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2.
Cell Rep Methods ; 1(7): 100104, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-35475000

RESUMO

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology.


Assuntos
Formaldeído , Humanos , Fixação de Tecidos/métodos , Inclusão em Parafina/métodos
3.
Mol Ecol Resour ; 20(4): 950-960, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32384204

RESUMO

Despite the importance of the geographical arrangement of populations for the inference of species boundaries, only a few approaches that integrate spatial information into species delimitation have thus far been developed. Persistent differentiation of sympatric groups of individuals is the best criterion for species status. Species delimitation becomes more prone to error if allopatric metapopulations are considered because it is often difficult to assess whether observed differences between allopatric metapopulations would be sufficient to prevent the fusion of these metapopulations upon contact. We propose a novel approach for testing the hypothesis that the multilocus genetic distances between individuals or populations belonging to two different candidate species are not larger than expected based on their geographical distances and the relationship of genetic and geographical distances within the candidate species. A rejection of this null hypothesis is an argument for classifying the two studied candidate species as distinct species. Case studies show that the proposed tests are suitable to distinguish between intra- and interspecific differentiation. The regression approach proposed here is more appropriate for testing species hypotheses with regard to isolation by distance than (partial) Mantel tests. Our tests assume a linear relationship between genetic and (transformed) geographical distances. This assumption can be compromised by a high genetic variability within populations as found in a case study with microsatellite markers.


Assuntos
Repetições de Microssatélites/genética , Plantas/genética , Variação Genética/genética , Geografia , Modelos Genéticos , Filogenia
4.
Dev Biol ; 447(2): 182-199, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30590018

RESUMO

The four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri show more divergence at the genomic level than humans compared to mice (Stein et al., 2003; Cutter et al., 2006, 2008). However, the behavior and anatomy of these nematodes are very similar. We present a detailed analysis of the embryonic development of these species using 4D-microscopic analyses of embryos including lineage analysis, terminal differentiation patterns and bioinformatical quantifications of cell behavior. Further functional experiments support the notion that the early development of all four species depends on identical induction patterns. Based on our results, the embryonic development of all four Caenorhabditis species are nearly identical, suggesting that an apparently optimal program to construct the body plan of nematodes has been conserved for at least 20 million years. This contrasts the levels of divergence between the genomes and the protein orthologs of the Caenorhabditis species, which is comparable to the level of divergence between mouse and human. This indicates an intricate relationship between the structure of genomes and the morphology of animals.


Assuntos
Caenorhabditis , Desenvolvimento Embrionário/fisiologia , Evolução Molecular , Genoma Helmíntico , Filogenia , Animais , Caenorhabditis/embriologia , Caenorhabditis/genética , Humanos , Camundongos , Especificidade da Espécie
5.
Commun Integr Biol ; 11(3): 1-7, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30214676

RESUMO

Various neurotransmitters influence neuronal migration in the developing zebrafish hindbrain. Migrating tegmental hindbrain nuclei neurons (THNs) are governed by depolarizing neurotransmitters (acetylcholine and glutamate), and glycine. In mature neurons, glycine binds to its receptor to hyperpolarize cells. This effect depends on the co-expression of the solute carrier KCC2. Immature precursors, however, typically express NKCC1 instead of KCC2, leading to membrane depolarization upon glycine receptor activation. As neuronal migration occurs in neurons after leaving the cell cycle and before terminal differentiation, we hypothesized that the switch from NKCC1 to KCC2 expression could alter the effect of glycine on THN migration. We tested this notion using in vivo cell tracking, overexpression of glycine receptor mutations and whole mount in situ hybridization. We summarize our findings in a speculative model, combining developmental age, glycine receptor strength and solute carrier expression to describe the effect of glycine on the migration of THNs.

6.
Cytometry A ; 93(9): 889-893, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30211969

RESUMO

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA-DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture-expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell. © 2018 International Society for Advancement of Cytometry.


