RESUMO
Usual interstitial pneumonia (UIP) is a histological pattern characteristic of idiopathic pulmonary fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection-coupled mass spectrometry, we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 nonfibrotic alveolar specimens as controls. The data were subjected to qualitative and quantitative analysis and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared with the nonfibrotic control. Furthermore, FF were positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of FF was predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that FF are the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This essential information will inform future mechanistic studies on fibrosis progression.
Assuntos
Fibrose Pulmonar Idiopática , Cicatriz/patologia , Colágeno , Matriz Extracelular/patologia , Fibrose , Humanos , Fibrose Pulmonar Idiopática/patologiaRESUMO
The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1- or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1- and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on â¼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2-like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.
Assuntos
Vesículas Extracelulares , Microscopia , Vesículas Extracelulares/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Macrófagos , ProteômicaRESUMO
INTRODUCTION: PD1/PD-L1 pathway targeting therapies are nowadays an established treatment option for patients with NSCLC. We assessed whether PD-L1 expression in NSCLC tumor cells was associated with specific clinical features or overall survival using four different clones. METHODS AND RESULTS: A retrospective study included formalin-fixed paraffin embedded (FFPE) surgical tumors from 482 patients. PD-L1 status was assessed with immunohistochemistry in tumor cells on tissue microarrays using clones 28-8, 22C3, SP263 and SP142. Associations with OS were assessed by Kaplan-Meier and multivariate Cox's regression analysis. Patients' median age: 68 years (39-86); histology: adenocarcinoma (AdCa) 61%, squamous-cell carcinoma (SqCC) 33%, and large cell carcinoma (LCC) 6%; p-stage: IA (46%), IB (30%), IIA (10%), IIB (11,4%), IIIA (1,2%), IIIB - IV (0,4%). PD-L1 positivity (≥1%) in NSCLC for clones 28-8, 22C3, SP263, SP142 was 41.5%, 34.2%, 42.7%, 10.4%, respectively (Pearson Chi-square p < 0.0001). PD-L1 expression was correlated with histology, tumor size and grading. Statistically significant association between PD-L1 expression and OS in NSCLC and Non-AdCa was observed with clone SP142 (log-rank p = 0.045 and p = 0.05, respectively). Statistically significant association between PD-L1 expression and OS in LCC was observed with clones 22C3 (log-rank p = 0.009) and SP263 (log-rank p = 0.050). CONCLUSIONS: Overexpression of the PD-L1 clone SP142 was associated with poor overall survival in NSCLC and Non-AdCa. Clones 22C3 and SP263 were associated with poor prognosis in LCC. PD-L1 status might serve as a prognostic marker in NSCLC.
Assuntos
Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Clonais/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Células Grandes/diagnóstico , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Células Clonais/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Imunoterapia/métodos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Prognóstico , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.
Assuntos
Macrófagos , Microscopia , Membrana Celular , Pulmão , MicrotúbulosRESUMO
AIMS: Lung transplant monitoring is usually performed with forceps transbronchial biopsies. These types of biopsy show limited reliability and a high degree of variability, owing to insufficient material and compression artefact, which lead to misinterpretation and, eventually, inappropriate treatment of the transplanted patients. The following study was undertaken to assess the diagnostic yield, histological quality and safety of cryobiopsy (CB) in comparison with conventional forceps biopsy (FB) for sampling lung tissue in transplant recipients. METHODS AND RESULTS: From January to December 2011, 81 consecutive transbronchial biopsies (41 FBs and 40 CBs) were indicated in single or bilateral lung transplantation recipients with clinical acute or chronic lung injury. Lung samples obtained by CB were larger (8.5 ± 6.5 mm in the FB group versus 22.1 ± 12.5 mm in the CB group; P < 0.0001) and had no crush artefacts (P = 0.002), allowing us to increase the diagnostic yield of acute (P = 0.0657) and chronic (P = 0.0053) cellular rejection. DISCUSSION: Transbronchial cryoprobe bronchoscopy allows the harvesting of larger and more expanded lung tissue samples, increasing the diagnostic yield in the monitoring of the lung allograft by means of a safe procedure.
