RESUMO
Cell shapes in tissues are affected by the biophysical interaction between cells. Tissue forces can influence specific cell features such as cell geometry and cell surface area. Here, we examined the 2-dimensional shape, size, and perimeter of pleural epithelial cells at various lung volumes. We demonstrated a 1.53-fold increase in 2-dimensional cell surface area and a 1.43-fold increase in cell perimeter at total lung capacity compared to residual lung volume. Consistent with previous results, close inspection of the pleura demonstrated wavy folds between pleural epithelial cells at all lung volumes. To investigate a potential explanation for the wavy folds, we developed a physical simulacrum suggested by D'Arcy Thompson in On Growth and Form. The simulacrum suggested that the wavy folds were the result of redundant cell membranes unable to contract. To test this hypothesis, we developed a numerical simulation to evaluate the impact of an increase in 2-dimensional cell surface area and cell perimeter on the shape of the cell-cell interface. Our simulation demonstrated that an increase in cell perimeter, rather than an increase in 2-dimensional cell surface area, had the most direct impact on the presence of wavy folds. We conclude that wavy folds between pleural epithelial cells reflects buckling forces arising from the excess cell perimeter necessary to accommodate visceral organ expansion.
Assuntos
Células Epiteliais , Pleura , Células Epiteliais/fisiologia , Células Epiteliais/citologia , Pleura/citologia , Pleura/fisiologia , Animais , Forma Celular/fisiologia , Humanos , Pulmão/citologia , Pulmão/fisiologia , Modelos Biológicos , Simulação por Computador , Fenômenos Biomecânicos/fisiologiaRESUMO
BACKGROUND: Changes in epithelial cell shape reflects optimal cell packing and the minimization of surface free energy, but also cell-cell interactions, cell proliferation, and cytoskeletal rearrangements. RESULTS: Here, we studied the structure of the rat pleura in the first 15 days after birth. After pleural isolation and image segmentation, the analysis demonstrated a progression of epithelial order from postnatal day 1 (P1) to P15. The cells with the largest surface area and greatest shape variability were observed at P1. In contrast, the cells with the smallest surface area and most shape consistency were observed at P15. A comparison of polygonal cell geometries demonstrated progressive optimization with an increase in the number of hexagons (six-sided) as well as five-sided and seven-sided polygons. Analysis of the epithelial organization with Voronoi tessellations and graphlet motif frequencies demonstrated a developmental path strikingly distinct from mathematical and natural reference paths. Graph Theory analysis of cell connectivity demonstrated a progressive decrease in network heterogeneity and clustering coefficient from P1 to P15. CONCLUSIONS: We conclude that the rat pleura undergoes a striking change in pleural structure from P1 to P15. Further, a geometric and network-based approach can provide a quantitative characterization of these developmental changes.
Assuntos
Pleura , Animais , Ratos , Pleura/citologia , Células Epiteliais/citologia , Forma Celular/fisiologia , Animais Recém-Nascidos , Ratos Sprague-DawleyRESUMO
Tissue engineering (TE) of long tracheal segments is conceptually appealing for patients with inoperable tracheal pathology. In tracheal TE, stem cells isolated from bone marrow or adipose tissue have been employed, but the ideal cell source has yet to be determined. When considering the origin of stem cells, cells isolated from a source embryonically related to the trachea may be more similar. In this study, we investigated the feasibility of isolating progenitor cells from pleura and pericard as an alternative cells source for tracheal tissue engineering. Porcine progenitor cells were isolated from pleura, pericard, trachea and adipose tissue and expanded in culture. Isolated cells were characterized by PCR, RNA sequencing, differentiation assays and cell survival assays and were compared to trachea and adipose-derived progenitor cells. Progenitor-like cells were successfully isolated and expanded from pericard and pleura as indicated by gene expression and functional analyses. Gene expression analysis and RNA sequencing showed a stem cell signature indicating multipotency, albeit that subtle differences between different cell sources were visible. Functional analysis revealed that these cells were able to differentiate towards chondrogenic, osteogenic and adipogenic lineages. Isolation of progenitor cells from pericard and pleura with stem cell features is feasible. Although functional differences with adipose-derived stem cells were limited, based on their gene expression, pericard- and pleura-derived stem cells may represent a superior autologous cell source for cell seeding in tracheal tissue engineering.
