RESUMO
Amyloid-ß (Aß) is the most prominent protein in Alzheimer's disease (AD) senile plaques. In addition, Aß interacts with a variety of Aß-associated proteins (AAPs), some of which can form complexes with Aß and influence its clearance, aggregation or toxicity. Identification of novel AAPs may shed new light on the pathophysiology of AD and the metabolic fate of Aß. In this study, we aimed to identify new AAPs by searching for proteins that may form soluble complexes with Aß in CSF, using a proteomics approach. We identified the secreted Wnt pathway protein Dickkopf-related protein 3 (Dkk-3) as a potential Aß-associated protein. Using immunohistochemistry on human AD brain tissue, we observed that (i) Dkk-3 co-localizes with Aß in the brain, both in diffuse and classic plaques. (ii) Dkk-3 is expressed in neurons and in blood vessel walls in the brain and (iii) is secreted by leptomeningeal smooth muscle cells in vitro. Finally, measurements using ELISA revealed that (iv) Dkk-3 protein is abundantly present in both cerebrospinal fluid and serum, but its levels are similar in non-demented controls and patients with AD, Lewy body dementia, and frontotemporal dementia. Our study demonstrates that Dkk-3 is a hitherto unidentified Aß-associated protein which, given its relatively high cerebral concentrations and co-localization with Aß, is potentially involved in AD pathology. In this study, we propose that Dickkopf-related protein-3 (Dkk-3) might be a novel Amyloid-ß (Aß) associated protein. We demonstrate that Dkk-3 is expressed in the brain, especially in vessel walls, and co-localizes with Aß in senile plaques. Furthermore, Dkk-3 levels in cerebrospinal fluid strongly correlate with Aß40 levels, but were not suitable to discriminate non-demented controls and patients with dementia.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Quimiocinas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , ProteômicaRESUMO
We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease.
Assuntos
Genes Recessivos , Mutação , Fosfolipases/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Sequência de Bases , Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Fácies , Feminino , Ordem dos Genes , Genótipo , Humanos , Imageamento por Ressonância Magnética , Masculino , Neuroimagem , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/diagnóstico , Adulto JovemRESUMO
Vasopressin-regulated expression and insertion of aquaporin-2 channels in the luminal membrane of renal principal cells is essential for urine concentration. Lithium affects urine concentrating ability, and approximately 20% of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder characterized by polyuria and polydipsia. Lithium-induced NDI is caused by aquaporin-2 downregulation and a reduced ratio of principal/intercalated cells, yet lithium induces principal cell proliferation. Here, we studied how lithium-induced principal cell proliferation can lead to a reduced ratio of principal/intercalated cells using two-dimensional and three-dimensional polarized cultures of mouse renal collecting duct cells and mice treated with clinically relevant lithium concentrations. DNA image cytometry and immunoblotting revealed that lithium initiated proliferation of mouse renal collecting duct cells but also increased the G2/S ratio, indicating G2/M phase arrest. In mice, treatment with lithium for 4, 7, 10, or 13 days led to features of NDI and an increase in the number of principal cells expressing PCNA in the papilla. Remarkably, 30%-40% of the PCNA-positive principal cells also expressed pHistone-H3, a late G2/M phase marker detected in approximately 20% of cells during undisturbed proliferation. Our data reveal that lithium treatment initiates proliferation of renal principal cells but that a significant percentage of these cells are arrested in the late G2 phase, which explains the reduced principal/intercalated cell ratio and may identify the molecular pathway underlying the development of lithium-induced renal fibrosis.
