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Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.
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Doenças do Cão , Melanoma , Neoplasias Bucais , Células Neoplásicas Circulantes , Cães , Animais , Doenças do Cão/patologia , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Projetos Piloto , Células Neoplásicas Circulantes/patologia , Neoplasias Bucais/veterinária , Neoplasias Bucais/patologia , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/sangue , Melanoma/veterinária , Melanoma/patologia , Melanoma/sangue , Melanoma/diagnóstico , Masculino , Feminino , Imuno-Histoquímica/veterinária , Biomarcadores Tumorais/sangueRESUMO
Infection by the root-knot nematode (RKN), Meloidogyne incognita, impacts crop productivity worldwide, including parsley cultures (Petroselinum crispum). Meloidogyne infection involves a complex relationship between the pathogen and the host plant tissues, leading to the formation of galls and feeding sites that disorganize the vascular system, affecting the development of cultures. Herein, we sought to evaluate the impact of RKN on the agronomic traits, histology, and cell wall components of parsley, with emphasis on giant cell formation. The study consisted of two treatments: (i) control, where 50 individuals of parsley grew without M. incognita inoculation; and (ii) inoculated plants, where 50 individuals were exposed to juveniles (J2) of M. incognita. Meloidogyne incognita infection affected the development of parsley, reducing the growth of some agronomical characteristics such as root weight and shoot weight and height. Giant cell formation was noticed at 18 days after inoculation, promoting disorganization of the vascular system. Epitopes of HGs detected in giant cells reveal the continuous capacity of giant cells to elongate under the stimulus of RKN, essential processes for feeding site establishment. In addition, the detection of epitopes of HGs with low and high methyl-esterified groups indicates the PMEs activity despite biotic stress.
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Petroselinum , Tylenchoidea , Humanos , Animais , Parede CelularRESUMO
Introducción: la citología permite examinar células de un tejido de manera mínimamente invasiva, sin embargo, la capacidad de realizar técnicas complementarias como la inmunocitoquímica (ICQ) no está exenta de dificultades. Es el objetivo de nuestro trabajo presentar una metodología que permita la utilización de ICQ automatizada asociada a un análisis automatizado mediante técnica de patología digital. Métodos: se incluyeron 5 sujetos sanos y se obtuvieron muestras de superficie ocular utilizando un citocepillo. La muestra fue procesada de manera automatizada mediante citología en fase líquida. Posteriormente se realizó ICQ automatizada para detectar la positividad nuclear del receptor de vitamina D. Para la evaluación, se utilizaron dos métodos: cuantificación directa bajo microscopio de luz y análisis automatizado usando analizador de imágenes en las diapositivas digitales obtenidas con un Scanner. El porcentaje de positividad encontrado con ambos métodos fueron comparados utilizando la prueba de Kappa. Resultados: todas las muestras presentaron una celularidad adecuada. En todos los casos fue posible realizar ICQ automatizada, más aún, todas las muestras presentaron una calidad óptima. Al comparar ambos métodos (manual versus automatizado) se observó un nivel de acuerdo sustancial (Kappa=0,69). Conclusiones: la metodología presentada en este manuscrito permite la evaluación automatizada de marcadores inmunohistoquímicos de la superficie ocular de manera mínimamente invasiva, siendo similar al conteo manual, pero más objetivo y reproducible. Esta técnica podría ser útil para el estudio proteómico en patologías como la enfermedad por ojo seco.