Assuntos
Células-Tronco Multipotentes/citologia , Pericárdio/citologia , Pleura/citologia , Traqueia/citologia , Adipócitos/citologia , Adipogenia/fisiologia , Tecido Adiposo/citologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/fisiologia , Células-Tronco/citologia , Suínos , Engenharia Tecidual/métodosRESUMO
Birt-Hogg-Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms "cell-cell adhesion junction" and "cell adhesion molecule binding". Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.
Assuntos
Síndrome de Birt-Hogg-Dubé/patologia , Líquido da Lavagem Broncoalveolar/citologia , Haploinsuficiência , Pleura/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Apoptose , Síndrome de Birt-Hogg-Dubé/genética , Movimento Celular , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Pleura/patologia , Cultura Primária de Células , Adulto JovemRESUMO
The mechanisms of chemical pleurodesis are still not fully explained. We aimed to evaluate the feasibility of using primary biopsy-derived human mesothelial cells to establish an in vitro culture and to assess the response of pleural mesothelial cells to different sclerosing agents. Talc, povidone-iodine, doxycycline, and TGF-ß were used at different doses to stimulate pleural mesothelial cells. After 6 and 24 h, mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, TGF-ß, MCP-1, IL-17A, and MMP9 was measured in cultured cells, and the protein level of IL-1ß, IL-6, and IL-8 was measured in the culture supernatant. The most pronounced response was observed after talc exposure. It was expressed as an increase in IL-1ß concentration in culture supernatant after 24 h of higher talc dose stimulation compared to 6 h of stimulation (17.14 pg/ml [11.96-33.32 pg/ml] vs. 1.84 pg/ml [1.81-1.90 pg/ml], p = 0.02). We showed that culture pleural mesothelial cells isolated from pleura biopsy specimens is feasible. Inflammatory responses of mesothelial cells to different sclerosants were highly variable with no consistent pattern of mesothelium reaction neither in terms of different sclerosing agents nor in the time of the most significant reaction. We demonstrated that pro-inflammatory mesothelial response includes an increase in IL-1ß mRNA expression and protein production. This may suggest the role of IL-1ß in the formation and maintenance of the inflammatory response during pleurodesis.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Pleura/citologia , Povidona-Iodo/farmacologia , Cultura Primária de Células/métodos , Soluções Esclerosantes/farmacologia , Linhagem Celular , Células Cultivadas , Citocinas/genética , Células Epiteliais/metabolismo , Humanos , Pleurodese/métodos , Talco/toxicidadeRESUMO
ABSTRACT: We evaluated the capacity of the XN-350 instrument to analyze 3 different types of body fluid samples under "body fluid mode."The performance of XN-350 was evaluated in terms of precision, carryover, limit of blank, limit of detection, limit of quantification, and linearity. Cell enumeration and differential data produced by the XN-350 were compared to manual chamber counting results in 63 cerebrospinal fluid (CSF), 51 ascitic fluid, and 51 pleural fluid (PF) samples. Comparisons between XN-350 versus Cytospin data were also performed in PF samples.The precision, carry-over, limit of blank, and linearity of the XN-350 were acceptable. The limits of detection for white blood cells (WBCs) and red blood cells were 1.0/µL, and 1,000.0/µL, respectively; the corresponding limits of quantitation (LOQs) were 5.0/µL and 2,000.0/µL, respectively. The XN-350's cell enumeration and differential counting correlated well with those of manual chamber counting for all 3 sample types (except for differential counting in CSF samples), particularly parameters involving monocytes (râ=â0.33) and mononuclear cells (MO- body fluid [BF]; râ=â0.26), as well as total cell (TC-BF) enumeration (râ=â0.50) and WBC-BF (râ=â0.50) in PF samples. The MO-BF in CSF samples differed significantly from manual chamber counting results, but neither TC-BF nor WBC-BF in PF samples did. The XN-350 also showed good correlations with Cytospin analyses for differential counting of neutrophils, lymphocytes, and monocytes in PF samples. The differential counting of eosinophils via the XN-350 and Cytospin were not significantly correlated, but the difference between them was not significant.The XN-350 is an acceptable alternative to manual fluid analysis. Samples with low cellularity around the LOQ should be checked manually. Moreover, manual differential counting should be performed on CSF samples, particularity those with low cell numbers.