Assuntos
Antimaníacos/efeitos adversos , Diabetes Insípido Nefrogênico/induzido quimicamente , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Lítio/efeitos adversos , Animais , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Diabetes Insípido Nefrogênico/enzimologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases/metabolismoRESUMO
The tumour microenvironment has been shown to be a valuable source of prognostic information for different cancer types. This holds in particular for triple negative breast cancer (TNBC), a breast cancer subtype for which currently no prognostic biomarkers are established. Although different methods to assess tumour infiltrating lymphocytes (TILs) have been published, it remains unclear which method (marker, region) yields the most optimal prognostic information. In addition, to date, no objective TILs assessment methods are available. For this proof of concept study, a subset of our previously described TNBC cohort (n = 94) was stained for CD3, CD8 and FOXP3 using multiplex immunohistochemistry and subsequently imaged by a multispectral imaging system. Advanced whole-slide image analysis algorithms, including convolutional neural networks (CNN) were used to register unmixed multispectral images and corresponding H&E sections, to segment the different tissue compartments (tumour, stroma) and to detect all individual positive lymphocytes. Densities of positive lymphocytes were analysed in different regions within the tumour and its neighbouring environment and correlated to relapse free survival (RFS) and overall survival (OS). We found that for all TILs markers the presence of a high density of positive cells correlated with an improved survival. None of the TILs markers was superior to the others. The results of TILs assessment in the various regions did not show marked differences between each other. The negative correlation between TILs and survival in our cohort are in line with previous studies. Our results provide directions for optimizing TILs assessment methodology.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Inteligência Artificial , Biomarcadores Tumorais/análise , Neoplasias da Mama/mortalidade , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Mastectomia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Países Baixos , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/mortalidade , Microambiente TumoralRESUMO
Digital pathology with whole-slide imaging (WSI) has a large potential to make the process of expert consultation and expert panel diagnosis more rapid and more efficient. However, comparison with the current methods is necessary for validation of the technique. In this study, we determined if digital assessment of whole-slide images of hematopathology specimens with a focus on the assessment of lymphoma can be used for consultation and panel diagnostics. Ninety-three histological specimens with a suspicion for lymphoma were assessed both with conventional microscopy and digital microscopy with a wash out period between assessments. A consensus diagnosis was based on full concordance between the pathologists or, in case of discordances, was reached at a joint session at a multi-headed microscope. In 81% of the cases, there was a full concordance between digital and light microscopical assessment for all three pathologists. Discordances between conventional microscopy and digital pathology were present in 3% of assessments. In comparison with the consensus diagnosis, discordant diagnoses were made in 5 cases with digital microscopy and in 3 cases with light microscopy. The reported level of confidence and need for additional investigations were similar between assessment by conventional and by digital microscopy. In conclusion, the performance of assessment by digital pathology is in general comparable with that of conventional light microscopy and pathologists feel confident using digital pathology for this subspecialty.
Assuntos
Interpretação de Imagem Assistida por Computador , Linfoma/patologia , Microscopia , Consulta Remota , Adulto , Idoso , Consenso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
AIMS: Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. METHODS AND RESULTS: Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker gamma-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. CONCLUSIONS: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.
Assuntos
Biomarcadores Tumorais/análise , DNA de Neoplasias/genética , Neoplasias Bucais/química , Neoplasias Bucais/genética , Ploidias , Lesões Pré-Cancerosas/química , Lesões Pré-Cancerosas/genética , Aneuploidia , Anticorpos Antinucleares , Anticorpos Monoclonais , Carcinoma in Situ/química , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , DNA de Neoplasias/análise , Histonas/análise , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Antígeno Ki-67/análise , Neoplasias Bucais/diagnóstico , Inclusão em Parafina , Lesões Pré-Cancerosas/diagnóstico , Estudos RetrospectivosRESUMO
AIMS: The aetiology of vulvar squamous cell carcinomas (SCC) that are not causally associated with high-risk human papillomavirus remains largely elusive. The aim of this study was to analyse the inflammatory response in its presumed precursor lesions, lichen sclerosus (LS) and differentiated vulvar intraepithelial neoplasia (dVIN), and provide evidence that dVIN is a likely precursor of vulvar SCC. METHODS AND RESULTS: Immunohistochemical analyses for CD4+, CD8+, CD20+, CD68+, S100+ and tryptase-positive immune cells were performed and quantified in LS (n = 7), dVIN (n = 19), SCC (n = 11), and normal vulvar tissue (n = 8). The subepithelial inflammatory response in dVIN and SCC was comparable, but absent in LS. Abundant intraepithelial mast cells were observed in dVIN only, and confirmed by electron microscopy, toluidine blue staining and cKIT expression. Adjacent keratinocytes displayed increased proliferation as determined by MIB-1 positivity. Electron microscopy revealed intraepithelial mast cell degranulation. Intraepithelial mast cells were not or infrequently observed in vulvar hyperplasia (n = 13), condylomata acuminata (n = 5), keratinocytic intraepidermal neoplasia of sun-exposed skin (n = 15), epidermal hyperplasia of head and neck (n = 12), and psoriasis (n = 3). CONCLUSIONS: These data indicate that dVIN can be recognized by intraepithelial mast cells and that they might promote the progression of dVIN to SCC.