Assuntos
5'-Nucleotidase/metabolismo , Células da Medula Óssea/citologia , Medula Óssea/fisiologia , Endoglina/metabolismo , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Antígenos Thy-1/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Biomarcadores/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Placenta/metabolismo , Gravidez
7.
J Neuroinflammation ; 15(1): 160, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29801453

RESUMO

BACKGROUND: The gold standard in cerebrospinal fluid (CSF) cell immunophenotyping is flow cytometry. Nevertheless, the small amount of CSF cells and the invasive character of lumbar puncture limit the spectrum of possible investigation. Chipcytometry, a modified approach to slide-based cytometry, might be a useful tool for CSF analysis due to the possibility of iterative staining, imaging, and bleaching cycles. The aim of this study was to compare flow cytometric leukocyte subset analysis with Chipcytometry comparing the percentage distribution of distinct cell populations and the T-cell CD4:CD8 ratio. Moreover, this study investigated the interpretability of chips loaded with CSF cells and examined the applicability of Chipcytometry in clinical practice. METHODS: 375 CSF samples from 364 patients were analyzed by Chipcytometry using an automated upright microscope. Cell surface molecules were stained using fluorescence-labeled monoclonal antibodies. For cross-validation experiments, flow cytometry data of six patients were analyzed and matched with Chipcytometry data. RESULTS: Our experiments showed a better agreement examined by Bland-Altman analysis for samples with CSF pleocytosis than for normocellular CSF samples. Data were more consistent for B cells and CD4:CD8 ratio than for T cells and monocytes. Advantages of Chipcytometry compared to flow cytometry are that cells once fixated can be analyzed for up to 20 months with additional markers at any time. The clinical application of Chipcytometry is demonstrated by two illustrative case reports. However, the low amount of CSF cells limits the analysis of normocellular CSF samples, as in our cohort only 11.7% of respectively loaded chips had sufficient cell density for further investigation compared to 59.8% of all chips loaded with samples with elevated cell counts (≥ 5/µl). Varying centrifuge settings, tube materials and resuspension technique were not able to increase the cell yield. CONCLUSION: In summary, the results demonstrate the great potential of Chipcytometry of CSF cells for both scientific questions and routine diagnostic. A new chip design optimized to meet the requirements of CSF would greatly enhance the value of this method. Cross-validation results need to be confirmed in a larger cohort.


Assuntos
Líquido Cefalorraquidiano/citologia , Citocinas/metabolismo , Encefalite/líquido cefalorraquidiano , Encefalite/patologia , Imunofenotipagem/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Citocinas/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Serial de Proteínas/métodos , Adulto Jovem
8.
PLoS Biol ; 16(1): e2002226, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29300740

RESUMO

Neuronal migration during embryonic development contributes to functional brain circuitry. Many neurons migrate in morphologically distinct stages that coincide with differentiation, requiring tight spatial regulation. It had been proposed that neurotransmitter-mediated activity could exert this control. Here, we demonstrate that intracellular calcium transients occur in cerebellar neurons of zebrafish embryos during migration. We show that depolarization increases and hyperpolarization reduces the speed of tegmental hindbrain neurons using optogenetic tools and advanced track analysis optimized for in vivo migration. Finally, we introduce a compound screening assay to identify acetylcholine (ACh), glutamate, and glycine as regulators of migration, which act regionally along the neurons' route. We summarize our findings in a model describing how different neurotransmitters spatially interact to control neuronal migration. The high evolutionary conservation of the cerebellum and hindbrain makes it likely that polarization state-driven motility constitutes an important principle in building a functional brain.


Assuntos
Movimento Celular/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Acetilcolina/metabolismo , Animais , Encéfalo , Mapeamento Encefálico , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Cerebelo/fisiologia , Desenvolvimento Embrionário/fisiologia , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Neurotransmissores/metabolismo , Optogenética/métodos , Peixe-Zebra/embriologia
9.
J Allergy Clin Immunol ; 141(2): 685-696.e6, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28601684

RESUMO

BACKGROUND: Allergic asthma is a chronic lung disease resulting from inappropriate immune responses to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. OBJECTIVE: We analyzed the mechanisms of perinatal tolerance induction to allergens, with particular focus on the role of B cells in preconception and early intrauterine immune priming. METHODS: Wild-type (WT) and B cell-deficient mice received ovalbumin (OVA) intranasally before mating. Their offspring were analyzed in a murine model of allergic airway inflammation. RESULTS: Although antigen application before conception protected WT progeny from allergy, it aggravated allergic airway inflammation in B cell-deficient offspring. B-cell transfer restored protection, demonstrating the crucial role of B cells in perinatal tolerance induction. Effective diaplacentar allergen transfer was detectable in pregnant WT mice but not in pregnant B-cell knockout dams, and antigen concentrations in WT amniotic fluid (AF) were higher than in IgG-free AF of B cell-deficient dams. Application of OVA/IgG immune complexes during pregnancy boosted OVA uptake by fetal dendritic cells (DCs). Fetal DCs in human subjects and mice expressed strikingly higher levels of Fcγ receptors compared with DCs from adults and were highly efficient in taking up OVA/IgG immune complexes. Moreover, murine fetal DCs effectively primed antigen-specific forkhead box P3+ regulatory T cells after in vitro coincubation with OVA/IgG-containing AF. CONCLUSION: Our data support a decisive role for B cells and immunoglobulins during in utero tolerance priming. These findings improve the understanding of perinatal immunity and might support the development of effective primary prevention strategies for allergy and asthma in the future.


