlight, cardiovascular risk factors, and diet is more controversial. Smoking is the risk factor most consistently associated with AMD. Current smokers are exposed to a two to three times higher risk of AMD than non-smokers and the risk increases with intensity of smoking. Moderate alcohol consumption is unlikely to increase the risk of AMD. Optometrists as front-line informers and educators of ocular health play a significant role in increasing public awareness of the risks of AMD. Cessation of smoking, the use of eye protection in high light conditions, dietary changes, and regular use of dietary supplements should all be considered to reduce the lifetime risk of AMD.

Overview of risk factors for age-related macular degeneration (AMD)

Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make life style choices that may reduce the risk of disease. This review discusses the role of genetics, sunlight, diet, cardiovascular factors, smoking, and alcohol as possible risk factors for AMD. Genetics plays a more significant role in AMD than previously thought, especially in younger patients, histocompatibility locus antigen (HLA) and complement system genes being the most significant. Whether the risk of AMD is increased by exposure to sunlight, cardiovascular risk factors, and diet is more controversial. Smoking is the risk factor most consistently associated with AMD. Current smokers are exposed to a two to three times higher risk of AMD than non-smokers and the risk increases with intensity of smoking. Moderate alcohol consumption is unlikely to increase the risk of AMD. Optometrists as front-line informers and educators of ocular health play a significant role in increasing public awareness of the risks of AMD. Cessation of smoking, the use of eye protection in high light conditions, dietary changes, and regular use of dietary supplements should all be considered to reduce the lifetime risk of AMD.

Overview of risk factors for age-related macular degeneration (AMD)

Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make life style choices that may reduce the risk of disease. This review discusses the role of genetics, sunlight, diet, cardiovascular factors, smoking, and alcohol as possible risk factors for AMD. Genetics plays a more significant role in AMD than previously thought, especially in younger patients, histocompatibility locus antigen (HLA) and complement system genes being the most significant. Whether the risk of AMD is increased by exposure to sunlight, cardiovascular risk factors, and diet is more controversial. Smoking is the risk factor most consistently associated with AMD. Current smokers are exposed to a two to three times higher risk of AMD than non-smokers and the risk increases with intensity of smoking. Moderate alcohol consumption is unlikely to increase the risk of AMD. Optometrists as front-line informers and educators of ocular health play a significant role in increasing public awareness of the risks of AMD. Cessation of smoking, the use of eye protection in high light conditions, dietary changes, and regular use of dietary supplements should all be considered to reduce the lifetime risk of AMD.

Forward-in-Time, Spatially Explicit Modeling Software to Simulate Genetic Lineages Under Selection

SELECTOR is a software package for studying the evolution of multiallelic genes under balancing or positive selection while simulating complex evolutionary scenarios that integrate demographic growth and migration in a spatially explicit population framework. Parameters can be varied both in space and time to account for geographical, environmental, and cultural heterogeneity. SELECTOR can be used within an approximate Bayesian computation estimation framework. We first describe the principles of SELECTOR and validate the algorithms by comparing its outputs for simple models with theoretical expectations. Then, we show how it can be used to investigate genetic differentiation of loci under balancing selection in interconnected demes with spatially heterogeneous gene flow. We identify situations in which balancing selection reduces genetic differentiation between population groups compared with neutrality and explain conflicting outcomes observed for human leukocyte antigen loci. These results and three previously published applications demonstrate that SELECTOR is efficient and robust for building insight into human settlement history and evolution.

Doença enxerto-versus hospedeiro: determinação da expressão de genes e alterações de vias associadas ao sistema imunológico usando metodos da biofísica

O transplante de medula óssea (TMO) é um procedimento terapêutico importante em casos relacionados à pacientes com leucemia ou linfoma. Em decorrência desse processo, uma reação conhecida como doença enxerto-versus-hospedeiro (GVHD) pode ocorrer em pacientes susceptíveis como conseqüência da presença de células imunocompetentes do doador. Entretanto, não existe um modelo para descrever completamente as ações relacionadas ao mecanismo imunológico da GVHD desde a fase que inicializa a doença até a fase efetora. O Objetivo geral deste estudo é a investigação da resposta imunológica considerando-se o sistema HLA (antígenos leucocitários humano) em pacientes que desenvolveram a GVHD em decorrência do TMO. O National Cancer Institute (NCI) – Pathway interaction Database e Reactome foram usados como bases de dados com o objetivo de se estudar a expressão de genes e vias relacionados às Classes I e II do sistema HLA (antígenos leucocitários humano). O estudo considerou a mudança de expressão de genes relacionados às 17 vias do sistema imunológico com potencialidade para se expressar em pacientes que desenvolveram a GVHD associada à TMO. Dados referentes aos transcriptomas foram obtidos utilizando-se a plataforma GPL570 Affymetrix Genoma Humano U133 Plus. A atividade relativa foi usada para determinar as alterações das vias em amostras de GVHD em relação ao controle. As análises foram realizadas utilizando-se o software Via Complex e Bioconductor. Observou-se aumento significativo da expressão de genes ralacionados às vias do sistema imune adaptativo, antígenos associados às Classe I e II do HLA, fosforilação de CD3 e CD247, sinalização dos receptores de células T em CD4+ nativas e ativação de NF-kapa β nas células B. Também observou-se alterações significativas na mudança de expressão dos genes associados às vias relacionadas à super família de moléculas B7:CD28\CTLA-4 quando comparadas ao controle. Isso pode indicar a necessidade de geração de um segundo sinal co-estimulador em GVHD, acionado pelas moléculas dessa super família. O aumento da expressão do gene CD69 nas amostras experimentais caracteriza a ativação celular e, portanto, a sinalização de estímulos em GVHD. Os achados obtidos neste estudo contribuem para melhor elucidar o mecanismo imunopatogênico associado à GVHD. P