Introduction: Cytology tests use small amounts of tissue samples for diagnosis as a minimally invasive technique; however, the ability to perform complementary methods such as immunocytochemistry (ICC) is not without difficulties. The aim of our work is to present a method that allows the use of automated ICC associated with an automated image analysis using digital pathology. Methods: Five healthy subjects were included, and ocular surface samples were obtained using a cytobrush. The sample was processed as liquid-based cytology. Automated ICC was subsequently performed to detect vitamin D receptor nuclear positivity. Two methods were used for evaluation: manual counting under a light microscope and automated analysis using an image analyzer on digitized slides. The percentage of positivity found in both methods was compared using the Kappa test. Results: All samples presented adequate cellularity. In all cases, it was possible to perform automated ICC; moreover, all samples presented optimal quality. When comparing both methods (manual versus automated), a substantial level of agreement was seen (Kappa=0.69). Conclusions. The method presented in this manuscript allows the minimally invasive automated evaluation of ocular surface ICC markers, being like manual counting but more objective and reproducible. This technique could be useful for proteomic study in pathologies such as dry eye disease.
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The red-handed howler monkey (Alouatta belzebul) is one of the 35 threatened Brazilian primate species found in two highly endangered Brazilian biomes. Their Amazonian native populations have been declining due to exponential deforestation associated with human activities, especially the construction of dams. The studied population (n = 27) was located in the Belo Monte dam Area of Influence. For the first time, we presented hematological parameters and the basic profile of T (CD3) and B (BSAP PAX5) cells by immunocytochemistry. The results supported the hypothesis that the immuno-hematological profile is influenced by sex, age, and season. Eosinophils were significantly higher in females (p = 0.03), monocytes statistically greater in juveniles (p = 0.04), and total plasma protein increased significantly (p > 0.001) during the dry season. Furthermore, adults showed a statistically higher average absolute number of B lymphocytes than young individuals (p = 0.03), in contrast to T lymphocytes. Even without knowing the full history of antigenic exposure, these results not only contribute to elucidating the boundaries between health and disease but may help lay the groundwork for future research into the effects of anthropogenic stress on immune activation.
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Alouatta , Feminino , Humanos , Animais , Alouatta/fisiologia , Ecossistema , Primatas , Estações do AnoRESUMO
INTRODUCTION: In women, most malignant effusions are from breast and ovary primary carcinomas that have metastasized to body cavity fluids (pleural, peritoneal and pericardial). When carcinoma is diagnosed in effusions, it is not possible to identify its site of origin solely by cytology (morphology); therefore, immunocytochemistry is used as a complementary method. There are no immunocytochemical markers with 100% sensitivity and specificity for identifying carcinoma primary site. The markers most used are TTF-1 for the lung, GATA-3 for the breast, and PAX-8 for the ovary. The aim of this study was to evaluate the sensitivity and specificity of a panel including these markers for detecting the primary site of carcinoma in effusions. METHODS: Samples of pleural, pericardial, and peritoneal effusions and peritoneal washings with carcinoma of known primary site from women (n = 60) and men (n = 18) were prepared by using the cell block method, and immunocytochemistry was performed to evaluate the expression of primary site markers (TTF-1, PAX-8, and GATA-3). RESULTS: In women, the breast was the most frequent primary site of metastatic carcinoma to both pleural and pericardial cavities, followed by the lung, whereas the ovary was the most frequent primary site of carcinoma within peritoneal effusions and washings, followed by the gastrointestinal tract (stomach or intestine). The expected profiles for carcinomas of the most common primary sites were: breast (GATA-3 (+), PAX-8 (-), TTF-1 (-)), ovary (PAX-8 (+), GATA-3 (-), TTF-1 (-)), lung (TTF-1 (+), PAX-8 (-) GATA-3 (-)) and gastrointestinal tract (PAX-8 (-), GATA-3 (-), TTF-1 (-)). These were observed in 88.23% (45/51) of women's samples with carcinoma from these primary sites. By using TTF-1 as the sole primary site marker, 6.25% of carcinomas of primary site other than the lung would have been misdiagnosed. CONCLUSION: An initial panel of markers including GATA-3, PAX-8, and TTF-1 allows, with high sensitivity and specificity, the identification or exclusion of frequent primary sites of carcinoma in effusions from women. Our results highlight the importance of using a panel of markers to avoid misidentification of the primary site of tumor.