Assuntos
Líquidos Corporais/química , Líquidos Corporais/citologia , Técnicas Citológicas/métodos , Testes Hematológicos/métodos , Microscopia/métodos , Líquido Ascítico/química , Líquido Ascítico/citologia , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/citologia , Humanos , Pleura/citologia , Pleura/metabolismo , Reprodutibilidade dos TestesRESUMO
The health hazard represented by the exposure to asbestos may also concern other minerals with asbestos-like crystal habit. One of these potentially hazardous minerals is fibrous glaucophane. Fibrous glaucophane is a major component of blueschist rocks of California (USA) currently mined for construction purposes. Dust generated by the excavation activities might potentially expose workers and the general public. The aim of this study was to determine whether fibrous glaucophane induces in vitro toxicity effects on lung cells by assessing the biological responses of cultured human pleural mesothelial cells (Met-5A) and THP-1 derived macrophages exposed for 24 h and 48 h to glaucophane fibres. Crocidolite asbestos was tested for comparison. The experimental configuration of the in vitro tests included a cell culture without fibres (i.e., control), cell cultures treated with 50 µg/mL (i.e., 15.6 µg/cm2) of crocidolite fibres and 25-50-100 µg/mL (i.e., 7.8-15.6-31.2 µg/cm2) of glaucophane fibres. Results showed that fibrous glaucophane may induce a decrease in cell viability and an increase in extra-cellular lactate dehydrogenase release in the tested cell cultures in a concentration dependent mode. Moreover, it was found that fibrous glaucophane has a potency to cause oxidative stress. The biological reactivity of fibrous glaucophane confirms that it is a toxic agent and, although it apparently induces lower toxic effects compared to crocidolite, exposure to this fibre may be responsible for the development of lung diseases in exposed unprotected workers and population.
Assuntos
Amiantos Anfibólicos/toxicidade , Asbesto Crocidolita/toxicidade , Macrófagos/efeitos dos fármacos , Pleura/efeitos dos fármacos , Amiantos Anfibólicos/administração & dosagem , Asbesto Crocidolita/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/patologia , Minerais/administração & dosagem , Minerais/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Pleura/citologia , Fatores de TempoRESUMO
Mesothelioma is a cancer of the lung pleura primarily associated with inhalation of asbestos fibers. Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials that pose a potential risk for mesothelioma due to properties that are similar to asbestos. Inhaled MWCNTs migrate to the pleura in rodents and some types cause mesothelioma. Like asbestos, there is a diversity of MWCNT types. We investigated the neoplastic potential of tangled (tMWCNT) versus rigid (rMWCNT) after chronic exposure using serial passages of rat mesothelial cells in vitro. Normal rat mesothelial (NRM2) cells were exposed to tMWCNTs or rMWCNTs for 45 weeks over 85 passages to determine if exposure resulted in transformation to a neoplastic phenotype. Rat mesothelioma (ME1) cells were used as a positive control. Osteopontin (OPN) mRNA was assayed as a biomarker of transformation by real time quantitative polymerase chain reaction (qPCR) and transformation was determined by a cell invasion assay. Exposure to rMWCNTs, but not tMWCNTs, resulted in transformation of NRM2 cells into an invasive phenotype that was similar to ME1 cells. Moreover, exposure of NRM2 cells to rMWCNTs increased OPN mRNA that correlated with cellular transformation. These data suggest that OPN is a potential biomarker that should be further investigated to screen the carcinogenicity of MWCNTs in vitro.
