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1.
Proc Natl Acad Sci U S A ; 117(26): 14779-14789, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32561645

RESUMO

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.


Assuntos
Contagem de Células Sanguíneas/métodos , Microscopia Ultravioleta/métodos , Imagem Molecular/métodos , Contagem de Células Sanguíneas/instrumentação , Células Sanguíneas/classificação , Células Sanguíneas/citologia , Desenho de Equipamento , Humanos , Microscopia Ultravioleta/instrumentação , Imagem Molecular/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito
2.
IEEE J Biomed Health Inform ; 24(1): 160-170, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892256

RESUMO

Cell classification, especially that of white blood cells, plays a very important role in the field of diagnosis and control of major diseases. Compared to traditional optical microscopic imaging, hyperspectral imagery, combined with both spatial and spectral information, provides more wealthy information for recognizing cells. In this paper, a novel blood cell classification framework, which combines a modulated Gabor wavelet and deep convolutional neural network (CNN) kernels, named as MGCNN, is proposed based on medical hyperspectral imaging. For each convolutional layer, multi-scale and orientation Gabor operators are taken dot product with initial CNN kernels. The essence is to transform the convolutional kernels into the frequency domain to learn features. By combining characteristics of Gabor wavelets, the features learned by modulated kernels at different frequencies and orientations are more representative and discriminative. Experimental results demonstrate that the proposed model can achieve better classification performance than traditional CNNs and widely used support vector machine approaches, especially as training small-sample-size situations.


Assuntos
Células Sanguíneas/classificação , Células Sanguíneas/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Redes Neurais de Computação , Algoritmos , Técnicas Citológicas/métodos , Humanos , Análise de Ondaletas
3.
Nucleic Acids Res ; 47(1): e4, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30256981

RESUMO

Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Células Sanguíneas/classificação , Linhagem da Célula/genética , DNA/classificação , Humanos , Leucócitos Mononucleares/citologia , Análise de Célula Única/métodos
4.
Proc Natl Acad Sci U S A ; 115(32): E7568-E7577, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038005

RESUMO

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems.


Assuntos
Anopheles/genética , Células Sanguíneas/classificação , Plasticidade Celular/genética , Malária/transmissão , Mosquitos Vetores/genética , Animais , Animais Geneticamente Modificados , Anopheles/imunologia , Células Sanguíneas/imunologia , Comunicação Celular/genética , Conjuntos de Dados como Assunto , Feminino , Genômica/métodos , Mosquitos Vetores/imunologia , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma
5.
J Clin Lab Anal ; 32(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28220972

RESUMO

BACKGROUND: Morphological characteristics of blood cells are still qualitatively defined. So a texture analysis (Tx) method using gray level co-occurrence matrices (GLCMs; CM-Tx method) was applied to images of erythrocyte precursor cells (EPCs) for quantitatively distinguishing four types of EPC stages: proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. METHODS: Fifty-five images of four types of EPCs were downloaded from an atlas uploaded by the Blood Cell Morphology Standardization Subcommittee (BCMSS) of the Japanese Society of Laboratory Hematology (JSLH). Using in-house programs, two types of GLCMs-(R: d=1, θ=0°) and (U: d=1, θ=270°)-and nine types of texture distinction index (TDI) were calculated with images removed outer part of cell. RESULTS: Three binary decision trees were sequentially divided among four types of EPC with the sum average of GLCM (U), the contrast of GLCM (R), and the sum average of GLCM (U). The average concordance rate (sensitivity) of CM-Tx method with the judgments of eleven experts in the BCMSS of the JSLH was 95.8% (87.5-100.0), and the average specificity was 97.6% (92.5-100.0). CONCLUSIONS: The CM-Tx method is an effective tool for quantitative distinction of EPC with their morphological features.