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The basic goal of all histological techniques is the identification of tissue components, both normal and pathologic. In the case of any immunodetection technique we expect: specific detection of the antigen with a mark strong enough to be easily detected; and a clear preservation of histological and cytological details of the environment. To achieve these expectations the technical procedures employed must be clearly understood so we can control the previous methodological steps, avoiding any failure and/or complication that could seriously affect the results of our study. In this chapter, we review the basic concepts related to representability of the sample, fixation process, and all procedures carried out before performing the immunohistochemical technique on paraffin sections, which constitute what we know as sample preparation. We describe and analyze the key events so the pathologist and/or the researcher can control the variables in order to obtain the best results in an immunodetection procedure.
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Imuno-Histoquímica , Antígenos , Inclusão em Parafina , Fixação de TecidosRESUMO
One of the biggest dilemmas facing a cytopathology slide is the differential diagnosis of follicular thyroid lesions, grouped as follicular pattern lesions, which include goiter, follicular adenoma and follicular carcinoma, follicular variant of papillary thyroid carcinoma and non-invasive follicular thyroid neoplasm with papillary like nuclear features. Such lesions share many characteristics, which makes the proper identification of malignant follicular lesions a challenge. The cytology obtained through fine needle aspiration puncture is the most effective standard method for diagnosis of thyroid nodules, but its diagnostic efficacy clearly decreases in lesions of thyroid follicular pattern. Thus, a series of auxiliary tools for diagnoses, such as morphometry and nuclear texture analysis, have been increasingly used in the pathologist's practice, as an objective and reproducible tool. These are techniques, which depend on the incorporation of software to digital image analysis and can add accuracy to classical morphological analysis and immunohistochemistry in the evaluation of follicular pattern lesions. In addition to immunocytochemistry and molecular techniques, morphometry allows the estimation of parameters identified in individual cells and represents a tool that, based on quantitative parameters, translates reliable parameters for objective classification of the malignancy. This study aims to review the nuclear characteristics and their role in the diagnosis of follicular thyroid lesions.
Um dos maiores dilemas diante de uma lâmina de citopatologia é o diagnóstico diferencial de lesões foliculares da tiroide agrupadas como lesões de padrão folicular e que incluem; bócio, adenoma e carcinoma foliculares, carcinoma papilífero variante folicular e a neoplasia folicular não invasiva com características nucleares papilares (Uno de los mayores dilemas que presenta una muestra de citopatología es el diagnóstico diferencial de las lesiones foliculares tiroideas reunidas como lesiones de patrón folicular, que incluyen: bocio, adenoma folicular, carcinoma folicular, variante folicular del carcinoma papilar y la neoplasia folicular no invasiva con características nucleares de tipo papilar). Tais lesões compartilham muitas características, o que faz com que a identificação adequada de lesões foliculares malignas represente um desafio. A citologia obtida através de punção aspirativa por agulha fina é o método padrão mais efetivo para diagnóstico em nódulos de tiroide, mas sua eficácia diagnóstica diminui nitidamente em lesões de padrão folicular da tiroide (La citología por punción y aspiración con aguja fina es el método estándar más eficaz para el diagnóstico de los nódulos tiroideos, pero su eficacia diagnóstica se ve notablemente reducida en las lesiones de patrón folicular de la tiroides). Assim, uma série de ferramentas auxiliares ao diagnóstico, como a morfometria e a análise de textura nuclear, têm sido utilizadas cada vez mais na prática do patologista, como ferramenta objetiva e reproduzível. São técnicas que dependem da incorporação de softwares para análise digital de imagens e podem agregar acurácia à análise morfológica clássica e à imunohistoquímica na avaliação de lesões de padrão folicular (para el análisis de imágenes digitales y puede agregar precisión al análisis morfológico clásico y la inmunohistoquímica en la evaluación de lesiones de patrón folicular). Somando-se à imunocitoquímica e às técnicas moleculares, a morfometria permite a estimativa de parâmetros identificados em células individuais e representam uma ferramenta que, a partir de parâmetros quantitativos, traduz parâmetros confiáveis para classificação objetiva de malignidade. O objetivo deste estudo é rever as características nucleares e seu papel no diagnóstico de lesões foliculares da tiroide (es revisar las características nucleares y su papel en el diagnóstico de las lesiones foliculares tiroideas).