Assuntos
Transformação Celular Neoplásica/genética , Células Epiteliais/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Osteopontina/genética , Animais , Biomarcadores , Linhagem Celular , Células Epiteliais/metabolismo , Masculino , Mesotelioma/genética , Pleura/citologia , RNA Mensageiro , Ratos Endogâmicos F344RESUMO
To elucidate the role of sialomucin in friction reduction, we investigated the sliding friction of pleural mesothelial cells monolayers cultured on fibrine gel. These measurements were performed on normal (4/4 RM-4) and on tumor (CARM-L1 TG3) cell lines. The effect of treatment with neuraminidase, which removes sialic acid from sialomucin, and of dexamethasone, which has shown to increase sialomucin expression, were also assessed. Furthermore, the expression of the main form of cell-surface-associated mucin (MUC1) present in the mesothelium, was assessed by western blot and immunofluorescence, under different experimental conditions. Expression of MUC1 was not significantly different in the two cell lines. Moreover, dexamethasone did not increase the expression of MUC1. Coefficient of kinetic friction (µ) was significantly higher in tumor cells than in normal cells. Neuraminidase increased µ in both cell lines. These results suggest that sialomucin may play a role in reducing the friction of pleural mesothelial cells.
Assuntos
Técnicas de Cultura de Células/métodos , Epitélio , Lubrificação , Mucina-1 , Sialomucinas , Linhagem Celular Tumoral , Células Cultivadas , Fricção/efeitos dos fármacos , Humanos , Mucina-1/efeitos dos fármacos , Mucina-1/metabolismo , Pleura/citologia , Sialomucinas/metabolismo , Sialomucinas/farmacologiaRESUMO
Malignant mesothelioma is an infrequent tumor that initiates from the mesothelial cells lining of body cavities. The great majority of mesotheliomas originate in the pleural cavity, while the remaining cases initiate in the peritoneal cavity, in the pericardial cavity or on the tunica vaginalis. Usually, mesotheliomas grow in a diffuse pattern and tend to enclose and compress the organs in the various body cavities. Mesothelioma incidence is increasing worldwide and still today, the prognosis is very poor, with a reported median survival of approximately one year from presentation. Thus, the development of alternative and more effective therapies is currently an urgent requirement. The aim of this review article was to describe recent findings about the anti-cancer activity of curcumin and some of its derivatives on mesotheliomas. The potential clinical implications of these findings are discussed.
Assuntos
Antineoplásicos/uso terapêutico , Curcumina/uso terapêutico , Mesotelioma Maligno/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Humanos , Mesotelioma Maligno/patologia , Compostos Fitoquímicos/uso terapêutico , Pleura/citologia , Pleura/patologia , Neoplasias Pleurais/patologia , PrognósticoRESUMO
In this study we have utilized an optical clearing method to allow visualization of a heretofore undescribed subpleural acinar structural organization in the mammalian lung. The clearing method enables visualization of the lung structure deep below the visceral pleura in intact inflated lungs. In addition to confirming previous observations that the immediate subpleural alveoli are uniform in appearance, we document for the first time that the subpleural lung parenchyma is much more uniformly organized than the internal parenchyma. Specifically, we report that below the surface layer of alveoli, there is a striking parallel arrangement of alveolar ducts that all run perpendicular to the visceral pleural surface. A three dimensional visualization of alveolar ducts allowed for a calculation of the average inner to outer duct diameter ratio of 0.53 in these subpleural ducts. This unique, self-organizing parallel duct structure likely impacts both elastic recoil and the transmission of tethering forces in healthy and diseased lungs.