Assuntos
Células Sanguíneas/citologia , Células da Medula Óssea/citologia , Técnicas Citológicas/métodos , Processamento de Imagem Assistida por Computador/métodos , Células Sanguíneas/classificação , Células da Medula Óssea/classificação , Humanos , Microscopia
6.
Bioinformatics ; 33(21): 3423-3430, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29036374

RESUMO

Motivation: Mass cytometry or CyTOF is an emerging technology for high-dimensional multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry. New methods for analyzing CyTOF data attempt to improve automation, scalability, performance and interpretation of data generated in large studies. Assigning individual cells into discrete groups of cell types (gating) involves time-consuming sequential manual steps, untenable for larger studies. Results: We introduce DeepCyTOF, a standardization approach for gating, based on deep learning techniques. DeepCyTOF requires labeled cells from only a single sample. It is based on domain adaptation principles and is a generalization of previous work that allows us to calibrate between a target distribution and a source distribution in an unsupervised manner. We show that DeepCyTOF is highly concordant (98%) with cell classification obtained by individual manual gating of each sample when applied to a collection of 16 biological replicates of primary immune blood cells, even when measured across several instruments. Further, DeepCyTOF achieves very high accuracy on the semi-automated gating challenge of the FlowCAP-I competition as well as two CyTOF datasets generated from primary immune blood cells: (i) 14 subjects with a history of infection with West Nile virus (WNV), (ii) 34 healthy subjects of different ages. We conclude that deep learning in general, and DeepCyTOF specifically, offers a powerful computational approach for semi-automated gating of CyTOF and flow cytometry data. Availability and implementation: Our codes and data are publicly available at https://github.com/KlugerLab/deepcytof.git. Contact: yuval.kluger@yale.edu. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Biologia Computacional/métodos , Citometria de Fluxo/normas , Aprendizado de Máquina , Análise de Célula Única/normas , Células Sanguíneas/classificação , Calibragem/normas , Separação Celular/normas , Humanos , Padrões de Referência , Reprodutibilidade dos Testes
7.
Sci Rep ; 7: 40942, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28106145

RESUMO

Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1- cDC (Lin-CD172+ CD1-CD4-) and CD1+ cDC (Lin-CD172a+ CD1+ CD4-), and characterised by phenotypic and functional analyses. CD1+ cDC were distinct from CD1- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1+ cDC produced IL-8 and IL-10 while CD1- cDC secreted IFN-α, IL-12 and TNF-α. CD1- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1- and CD1+ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.


Assuntos
Antígenos CD1/análise , Células Sanguíneas/química , Células Sanguíneas/imunologia , Células Dendríticas/química , Células Dendríticas/imunologia , Animais , Antígenos de Superfície/análise , Células Sanguíneas/classificação , Citocinas/metabolismo , Células Dendríticas/classificação , Citometria de Fluxo , Perfilação da Expressão Gênica , Suínos , Doenças dos Suínos
8.
Epigenetics ; 11(9): 690-698, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27494297

RESUMO

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.


Assuntos
Células Sanguíneas/citologia , Metilação de DNA , Sangue Fetal/citologia , Citometria de Fluxo/normas , Adulto , Células Sanguíneas/classificação , Células Sanguíneas/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Padrões de Referência
9.
J Anim Sci ; 93(3): 892-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26020867

RESUMO

The cost of feed is a serious issue in the pork industry, contributing about 65 to 75% of the total production cost. To prevent economic losses and decreased productivity of the herd, it is important to select for animals that eat less for the same lean gain, or more efficient animals. Residual feed intake (RFI) is the difference between observed feed intake and expected feed intake based on estimated maintenance and production requirements. Selection for decreased RFI, or more efficient animals, is a potential solution to higher feed costs in pig production. However, animals that are highly selected for decreased RFI may have reduced energy input to the immune system and fail to withstand diseases and stressors after infection that negatively impact profitability. The objective of this study was to evaluate differences in circulating blood cell profiles at a young age between 2 lines of Yorkshire pigs that were divergently selected for RFI as well as the heritability of these traits, to investigate effects of selection for RFI on immune system parameters, and to identify potential biomarkers for feed efficiency. Previous work has shown that the 2 lines had diverged for IGF-1 in serum in young pigs and, therefore, this stage was investigated for other potential physiological differences. Blood samples were drawn for a complete blood count (CBC) analysis from 517 gilts and barrows, ages 35 to 42 d, across the 2 lines. In general, the low-RFI line had lower numbers of specific types of white blood cells but higher hemoglobin concentration and red blood cell volume compared to the high-RFI line. No significant correlations were found between CBC traits and RFI across and within the lines (0.05 < < 0.1). Of the 15 CBC traits that were measured, 3 were highly heritable (0.56 < < 0.62), 9 were moderately heritable (0.12 < < 0.47), and 3 were lowly heritable ( < 0.12), suggesting a substantial genetic component for CBC traits and that selection for CBC traits could be effective. Our results also show that selection for RFI has significantly impacted the number of circulating blood cells. In this experiment, we studied only healthy animals that were not under known pathogen challenge; therefore, our results cannot be directly applied to a disease challenge situation. Future work will be to challenge the animals and determine the effect of challenge on CBC levels.