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Câncer Papilífero da Tireoide , Glândula Tireoide , Imuno-Histoquímica , Adenoma , Carcinoma Papilar, Variante Folicular , Biologia CelularRESUMO
Super-host plants are elegant models to evaluate the peculiarities of gall structural and nutritional profiles due to the stimuli of distinct gall inducers in temporal and spatial perspectives. Galls induced by congeneric insects, Lopesia spp. (Diptera, Cecidomyiidae) on the same host plant, Mimosa gemmulata Barneby (Fabaceae) were analyzed to estimate if variations of 1 or 2 months in gall lifespans may result in differences over the accumulation of nutritional resources, and their compartmentalization both in cell walls and protoplasm. Mimosa gemmulata hosts four Lopesia-induced galls: the lenticular bivalve-shaped gall (LG) with a 2-month life cycle, the brown lanceolate bivalve-shaped gall (BLG) and the green lanceolate bivalve-shaped gall (GLG) with 3 month-life cycles, and the globoid bivalve-shaped gall (GG) with a 4 month-life cycle. The comparisons among the four Lopesia galls, using anatomical, histometric, histochemical, and immunocytochemical tools, have demonstrated that the longest lifespan of the GG related to its highest increment in structural and nutritional traits compared with the LG, GLG, and BLG. The differences among the tissue stratification and cell wall thickness of the galls with the 2-month and the 3-month lifespans were subtle. However, the GG had thicker cell walls and higher stratification of the common storage tissue, schlerenchymatic layers and typical nutritive tissue than the other three gall morphospecies. The higher tissue thickness of the GG was followed by the formation of a bidirectional gradient of carbohydrates in the protoplasm, and the detection of xyloglucans in cell walls. Current data supported the presumption that the longest the lifespan, the highest the impact over the structural and nutritional metabolism of the Lopesia galls associated to M. gemmulata.
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3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.
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Alginatos/química , Técnicas de Cultura de Células , Imunofluorescência , Hepatócitos/efeitos dos fármacos , Microscopia Confocal , Testes de Toxicidade , Biomarcadores/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Esferoides CelularesRESUMO
Different species of the genus Pouteria have been used in folk medicine for the treatment of inflammation, fever, ulcers, diabetes, and diarrhea. We analyzed the phytochemical profile of the hydroethanolic extract from Pouteria ramiflora leaves by electrospray ionization ion trap tandem mass spectrometry and high-performance liquid chromatography-diode array detection, and examined whether it alone and in combination with cisplatin interfered with cell proliferation and death processes in HepG2 (human hepatocellular carcinoma) and FGH (human gingival fibroblasts) cells. Five compounds were identified in the extract: gallic acid, myricetin-3-O-α-l-arabinopyranoside, quercetin-3-O-ß-d-galactopyranoside, myricetin-3-O-α-l-rhamnopyranoside, and myricetin-3-O-ß-d-galactopyranoside. The extract was cytotoxic to both cell lines by inducing apoptotic cell death and acted in synergy with cisplatin; such effect was stronger in HepG2 cells than in FGH cells, demonstrating some selectivity to tumor cells. In HepG2 cells, the extract exerted antiproliferative effect mediated by induction of cell cycle arrest at the S and G2/M phases. Association of the extract with cisplatin enhanced the latter's antiproliferative effect, arrested the cell cycle at the S phase by CDK2 modulation, and reduced the number of anti-cyclin D1-stained HepG2 cells. Simultaneous treatment with the extract and cisplatin increased the latter's cytotoxicity, apoptotic cell death, and BAX expression in HepG2 cells. Altogether, the results reported herein indicate that P. ramiflora extract is a possible adjuvant to cancer therapy, which can circumvent the cisplatin-mediated resistance mechanisms in cancer cells.