Assuntos
Asma/imunologia , Linfócitos B/imunologia , Tolerância Imunológica , Troca Materno-Fetal/imunologia , Animais , Asma/genética , Asma/patologia , Asma/prevenção & controle , Linfócitos B/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Imunoglobulina G/imunologia , Troca Materno-Fetal/genética , Camundongos , Camundongos Transgênicos , Gravidez
10.
Stat Methods Med Res ; 27(11): 3492-3504, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-28535712

RESUMO

Many studies measure the same type of information longitudinally on the same subject at multiple time points, and clustering of such functional data has many important applications. We propose a novel and easy method to implement dissimilarity measure for functional data clustering based on smoothing splines and smoothing parameter commutation. This method handles data observed at regular or irregular time points in the same way. We measure the dissimilarity between subjects based on varying curve estimates with pairwise commutation of smoothing parameters. The intuition is that smoothing parameters of smoothing splines reflect the inverse of the signal-to-noise ratios and that when applying an identical smoothing parameter the smoothed curves for two similar subjects are expected to be close. Our method takes into account the estimation uncertainty using smoothing parameter commutation and is not strongly affected by outliers. It can also be used for outlier detection. The effectiveness of our proposal is shown by simulations comparing it to other dissimilarity measures and by a real application to methadone dosage maintenance levels.


Assuntos
Análise por Conglomerados , Confiabilidade dos Dados , Estudos Longitudinais , Algoritmos , Viés , Humanos , Metadona/administração & dosagem , Tratamento de Substituição de Opiáceos
11.
Eur J Immunol ; 46(7): 1656-68, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27130185

RESUMO

Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.


Assuntos
Fatores de Transcrição Forkhead/deficiência , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Contagem de Células , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citocinas/metabolismo , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Mielopoese/genética , Mielopoese/imunologia , Baço/imunologia , Baço/metabolismo , Baço/patologia
12.
J Immunol ; 196(8): 3245-52, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26962231

RESUMO

Autoreactivity may play a critical role in the chronification of atopic dermatitis (AD). Several studies showed that AD patients produce IgE Abs specific for autoantigens, and we described Th as well as CD8(+) T cells specific for the autoallergen Hom s 2, the α-chain of the nascent polypeptide-associated complex (α-NAC). This study aimed to investigate the frequency and inflammatory phenotype of autoallergen-specific CD8(+) T cells. CD8(+) T cell immunodominant epitopes of α-NAC were mapped by applying prediction softwares, and binding affinity was confirmed by stabilization of empty MHC complexes. MHC class I tetramers were assembled and binding cells were analyzed directly ex vivo by flow cytometry and in terms of single-cell assessment by ChipCytometry. We report significantly elevated numbers of α-NAC-specific peripheral T cells in sensitized patients compared with nonatopic controls. These cells secrete IL-4 and IFN-γ, and surface markers revealed significantly elevated frequencies of circulating terminally differentiated α-NAC-specific CD8(+) T cells in patients with AD compared with nonatopic donors. The observed phenotype of α-NAC-specific CD8(+) T cells indicates a role in the pathogenesis of AD.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Dermatite Atópica/imunologia , Memória Imunológica/imunologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Chaperonas Moleculares/imunologia , Adulto , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Humanos , Imunoglobulina E/imunologia , Ligação Proteica/fisiologia
13.
PLoS Genet ; 11(10): e1005624, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26505631

RESUMO

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.