Assuntos
Ração Animal , Células Sanguíneas/citologia , Ingestão de Alimentos/genética , Seleção Genética/genética , Suínos/sangue , Suínos/genética , Envelhecimento/sangue , Ração Animal/economia , Criação de Animais Domésticos/economia , Criação de Animais Domésticos/métodos , Animais , Contagem de Células Sanguíneas , Células Sanguíneas/classificação , Células Sanguíneas/fisiologia , Ingestão de Alimentos/fisiologia , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Feminino , Sistema Imunitário/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Fenótipo , Suínos/fisiologia
10.
J Lab Autom ; 20(6): 670-5, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25925737

RESUMO

Differential counting of peripheral blood cells is an important diagnostic tool. However, manual morphological analysis using the microscope is time-consuming and requires highly trained personnel. The digital microscope is capable of performing an automated peripheral blood cell differential, which is as reliable as manual classification by experienced laboratory technicians. To date, information concerning the interlaboratory variation and quality of cell classification by independently operated digital microscopy systems is limited. We compared four independently operated digital microscope systems for their ability in classifying the five main peripheral blood cell classes and detection of blast cells in 200 randomly selected samples. Set against the averaged results, the R(2) values for neutrophils ranged between 0.90 and 0.96, for lymphocytes between 0.83 and 0.94, for monocytes between 0.77 and 0.82, for eosinophils between 0.70 and 0.78, and for blast cells between 0.94 and 0.99. The R(2) values for the basophils were between 0.28 and 0.34. This study shows that independently operated digital microscopy systems yield reproducible preclassification results when determining the percentages of neutrophils, eosinophils, lymphocytes, monocytes, and blast cells in a peripheral blood smear. Detection of basophils was hampered by the low incidence of this cell class in the samples.


Assuntos
Células Sanguíneas/classificação , Células Sanguíneas/citologia , Citometria por Imagem/métodos , Microscopia/métodos , Humanos , Citometria por Imagem/instrumentação , Processamento de Imagem Assistida por Computador , Microscopia/instrumentação , Reprodutibilidade dos Testes
11.
J Vet Med Sci ; 76(5): 693-704, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24476851

RESUMO

Peripheral blood cells from ayu, Plecoglossus altivelis altivelis, were separated using a density gradient. Blood cells were then smeared using Shandon Cytospin and subjected to cytochemical staining. Blood cells were categorized based on morphological and cytochemical characteristics, and the density fractionation range and nucleus area/cell area ratio were observed. Lymphocytes are distinguished from neutrophils by their basophilic cytoplasm and Golgi-like field. The features of chromatin in thrombocytes are different from those of lymphocytes or neutrophils, but some small neutrophils have similar chromatin. Therefore, it is necessary to perform peroxidase staining to distinguish small neutrophils from thrombocytes. Basophils have large basophilic granules in cytoplasm. Based on density fractionation of blood cells, thrombocytes in the low-density area were separated from other blood cells. Identification of peripheral blood cells from ayu was possible with these staining methods. Monocytes/macrophages from spleen are specifically positive for esterase staining by α-naphthyl butyrate. As a result, thrombocytes, lymphocytes, neutrophils, basophils and monocytes/macrophages were identified in smears from peripheral blood or spleen tissue. In this paper, we confirmed that the peripheral blood corpuscles of ayu are able to be identified using the present staining methods.


Assuntos
Células Sanguíneas/química , Células Sanguíneas/citologia , Osmeriformes/sangue , Animais , Aquicultura/métodos , Células Sanguíneas/classificação , Fracionamento Celular/veterinária , Cromatina/química , Técnicas Citológicas/veterinária , Processamento de Imagem Assistida por Computador , Japão , Coloração e Rotulagem/veterinária
12.
Pesqui. vet. bras ; 33(9): 1151-1154, set. 2013. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-694066

RESUMO

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.


