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1.
J Forensic Leg Med ; 82: 102228, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34388443

RESUMO

Even if the Amussat's sign is known since the mid-19th century, few studies have been made in order to assess its real occurrence. In particular, the histopathologic examination of the Amussat's sign lacks in the medicolegal literature. The review of the literature shows indeed a significant range of variability (from 1.1 % up to 25 %) regarding the macroscopic detection of the Amussat's sign. In this study, the authors report that the identification of a vital Amussat's sign is important and may require the immunohistochemical staining for the Glycophorin A (a marker of vital reaction). The victim was a 63-year-old man, who was found suspended from the staircase with a rope. Both the carotid arteries were opened in situ by using fine scissors with blunt tips. A horizontal lesion (length 4 mm) of the intima of the left common carotid artery was documented. A sample was obtained; then, a standard post-fixative histopathologic examination and immunohistochemical staining for the Glycophorin A were carried out. The standard histopathologic examination only revealed the intimal laceration with a poor hemorrhagic infiltration. However, the immunohistochemical staining for the Glycophorin A allowed the clear identification of the hemorrhagic infiltration, which was documented both in the intimal laceration and in the periadventitial soft tissues. The immunohistochemical staining for the Glycophorin A can identify the vitality of an Amussat's sign. When an Amussat's sign is documented, the Glycophorin A may therefore help the forensic pathologist to differentiate a hanging death from a postmortem suspension of the body.


Assuntos
Artérias Carótidas/patologia , Glicoforinas/sangue , Túnica Íntima/patologia , Hemorragia/patologia , Humanos , Imuno-Histoquímica/métodos , Lacerações/sangue , Masculino , Pessoa de Meia-Idade , Mudanças Depois da Morte , Suicídio
2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204207

RESUMO

ANCA-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). While systemic vasculitis is a hallmark of all AAV, GPA is characterized by extravascular granulomatous inflammation, preferentially affecting the respiratory tract. The mechanisms underlying the emergence of neutrophilic microabscesses; the appearance of multinucleated giant cells; and subsequent granuloma formation, finally leading to scarred or destroyed tissue in GPA, are still incompletely understood. This review summarizes findings describing the presence and function of molecules and cells contributing to granulomatous inflammation in the respiratory tract and to renal inflammation observed in GPA. In addition, factors affecting or promoting the development of granulomatous inflammation such as microbial infections, the nasal microbiome, and the release of damage-associated molecular patterns (DAMP) are discussed. Further, on the basis of numerous results, we argue that, in situ, various ways of exposure linked with a high number of infiltrating proteinase 3 (PR3)- and myeloperoxidase (MPO)-expressing leukocytes lower the threshold for the presentation of an altered PR3 and possibly also of MPO, provoking the local development of ANCA autoimmune responses, aided by the formation of ectopic lymphoid structures. Although extravascular granulomatous inflammation is unique to GPA, similar molecular and cellular patterns can be found in both the respiratory tract and kidney tissue of GPA and MPA patients; for example, the antimicrobial peptide LL37, CD163+ macrophages, or regulatory T cells. Therefore, we postulate that granulomatous inflammation in GPA or PR3-AAV is intertwined with autoimmune and destructive mechanisms also seen at other sites.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/etiologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Suscetibilidade a Doenças , Granulomatose com Poliangiite/etiologia , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/diagnóstico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/terapia , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade , Biomarcadores , Movimento Celular/imunologia , Gerenciamento Clínico , Granulomatose com Poliangiite/diagnóstico , Granulomatose com Poliangiite/metabolismo , Granulomatose com Poliangiite/terapia , Humanos , Imunidade Inata , Imuno-Histoquímica/métodos , Especificidade de Órgãos/imunologia
3.
Methods Mol Biol ; 2350: 105-123, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331282