Assuntos
Carcinoma de Células Escamosas/imunologia , Mastócitos/citologia , Lesões Pré-Cancerosas/imunologia , Neoplasias Vulvares/imunologia , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Mastócitos/imunologia , Mastócitos/ultraestrutura , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/ultraestrutura , Neoplasias Vulvares/patologia , Neoplasias Vulvares/ultraestruturaRESUMO
OBJECTIVE: The objective of the study was to quantify vessel type and density in lichen sclerosus (LS) to find a marker for its malignant potential. STUDY DESIGN: Quantitative analysis was performed on paraffin-embedded tissue samples of 28 patients with LS (7 adjacent to vulvar squamous cell carcinoma, 21 solitary) and immunohistochemical staining for CD34 (vascular and lymphangiogenic lymph endothelial cells), D2-40 (lymphatic-specific marker), and alpha-SMA (pericyte marker). Electron microscopy was performed on fresh tissue. RESULTS: No significant differences in vessel density or other vessel parameters could be demonstrated between the 2 groups. In hyalinized lesions, vessel diameter, and alpha-SMA positivity was reduced compared with nonhyalinized lesions. Electron microscopy revealed detachment of pericytes from vascular endothelial cells and increased thickening of basement membrane, whereas endothelial cell function did not appear strongly impaired. CONCLUSION: Malignant potential of LS cannot be predicted by vessel characteristics. Hyalinization in LS is associated with pericyte detachment from the basal lamina of vascular endothelial cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Lesões Pré-Cancerosas/patologia , Líquen Escleroso Vulvar/patologia , Neoplasias Vulvares/patologia , Biópsia por Agulha , Vasos Sanguíneos/patologia , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Imuno-Histoquímica , Vasos Linfáticos/patologia , Microscopia Eletrônica , Inclusão em Parafina , Probabilidade , Prognóstico , Estatísticas não Paramétricas , Vulva/patologia , Vulva/ultraestruturaRESUMO
Breast cancer treatment depends on human epidermal growth factor receptor-2 (HER2) status, which is often determined using dual probe fluorescence in situ hybridisation (FISH). Hereby, also loss and gain of the centromere of chromosome 17 (CEP17) can be observed (HER2 is located on chromosome 17). CEP17 gain can lead to difficulty in interpretation of HER2 status, since this might represent true polysomy. With this study we investigated whether isolated polysomy is present and how this effects HER2 status in six breast cancer cell lines and 97 breast cancer cases, using HER2 FISH and immunohistochemistry, DNA ploidy assessment and multiplex ligation dependent probe amplification. We observed no isolated polysomy of chromosome 17 in any cell line. However, FISH analysis did show CEP17 gain in five of six cell lines, which reflected gains of the whole chromosome in metaphase spreads and aneuploidy with gain of multiple chromosomes in all these cases. In patients' samples, gain of CEP17 indeed correlated with aneuploidy of the tumour (91.1%; p < 0.001). Our results indicate that CEP17 gain is not due to isolated polysomy, but rather due to widespread aneuploidy with gain of multiple chromosomes. As aneuploidy is associated with poor clinical outcome, irrespective of tumour grade, this could improve future therapeutic decision making.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Centrômero/química , Cromossomos Humanos Par 17/química , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Feminino , Duplicação Gênica , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Ploidias , PrognósticoRESUMO
Manual counting of mitotic tumor cells in tissue sections constitutes one of the strongest prognostic markers for breast cancer. This procedure, however, is time-consuming and error-prone. We developed a method to automatically detect mitotic figures in breast cancer tissue sections based on convolutional neural networks (CNNs). Application of CNNs to hematoxylin and eosin (H&E) stained histological tissue sections is hampered by: (1) noisy and expensive reference standards established by pathologists, (2) lack of generalization due to staining variation across laboratories, and (3) high computational requirements needed to process gigapixel whole-slide images (WSIs). In this paper, we present a method to train and evaluate CNNs to specifically solve these issues in the context of mitosis detection in breast cancer WSIs. First, by combining image analysis of mitotic activity in phosphohistone-H3 (PHH3) restained slides and