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Rationale: Human induced pluripotent stem cell-derived endothelial cells can be candidates for engineering therapeutic vascular grafts. Methods: Here, we studied the role of three-dimensional culture on their characteristics and function both in vitro and in vivo. Results: We found that differentiated hPSC-EC can re-populate decellularized biomatrices; they remain viable, undergo maturation and arterial/venous specification. Human PSC-EC develop antifibrotic, vasoactive and anti-inflammatory properties during recellularization. In vivo, a robust increase in perfusion was detected at the engraftment sites after subcutaneous implantation of an hPSC-EC-laden hydrogel in rats. Histology confirmed survival and formation of capillary-like structures, suggesting the incorporation of hPSC-EC into host microvasculature. In a canine model, hiPSC-EC-seeded onto decellularised vascular segments were functional as aortic grafts. Similarly, we showed the retention and maturation of hiPSC-EC and dynamic remodelling of the vessel wall with good maintenance of vascular patency. Conclusions: A combination of hPSC-EC and biomatrices may be a promising approach to repair ischemic tissues.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Prótese Vascular , Diferenciação Celular , Cães , Células Endoteliais/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RatosRESUMO
Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 µm (mean±S.D.: 1.17 ± 0.34 µm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.
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Nuclear estrogen receptor (ER), progesterone receptor (PR) and Ki-67 protein positive tumor cell fractions are semiquantitatively assessed in breast cancer for prognostic and predictive purposes. These biomarkers are usually revealed using immunoperoxidase methods resulting in diverse signal intensity and frequent inhomogeneity in tumor cell nuclei, which are routinely scored and interpreted by a pathologist during conventional light-microscopic examination. In the last decade digital pathology-based whole slide scanning and image analysis algorithms have shown tremendous development to support pathologists in this diagnostic process, which can directly influence patient selection for targeted- and chemotherapy. We have developed an image analysis algorithm optimized for whole slide quantification of nuclear immunostaining signals of ER, PR, and Ki-67 proteins in breast cancers. In this study, we tested the consistency and reliability of this system both in a series of brightfield and DAPI stained fluorescent samples. Our method allows the separation of overlapping cells and signals, reliable detection of vesicular nuclei and background compensation, especially in FISH stained slides. Detection accuracy and the processing speeds were validated in routinely immunostained breast cancer samples of varying reaction intensities and image qualities. Our technique supported automated nuclear signal detection with excellent efficacy: Precision Rate/Positive Predictive Value was 90.23 ± 4.29%, while Recall Rate/Sensitivity was 88.23 ± 4.84%. These factors and average counting speed of our algorithm were compared with two other open source applications (QuPath and CellProfiler) and resulted in 6-7% higher Recall Rate, while 4- to 30-fold higher processing speed. In conclusion, our image analysis algorithm can reliably detect and count nuclear signals in digital whole slides or any selected large areas i.e. hot spots, thus can support pathologists in assessing clinically important nuclear biomarkers with less intra- and interlaboratory bias inherent of empirical scoring. © 2017 International Society for Advancement of Cytometry.
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Algoritmos , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Antígeno Ki-67/genética , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Automação Laboratorial , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Núcleo Celular/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Expressão Gênica , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente , Sensibilidade e EspecificidadeRESUMO
HER2-positive breast cancers usually benefit from anti-HER2 therapy, thus, HER2 evaluation became inevitable for patient selection. HER2-negative (IHC 0, 1+) and strong positive (IHC 3+) cases can easily be interpreted with immunohistochemistry, but equivocal (IHC 2+) cases require further analysis of HER2 gene amplification using in situ hybridization. Our study aimed to validate digital pathology and automated image analysis for unbiased evaluation of HER2 immunostains. We developed an image segmentation algorithm for analyzing HER2-immunostaining (4B5 clone) in tissue microarrays of breast cancers. Two pathologists assessed 309 microscopic regions of at least 100 tumor cells each--representing all HER2 positivity groups--according to international guidelines either semi-quantitatively or by using the MembraneQuant software. Scoring results were statistically correlated with each other and with FISH data, and almost perfect agreement was found (inter-method Cohen's kappa = 0.872, Spearman-rho = 0.928). When clinical relevance (scoring disagreement that may define erroneous treatment selection) was examined high agreement was found (quadratic weighted kappa = 0.967). Image analysis classified cases with excellent correlation with visual evaluation, therefore, MembraneQuant software proved to be a reliable tool for assessing HER2 immunoreactions and supporting better targeting anti-HER2 therapy. As digital analysis of immunomorphological markers allows permanent archiving, standardization and accurate reviewing of results, it supports quality assurance initiatives in diagnostic pathology--especially of equivocal cases which are hard to interpret.
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Algoritmos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Monitoramento de Medicamentos , Processamento de Imagem Assistida por Computador/métodos , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos , Automação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/patologia , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Software , Análise Serial de TecidosRESUMO
The anatomical organization of the peripheral and central sensory structures of the earthworm Lumbricus terrestris was investigated applying a fluorescent carbocyanine dye (DiI) as a neuronal tracer. Using whole-mount preparations and confocal laser scanning microscopy, the pattern of primary sensory cells and pathways of their processes were traced and reconstructed in three-dimensions. Our study shows that a ventral nerve cord ganglion receives sensory fibers from at least two adjacent segments suggesting that the peripheral nervous system is not segmental in its arrangement and the receptive-fields of the body wall overlap in earthworms. Furthermore, our result suggests an integrative function of the basiepidermal plexus consists of sensory and motor fibers.