RESUMO

Early detection of malignant tumors, micrometastases, and disseminated tumor cells is one of the effective way of fighting cancer. Among the many existing imaging methods like computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), optical imaging with fluorescent probes is one of the most promising alternatives because it is fast, inexpensive, safe, sensitive, and specific. However, traditional fluorescent probes, based on organic fluorescent dyes, suffer from the low signal-to-noise ratio. Furthermore, conventional organic fluorescent dyes are unsuitable for deep tissue imaging because of the strong visible light absorption by biological tissues. The use of fluorescent semiconductor nanocrystals, or quantum dots (QDs), may overcome this limitation due to their large multiphoton cross section, which ensures efficient imaging of thick tissue sections inaccessible with conventional fluorescent probes. Moreover, the lower photobleaching and higher brightness of fluorescence signals from QDs ensures a much better discrimination of positive signals from the background. The use of fluorescent nanoprobes based on QDs conjugated to uniformly oriented high-affinity single-domain antibodies (sdAbs) may significantly increase the sensitivity and specificity due to better recognition of analytes and deeper penetration into tissues due to small size of such nanoprobes.Here, we describe a protocol for the fabrication of nanoprobes based on sdAbs and QDs, preparation of experimental xenograft mouse models for quality control, and multiphoton imaging of deep-tissue solid tumors, micrometastases, and disseminated tumor cells.


Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Pontos Quânticos , Anticorpos de Domínio Único , Linhagem Celular Tumoral , Imunofluorescência/normas , Humanos , Imunoconjugados/química , Imuno-Histoquímica/métodos , Sondas Moleculares , Imagem Multimodal/métodos , Nanopartículas , Micrometástase de Neoplasia , Imagem Óptica/métodos
4.
Methods Mol Biol ; 2350: 267-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331291

RESUMO

The UltraPlex method for multiplexed two-dimensional fluorescent immunohistochemistry is described, in which hapten tags conjugated to primary antibodies facilitate multiplexed imaging of four or more antigens per tissue section at once. Anti-hapten secondary antibodies labeled with fluorophores provide amplified signal for detection, which is accomplished using a standard fluorescent microscope or digital slide scanner. The protocol is rapid and straightforward and utilizes conventionally prepared tissue samples. The resulting staining is highly sensitive and specific, enabling high-resolution imaging of multiple cellular subtypes within tissue samples. Tumor cells and tumor-infiltrating lymphocytes are presented as examples. Multiple 4-plex-stained tissue samples can be digitally overlaid to create 8-plex (or more) high-content images, enabling visualization of distribution of complex cellular subtypes across tissues.


Assuntos
Imunofluorescência , Haptenos , Imuno-Histoquímica/métodos , Biomarcadores , Biomarcadores Tumorais , Análise de Dados , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Coloração e Rotulagem
5.
Methods Mol Biol ; 2350: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331294

RESUMO

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.


Assuntos
Biomarcadores , Matriz Extracelular/metabolismo , Histocitoquímica/métodos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Glicosilação , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Pesquisa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fluxo de Trabalho
6.
Methods Mol Biol ; 2319: 61-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331243

RESUMO

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.


Assuntos
Células Endoteliais/citologia , Imuno-Histoquímica/métodos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Pele/citologia , Coloração e Rotulagem/métodos , Animais , Células Endoteliais/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Pele/crescimento & desenvolvimento , Pele/metabolismo
7.
Anticancer Res ; 41(7): 3439-3448, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230139

RESUMO

BACKGROUND/AIM: The role of immune cells and PD-L1 in cutaneous squamous carcinogenesis is unclear. This study examines T-cell populations, Langerhans cells (LCs) and PD-L1 in invasive squamous cell carcinoma (inSCC), adjacent precursors and normal skin (NS) to investigate their participation in tumorigenesis. MATERIALS AND METHODS: Cases of cutaneous inSCC with adjacent precursors (n=125) were selected. In situ SCC (isSCC) and actinic keratosis (AK) were observed in 53 and 123 cases, respectively, whereas NS was present in 123 lesions. Immunohistochemistry was performed for CD3, CD8, Foxp3, CD1a and PD-L1. RESULTS: T-cells, LCs and PD-L1 gradually increase during the evolution from AK to isSCC and inSCC, with statistical significance between all lesions, except for CD3+ and CD8+ cells between isSCC and inSCC. Epithelial PD-L1 expression correlates with tumor diameter and thickness. CONCLUSION: The progressive increase of T-cells, LCs and PD-L1 in cutaneous squamous carcinogenesis provides rationale for immunotherapy and identification of predictive biomarkers.


