RESUMO
Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.
Assuntos
DNA Mitocondrial , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Humanos , Animais , Armadilhas Extracelulares/metabolismo , Membrana Celular/metabolismo , Eosinófilos/metabolismo , Metilação de DNA , Ilhas de CpG , Bicamadas Lipídicas/metabolismoRESUMO
Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 µm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).
Assuntos
Microplásticos , Poluentes Químicos da Água , Plásticos/análise , Análise Espectral Raman , Fluxo de Trabalho , Microscopia Eletrônica , Monitoramento Ambiental , Poluentes Químicos da Água/análiseRESUMO
Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.
RESUMO
Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5'-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin ß4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air-liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.
Assuntos
5'-Nucleotidase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Epiteliais/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Células-Tronco Mesenquimais/metabolismo , CamundongosRESUMO
With substantial progress of nanotechnology, there is rising concern about possible adverse health effects related to inhalation of nanomaterials, such as multi-walled carbon nanotubes (MWCNT). In particular, individuals with chronic respiratory disorders, such as chronic obstructive pulmonary disease (COPD), may potentially be more susceptible to adverse health effects related to inhaled MWCNT. Hazard assessment of such inhaled nanomaterials therefore requires timely clarification. This was assessed in this study using a mouse model of COPD by exposing animals to 0.08 µg/cm2 of MWCNT administered by intratracheal instillation. Treatment with MWCNT induced an accumulation of alveolar macrophages (AMφ) in bronchoalveolar lavage fluid (BALF) in COPD mice that increased from 24 h to 7 d. In COPD mice, MWCNT induced a dynamic shift in macrophage polarization as measured by expression of CD38 and CD206, and increased AMφ and lung parenchyma macrophage (LPMΦ) activation with upregulation of co-stimulatory markers CD40 and CD80. Moreover, MWCNT treatment increased the frequencies of pulmonary dendritic cells (DC), leading to an expansion of the CD11b+CD103- DC subset. Although MWCNT did not trigger lung functional or structural changes, they induced an increased expression of the muc5AC transcript in mice with COPD. Our data provide initial evidence that inhaled MWCNT affect the pulmonary mucosal immune system by altering the numbers, phenotype, and activation status of antigen-presenting cell populations. Extrapolating these in vivo mouse findings to human pulmonary MWCNT exposure, caution is warranted in limiting exposure when handling inhalable nanofibers.
Assuntos
Células Dendríticas/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Exposição por Inalação , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Masculino , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/química , Doença Pulmonar Obstrutiva Crônica/imunologiaRESUMO
The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Ligação Proteica , Transporte Proteico , Trypanosoma/metabolismo , Trypanosoma/fisiologia , Trypanosoma brucei brucei/fisiologiaRESUMO
BACKGROUND: The aim of this study was to investigate the influence of age on the ultrastructure of venous valve morphology in patients with C2 classified chronic venous disorders according to the CEAP classification. PATIENTS AND METHODS: The study population consisted of 16 consecutive patients with varicose veins (C2). The mean age was 49.8 years (30-66). The (pre-) terminal valve including the vessel wall was harvested within the proximal 2 centimetres of the great saphenous vein. The mean thickness (volume-to-surface ratio = V/S ratio) of elastin, collagen, endothelium and of the entire valve was determined. A blinded morphologist performed the examination by transmission electron microscopy and stereology. Analyses by Pearson's product moment correlation, Kendall's tau and Spearman's rank correlation were performed to investigate whether there is a correlation between age and the ultrastructural morphology. RESULTS: Stereological analysis of the valves demonstrated a mean V/S ratio (signifying a thickness estimation) for elastin of 0.87 µm3/µm2, for collagen of 18.0 µm3/µm2, for endothelium of 0.65 µm3/µm2, and for the entire valve of 25.2 µm³/µm². Statistical analyses showed no statistically significant correlation between age and the ultrastructural morphology in this patient group. CONCLUSIONS: The ultrastructural morphology of the venous valves in chronic venous disorders may not depend on age in patients presenting with C2 disease. This conclusion may or may not apply to all C classes as we investigated a homogenous group of patients with C2 limbs.
Assuntos
Microscopia Eletrônica de Transmissão , Veia Safena/ultraestrutura , Varizes/patologia , Válvulas Venosas/ultraestrutura , Fatores Etários , Biópsia , Doença Crônica , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Veia Safena/cirurgia , Varizes/cirurgia , Válvulas Venosas/cirurgiaRESUMO
Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.