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Pouteria , Apoptose , Proliferação de Células , Cisplatino/farmacologia , Células Hep G2 , Humanos , Compostos Fitoquímicos , Extratos Vegetais/farmacologiaRESUMO
Most seeds store reserves, which mobilization after germination is complex and diversified among plant species. Information on the reserve mobilization in recalcitrant seeds (i.e., intolerant to desiccation) is scarce. The aim of this work was to characterize the dynamics of reserve mobilization and the degradation pattern of the endospermic cell walls in the recalcitrant seeds of the neotropical palm Mauritia flexuosa. Biometric, anatomical, histochemical, ultrastructural and immunocytochemistry assessments were performed in the endosperm and haustorium (structure of the seedling involved in reserve mobilization), during germination and throughout seedling development. Endo-ß-mannanase activity was assessed. The main reserves stored in the seeds are mucilage in the living protoplast and, mainly, heteromannans in the thick cell walls of the endosperm cells. The reserve mobilization extends for about 180 days, in four phases. During germination, the embryonic reserves are catabolized, which induces the mobilization of the endosperm by establishing the flow of water and carrying substances to the haustorium. After germination, the cells of the endosperm actively control the process of their degradation, which results in the formation of the digestion zone. The growth of the haustorium promotes the crushing of endospermic cells and facilitates the entry of substances via the apoplastic route. The pattern of degradation of endospermic cells involves three phases: 1) mobilization of the vacuolar content by symplastic route; 2) increased vacuole turgor pressure, directing the content of the cytoplasm to the cell walls; 3) degradation of cell walls.
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Arecaceae , Parede Celular/química , Germinação , Mananas/química , Sementes/fisiologia , Sementes/químicaRESUMO
The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti-Vasa, anti-GFRα1, and anti-OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.
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BACKGROUND: Considering the potential of p16 as a marker for diagnosis, prognosis and therapeutic response, the aim of this study was to assess its presence, via immunocytochemistry, in metastatic carcinoma of different primary sites and histological types obtained from effusions and peritoneal washings. A total of 118 samples including 85 of metastatic carcinoma and 33 samples of benign effusion/peritoneal washing were prepared by the plasma/thromboplastin method. Immunocytochemistry reactions were performed on cell block sections using antibodies against p16, claudin-4, MOC-31, calretinin, HBME and CD68. RESULTS: P16 overexpression was observed in 88.23% of all carcinoma samples. All cervix adenocarcinoma samples showed p16 overexpression. Overexpression in adenocarcinomas of ovary, lung and breast was observed in 93.75, 93.10 and 75% of the samples, respectively. Overexpression was observed in all different histological types analyzed: small cell carcinoma (lung), squamous cell carcinoma (cervical) and urothelial carcinoma (bladder). The specificity of p16 for carcinoma detection was of 96.96%. CONCLUSION: Overexpression of p16 was observed in most metastatic carcinoma, from different primary sites and histological types, obtained from effusions and peritoneal washings. Due to its high frequency of overexpression in metastatic carcinoma, p16 may play a possible role in tumor progression and it may be considered as a complementary diagnostic marker depending on histological type and primary site of carcinoma.
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Líquido Ascítico/química , Biomarcadores Tumorais/análise , Carcinoma/diagnóstico , Carcinoma/secundário , Inibidor p16 de Quinase Dependente de Ciclina/análise , Neoplasias/diagnóstico , Neoplasias/patologia , Derrame Pericárdico/química , Derrame Pleural Maligno/química , Antígenos CD/análise , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Biomarcadores Tumorais/imunologia , Calbindina 2/análise , Calbindina 2/imunologia , Claudina-4/análise , Claudina-4/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Molécula de Adesão da Célula Epitelial , Humanos , PrognósticoRESUMO
Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.(AU)
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Animais , Sulfatases/análise , Sulfatases/imunologia , Galinhas/genética , Galinhas/imunologia , Células Satélites de Músculo EsqueléticoRESUMO
Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.