registration, we built a reference standard for mitosis detection in entire H&E WSIs requiring minimal manual annotation effort. Second, we designed a data augmentation strategy that creates diverse and realistic H&E stain variations by modifying the hematoxylin and eosin color channels directly. Using it during training combined with network ensembling resulted in a stain invariant mitosis detector. Third, we applied knowledge distillation to reduce the computational requirements of the mitosis detection ensemble with a negligible loss of performance. The system was trained in a single-center cohort and evaluated in an independent multicenter cohort from The Cancer Genome Atlas on the three tasks of the Tumor Proliferation Assessment Challenge (TUPAC). We obtained a performance within the top-3 best methods for most of the tasks of the challenge.
RESUMO
The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the epsilon2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-beta1-40 peptide (Abeta1-40) with the 22Glu--> Gln "Dutch" mutation caused pericyte death in vitro by a mechanism that involves Abeta fibril-like assembly at the cell surface. It is known that ApoE binds to Abeta and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Abeta-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an epsilon4/epsilon4 genotype were more vulnerable to Abeta than cultures with an epsilon3/epsilon3 or epsilon3/epsilon4 genotype. The one cell culture with the epsilon2/epsilon3 genotype was relatively resistant to Abeta compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Abeta-mediated toxicity of cerebrovascular cells and, in addition, ApoE epsilon2/epsilon3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with epsilon4/epsilon4 cells. Thus, the disparity between ApoE genotype and Abeta-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Abeta cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to Abeta and thereby modify Abeta-mediated cell death.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Apolipoproteínas E/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Pericitos/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Análise de Variância , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/genética , Northern Blotting/métodos , Western Blotting/métodos , Encéfalo/citologia , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Masculino , Microscopia Imunoeletrônica/métodos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Pericitos/metabolismo , Pericitos/ultraestrutura , RNA Mensageiro/metabolismo , Transfecção/métodosRESUMO
Small heat shock proteins Hsp20 and HspB2/B3 co-localize with Abeta deposition in senile plaques and cerebral amyloid angiopathy in Alzheimer's disease brains, respectively. It was the aim of our study to investigate if these and other sHsps bind to wild-type Abeta1-42 or the more toxic Abeta1-40 carrying the 'Dutch' mutation (22Glu-->Gln) (D-Abeta1-40), affect Abeta aggregation and thereby influence Abeta cytotoxicity. Binding affinity between sHsps and Abeta was investigated by surface plasmon resonance. Abeta aggregation was studied by using circular dichroism spectroscopy and electron microscopy. Furthermore, we used cultured cerebrovascular cells to investigate the effects of sHsps on Abeta-mediated cytotoxicity. Hsp20, Hsp27 and alphaB-crystallin, but not HspB2/B3, bound to Abeta (both D-Abeta1-40 and Abeta1-42) and reduced or completely inhibited aggregation of D-Abeta1-40 into mature fibrils but did not affect Abeta1-42 aggregation. Furthermore, these sHsps were effective inhibitors of the cerebrovascular toxicity of Abeta (both D-Abeta1-40 and Abeta1-42) in vitro. Binding affinity of the sHsps to D-Abeta1-40 correlated to the degree of inhibition of Abeta-mediated cytotoxicity and the potential to reduce Abeta beta-sheet and fibril formation. With Abeta1-42, a similar correlation between binding affinity and cytotoxicity was observed, but not with its aggregation state. In conclusion, sHsps may regulate Abeta aggregation and serve as antagonists of the biological action of Abeta, but the extent of their interaction depends on the type of sHsp and Abeta peptide.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Angiopatia Amiloide Cerebral/metabolismo , Artérias Cerebrais/metabolismo , Proteínas de Choque Térmico/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Células Cultivadas , Angiopatia Amiloide Cerebral/fisiopatologia , Artérias Cerebrais/fisiopatologia , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP20/farmacologia , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/farmacologia , Humanos , Chaperonas Moleculares , Mutação/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Ligação Proteica/genética , alfa-Cristalinas/metabolismo , alfa-Cristalinas/farmacologiaRESUMO
