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1.
Cells ; 13(1)2023 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-38201244

RESUMO

BACKGROUND: The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration. METHODS: We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization. RESULTS: Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created. CONCLUSIONS: A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process-especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases-has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing.


Assuntos
Consolidação da Fratura , Fraturas Ósseas , Animais , Camundongos , Osteócitos , Osteoblastos , Regeneração Óssea
2.
J Breath Res ; 14(4): 046012, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021213

RESUMO

Breath analysis holds promise for non-invasive in vivo monitoring of disease related processes. However, physiological parameters may considerably affect profiles of exhaled volatile organic substances (VOCs). Volatile substances can be released via alveoli, bronchial mucosa or from the upper airways. The aim of this study was the systematic investigation of the influence of different sampling sites in the respiratory tract on VOC concentration profiles by means of a novel experimental setup. After ethical approval, breath samples were collected from 25 patients undergoing bronchoscopy for endobronchial ultrasound or bronchoscopic lung volume reduction from different sites in the airways. All patients had total intravenous anaesthesia under pressure-controlled ventilation. If necessary, respiratory parameters were adjusted to keep PETCO2 = 35-45 mm Hg. 30 ml gas were withdrawn at six sampling sites by means of gastight glass syringes: S1 = Room air, S2 = Inspiration, S3 = Endotracheal tube, S4 = Trachea, S5 = Right B6 segment, S6 = Left B6 segment (S4-S6 through the bronchoscope channel). 10 ml were used for VOC analysis, 20 ml for PCO2 determination. Samples were preconcentrated by solid-phase micro-extraction (SPME) and analysed by gas chromatography-mass spectrometry (GC-MS). PCO2 was determined in a conventional blood gas analyser. Statistically significant differences in substance concentrations for acetone, isoprene, 2-methyl-pentane and n-hexane could be observed between different sampling sites. Increasing substance concentrations were determined for acetone (15.3%), 2-methyl-pentane (11.4%) and n-hexane (19.3%) when passing from distal to proximal sampling sites. In contrast, isoprene concentrations decreased by 9.9% from proximal to more distal sampling sites. Blank bronchoscope measurements did not show any contaminations. Increased substance concentrations in the proximal respiratory tract may be explained through substance excretion from bronchial mucosa while decreased concentrations could result from absorption or reaction processes. Spatial mapping of VOC profiles can provide novel insights into substance specific exhalation kinetics and mechanisms.


Assuntos
Testes Respiratórios/métodos , Broncoscopia , Expiração , Manejo de Espécimes , Compostos Orgânicos Voláteis/análise , Dióxido de Carbono/química , Feminino , Humanos , Limite de Detecção , Pulmão/química , Masculino , Pessoa de Meia-Idade , Pressão Parcial
3.
Int J Mol Sci ; 22(1)2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33383915

RESUMO

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology.


Assuntos
Células da Medula Óssea/metabolismo , Remodelação Óssea , Animais , Biomarcadores , Medula Óssea/metabolismo , Medula Óssea/patologia , Diáfises , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Imunofenotipagem , Camundongos , Osteoblastos/imunologia , Osteoblastos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Quimeras de Transplante , Transplante Homólogo
4.
Sci Rep ; 9(1): 18894, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827195

RESUMO

Influenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such as Streptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processes in vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processes in vitro. In this study we analysed VOCs from headspace above (co)-infected human cells by using a customized sampling system. For investigating the influenza A mono-infection and the viral-bacterial co-infection in vitro, we analysed VOCs from Detroit cells inoculated with influenza A virus and S. pyogenes by means of needle-trap micro-extraction (NTME) and gas chromatography mass spectrometry (GC-MS). Besides the determination of microbiological data such as cell count, cytokines, virus load and bacterial load, emissions from cell medium, uninfected cells and bacteria mono-infected cells were analysed. Significant differences in emitted VOC concentrations were identified between non-infected and infected cells. After inoculation with S. pyogenes, bacterial infection was mirrored by increased emissions of acetaldehyde and propanal. N-propyl acetate was linked to viral infection. Non-destructive monitoring of infections by means of VOC analysis may open a new window for infection research and clinical applications. VOC analysis could enable early recognition of pathogen presence and in-depth understanding of their etiopathology.


Assuntos
Vírus da Influenza A , Influenza Humana/metabolismo , Odorantes/análise , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes , Compostos Orgânicos Voláteis/análise , Linhagem Celular Tumoral , Coinfecção , Cromatografia Gasosa-Espectrometria de Massas , Humanos
5.
Sci Rep ; 8(1): 14857, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30291257

RESUMO

Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.


Assuntos
Testes Respiratórios/instrumentação , Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/diagnóstico , Suínos/virologia , Compostos Orgânicos Voláteis/análise , Animais , Desenho de Equipamento , Infecções por Orthomyxoviridae/diagnóstico , Respiração , Doenças dos Suínos/virologia
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