O objetivo deste estudo foi isolar, cultivar e caracterizar as células mesenquimais multipotentes estromais derivadas do sangue periférico (SpCTMs) equino. O sangue periférico foi coletado, seguido do isolamento das células mononucleadas utilizando o reagente de gradiente de densidade e o cultivo das células aderentes. Os anticorpos monoclonais mouse anti-horse CD13, mouse anti-horse CD44 e mouse anti-rat CD90 foram utilizados para a caracterização imunofenotípica da superfície das SpCTMs. Estas células também foram cultivadas utilizando meio de cultura específico para a diferenciação adipogênica e condrogênica. Não houve expressão do marcador CD13, mas os marcadores CD44 e CD90 foram expressos em todas as passagens testadas. Após 14 dias da diferenciação das células em adipócitos, gotículas de lipídeos foram observados através da coloração com Oil Red O. Vinte e um dias após a diferenciação condrogênica, as células foram coradas com o Alcian Blue. Embora a técnica de isolamento destas células necessite ser otimizada, o presente estudo demonstra a caracterização parcial das SpCTMs, classificando-as como um tipo de células progenitoras promissoras para o uso na terapia celular em equinos.


Assuntos
Animais , Adulto , Cavalos/sangue , Células-Tronco Mesenquimais/citologia , Células Sanguíneas/classificação , Células-Tronco Multipotentes/fisiologia , Imunofenotipagem/veterinária
13.
Parasit Vectors ; 6: 93, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23578139

RESUMO

BACKGROUND: Two biological forms of the mosquito Culex pipiens s.s., denoted pipiens and molestus, display behavioural differences that may affect their role as vectors of arboviruses. In this study, the feeding patterns of molestus and pipiens forms were investigated in Comporta (Portugal), where high levels of inter-form admixture have been recorded. METHODS: Indoor and outdoor mosquito collections were performed in the summer of 2010. Collected Cx. pipiens s.l. females were molecularly identified to species and form by PCR and genotyped for six microsatellites. The source of the blood meal in post-fed females was determined by ELISA and mitochondrial DNA sequencing. RESULTS: The distribution of the forms differed according to the collection method. The molestus form was present only in indoor collections, whereas pipiens and admixed individuals were sampled both indoors and outdoors. In both forms, over 90% of blood meals were made on avian hosts. These included blood meals taken from Passeriformes (Passer domesticus and Turdus merula) by females caught resting inside domestic shelters. CONCLUSION: Genetic structure and blood meal analyses suggest the presence of a bird biting molestus population in the study area. Both forms were found to rest indoors, mainly in avian shelters, but at least a proportion of females of the pipiens form may bite outdoors in sylvan habitats and then search for anthropogenic resting sites to complete their gonotrophic cycle. This behaviour may potentiate the accidental transmission of arboviruses to humans in the region.


Assuntos
Células Sanguíneas/classificação , Culex/fisiologia , Insetos Vetores , Animais , Aves/parasitologia , Culex/classificação , Culex/genética , Comportamento Alimentar , Feminino , Genótipo , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Portugal
14.
Infect Genet Evol ; 16: 122-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23352890

RESUMO

Blood meal analysis (BMA) is a useful tool for epidemiologists and vector ecologists to assess which vector species are critical to disease transmission. In most current BMA assays vertebrate primers amplify DNA from a blood meal, commonly an abundant mitochondrial (mtDNA) locus, which is then sequenced and compared to known sequences in GenBank to identify its source. This technique, however, is time consuming and costly as each individual sample must be sequenced for species identification and mixed blood meals cloned prior to sequencing. Further, we found that several standard BMA vertebrate primers match sequences of the mtDNA of the Asian tiger mosquito, Aedes albopictus, making their use for blood meal identification in this species impossible. Because of the importance of Ae. albopictus as a vector of dengue and chikungunya viruses to humans, we designed a rapid assay that allows easy identification of human blood meals as well as mixed meals between human and nonhuman mammals. The assay consists of a nested PCR targeting the cytochrome b (cytb) mtDNA locus with a blocking primer in the internal PCR. The blocking primer has a 3' inverted dT modification that when used with the Stoffel Taq fragment prevents amplification of nuclear cytochrome b pseudogenes in humans and allows for the continued use of cytb in BMA studies, as it is one of the most species-rich loci in GenBank. We used our assay to examine 164 blooded specimens of Ae. albopictus from suburban coastal New Jersey and found 62% had obtained blood from humans with 7.6% mixes between human and another mammal species. We also confirmed the efficiency of our assay by comparing it with standard BMA primers on a subset of 62 blooded Ae. albopictus. While this assay was designed for use in Ae. albopictus, it will have broader application in other anthropophilic mosquitoes.