Assuntos
Caenorhabditis elegans/genética , Divisão Celular/genética , Proteínas de Ligação ao GTP/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Adesão Celular/genética , AMP Cíclico/genética , Embrião não Mamífero , Proteínas de Ligação ao GTP/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transdução de Sinais
14.
J Res Spec Educ Needs ; 15(1): 25-36, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26097431

RESUMO

Much relevant internet-mediated information is inaccessible to people with learning disabilities because of difficulties in navigating the web. This paper reports on the methods undertaken to determine how information can be optimally presented for this cohort. Qualitative work is outlined where attributes relating to site layout affecting usability were elicited. A study comparing web sites of different design layouts exhibiting these attributes is discussed, with the emphasis on methodology. Eight interfaces were compared using various combinations of menu position (vertical or horizontal), text size and the absence or presence of images to determine which attributes of a site have the greatest performance impact. Study participants were also asked for their preferences, via a 'smiley-face' rating scale and simple interviews. 'Acquiescence bias' was minimised by avoiding polar ('yes/no') interrogatives, achieved by asking participants to compare layouts (such as horizontal versus vertical menu), with reasons coaxed from those able to articulate them. Preferred designs were for large text and images. This was the reverse of those facilitating fastest retrieval times, a discrepancy due to preferences being judged on aesthetic considerations. Design recommendations that reconcile preference and performance findings are offered. These include using a horizontal menu, juxtaposing images and text, and reducing text from sentences to phrases, thus facilitating preferred large text without increasing task times.

16.
Eur J Immunol ; 45(1): 214-24, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25329762

RESUMO

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high-content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T-cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T-cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.


Assuntos
Envelhecimento/imunologia , Imunidade Inata , Macrófagos Peritoneais/imunologia , Linfócitos T/imunologia , Transcriptoma/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígeno B7-1/deficiência , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/deficiência , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Proliferação de Células , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunofenotipagem , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Cultura Primária de Células , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos
17.
J Nutr ; 144(8): 1274-82, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24872222

RESUMO

Principal component analysis (PCA) and cluster analysis are used frequently to derive dietary patterns. Decisions on how many patterns to extract are primarily based on subjective criteria, whereas different solutions vary in their food-group composition and perhaps association with disease outcome. Literature on reliability of dietary patterns is scarce, and previous studies validated only 1 preselected solution. Therefore, we assessed reliability of different pattern solutions ranging from 2 to 6 patterns, derived from the aforementioned methods. A validated food frequency questionnaire was administered at baseline (1993-1997) to 39,678 participants in the European Prospective Investigation into Cancer and Nutrition-The Netherlands (EPIC-NL) cohort. Food items were grouped into 31 food groups for dietary pattern analysis. The cohort was randomly divided into 2 halves, and dietary pattern solutions derived in 1 sample through PCA were replicated through confirmatory factor analysis in sample 2. For cluster analysis, cluster stability and split-half reproducibility were assessed for various solutions. With PCA, we found the 3-component solution to be best replicated, although all solutions contained ≥1 poorly confirmed component. No quantitative criterion was in agreement with the results. Associations with disease outcome (coronary heart disease) differed between the component solutions. For all cluster solutions, stability was excellent and deviations between samples was negligible, indicating good reproducibility. All quantitative criteria identified the 2-cluster solution as optimal. Associations with disease outcome were comparable for different cluster solutions. In conclusion, reliability of obtained dietary patterns differed considerably for different solutions using PCA, whereas cluster analysis derived generally stable, reproducible clusters across different solutions. Quantitative criteria for determining the number of patterns to retain were valuable for cluster analysis but not for PCA. Associations with disease risk were influenced by the number of patterns that are retained, especially when using PCA. Therefore, studies on associations between dietary patterns and disease risk should report reasons to choose the number of retained patterns.


Assuntos
Doença das Coronárias/epidemiologia , Dieta , Comportamento Alimentar , Adulto , Idoso , Doença Crônica , Análise por Conglomerados , Ingestão de Energia , Feminino , Humanos , Incidência , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Atividade Motora , Países Baixos , Avaliação Nutricional , Análise de Componente Principal , Estudos Prospectivos , Reprodutibilidade dos Testes , Fatores de Risco , Inquéritos e Questionários , Adulto Jovem
18.
JAMA Neurol ; 71(7): 905-12, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24818670

