26 research outputs found
Precision cut lung slices (PCLS) for respiratory sensitisation potential testing of allergens and irritants
No full textPrecision cut lung slices (PCLS) as a tool to predict effective LPS concentrations for in vivo use in common marmoset monkeys
No full textCharacterization of local respiratory irritation and inflammation after acute exposure to biological and chemical substances in PCLS
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LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing.
No full textIncreasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs
Assessment of lung toxicity induced by chemical allergens in human precision-cut lung slices
No full textAbstract A4662Introduction: A number of low-molecular weight (LMW) chemicals at workplaces are involved in the development of occupational asthma. There are no currently accepted and validated test methods to identify chemicals with the potential to cause respiratory sensitization. Risk assessment is normally performed in animal experiments, however, in the context of REACH and its principle of 3Rs there is an increasing public demand for alternative methods. Human precision cut lung slices (PCLS) is an ex vivo approach in which all relevant cell types of lung tissue are present in their natural position. The aim of the study was to analyse chemical-induced inflammation and irritation by assessing a variety of immunotoxic endpoints in human PCLS. Methods: Human PCLS were prepared from tumor-free tissue from resected lung lobes of cancer patients. PCLS were incubated with 20 substances in DMEM under standard submerged cell culture conditions. PCLS without test substances were incubated as controls. After 24 hours incubation, induced toxicity was assessed by vitality staining and determination of enzymatic activity using WST-1 assay. EC25 with respect to vehicle controls were calculated for all chemicals. Cytokine contents were detected with MSD technology and ELISA. Results: Individual EC25 values correlated significantly with data published for in vitro approaches with THP-1 and NCTC cell lines (r2 = 0.87 and 0.83, respectively) (Mitjans et al. 386-95;Corsini et al. 789-96). Furthermore EC25 of human PCLS correlated with LD50 data published for in vivo rat inhalation toxicity with r of 0.53 (p value of 0.08). Subtoxic concentrations were used to investigate cytokine pattern for the differentiation between allergen and non-allergen. Respiratory allergens trimellitic anhydride and ammonium hexachloroplatinate increased the expression of the cytokines TNF? and IL-1? significantly while contact allergens (cinnamaldehyde, 2-bromo-2-glutaronitrile) and controls (phenol, SLS) failed to induce these cytokines to the same extent. Conclusion: The toxicity of chemicals in human PCLS resembles the in vitro situation very closely and also correlates with LD50 values from in vivo studies. The pro-inflammatory effects could be shown for some respiratory allergens. However, some respiratory allergens like glutaraldehyde did not induce cytokine due to the chemical properties. Altogether, PCLS could be regarded as an ex vivo immunotoxicity model, displaying chemical-induced inflammation and irritation on all relevant cell types of lung tissue.18
Acute Cigarette Smoke Exposure Induces Cytotoxicity And Inflammation In Living Tissue Of Precision-Cut Lung Slices
No full textDetermination of genotoxicity by the Comet assay applied to murine precision-cut lung slices
No full textS.798-803Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay. Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate 'EMS' (0.03-0.4%) and formalin (0.5-5 mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4 × 105 and 6.7 × 105 cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24 µm to 75 µm. In contrast, formalin resulted in a significant induction of DNA cross-links. The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future.27Nr.
Assessment Of Lung Toxicity Induced By Chemical Allergens In Human Precision-Cut Lung Slices
No full textAcute cigarette smoke exposure induces cytotoxicity and inflammation in living tissue of precision-cut lung slices
No full textAbstract A6054Introduction: Chronic obstructive pulmonary disease (COPD) is a severe lung disease with high mortality and increasing prevalence. It is characterized by profound damage of lung parenchyma due to cigarette smoke-induced stress. To understand the underlying mechanisms the need emerged to develop human relevant models. Lipopolysaccharide (LPS) is a widely used model compound which does not directly induce oxidative stress due to lacking radicals. It therefore can not reflect cigarette smoke-induced toxicity in the lung. The aim of this study is to establish precision-cut lung slices (PCLS) as a relevant toxicity model by using cigarette smoke and cigarette smoke condensate in comparison to LPS as model compounds. Methods: Murine PCLS were prepared and exposed submerged to LPS and cigarette smoke condensate, and to cigarette mainstream smoke using the P.R.I.T.® air-liquid interface (ALI) culturing and exposure system. Induced toxicity was assessed by LIVE/DEAD® vitality staining and determination of metabolic activity using the WST-1 assay. Pro-inflammatory immune responses connected to toxic and subtoxic doses were quantified using ELISA. Additionally, therapeutic intervention with dexamethasone and roflumilast was assessed. Results: Concentration dependent toxicity could be shown for both cigarette smoke condensate and cigarette smoke with EC50 of 158 µg/mL and 0.255 µg/cm respectively. This is less compared to data published for A549 with ED50 ranging from 0.330 to 1.78 µg/cm3. Subtoxic and toxic doses of cigarette condensate induced a significant release of tumor necrosis factor (TNF)-a and interleukin (IL)-1a. Release of TNF-a and IL-1a was significantly inhibited by dexamethasone and roflumilast. Conclusion: PCLS represent a promising model to reflect the toxic aspects of cigarette smoke-induced tissue damage occurring in COPD. It furthermore can be used to study pharmacological intervention.18
Establishment Of A Tiered Model Of Airway Inflammation In Marmoset Monkey For Preclinical Pharmaceutical Testing
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