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Animais , Células Satélites de Músculo Esquelético , Galinhas/genética , Galinhas/imunologia , Sulfatases/análise , Sulfatases/imunologiaRESUMO
Epithelial cell adhesion molecule (EpCAM) has been used as diagnostic/prognostic marker and therapeutic target. The aim of the present study was to compare immunoreactivity of antibodies against distinct epitopes in the ectodomain of EpCAM for detection of carcinoma from different primary sites and of different histological types in effusions and peritoneal wash. Two antibodies against epitopes in the EGF-like domain I (clones Moc-31 and Ber-EP4) and one antibody against the epitope in the cysteine-poor region (158210) of EpCAM were used (all commercially available). Independently of the clone used, EpCAM overexpression was observed in almost all samples when all the adenocarcinoma samples were analyzed together. By using Moc-31, EpCAM overexpression was observed in all samples of adenocarcinoma. Absence of EpCAM overexpression was observed in a few adenocarcinoma samples at some sites of tumor origin, including ovary, breast and stomach, when Ber-EP4 and 158210 were used. Regarding carcinomas aside from adenocarcinomas, histological types, such as squamous cell, urothelial and small cell carcinoma showed different degrees of EpCAM expression according to the antibody used. In squamous cell carcinoma, overexpression was observed only with the clone 158210. It was concluded that, overall, most samples of metastatic carcinoma from effusions showed overexpression of EpCAM. However, there are significant variations in its detection according to the primary site, histological type of the carcinoma and depending on the antibody used. Thus, the use of more than one type of anti-EpCAM antibody would increase the chance of its detection in metastatic carcinoma effusion.
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Background: Mycoplasmosis is an infectious disease caused by Mycoplasma gallisepticum (MG), usually seen in therespiratory system of chickens, chick and turkeys, that causing great economic loss. The disease is characterized by respiratory system lesions such as sinusitis, tracheitis, airsacculitis, pneumonia and other symptoms such as loss of yield, arthritis,tenosynovitis. In this study, it was aimed to investigate diagnose of the disease by pathologic and molecular techniquesin hens that naturally infected with MG as well as the usability of immunocytochemical (ICC) method in diagnose of thedisease.Materials, Methods & Results: For this purpose, 98 hens were collected from 10 different coops that serologically positive.After necropsy, routine pathological procedures were performed to samples taken from nose, sinus, larynx, trachea, lungand air sacs. Scraping samples taken from lungs and tracheas were evaluated by ICC. Immunohistochemical (IHC) stainingwas performed to samples taken from nose, sinus, larynx, trachea, lung and air sacs. Indirect immunoperoxidase methodwas applied in the both IHC and ICC staining. Rabbit polyclonal anti MG antibody was used as primer antibody in theIHC and ICC staining. Additionally, culture and PCR techniques were applied to tracheas of all hens for MG. The GPO3and MGSO genes were made for PCR analysis. In the tracheal examinations, 23 cases were positive for PCR, 17 casesICC positive, 16 cases IHC positive and 10 culture samples found positive. All of culture positive cases were also positivefor other three methods. When findings in all organs were evaluated, in 37 cases were detected positive by IHC (38%) and23 cases were positive by ICC (23.5%). In the IHC positive cases, the first order was trachea in 16 cases followed by in 11cases in sinus, in 8 cases in lung, in 6 cases air sac and 4 cases in nose, respectively. In 8 cases, IHC positivity was foundin at least two organs...(AU)
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Animais , Mycoplasma gallisepticum/isolamento & purificação , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/veterinária , Aves Domésticas/virologia , Imuno-Histoquímica/veterinária , Reação em Cadeia da Polimerase/veterináriaRESUMO
The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35S-cysteine and 3H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.