Assuntos
Pleura/citologia , Alvéolos Pulmonares/citologia , Animais , Processamento de Imagem Assistida por Computador , Masculino , Camundongos Endogâmicos BALB CRESUMO
Purpose: Although the isolation of rat and mouse mesothelial cells has previously been reported, most mesothelial cells used for experimental studies are obtained from peritoneal cells. Here, we describe an optimized method for the isolation and in vitro propagation of rodent pleural mesothelial cells without the requirement for specialized surgical techniques. Materials and Methods: To harvest pleural mesothelial cells, the pleural space of 8-9-week-old rats or older mice was filled with 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) buffer for 20 min at 37 °C. Cells were then harvested, and incubated at 37 °C in a humidified atmosphere with 5% CO2. Immunofluorescence analysis of plated pleural mesothelial cells was performed using Alexa 546 (calretinin). To investigate optimal proliferation conditions, medium enriched with various concentrations of fetal calf serum (FCS) was used for pleural mesothelial cell proliferation. Results: By day 10, confluent cell cultures were established, and the cells displayed an obvious cobblestone morphology. Immunofluorescence analysis of the cells demonstrated that all stained positive for Alexa 546 (calretinin) expression. Mesothelial cells grew better in medium containing 20% FCS than with 10% FCS. Conclusions: This is a simple procedure for the efficient collection of primary pleural mesothelial cells, which were obtained in defined culture conditions from the euthanized rodent thoracic cavity using trypsin-EDTA treatment. The ability to easily culture and maintain identifiable pleural mesothelial cells from rodents will be helpful for future experiments using these cells.
Assuntos
Pleura/citologia , Cultura Primária de Células , Animais , Camundongos , RatosRESUMO
BACKGROUND: Pleural fibrosis is defined as excessive depositions of matrix components that result in pleural tissue architecture destruction and dysfunction. In severe cases, the progression of pleural fibrosis leads to lung entrapment, resulting in dyspnea and respiratory failure. However, the mechanism of pleural fibrosis is poorly understood. METHODS: miR-4739 levels were detected by miRNA array and real-time PCR. Real-time PCR, western blotting and immunofluorescence were used to identify the expression profile of indicators related to fibrosis. Target gene of miR-4739 and promoter activity assay was measured by using dual-luciferase reporter assay system. In vivo, pleural fibrosis was evaluated by Masson staining and miR-4739 level was detected by In situ hybridization histochemistry. FINDINGS: We found that bleomycin induced up-regulation of miR-4739 in pleural mesothelial cells (PMCs). Over-regulated miR-4739 mediated mesothelial-mesenchymal transition and increased collagen-I synthesis in PMCs. Investigation on the clinical specimens revealed that high levels of miR-4739 and low levels of bone morphogenetic protein 7 (BMP-7) associated with pleural fibrosis in patients. Then we next identified that miR-4739 targeted and down-regulated BMP-7 which further resulted in unbalance between Smad1/5/9 and Smad2/3 signaling. Lastly, in vivo studies revealed that miR-4739 over-expression induced pleural fibrosis, and exogenous BMP-7 prevented pleural fibrosis in mice. INTERPRETATION: Our data indicated that miR-4739 targets BMP-7 which mediates pleural fibrosis. The miR-4739/BMP-7 axis is a promising therapeutic target for the disease. FUND: The National Natural Science Foundation of China.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Bleomicina/farmacologia , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/genética , Colágeno Tipo I/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pleura/citologia , Regiões Promotoras Genéticas , Ratos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
In the present study, we sought to elucidate the mechanisms by which monocytes migrate into the pleural space in the presence of anaphylatoxins in tuberculous pleural effusion (TPE). Monocytes in both pleural effusion and blood were counted, and their phenotypic characteristics were analyzed. Activation of the complement system was detected in TPE. The effects of Mpt64 and anaphylatoxins on the production of chemokines in pleural mesothelial cells (PMCs) were measured. The chemoattractant activity of chemokines produced by PMCs for monocytes was observed. Levels of CD14+CD16+ monocytes were significantly higher in TPE than in blood. Three pathways of the complement system were activated in TPE. C3a-C3aR1, C5a-C5aR1, CCL2-CCR2, CCL7-CCR2, and CX3CL1-CX3CR1 were coexpressed in PMCs and monocytes isolated from TPE. Moreover, we initially found that Mpt64 stimulated the expression of C3a and C5a in PMCs. C3a and C5a not only induced CCL2, CCL7, and CX3CL1 expression in PMCs but also stimulated production of IL-1ß, IL-17, and IL-27 in monocytes. C3a and C5a stimulated PMCs to secrete CCL2, CCL7, and CX3CL1, which recruited CD14+CD16+ monocytes to the pleural cavity. As a result, the infiltration of CD14+CD16+ monocytes engaged in the pathogenesis of TPE by excessive production of inflammatory cytokines.