Variations in the color and intensity of hematoxylin and eosin (H&E) stained histological slides can potentially hamper the effectiveness of quantitative image analysis. This paper presents a fully automated algorithm for standardization of whole-slide histopathological images to reduce the effect of these variations. The proposed algorithm, called whole-slide image color standardizer (WSICS), utilizes color and spatial information to classify the image pixels into different stain components. The chromatic and density distributions for each of the stain components in the hue-saturation-density color model are aligned to match the corresponding distributions from a template whole-slide image (WSI). The performance of the WSICS algorithm was evaluated on two datasets. The first originated from 125 H&E stained WSIs of lymph nodes, sampled from 3 patients, and stained in 5 different laboratories on different days of the week. The second comprised 30 H&E stained WSIs of rat liver sections. The result of qualitative and quantitative evaluations using the first dataset demonstrate that the WSICS algorithm outperforms competing methods in terms of achieving color constancy. The WSICS algorithm consistently yields the smallest standard deviation and coefficient of variation of the normalized median intensity measure. Using the second dataset, we evaluated the impact of our algorithm on the performance of an already published necrosis quantification system. The performance of this system was significantly improved by utilizing the WSICS algorithm. The results of the empirical evaluations collectively demonstrate the potential contribution of the proposed standardization algorithm to improved diagnostic accuracy and consistency in computer-aided diagnosis for histopathology data.
Assuntos
Algoritmos , Interpretação de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/métodos , Coloração e Rotulagem/métodos , Humanos , Linfonodos/diagnóstico por imagemRESUMO
Amyloid-beta (Abeta) deposition in the cerebral arterial and capillary walls is one of the characteristics of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. In vitro, Abeta1-40, carrying the "Dutch" mutation (DAbeta1-40), induced reproducible degeneration of cultured human brain pericytes (HBP), by forming fibrils at the cell surface. Thus, this culture system provides an useful model to study the vascular pathology seen in Alzheimer's disease. In this study, we used this model to investigate the effects of insulin on Abeta-induced degeneration of HBP, as it has been mentioned previously that insulin is able to protect neurons against Abeta-induced cell-death. The toxic effect of DAbeta1-40 on HBP was inhibited by insulin in a dose-dependent matter. Insulin interacted with Abeta and inhibited fibril formation of Abeta in a cell-free assay, as well as at the cell surface of HBP. Our data indicate that the formation of a fibril network is essential for Abeta-induced cell death in HBP. Additionally, insulin may be involved in the regulation of Abeta fibrillization in AD.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Encéfalo/citologia , Morte Celular/efeitos dos fármacos , Insulina/farmacologia , Fragmentos de Peptídeos/farmacologia , Pericitos/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Western Blotting/métodos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Imunofluorescência/métodos , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Insulina/metabolismo , Microscopia Imunoeletrônica/métodos , Fragmentos de Peptídeos/metabolismo , Pericitos/metabolismo , Pericitos/ultraestrutura , Placa Amiloide/metabolismo , Placa Amiloide/ultraestrutura , Fatores de TempoRESUMO