Assuntos
Glicoproteínas/metabolismo , Órgão Subcomissural/fisiologia , Animais , Bovinos , Cisteína/metabolismo , Citoplasma/metabolismo , Epêndima/citologia , Epêndima/metabolismo , Galactose/metabolismo , Galectina 1/metabolismo , Glicoproteínas/ultraestrutura , Glicosilação , Masculino , Polissacarídeos/química , Polissacarídeos/metabolismo , Ratos Sprague-Dawley , Via Secretória , Coloração e Rotulagem , Órgão Subcomissural/ultraestrutura , Radioisótopos de Enxofre/metabolismo , Trítio/metabolismoRESUMO
The present study aimed to compare the expression of p63/p40 with smooth muscle actin (SMA) and vimentin (VIM) by myoepithelial cells in minor salivary gland tumors. Fifty-two formalin-fixed paraffin-embedded samples of minor salivary gland tumors derived from intercalated duct (pleomorphic adenoma [PA], adenoid cystic carcinoma [ACC], epithelial-myoepithelial carcinoma [EMC], polymorphous adenocarcinoma [PAC], and secretory carcinoma [SC]) and 3 samples of minor salivary gland tumors derived from excretory duct (mucoepidermoid carcinoma [MEC]) were evaluated by means of immunohistochemistry. The data were analyzed qualitatively. The results indicated that p63 and p40 expression were detected in myoepithelial cells present in PA, ACC, and EMC. However, both proteins were also observed in squamous areas of PA and all cases of MEC. SMA were noticed in some myoepithelial cells of PA, ACC, and EMC. Expression of SMA was negative in the other salivary gland tumors evaluated. VIM was constantly expressed by myoepithelial cells in PA, ACC, and EMC. VIM was also observed in cells of PAC and SC, but not in squamous areas of PA and MEC. In conclusion, p63 expression is almost comparable with VIM in detecting myoepithelial cells, an immunolabeling pattern not followed by p40, and consequently, caution has to be taken during the interpretation of salivary gland tumor exhibiting an p63/p40 phenotype in order to avoid a misdiagnosis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/patologia , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares Menores/patologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Actinas/análise , Actinas/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Carcinoma/diagnóstico , Diagnóstico Diferencial , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias das Glândulas Salivares/diagnóstico , Glândulas Salivares Menores/citologia , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análise , Vimentina/análise , Vimentina/metabolismo , Adulto JovemRESUMO
The cellular mechanisms of laticifer growth are of particular interest in plant biology but are commonly neglected. Using transmission electron microscopy and immunocytochemical methods, we recorded cytological differentiation and evaluated the cell wall involvement in the growth of articulated laticifers with intrusive growth in the mature embryo and plant shoot apex of Tabernaemontana catharinensis. The incorporation of adjacent meristematic cells into the laticifer system occurred in the embryo and plant shoot apex, and the incorporated cells acquired features of laticifer, confirming the laticifers' action-inducing mechanism. In the embryo, this was the main growth mechanism, and began with enlargement of the plasmodesmata and the formation of pores between laticifers and meristematic cells. In the plant shoot apex, it began with loose and disassembled walls and the reorientation of the cortical microtubules of the incorporated cell. Plasmodesmata were absent in these laticifers. There was stronger evidence of intrusive growth in undifferentiated portions of the plant shoot apex than in the embryo. The numerous plasmodesmata in laticifers of the embryo may have been related to the lower frequency of intrusive growth. Intrusive growth was associated with presence of arabinan (increasing wall flexibility and fluidity), and absence of galactan (avoiding wall stiffness), and callose (as a consequence of reduction in symplastic connections) in the laticifer walls. The abundance of low de-methyl-esterified homogalacturonan in the middle lamella and corners may reestablish cell-cell bonding in the laticifers. The cell wall features differed between embryo and plant shoot apex and are directly associated to laticifer growth mechanisms.