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Oligoquetos/anatomia & histologia , Oligoquetos/fisiologia , Animais , Anelídeos , Carbocianinas/metabolismo , Corantes Fluorescentes , Gânglios/anatomia & histologia , Microscopia Confocal , Sistema Nervoso Periférico/anatomia & histologia , Sistema Nervoso Periférico/fisiologiaRESUMO
BACKGROUND: The immunohistochemical detection of estrogen (ER) and progesterone (PR) receptors in breast cancer is routinely used for prognostic and predictive testing. Whole slide digitalization supported by dedicated software tools allows quantization of the image objects (e.g. cell membrane, nuclei) and an unbiased analysis of immunostaining results. Validation studies of image analysis applications for the detection of ER and PR in breast cancer specimens provided strong concordance between the pathologist's manual assessment of slides and scoring performed using different software applications. METHODS: The effectiveness of two connected semi-automated image analysis software (NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14) for determination of ER and PR status in formalin-fixed paraffin embedded breast cancer specimens immunostained with the automated Leica Bond Max system was studied. First the detection algorithm was calibrated to the scores provided an independent assessors (pathologist), using selected areas from 38 small digital slides (created from 16 cases) containing a mean number of 195 cells. Each cell was manually marked and scored according to the Allred-system combining frequency and intensity scores. The performance of the calibrated algorithm was tested on 16 cases (14 invasive ductal carcinoma, 2 invasive lobular carcinoma) against the pathologist's manual scoring of digital slides. RESULTS: The detection was calibrated to 87 percent object detection agreement and almost perfect Total Score agreement (Cohen's kappa 0.859, quadratic weighted kappa 0.986) from slight or moderate agreement at the start of the study, using the un-calibrated algorithm. The performance of the application was tested against the pathologist's manual scoring of digital slides on 53 regions of interest of 16 ER and PR slides covering all positivity ranges, and the quadratic weighted kappa provided almost perfect agreement (κ = 0.981) among the two scoring schemes. CONCLUSIONS: NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14 software application proved to be a reliable image analysis tool for pathologists testing ER and PR status in breast cancer.
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Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Software/normas , Algoritmos , Biomarcadores Tumorais/metabolismo , Calibragem , Diagnóstico por Imagem/métodos , Feminino , HumanosRESUMO
The regeneration of the ventral nerve cord ganglion and peripheral tissues was investigated by radioimmunoassay and immunohistochemistry in the model animal, Eisenia fetida (Annelida, Oligochaeta). It is now well-established that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic factor, playing important roles in the development of the nervous system in vertebrate animals. Based on the apparent evolutionary conservation of PACAP and on the several common mechanisms of vertebrate and invertebrate nervous regeneration, the question was raised whether PACAP has any role in the regeneration of the earthworm nervous system. As a first step, we studied the distribution, concentration, and time-course of PACAP-like immunoreactivity during caudal regeneration of both lost segments and the ventral nerve cord ganglia in E. fetida. A strong upregulation of PACAP-like immunoreactivity was observed in most tissues following injury as determined by radioimmunoassay and immunohistochemistry. Significant increases in the concentration of PACAP-like compounds were found in the body wall, alimentary canal, and in coelomocytes. The most characteristic morphological feature was the accumulation of immunolabeled neoblasts in the injured tissues, especially in the ventral nerve cord ganglion that initiates and mediates regeneration processes. Our present results show that PACAP/PACAP-like peptides accumulate in the regenerating tissues of the earthworm, suggesting trophic functions of these compounds in earthworm tissues similarly to vertebrate species.
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Oligoquetos/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Regeneração/fisiologia , Animais , Imuno-Histoquímica , Oligoquetos/anatomia & histologia , RadioimunoensaioRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at very early stages in the vertebrate nervous system, and its functions in the embryonic development have been shown by various studies. PACAP is an extremely conserved molecule in phylogeny; however, little is known about its presence and functions in invertebrates. Our previous studies have shown the occurrence of PACAP-like immunoreactivity in the invertebrate nervous system. The aim of this study was to investigate the presence and localization of PACAP-like compounds during the embryonic development of earthworms from cocoon deposition to hatching using immunological methods (radioimmunoassay, dot blot, immunohistochemistry). PACAP-like immunoreactive compounds were detected at very early stages of the embryonic development of the earthworm Eisenia fetida. No significant changes were observed during the early stages in the developing embryo, but a marked increase occurred before hatching. In contrast, during the embryonic development, the level of PACAP-like compounds gradually decreased in cocoon fluids. Immunohistochemistry revealed the presence of PACAP-like immunoreactive cell bodies and processes in the developing body wall, prostomium, pharyngeal wall, and central nervous system. Cells located in the body wall correspond to putative progenitor cells of primary sensory cells. In the present study, we also showed that the clitellum (reproductive organ) of sexually mature worms contained significantly higher levels of PACAP-like immunoreactivity than other regions of the same animals or the clitellar region of a non-reproducing animal. In summary, these observations provide a morphological basis and suggest a role of PACAP(-like peptides) in the reproductive and developmental functions of invertebrates.