Alzheimer's disease (AD) brains are characterized by the presence of senile plaques (SPs), which primarily consist of amyloid beta protein (Abeta). Besides Abeta, several other proteins with the ability to modulate amyloid fibril formation accumulate in SPs, e.g. heparan sulfate proteoglycans (HSPGs). Cerebellar SPs are predominantly of the diffuse type, whereas fibrillar SPs are rarely observed. Furthermore, because of the spatial separation of non-fibrillar and fibrillar SPs in the cerebellum, this brain region provides a model for the study of the association of Abeta-associated factors with various stages of SP formation. In the present study, we performed an immunohistochemical analysis to investigate the expression of the HSPG species agrin, perlecan, glypican-1 and the syndecans 1-3 as well as glycosaminoglycan side-chains in cerebellar SPs. We demonstrated that agrin and glypican-1 were expressed in both non-fibrillar and fibrillar cerebellar SPs, whereas the syndecans were only associated with fibrillar cerebellar SPs. Perlecan expression was absent in all cerebellar SPs. Since fibrillar and non-fibrillar SPs may develop independently in the cerebellum, it is likely that agrin, glypican-1 as well as heparan sulfate glycosaminoglycans may contribute to the formation of both cerebellar plaque types, whereas syndecan only seems to play a role in the generation of cerebellar fibrillar plaques.
Assuntos
Cerebelo/metabolismo , Heparitina Sulfato/metabolismo , Placa Amiloide/metabolismo , Proteoglicanas/metabolismo , Idoso , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Imuno-Histoquímica , MasculinoRESUMO
It has been suggested that the successive proteolytic events leading to the production of the amyloid-beta protein from its precursor may take place at different intracellular locations. Using cultured human leptomeningeal smooth muscle cells and brain pericytes, we modulated the intracellular localization of the amyloid-beta precursor protein (APP) to study possible effects on its processing. By using immunofluorescence and immunoelectron microscopy we demonstrated that, under normal conditions, the APP is found in small intracellular vesicles, some of which were characterized as lysosomes. Both the cytokine interferon-gamma and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-alpha (TNF-alpha), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-beta precursor protein was slightly reduced by interferon-gamma or chloroquine. Double-labeling and tracer molecule uptake experiments showed that the multivesicular body-like organelles were part of the endocytic pathway. Our findings suggest that the multivesicular body-like organelles function as an intermediate organelle in the intracellular trafficking of the APP. Accumulation of the APP in this organelle is reflected by its reduced secretion from the cell.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/irrigação sanguínea , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Organelas/metabolismo , Pericitos/metabolismo , Encéfalo/ultraestrutura , Células Cultivadas , Cloroquina/farmacologia , Endocitose , Endotélio Vascular/ultraestrutura , Imunofluorescência , Humanos , Interferon gama/farmacologia , Meninges/irrigação sanguínea , Microcirculação , Microscopia Imunoeletrônica , Músculo Liso Vascular/ultraestrutura , Organelas/efeitos dos fármacos , Pericitos/ultraestruturaRESUMO
PURPOSE: Recently, it was reported that tumor cells themselves generate channels and networks in three-dimensional culture and can be found lining channels (some containing red blood cells [RBCs]) in vivo, and they express endothelial or vascular genes in aggressive uveal melanoma. The implications of these data for current insights in the involvement of angiogenesis in tumor growth, metastasis and therapeutic intervention are considerable. Therefore, this possibility was investigated in the current study. METHODS: Thirty human uveal melanomas and 20 xenografts of human cutaneous melanoma were analyzed by Azan histochemistry and immunostaining of endothelial markers. Additionally, in xenografted tumors a tracer study was performed with confocal microscopy and immunoelectron microscopy. RESULTS: Lumina or spaces without endothelial lining containing RBCs were not detected in any lesion. Functional evaluation of the vasculature in xenografts demonstrated rapid tracer appearance both inside and outside blood vessels. Outside blood vessels it spread along matrix networks of arcs and back-to-back loops. Confocal microscopy showed that this extracellular matrix was deposited as stromal sheets around nests of tumor cells. Laminin immunostaining revealed that between sheets surrounding adjacent nests, spaces were present. These spaces were filled, however, with collagen and different types of cells, including cells stained for macrophage markers. CONCLUSIONS: Although no evident endothelium-free and RBC-containing channels were present in the tissues examined, there are fluid-conducting spaces in the form of stromal sheets between nests of tumor cells. In this stromal network, blood vessels are embedded. The authors postulate that this extracellular matrix tissue represents a fluid-conducting meshwork.