Assuntos
Biópsia/métodos , Broncoscopia/métodos , Rejeição de Enxerto/diagnóstico , Transplante de Pulmão/efeitos adversos , Adulto , Idoso , Biópsia/instrumentação , Broncoscopia/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I-III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis-necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis-necrosis score is also proposed.
Assuntos
Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Necrose/patologia , Gradação de Tumores/métodos , Neoplasias Pleurais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Núcleo Celular/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Masculino , Mesotelioma/mortalidade , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Pleurais/mortalidade , PrognósticoRESUMO
PURPOSE OF THE REVIEW: Idiopathic pleuroparenchymal fibroelastosis (IPPFE) is a rare fibrosing lung disease, affecting the visceral pleura and the subpleural parenchyma with an upper lobe predilection, included as a distinct clinicopathologic entity in the latest international multidisciplinary classification of the idiopathic interstitial pneumonias (IIP). We aim to summarize the current evidence on IPPFE, in terms of clinical features and potential treatments. RECENT FINDINGS: Overall, there is increasing awareness of PPFE in association with a separate ILD pattern. Although an agreed consensus on diagnosis has yet to be defined, a list of radiological and histopathological criteria has been proposed. Due to the unfavorable risk-benefit profile of surgical lung biopsy in a significant proportion of patients, a potential role for transbronchial lung cryobiopsy has been suggested. At present, lung transplantation remains the only curative option. SUMMARY: The increasing awareness of this condition among specialists has led to more frequent identification of IPPFE. Large international studies are needed to better characterize pathogenesis and pheno/endotypes of disease, a key step towards the development of effective treatments.
RESUMO
BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.
Assuntos
Pulmão/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Enfisema Pulmonar/fisiopatologia , Regeneração/fisiologia , Tretinoína/farmacologia , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/fisiologia , Linhagem Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/patologia , RNA Mensageiro/genética , Receptores do Ácido Retinoico/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
INTRODUCTION: The presence of ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (in â¼1% of non-small cell lung carcinomas) that are typically identified by fluorescence in situ hybridization (FISH), and so screening using immunohistochemical (IHC) staining would be both cost- and time-efficient. METHODS: A cohort of lung tumors negative for other common mutations related to targeted therapies were screened to assess the sensitivity and specificity of IHC staining in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken. RESULTS: IHC staining was 100% sensitive (95% confidence interval: 48-100) and 83% specific (95% confidence interval: 86-100) overall when an h-score higher than 100 was used. Patients with ROS1 gene rearrangements were younger and typically never-smokers, with the tumors all being adenocarcinomas with higher-grade architectural features and focal signet ring morphologic features (two of five). Four patients treated with crizotinib showed a partial response, with three also showing a partial response to pemetrexed. Three of four patients remain alive at 13, 27, and 31 months, respectively. CONCLUSION: IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.