Assuntos
Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Células de Langerhans/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Subpopulações de Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Células de Langerhans/imunologia , Células de Langerhans/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral/imunologia
8.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298861

RESUMO

The pathogenesis of hidradenitis suppurativa (HS) is yet to be fully understood. However, inflammation is a key element in the development of skin lesions. The aim of this study was to evaluate the expression of monocyte chemotactic protein-1-induced protein-1 (MCPIP1) in the skin of patients suffering from HS. Skin biopsies of 15 patients with HS and 15 healthy controls were obtained and processed for immunohistochemistry, western blot, and real time PCR. The highest mean MCPIP1 mRNA expression was found in the inflammatory lesional skin of HS patients. It was significantly higher than MCPIP1 mRNA expression in the biopsies from both healthy controls and non-lesional skin of HS patients. Western blot analysis indicated that expression of MCPIP1 was elevated within both lesional and non-lesional skin compared to the healthy control. The increased MCPIP1 mRNA and protein expression level in HS lesions may indicate its possible role in the disease pathogenesis.


Assuntos
Hidradenite Supurativa/metabolismo , Queratinócitos/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica/métodos , Inflamação/metabolismo , Masculino , RNA Mensageiro/metabolismo , Pele/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281183

RESUMO

Cryptorchidism in horses is a commonly occurring malformation. The molecular basis of this pathology is not fully known. In addition, the origins of high intratesticular estrogen levels in horses remain obscure. In order to investigate the role of the G-protein-coupled membrane estrogen receptor (GPER) and establish histological and biochemical cryptorchid testis status, healthy and cryptorchid horse testes were subjected to scanning electron microscopy analysis, histochemical staining for total protein (with naphthol blue black; NBB), acid content (with toluidine blue O; TBO), and polysaccharide content (with periodic acid-Schiff; PAS). The expression of GPER was analyzed by immunohistochemistry and Western blot. GPER-mediated intracellular cAMP and calcium (Ca2+) signaling were measured immunoenzymatically or colorimetrically. Our data revealed changes in the distribution of polysaccharide content but not the protein and acid content in the cryptorchid testis. Polysaccharides seemed to be partially translocated from the interstitial compartment to the seminiferous tubule compartment. Moreover, the markedly decreased expression of GPER and GPER downstream molecules, cAMP and Ca2+, suggests their potential role in testis pathology. Increased estrogen levels in cryptorchid conditions may be linked to disturbed GPER signaling. We postulate that GPER is a prominent key player in testis development and function and may be used as a new biomarker of horse testis in health and disease.


Assuntos
Criptorquidismo/veterinária , Doenças dos Cavalos/metabolismo , Receptores de Estrogênio/metabolismo , Testículo/metabolismo , Animais , Western Blotting/métodos , Criptorquidismo/metabolismo , Estrogênios/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cavalos , Imuno-Histoquímica/métodos , Masculino , Microscopia Eletrônica de Varredura/métodos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
10.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071338