Assuntos
Matriz Extracelular/patologia , Melanoma/irrigação sanguínea , Neovascularização Patológica/patologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Uveais/irrigação sanguínea , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Melanoma/metabolismo , Melanoma/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Imunoeletrônica , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/ultraestrutura , Transplante Heterólogo , Neoplasias Uveais/metabolismo , Neoplasias Uveais/ultraestruturaRESUMO
Amyloid-beta protein (A beta) deposition in the cerebral vascular walls is one of the key features of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). A beta(1-40) carrying the 'Dutch' mutation (HCHWA-D A beta(1-40)) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of A beta-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D A beta(1-40) on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with A beta, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of A beta binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade A beta and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of A beta at the cell surface of human brain pericytes is a crucial step in A beta-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Morte Celular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Pericitos/citologia , Pericitos/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Antioxidantes/farmacologia , Western Blotting , Encéfalo/citologia , Catalase/metabolismo , Catalase/farmacologia , Células Cultivadas , Cromanos/farmacologia , Glutationa/farmacologia , Humanos , Técnicas In Vitro , Microscopia Imunoeletrônica , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Pericitos/metabolismo , Ligação Proteica , Superóxido Dismutase/farmacologiaRESUMO
OBJECTIVES: The aim of the present study is to determine the prevalence of endometrial premalignancies in women diagnosed with epithelial ovarian cancer (EOC). METHODS: Endometrial and ovarian specimens of 186 patients with EOC were retrospectively selected using the nationwide pathology network and registry, and sections were comprehensively reviewed: 136 (73%) serous, 19 (10%) endometrioid, 15 (8%) mucinous, seven (4%) clear cell, and nine (5%) undifferentiated. Immunohistochemical phenotypes were compared for patients with serous EOC with concurrent endometrial pathology. RESULTS: In 31%, endometrial (pre)malignancy was found: carcinoma in 3%, endometrial intraepithelial carcinoma (EIC) in 4%, and atypical hyperplasia in 24%. Atypical hyperplasia was found in 47% of endometrioid EOCs but in 7% to 33% of other subtypes. Body mass index was higher concurrent to atypical hyperplasia (P=.001). Serous EOC and EIC immunophenotypes were comparable, whereas atypical hyperplasia was expressed differently. CONCLUSIONS: Apart from synchronous endometrial carcinoma, endometrial premalignancies should be taken into account when determining optimal treatment for women diagnosed with EOC.
Assuntos
Endométrio/metabolismo , Hiperplasia/epidemiologia , Neoplasias Epiteliais e Glandulares/epidemiologia , Neoplasias Ovarianas/epidemiologia , Adulto , Idoso , Carcinoma Epitelial do Ovário , Feminino , Humanos , Hiperplasia/patologia , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/terapia , Lesões Pré-Cancerosas/patologia , Prevalência , Estudos RetrospectivosRESUMO
Targeted carrier systems (e.g., liposomes or nanoparticles) are used to specifically deliver drugs to a site of interest. Site-direction can be achieved by attachment of targeting molecules, such as peptides, DNA/RNA, or antibodies, to the surface of the carrier. Here, the formation of polymersomes with tumor-targeting potential is described. A single-domain antibody (A12) that specifically targets PlexinD1 (a transmembrane protein overexpressed in tumor vasculature) is equipped with an azide-functionality using expressed protein ligation. This azide-containing A12 can subsequently be attached to BCN-functionalized polymersomes using a strain-promoted azide alkyne cycloaddition, thereby forming polymersomes with tumor-targeting potential.