Assuntos
Rearranjo Gênico , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Crizotinibe , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/química , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/análise , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/análise , Pirazóis/uso terapêutico , Piridinas/uso terapêuticoAssuntos
Hipertensão Pulmonar/diagnóstico , Neoplasias Pulmonares/diagnóstico , Microangiopatias Trombóticas/diagnóstico , Adulto , Feminino , Humanos , Hipertensão Pulmonar/complicações , Imuno-Histoquímica , Neoplasias Pulmonares/complicações , Masculino , Pessoa de Meia-Idade , Prognóstico , Microangiopatias Trombóticas/complicações , Tomografia Computadorizada por Raios X/métodos , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: Transbronchial lung biopsy (TBLB) is required for evaluation in selected patients with interstitial lung disease (ILD). The diagnostic yield of histopathologic assessment is variable and is influenced by factors such as the size of samples and the presence of crush artefacts left by conventional biopsy forceps. We compared the diagnostic yield and safety of TBLB with cryoprobe sampling versus conventional forceps sampling. METHODS: This randomized clinical trial analysed data for 77 patients undergoing TBLB for evaluation of ILD; patients were assigned to either a conventional-forceps group or a cryoprobe group. Two pathologists assessed the tissue samples and agreed on histopathologic diagnoses. We also compared the duration of procedures, complications and sample-quality variables. RESULTS: The most frequent diagnosis observed in the cryoprobe group was non-specific interstitial pneumonia. Histopathologic diagnoses were identified in more cases in the cryoprobe group (74.4%) than in the conventional-forceps group (34.1%) (P < 0.001), and the diagnostic yield was higher in the cryoprobe group (51.3% vs 29.1% in the conventional forceps group; P = 0.038). A larger mean area of tissue was harvested by cryoprobe (14.7 ± 11 mm(2) ) than by conventional forceps (3.3 ± 4.1 mm(2)) (P < 0.001). More grade 2 bleeding (not statistically significant) occurred in the cryoprobe group (56.4%) than in the conventional-forceps group (34.2%). No differences in other complications were observed. CONCLUSIONS: TBLB by cryoprobe is safe and potentially useful in the diagnosis of ILD. Larger multisite randomized trials are required to confirm the potential benefits of this procedure. Clinical trial registration at ClinicalTrials.gov: NCT01064609.
Assuntos
Biópsia/métodos , Broncoscopia/instrumentação , Criopreservação/instrumentação , Técnicas Histológicas/instrumentação , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/patologia , Idoso , Biópsia/efeitos adversos , Biópsia/instrumentação , Broncoscopia/efeitos adversos , Broncoscopia/métodos , Criopreservação/métodos , Feminino , Hemorragia/epidemiologia , Hemorragia/etiologia , Técnicas Histológicas/métodos , Humanos , Incidência , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Reprodutibilidade dos Testes , Instrumentos CirúrgicosAssuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/cirurgia , Transplante de Pulmão , Pulmão/patologia , Pulmão/cirurgia , Linfangioleiomiomatose/cirurgia , Miócitos de Músculo Liso/patologia , Sirolimo/farmacologia , Biópsia , Bronquiolite/patologia , Contagem de Células , Feminino , Humanos , Imunossupressores/farmacologia , Pulmão/efeitos dos fármacos , Doenças Pulmonares Intersticiais/patologia , Transplante de Pulmão/imunologia , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND: In COPD, although histological lesions at both the small airways (wall thickening and tissue remodeling) and lung parenchyma (emphysematous destruction) are definitely different, the inflammatory cells involved in both processes are the same. Our study aims to determine if these histopathological phenotypes are related to two different lymphocyte profiles. METHODS: Distribution and cell density of CD3(+), CD4(+), CD8(+) and B lymphocytes were compared in small airways and parenchymal interstitium of 9 non-smokers, 18 smokers without COPD, 16 smokers with moderate COPD and 16 patients with very severe COPD undergoing lung transplantation. Spatial distribution of lymphocytes in periemphysematous parenchyma was also assessed. RESULTS: CD3(+) and B cell densities were significantly higher in small airways than parenchyma interstitium of very severe COPD patients. Furthermore, CD8(+) cells were increased in the epithelium of airways of moderate COPD patients compared to non-smokers. Although CD8(+) cell density was increased in parenchyma of COPD patients, CD8(+) and B cell densities were similar when comparing periemphysematous and non-emphysematous alveolar interstitium. CONCLUSIONS: In COPD, it is true that the small airways' wall shows a clear inflammatory pattern, with a high mononuclear infiltration and tissue remodeling. However, parenchymal interstitium shows a milder CD8(+) infiltration which, moreover, is not spatially related to emphysematous destroyed areas.