RESUMO

IMPORTANCE: Cerebrospinal fluid (CSF) is the compartment in closest proximity to the central nervous system (CNS) parenchyma and might reflect immune pathology in inflammatory CNS disorders like multiple sclerosis (MS). Multiparameter flow cytometry is used to characterize immunological alterations in the CSF of patients with MS. OBJECTIVES: To present a comprehensive review of the cellular alterations in CSF that distinguish MS from physiological conditions and other CNS disorders; integrate relevant findings into a model of leukocyte trafficking in the CNS; highlight treatment-related changes in leukocyte subsets; and evaluate the potential of CSF immunophenotyping in the search of novel biomarkers in MS. EVIDENCE REVIEW: We searched MEDLINE articles published between 1980 and 2013 that include the flow cytometric characterization of leukocyte subsets in the CSF of patients with MS. FINDINGS: All of the articles have shown CSF pleocytosis in MS. Interesting results include CSF enrichment of helper T cells (subtypes TH1 and TH17) and regulatory T cells, as well as intrathecal B-cell differentiation resulting in the generation of antibody-producing plasmablasts and plasma cells. Other leukocyte populations, including natural killer cells, monocytes, and dendritic cells, show alterations as well. Characterization of CSF cells increases our understanding of MS pathogenesis and may provide useful biomarkers for individual prognosis and treatment decisions. However, validation in controlled settings is lacking in most cases. CONCLUSIONS AND RELEVANCE: With the advent of more sophisticated approaches, immunophenotyping of CSF cells in MS might become increasingly important to correlate cellular subsets with different stages of disease activity and remission. An assessment of CSF cell numbers and composition should be incorporated into clinical trials.


Assuntos
Biomarcadores/líquido cefalorraquidiano , Imunofenotipagem/métodos , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Humanos , Esclerose Múltipla/patologia
19.
Cytometry A ; 85(7): 580-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24733663

RESUMO

During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil-directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry-based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single-cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May-Grünwald-Giemsa-stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123(+) HLA-DR(-) CD3(-) CD14(-) CD19(-) CD20(-) CD56(-) cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cell's activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.


Assuntos
Asma/sangue , Asma/imunologia , Basófilos/citologia , Líquido da Lavagem Broncoalveolar/citologia , Citometria de Fluxo/métodos , Adulto , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antígenos CD/análise , Contagem de Células Sanguíneas , Broncoscopia , Feminino , Humanos , Pulmão/citologia , Masculino , Pyroglyphidae/imunologia , Rinite Alérgica Sazonal/imunologia , Testes Cutâneos
20.
J Allergy Clin Immunol ; 133(1): 172-80.e1-10, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24012209

RESUMO

BACKGROUND: Primary antibody deficiencies represent the most prevalent, although very heterogeneous, group of inborn immunodeficiencies, with a puzzling complexity of cellular and molecular processes involved in disease pathogenesis. OBJECTIVE: We aimed to study in detail the kinetics of CD40 ligand/IL-21-induced B-cell differentiation to define new biomarker sets for further research into primary antibody deficiencies. METHODS: We applied high-content screening methods to monitor B-cell activation on the cellular (chip cytometry) and transcriptomic (RNA microarray) levels. RESULTS: The complete activation process, including stepwise changes in protein and RNA expression patterns, entry into the cell cycle, proliferation and expression of activation-induced cytidine deaminase (AID), DNA repair enzymes, and post-class-switch expression of IgA and IgG, was successfully monitored during in vitro differentiation. We identified a number of unknown pathways engaged during B-cell activation, such as CXCL9/CXCL10 secretion by B cells. Finally, we evaluated a deduced set of biomarkers on a group of 18 patients with putative or proved intrinsic B-cell defects recruited from the European Society for Immunodeficiencies database and successfully predicted 2 AID defects and 1 DNA repair defect. Complete absence of class-switched B cells was a sensitive predictor of AID deficiency and should be further evaluated as a diagnostic biomarker. CONCLUSION: The biomarkers found in this study could be used to further study the complex process of B-cell activation and to understand conditions that lead to the development of primary antibody deficiencies.


Assuntos
Linfócitos B/imunologia , Síndromes de Imunodeficiência/diagnóstico , Ativação Linfocitária/genética , Adolescente , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Criança , Feminino , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Citometria por Imagem , Switching de Imunoglobulina , Síndromes de Imunodeficiência/imunologia , Recém-Nascido , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , RNA Mensageiro/análise , Transcriptoma/imunologia , Adulto Jovem
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