Assuntos
Aedes/fisiologia , Células Sanguíneas/química , DNA/classificação , DNA/isolamento & purificação , Conteúdo Gastrointestinal/química , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Células Sanguíneas/classificação , Gatos , DNA/análise , DNA/química , Cães , Comportamento Alimentar , Humanos , Dados de Sequência Molecular
15.
Clin Implant Dent Relat Res ; 15(2): 262-70, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21834861

RESUMO

BACKGROUND: The relationship between the immune response and red and white blood cell homeostasis is cited in literature, but no studies regarding the balance of these cell populations following maxillary bone-graft surgeries can be found. AIM: The aim of this study was to evaluate the possible impairments in the blood cell balance following fresh-frozen allogeneic bone-graft augmentation procedures in patients who needed maxillary reconstruction prior to implants. MATERIAL AND METHODS: From 33 patients elected to onlay bone grafting procedures, 20 were treated with fresh-frozen bone allografts and 13 with autologous bone grafts. Five blood samples were collected from each patient in a 6-month period (baseline: 14, 30, 90, and 180 days postsurgery), and the hematological parameters (erythrogram, leukogram, and platelets count) were accessed. RESULTS: All evaluated parameters were within the reference values accepted as normal, and significant differences were found for the eosinophils count when comparing the treatments (30 days, p = .035) and when comparing different periods of evaluation (allograft-treated group, baseline × 180 days, p ≤ .05 and 90 × 180 days, p ≤ .01; autograft-treated group, 30 × 90 days, p ≤ .05 and 30 × 180 days, p ≤ .05). CONCLUSIONS: Both autologous and fresh-frozen allogeneic bone grafts did not cause any impairment in the red and white blood cell balance, based on quantitative hemogram analysis, in patients subjected to maxillary reconstruction.


Assuntos
Aloenxertos/transplante , Aumento do Rebordo Alveolar/métodos , Contagem de Células Sanguíneas/classificação , Células Sanguíneas/classificação , Transplante Ósseo/métodos , Maxila/cirurgia , Adulto , Idoso , Autoenxertos/transplante , Criopreservação/métodos , Eosinófilos/patologia , Contagem de Eritrócitos , Índices de Eritrócitos , Feminino , Seguimentos , Hematócrito , Hemoglobinas/análise , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Contagem de Plaquetas
16.
J Neuroinflammation ; 9: 215, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22978757

RESUMO

BACKGROUND: Numerous cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS), but studies are often limited to whole blood (WB) or peripheral blood mononuclear cells (PBMCs), thereby omitting important information about the cellular origin of the cytokines. Knowledge about the relation between blood and cerebrospinal fluid (CSF) cell expression of cytokines and the cellular source of CSF cytokines is even more scarce. METHODS: We studied gene expression of a broad panel of cytokines in WB from relapsing-remitting multiple sclerosis (RRMS) patients in remission and healthy controls (HCs). Subsequently we determined the gene expression of the dysregulated cytokines in isolated PBMC subsets (CD4+, CD8+T-cells, NK-cells, B-cells, monocytes and dendritic cells) from RRMS patients and HCs and in CSF-cells from RRMS patients in clinical relapse and non-inflammatory neurological controls (NIND). RESULTS: RRMS patients had increased expression of IFN-gamma (IFNG), interleukin (IL) 1-beta (IL1B), IL7, IL10, IL12A, IL15, IL23, IL27, lymphotoxin-alpha (LTA) and lymphotoxin-beta (LTB) in WB. In PBMC subsets the main sources of pro-inflammatory cytokines were T- and B-cells, whereas monocytes were the most prominent source of immunoregulatory cytokines. In CSF-cells, RRMS patients had increased expression of IFNG and CD19 and decreased expression of IL10 and CD14 compared to NINDs. CD19 expression correlated with expression of IFNG, IL7, IL12A, IL15 and LTA whereas CD14 expression correlated with IL10 expression. CONCLUSIONS: Using a systematic approach, we show that expression of pro-inflammatory cytokines in peripheral blood primarily originates from T- and B-cells, with an important exception of IFNG which is most strongly expressed by NK-cells. In CSF-cell studies, B-cells appear to be enriched in RRMS and associated with expression of pro-inflammatory cytokines; contrarily, monocytes are relatively scarce in CSF from RRMS patients and are associated with IL10 expression. Thus, our findings suggest a pathogenetic role of B-cells and an immunoregulatory role of monocytes in RRMS.