RESUMO

Although radiological diagnostics have been progressing, pathological diagnosis remains the most reliable method for diagnosing liver tumors. In some cases, definite pathological diagnosis cannot be obtained by histological evaluation alone, especially when the sample is a small biopsy; in such cases, immunohistochemical staining is very useful. Immunohistochemistry is the most frequently used technique for molecular pathological diagnosis due to its broad application, ease of performance and evaluation, and reasonable cost. The results occasionally reflect specific genetic mutations. The immunohistochemical markers of hepatocellular carcinoma include those of hepatocellular differentiation-such as hepatocyte paraffin 1 and arginase-1-and those of malignant hepatocytes-such as glypican-3, heat shock protein 70, and glutamine synthetase (GS). To classify the subtypes of hepatocellular adenoma, examination of several immunohistochemical markers, such as liver fatty acid-binding protein, GS, and serum amyloid A, is indispensable. Immunohistochemical staining for GS is also important for the diagnosis of focal nodular hyperplasia. The representative immunohistochemical markers of intrahepatic cholangiocarcinoma include cytokeratin (CK) 7 and CK19. In this article, we provide an overview of the application of immunohistochemistry in the pathological diagnosis of liver tumors referring to the association with genetic alterations. Furthermore, we aimed to explain the practical points in the differential diagnosis of liver tumors by immunohistochemical staining.


Assuntos
Adenoma de Células Hepáticas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Colangiocarcinoma/metabolismo , Imuno-Histoquímica/métodos , Neoplasias Hepáticas/metabolismo , Adenoma de Células Hepáticas/diagnóstico , Carcinoma Hepatocelular/diagnóstico , Colangiocarcinoma/diagnóstico , Diagnóstico Diferencial , Glipicanas/metabolismo , Humanos , Queratina-7/metabolismo , Neoplasias Hepáticas/diagnóstico
11.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
12.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065143

RESUMO

In humans, injuries and diseases can result in irreversible tissue or organ loss. This well-known fact has prompted several basic studies on organisms capable of adult regeneration, such as amphibians, bony fish, and invertebrates. These studies have provided important biological information and helped to develop regenerative medicine therapies, but important gaps concerning the regulation of tissue and organ regeneration remain to be elucidated. To this aim, new models for studying regenerative biology could prove helpful. Here, the description of the cephalic tentacle regeneration in the adult of the freshwater snail Pomacea canaliculata is presented. In this invasive mollusk, the whole tentacle is reconstructed within 3 months. Regenerating epithelial, connective, muscular and neural components are already recognizable 72 h post-amputation (hpa). Only in the early phases of regeneration, several hemocytes are retrieved in the forming blastema. In view of quantifying the hemocytes retrieved in regenerating organs, granular hemocytes present in the tentacle blastema at 12 hpa were counted, with a new and specific computer-assisted image analysis protocol. Since it can be applied in absence of specific cell markers and after a common hematoxylin-eosin staining, this protocol could prove helpful to evidence and count the hemocytes interspersed among regenerating tissues, helping to unveil the role of immune-related cells in sensory organ regeneration.


Assuntos
Hemócitos/citologia , Hemócitos/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Regeneração , Caramujos/fisiologia , Animais , Contagem de Células , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Especificidade de Órgãos
13.
Nat Commun ; 12(1): 3852, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158500

RESUMO

Vertebrate muscles and tendons are derived from distinct embryonic origins yet they must interact in order to facilitate muscle contraction and body movements. How robust muscle tendon junctions (MTJs) form to be able to withstand contraction forces is still not understood. Using techniques at a single cell resolution we reexamine the classical view of distinct identities for the tissues composing the musculoskeletal system. We identify fibroblasts that have switched on a myogenic program and demonstrate these dual identity cells fuse into the developing muscle fibers along the MTJs facilitating the introduction of fibroblast-specific transcripts into the elongating myofibers. We suggest this mechanism resulting in a hybrid muscle fiber, primarily along the fiber tips, enables a smooth transition from muscle fiber characteristics towards tendon features essential for forming robust MTJs. We propose that dual characteristics of junctional cells could be a common mechanism for generating stable interactions between tissues throughout the musculoskeletal system.


Assuntos
Fibroblastos/metabolismo , Junções Intercelulares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Tendões/metabolismo , Animais , Fusão Celular , Células Cultivadas , Fibroblastos/citologia , Expressão Gênica , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Muscular/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , RNA-Seq/métodos , Tendões/citologia
14.
Hum Pathol ; 114: 110-119, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33961839

RESUMO

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.