Assuntos
Anafilatoxinas/metabolismo , Monócitos/metabolismo , Derrame Pleural/patologia , Tuberculose Pulmonar/patologia , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CX3CL1/metabolismo , Complemento C3a/imunologia , Complemento C5a/imunologia , Células Epiteliais , Epitélio/patologia , Humanos , Pleura/citologia , Pleura/patologia , Derrame Pleural/microbiologiaRESUMO
BACKGROUND: Hematologic counters with dedicated body fluids mode allows these samples to be analyzed in an automated manner. Our aim was to evaluate the performance of the Sysmex XN-3000 and compare it with the Sysmex XE-5000, in use in our laboratory for cytological analysis of body fluids. METHODS: We studied 108 pleural and peritoneal fluids. Laboratory routine included manual and automated cell counts. The Sysmex XN-3000 validation protocol included precision, carryover, linearity studies, and comparison with traditional microscopic differential counts and with the analyzer in use. RESULTS: Sysmex XN-3000 met all the criteria for analytical quality with strong correlation with microscopy (r = 0.95 for MN and PMN) and agreement of 93% (kappa = 0.813, p < 0.0001). Comparison between both analyzers revealed no significant differences and strong correlation regarding WBC and RBC (r > 0.98), mononuclear and polymorphonuclear cells (r = 0.99). CONCLUSIONS: Sysmex XN-3000 showed strong correlation and agreement with traditional microscopy with an equivalent performance compared to the XE-5000.
Assuntos
Líquido Ascítico/citologia , Automação Laboratorial , Contagem de Células Sanguíneas/instrumentação , Líquidos Corporais/citologia , Pleura/citologia , Contagem de Células Sanguíneas/métodos , Contagem de Células/instrumentação , Contagem de Eritrócitos , Humanos , Contagem de Leucócitos , Microscopia/instrumentação , Microscopia/métodos , Neutrófilos/citologia , Reprodutibilidade dos TestesRESUMO
We report the case of a 41-year-old woman who presented with a unilateral exudative effusion with prominent eosinophils on pleural cytology. Carbimazole had been started 4 weeks prior to presentation. No immediate cause was identified on imaging or laboratory testing. The effusion persisted at 2-month follow-up. Further investigation at this time, including autoimmune serology was negative. At 2-month follow-up, the effusion was loculated on ultrasound imaging and had a low fluid pH on diagnostic aspiration, in keeping with an empyema. The patient received treatment for pleural empyema, including antibiotics, intercostal drain insertion and video-assisted thoracoscopic pleural biopsy. Carbimazole was stopped, and following treatment for the empyema, the effusion did not reaccumulate.This case illustrates the diagnostic difficulties that pleural effusions may present. It demonstrates that drug reactions should be considered in the differential diagnosis following thorough investigation for other potential causes and also describes the complications that may occur.