Assuntos
DNA de Cinetoplasto/biossíntese , DNA de Cinetoplasto/genética , DNA Mitocondrial/biossíntese , DNA Mitocondrial/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Animais , Genoma Mitocondrial , Genoma de Protozoário , Humanos , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/patogenicidadeRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1005586.].
RESUMO
Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.
Assuntos
Segregação de Cromossomos/fisiologia , Genoma Mitocondrial , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , DNA de Cinetoplasto/metabolismo , Flagelos , Imunofluorescência , Immunoblotting , Microscopia Eletrônica de Transmissão , Trypanosoma brucei brucei/ultraestruturaRESUMO
Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.
Assuntos
Remodelação das Vias Aéreas , Modelos Biológicos , Óxido Nítrico Sintase Tipo II/metabolismo , Alvéolos Pulmonares/metabolismo , Proteína D Associada a Surfactante Pulmonar/deficiência , Mecânica Respiratória , Animais , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Alvéolos Pulmonares/patologia , Proteína D Associada a Surfactante Pulmonar/metabolismoRESUMO
RATIONALE: Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis. OBJECTIVE: In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS). METHODS: Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd(-/-)) and iNOS single knockout mice (NOS2(-/-)) as well as wild type (WT) littermates. MEASUREMENTS AND RESULTS: DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd(-/-) demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS. CONCLUSION: iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.
Assuntos
Homeostase , Óxido Nítrico Sintase Tipo II/deficiência , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/patologia , Proteína D Associada a Surfactante Pulmonar/deficiência , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Expressão Gênica , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Ativação de Macrófagos , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Alvéolos Pulmonares/enzimologia , Enfisema Pulmonar/enzimologia , Enfisema Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/genética , Surfactantes Pulmonares/metabolismo , Testes de Função RespiratóriaRESUMO
The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70-80%) and granulocytes (20-30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionisation-mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.
Assuntos
Granulócitos/metabolismo , Hemócitos/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Plasmócitos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aranhas/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/metabolismo , Evolução Biológica , Proteínas Sanguíneas/metabolismo , Diferenciação Celular , Granulócitos/imunologia , Imunidade Celular , Imunidade Inata , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Imagens de Fantasmas , Plasmócitos/imunologiaRESUMO
INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 µm3/m2; in group B, 0.67 µm3/m2; in group C, 0.97 µm3/m2. The ratio was similar for collagen (A, 15.7 µm3/m2; B, 26.8 µm3/m2; C, 30.1 µm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.
Assuntos
Endotélio Vascular/ultraestrutura , Veia Safena/ultraestrutura , Varizes/patologia , Válvulas Venosas/ultraestrutura , Adulto , Idoso , Biomarcadores/metabolismo , Doença Crônica , Colágeno/metabolismo , Progressão da Doença , Elastina/metabolismo , Endotélio Vascular/metabolismo , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Veia Safena/metabolismo , Método Simples-Cego , Varizes/metabolismoRESUMO
We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 µm h(-1) healing rate).
Assuntos
Epitélio/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Cicatrização/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Desenho de Equipamento , Humanos , Fenótipo , Alvéolos Pulmonares/patologia , Regeneração/efeitos dos fármacos , Mucosa Respiratória/patologiaRESUMO
BACKGROUND: The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells. METHODS: NBM particles were precoated in various settings with EMD or human blood and analyzed for protein adsorption patterns via fluorescent imaging and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using fluorescent double-stranded DNA-binding dye. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding runt-related transcription factor 2, alkaline phosphatase (ALP), osteocalcin (OC), and collagen1α1 (COL1A1), and mineralization was assessed using red dye staining. RESULTS: Analysis of cell attachment and cell proliferation revealed significantly higher osteoblast and PDL cell attachment on EMD-coated surfaces when compared with control and blood-coated surfaces. EMD also stimulated release of growth factors and cytokines, including bone morphogenetic protein 2 and transforming growth factor ß1. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers, including COL1A1, ALP, and OC, in osteoblasts and PDL cells cultured on EMD-coated NBM particles. CONCLUSION: The present results suggest that 1) EMD enhances osteoblast and PDL cell attachment, proliferation, and differentiation on NBM particles, and 2) blood contamination of the grafting material before mixing with EMD may inhibit EMD adsorption.