Assuntos
COVID-19/patologia , Fígado/virologia , Pulmão/virologia , SARS-CoV-2/patogenicidade , Animais , Autopsia/métodos , COVID-19/virologia , Chlorocebus aethiops , Progressão da Doença , Humanos , Imuno-Histoquímica/métodos , Fígado/química , Fígado/patologia , Pulmão/patologia , RNA Viral/metabolismo , Células Vero/virologia , Carga Viral/métodos
15.
Methods Mol Biol ; 2325: 107-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053054

RESUMO

Tissue microarray (TMA) is a smart technical innovation recently imposed in pathology research. This technology provides a high-throughput analysis of multiple tissues at the same time. The technique allows faster analysis and considerably reducing costs for the staining because many small representative tissue samples from hundreds of different cases are assembled on a single histologic slide. This versatile technique may improve conventional microscopic techniques to detect and characterize cytotoxic T lymphocytes (CTL). Immunohistochemistry (IHC) may be effectively employed in CTL characterization to identify the location and distribution of target antigens in tissues by staining with a specific antibody. The antibody may be conjugated to either a fluorescent or enzymatic label, and the location of the label seen through a microscope approximates the position of the target antigen.This article summarizes the technical aspects of tissue microarray construction and sectioning, advantages, application, and limitations associated with immunohistochemistry and immunofluorescence.


Assuntos
Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Linfócitos T Citotóxicos/metabolismo , Análise Serial de Tecidos/métodos , Anticorpos , Antígenos , Corantes Fluorescentes , Humanos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos
16.
Lab Invest ; 101(8): 970-982, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34006891

RESUMO

Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n = 22) taken at 6 weeks post-transplantation. Patients were stratified for IFTA development (<10% versus ≥10%) from 6 weeks to 6 months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios, and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at 6 weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development 6 months post-transplantation (p < 0.05). CD3+CD8-/CD3+CD8+ ratios were higher in patients with <10% IFTA development (p < 0.05). We observed a high correlation between CD163+ and CD4+GATA3+ cell density (R = 0.74, p < 0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi spectrally imaged slides of kidney transplant biopsies.


Assuntos
Aprendizado Profundo , Imuno-Histoquímica/métodos , Transplante de Rim , Insuficiência Renal Crônica/patologia , Imunologia de Transplantes , Adulto , Idoso , Biópsia , Feminino , Humanos , Inflamação/patologia , Rim/citologia , Rim/diagnóstico por imagem , Rim/patologia , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/diagnóstico por imagem
17.
Methods Mol Biol ; 2318: 209-229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019292

RESUMO

Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell cycle arrest or cell death by apoptosis. Elevated and deregulated expression of the Myc protein promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry, microscopy, and immunoblotting, and in vivo using immunohistochemical staining, immunoblotting, and analysis of RNA expression.


Assuntos
Citometria de Fluxo/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Anexina A5/metabolismo , Apoptose/genética , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Proliferação de Células/genética , DNA/genética , Genes myc , Humanos , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo
18.
Methods Mol Biol ; 2318: 313-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019299

RESUMO

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.


Assuntos
Hibridização in Situ Fluorescente/métodos , Hibridização In Situ/métodos , Proteínas Proto-Oncogênicas c-myc/imunologia , Compostos Cromogênicos/química , DNA/genética , Sondas de DNA , Amplificação de Genes , Genes myc/genética , Genes myc/fisiologia , Humanos , Imuno-Histoquímica/métodos , Neoplasias , Inclusão em Parafina/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Translocação Genética
19.
Turk Neurosurg ; 31(4): 623-633, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33978223