Assuntos
Carbimazol/efeitos adversos , Empiema Pleural/patologia , Exsudatos e Transudatos/química , Pleura/patologia , Derrame Pleural/induzido quimicamente , Adulto , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Antitireóideos/efeitos adversos , Diagnóstico Diferencial , Empiema Pleural/diagnóstico por imagem , Empiema Pleural/tratamento farmacológico , Empiema Pleural/cirurgia , Eosinófilos/citologia , Eosinófilos/patologia , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/microbiologia , Feminino , Humanos , Pleura/citologia , Derrame Pleural/diagnóstico por imagem , Derrame Pleural/microbiologia , Streptococcus oralis/isolamento & purificação , Cirurgia Torácica Vídeoassistida/métodos , Tomografia Computadorizada por Raios X/métodos , Resultado do Tratamento , Ultrassonografia/métodosRESUMO
Background: Tumor immune microenvironment (TME) plays a key role in malignant pleural mesothelioma (MPM) pathogenesis and treatment outcome, supporting a role of immune checkpoint inhibitors as anticancer approach. This study retrospectively investigated TME and programmed death ligand 1 (PD-L1) expression in naïve MPM cases and their change under chemotherapy. Patients and methods: Diagnostic biopsies of MPM patients were collected from four Italian and one Slovenian cancer centers. Pathological assessment of necrosis, inflammation, grading, and mitosis was carried out. Ki-67, PD-L1 expression, and tumor infiltrating lymphocytes were detected by immunohistochemistry. When available, the same paired sample after chemotherapy was analyzed. Pathological features and clinical characteristics were correlated to overall survival. Results: TME and PD-L1 expression were assessed in 93 and 65 chemonaive MPM samples, respectively. Twenty-eight samples have not sufficient tumor tissue for PD-L1 expression. Sarcomatoid/biphasic samples were characterized by higher CD8+ T lymphocytes and PD-L1 expression on tumor cells, while epithelioid showed higher peritumoral CD4+ T and CD20+ B lymphocytes. Higher CD8+ T lymphocytes, CD68+ macrophages, and PD-L1 expression were associated with pathological features of aggressiveness (necrosis, grading, Ki-67). MPM cases characterized by higher CD8+ T-infiltrate showed lower response to chemotherapy and worse survival at univariate analysis. Patients stratification according to a combined score including CD8+ T lymphocytes, necrosis, mitosis, and proliferation index showed median overall survival of 11.3 months compared with 16.4 months in cases with high versus low combined score (P < 0.003). Subgroup exploratory analysis of 15 paired samples before and after chemotherapy showed a significant increase in cytotoxic T lymphocytes in MPM samples and PD-L1 expression in immune cells. Conclusions: TME enriched with cytotoxic T lymphocytes is associated with higher levels of macrophages and PD-L1 expression on tumor cells and with aggressive histopathological features, lower response to chemotherapy and shorter survival. The role of chemotherapy as a tumor immunogenicity inducer should be confirmed in a larger validation set.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Neoplasias Pleurais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Biópsia , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/imunologia , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/imunologia , Mesotelioma/mortalidade , Mesotelioma Maligno , Pessoa de Meia-Idade , Índice Mitótico , Pleura/citologia , Pleura/imunologia , Pleura/patologia , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/imunologia , Neoplasias Pleurais/mortalidade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Linfócitos T Citotóxicos/imunologia , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND AND OBJECTIVE: Parapneumonic effusion (PPE) is commonly caused by Gram-positive bacteria (GPB) and often presents with pleural loculation, which is characterized by overproduction of plasminogen activator inhibitor (PAI)-1. Lipoteichoic acid (LTA), a surface adhesion molecule of GPB, binds to the pleural mesothelium and triggers inflammation. However, the effects of LTA on PAI-1 expression in PPE and underlying mechanisms remain unclear. METHODS: Thirty consecutive patients with PPE were enrolled, including uncomplicated culture negative (CN, n = 11), Gram-negative bacteria (GNB, n = 7) and GPB (n = 12) groups stratified by pleural fluid characteristics and bacteriology, and the effusion PAI-1 levels were measured. In addition, human pleural mesothelial cells (PMC) were treated with LTA and the expression of PAI-1 and activation of signalling pathways were assayed. RESULTS: The median levels of PAI-1 were significantly higher in GPB (160.5 ng/mL) and GNB (117.0 ng/mL) groups than in the uncomplicated CN (58.0 ng/mL) group. In human PMC, LTA markedly upregulated PAI-1 mRNA and protein expression and enhanced elaboration of Toll-like receptor 2 (TLR2). Furthermore, LTA increased c-Jun N-terminal kinase (JNK) phosphorylation, induced activating transcription factor 2 (ATF2)/c-Jun nuclear translocation and activated PAI-1 promoter activity. Pretreatment with TLR2 siRNA significantly inhibited LTA-induced JNK phosphorylation and PAI-1 protein expression. CONCLUSION: Culture-positive PPE, especially that caused by GPB, has a significantly higher level of PAI-1 than uncomplicated CN PPE. LTA upregulates PAI-1 expression through activation of TLR2/JNK/activator protein 1 (AP-1) pathway in human PMC. Better understanding of the modulation of PAI-1 synthesis by LTA in PPE may provide potential therapies for infected pleural effusions.