Assuntos
Células Sanguíneas/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Esclerose Múltipla Recidivante-Remitente/metabolismo , Esclerose Múltipla Recidivante-Remitente/patologia , Adulto , Células Sanguíneas/classificação , Citocinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo
18.
Blood ; 119(15): 3431-9, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22374695

RESUMO

Delayed recovery of mature blood cells poses a serious, expensive, and often life-threatening problem for many stem cell transplantation recipients, particularly if heavily pretreated and serving as their own donor, or having a CB transplantation as the only therapeutic option. Importantly, the different cells required to ensure a rapid, as well as a permanent, hematopoietic recovery in these patients remain poorly defined. We now show that human CB and mobilized peripheral blood (mPB) collections contain cells that produce platelets and neutrophils within 3 weeks after being transplanted into sublethally irradiated NOD/scid-IL-2Rγc-null mice. The cells responsible for these 2 outputs are similarly distributed between the aldehyde dehydrogenase-positive and -negative subsets of lineage marker-negative CB and mPB cells, but their overall frequencies vary independently in individual samples. In addition, their total numbers can be seen to be much (> 30-fold) lower in a single "average" CB transplantation compared with a single "average" mPB transplantation (normalized for a similar weight of the recipient), consistent with the published differential performance in adult patients of these 2 transplantation products. Experimental testing confirmed the clinical relevance of the surrogate xenotransplantation assay for quantifying cells with rapid platelet regenerative activity, underscoring its potential for future applications.


Assuntos
Células Sanguíneas/citologia , Células Sanguíneas/fisiologia , Plaquetas/fisiologia , Transplante de Células-Tronco Hematopoéticas , Neutrófilos/fisiologia , Adulto , Animais , Células Sanguíneas/classificação , Humanos , Recém-Nascido , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Fenótipo , Contagem de Plaquetas , Transplante Heterólogo
19.
Blood ; 118(19): e149-55, 2011 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21931111

RESUMO

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.


Assuntos
Células Sanguíneas/citologia , Células Sanguíneas/imunologia , Separação Celular/métodos , Quimerismo , Citometria de Fluxo/métodos , Antígenos HLA/sangue , Alelos , Anticorpos Monoclonais , Células Sanguíneas/classificação , Sobrevivência Celular , Cromossomos Humanos Y/genética , Cromossomos Humanos Y/imunologia , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Antígenos HLA/genética , Humanos , Masculino , Reação em Cadeia da Polimerase , Gravidez
20.
BMC Bioinformatics ; 12: 6, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21208450

RESUMO

BACKGROUND: The Cell Ontology (CL) is an ontology for the representation of in vivo cell types. As biological ontologies such as the CL grow in complexity, they become increasingly difficult to use and maintain. By making the information in the ontology computable, we can use automated reasoners to detect errors and assist with classification. Here we report on the generation of computable definitions for the hematopoietic cell types in the CL. RESULTS: Computable definitions for over 340 CL classes have been created using a genus-differentia approach. These define cell types according to multiple axes of classification such as the protein complexes found on the surface of a cell type, the biological processes participated in by a cell type, or the phenotypic characteristics associated with a cell type. We employed automated reasoners to verify the ontology and to reveal mistakes in manual curation. The implementation of this process exposed areas in the ontology where new cell type classes were needed to accommodate species-specific expression of cellular markers. Our use of reasoners also inferred new relationships within the CL, and between the CL and the contributing ontologies. This restructured ontology can be used to identify immune cells by flow cytometry, supports sophisticated biological queries involving cells, and helps generate new hypotheses about cell function based on similarities to other cell types. CONCLUSION: Use of computable definitions enhances the development of the CL and supports the interoperability of OBO ontologies.


Assuntos
Células Sanguíneas/classificação , Biologia Computacional/métodos , Bases de Dados Factuais , Armazenamento e Recuperação da Informação/métodos , Vocabulário Controlado
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