Assuntos
Substitutos Ósseos/química , Materiais Revestidos Biocompatíveis/química , Proteínas do Esmalte Dentário/química , Minerais/química , Osteoblastos/fisiologia , Ligamento Periodontal/citologia , Adsorção , Fosfatase Alcalina/análise , Animais , Antraquinonas , Sangue , Proteína Morfogenética Óssea 2/análise , Calcificação Fisiológica/efeitos dos fármacos , Bovinos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Colágeno Tipo I/análise , Cadeia alfa 1 do Colágeno Tipo I , Corantes , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Proteínas do Esmalte Dentário/farmacologia , Corantes Fluorescentes , Humanos , Microscopia Eletrônica de Varredura , Osteoblastos/efeitos dos fármacos , Osteocalcina/análise , Ligamento Periodontal/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/análiseRESUMO
So far, little is known about the interaction of nanoparticles with lung cells, the entering of nanoparticles, and their transport through the blood stream to other organs. The entering and localization of different nanoparticles consisting of differing materials and of different charges were studied in human red blood cells. As these cells do not have any phagocytic receptors on their surface, and no actinmyosin system, we chose them as a model for nonphagocytic cells to study how nanoparticles penetrate cell membranes. We combined different microscopic techniques to visualize fine and nanoparticles in red blood cells: (I) fluorescent particles were analyzed by laser scanning microscopy combined with digital image restoration, (II) gold particles were analyzed by conventional transmission electron microscopy and energy filtering transmission electron microscopy, and (III) titanium dioxide particles were analyzed by energy filtering transmission electron microscopy. By using these differing microscopic techniques we were able to visualize and detect particles < or = 0.2 microm and nanoparticles in red blood cells. We found that the surface charge and the material of the particles did not influence their entering. These results suggest that particles may penetrate the red blood cell membrane by a still unknown mechanism different from phagocytosis and endocytosis.
Assuntos
Eritrócitos/efeitos dos fármacos , Microscopia/métodos , Nanopartículas , Eritrócitos/ultraestrutura , Ouro , Humanos , Microscopia Eletrônica de TransmissãoRESUMO
Glucocorticoids are often applied in neonatology and perinatology to fight the problems of respiratory distress and chronic lung disease. There are, however, many controversies regarding the adverse side effects and long-term clinical benefits of this therapeutic approach. In rats, glucocorticoids are known to seriously impair the formation of alveoli when applied during the first two postnatal weeks even at very low dosage. The current study investigates short-term and long-term glucocorticoid effects on the rat lung by means of morphologic and morphometric observations at light and electron microscopic levels. Application of a high-dosage protocol for only few days resulted in a marked acceleration of lung development with a precocious microvascular maturation resulting in single capillary network septa in the first 4 postnatal days. By postnatal d 10, the lung morphologic phenotype showed a step back in the maturational state, with an increased number of septa with double capillary layer, followed by an exceptional second round of the alveolarization process. As a result of this process, there was an almost complete recovery in the parenchymal lung structure by postnatal d 36, and by d 60, there were virtually no qualitative or quantitative differences between experimental and control rats. These findings indicate that both dosage and duration of glucocorticoid therapy in the early postnatal period are very critical with respect to lung development and maturation and that a careful therapeutic strategy can minimize late sequelae of treatment.
Assuntos
Animais Recém-Nascidos , Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Pulmão/crescimento & desenvolvimento , Pulmão/ultraestrutura , Microscopia Eletrônica , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: Glucocorticoids (GC) are successfully applied in neonatology to improve lung maturation in preterm born babies. Animal studies show that GC can also impair lung development. In this investigation, we used a new approach based on digital image analysis. Microscopic images of lung parenchyma were skeletonised and the geometrical properties of the septal network characterised by analysing the 'skeletal' parameters. Inhibition of the process of alveolarisation after extensive administration of small doses of GC in newborn rats was confirmed by significant changes in the 'skeletal' parameters. The induced structural changes in the lung parenchyma were still present after 60 days in adult rats, clearly indicating a long lasting or even definitive impairment of lung development and maturation caused by GC. CONCLUSION: digital image analysis and skeletonisation proved to be a highly suited approach to assess structural changes in lung parenchyma.