Assuntos
Células Epiteliais/metabolismo , Lipopolissacarídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Derrame Pleural/metabolismo , Ácidos Teicoicos/farmacologia , Fator 2 Ativador da Transcrição/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cultura de Células , Feminino , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Pleura/citologia , Derrame Pleural/microbiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Pleural fibrosis can dramatically lower the quality of life. Numerous studies have reported that epithelial-mesenchymal transition (EMT) regulated by transforming growth factor-ß (TGF-ß) is involved in fibrosis. However, the molecular mechanism is inadequately understood. Fibroblast-specific protein-1 (S100A4) is a target of TGF-ß signaling. In our previous study, we have reported that S100A4 is highly expressed in pleural fibrosis. Thus, we suggest that S100A4 took part in the TGF-ß-induced EMT in pleural fibrosis. In this study, we determined the expression of S100A4 and EMT-related markers in Met-5A cells (pleural mesothelial cells) treated with TGF-ß or TGF-ß inhibitor by real-time PCR and western blot. In order to explore the role of S100A4, we used siRNA to knock down the expression of S100A4 in cell model. We found that the expression of epithelial cell marker was decreased and the mesenchymal cell marker increased with S100A4 upregulation after treatment with TGF-ß. Moreover, the changes of EMT-related event were restricted when the expression of S100A4 was knocked down. Conversely, S100A4 can partially rescue the EMT-related expression changes induced by TGF-ß inhibitor. These findings suggest that S100A4 expression is induced by the TGF-ß pathway, and silencing S100A4 expression can inhibit the process of TGF-ß-induced EMT.
Assuntos
Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Pleura/citologia , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Pirazóis/farmacologia , Quinolinas/farmacologiaRESUMO
PURPOSE: The pleural covering technique, i.e., wrapping a part of or the entire surface of the lung with oxidized regenerative cellulose (ORC), reinforces visceral pleura through pleural thickening for patients with pneumothorax and cystic lung diseases. However, it remains undetermined how ORC induces pleural thickening. METHODS: A histopathological examination was performed for lung specimens from patients who had recurrent pneumothoraces after pleural covering and re-operation (n = 5). To evaluate the influence of ORC on the pleura in vitro, we used MeT-5A cells (a human pleural mesothelial cell line). RESULTS: Pleural thickening was confirmed in all lung specimens examined. Three months after covering, the thickened pleura showed inflammatory cell infiltration, proliferation of myofibroblasts, and expression of fibronectin and TGF-ß. However, after 1 year, those findings virtually disappeared, and the thickened pleura was composed mainly of abundant collagen. When MeT-5A cells were cultured in ORC-immersed medium, their morphology changed from a cobblestone to spindle-shaped appearance. The expression of E-cadherin decreased, whereas that of N-cadherin, α-smooth muscle actin, and fibronectin increased, suggesting mesothelial-mesenchymal transition (Meso-MT). CONCLUSIONS: Our results suggest that Meso-MT may be involved as a mechanism of pleural thickening induced by pleural covering with ORC.