RESUMO

AIM: To investigate neurogenesis in both adult and 3-week-old genetic absence epilepsy rats from Strasbourg (GAERS) to determine if newly formed neurons within the dentate gyrus (DG) form synaptic contacts with GABAergic (gamma aminobutyric acid) and glutamatergic nerve terminals and compared to the control (non-GAERS) Wistar rats. MATERIAL AND METHODS: Brain tissue was processed for electron microscopic assessment. Thin sections from the hippocampal DG were double-labelled for anti-GABA or anti-VGLUT1 (vesicular glutamate transporter 1) and anti-doublecortin (DCX) antibodies using immunogold methodology and examined with the transmission electron microscope for morphological changes and to quantify the density of gold labeling. RESULTS: DCX immunoreactivity was demonstrated within axon terminals, dendrites and somata in all groups. DCX and GABA or VGLUT1 were found to be co-localized in the axon terminals in all groups. We observed that DCX-immunoreactive (-ir) profiles formed synaptic contacts with GABAergic and glutamatergic terminals. The percentage of DCX labeling in dendrites, compared to axons, and the percentage of DCX-ir terminal profiles forming asymmetrical synapses, compared to those forming symmetrical synapses, were increased in all groups compared to the control group. DCX immunoreactivity in the 21-day-old GAERS group was found to be increased compared to the Wistar group. CONCLUSION: We conclude that newly born neurons are incorporated into the local hippocampal network in both the GAERS and the control Wistar rats. The results suggest that the neurogenesis taking place in the hippocampus may also be involved in the mechanism underlying absence seizures in GAERS.


Assuntos
Epilepsia Tipo Ausência/genética , Epilepsia Tipo Ausência/fisiopatologia , Neurogênese/fisiologia , Animais , Epilepsia Tipo Ausência/diagnóstico , Epilepsia Tipo Ausência/metabolismo , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Imuno-Histoquímica/métodos , Masculino , Microscopia Eletrônica/métodos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Ratos , Ratos Transgênicos , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/metabolismo
20.
Cancer Sci ; 112(7): 2895-2904, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33931909

RESUMO

Several therapeutic regimens, including neoadjuvant chemoradiation therapy (NACRT), have been reported to serve as anticancer immune effectors. However, there remain insufficient data regarding the immune response after NACRT in pancreatic ductal adenocarcinoma (PDAC) patients. Data from 40 PDAC patients that underwent surgical resection after NACRT (NACRT group) and 30 PDAC patients that underwent upfront surgery (US group) were analyzed to examine alterations in immune cell counts/distribution using a multiplexed fluorescent immunohistochemistry system. All immune cells were more abundant in the cancer stroma than in the cancer cell nest regardless of preoperative therapy. Although the stromal counts of CD4+ T cells, CD20+ B cells, and Foxp3+ T cells in the NACRT group were drastically decreased in comparison with those of the US group, counts of these cell types in the cancer cell nest were not significantly different between the two groups. In contrast, CD204+ macrophage counts in the cancer stroma were similar between the NACRT and US groups, while those in the cancer cell nests were significantly reduced in the NACRT group. Following multivariate analysis, only a high CD204+ macrophage count in the cancer cell nest remained an independent predictor of shorter relapse-free survival (odds ratio = 2.37; P = .033). NACRT for PDAC decreased overall immune cell counts, but these changes were heterogeneous within the cancer cell nests and cancer stroma. The CD204+ macrophage count in the cancer cell nest is an independent predictor of early disease recurrence in PDAC patients after NACRT.


Assuntos
Carcinoma Ductal Pancreático/terapia , Quimiorradioterapia Adjuvante , Imunidade Celular , Neoplasias Pancreáticas/terapia , Microambiente Tumoral/imunologia , Idoso , Antígenos CD20 , Linfócitos B/imunologia , Contagem de Linfócito CD4 , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/cirurgia , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Imuno-Histoquímica/métodos , Contagem de Linfócitos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Terapia Neoadjuvante/métodos , Recidiva Local de Neoplasia/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/cirurgia , Cuidados Pré-Operatórios
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