Establishment of 3D cell culture systems with decellularized lung-derived extracellular matrix hydrogel scaffold.

Decellularized tissue hydrogels, especially that mimic the native tissue, have a high potential for tissue engineering, three-dimensional (3D) cell culture, bioprinting, and therapeutic agent encapsulation due to their excellent biocompatibility and ability to facilitate the growth of cells. It is important to note that the decellularization process significantly affects the structural integrity and properties of the extracellular matrix, which in turn shapes the characteristics of the resulting hydrogels at the macromolecular level. Therefore, our study aims to identify an effective chemical decellularization method for sheep lung tissue, using a mixing/agitation technique with a range of detergents, including commonly [Sodium dodecyl sulfate (SDS), Triton X-100, and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate] (CHAPS), and rarely used (sodium cholate hydrate, NP-40, and 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate) (ASB-14). After the effectiveness of the used detergents on decellularization was determined by histological and biochemical methods, lung derived decellularized extracellular matrix was converted into hydrogel. We investigated the interactions between lung cells and decellularized extracellular matrix using proliferation assay, scanning electron microscopy, and immunofluorescence microscopy methods on BEAS-2B cells in air-liquid interface. Notably, this study emphasizes the effectiveness of ASB-14 in the decellularization process, showcasing its crucial role in removing cellular components while preserving vital extracellular matrix biological macromolecules, including glycosaminoglycans, collagen, and elastin. The resulting hydrogels demonstrated favorable mechanical properties and are compatible with both cell-cell and cell-extracellular matrix interactions.

Oxidative stress stimulation leads to cell-specific oxidant and antioxidant responses in airway resident and inflammatory cells.

AIMS: The imbalance between reactive oxygen species (ROS) and the antioxidant response has been linked to various airway diseases, including asthma. However, knowledge on cell-specific responses of the airway resident and inflammatory cells against increased oxidant stress is very limited. We aim to better understand the cell-specific antioxidant response that contributes to the pathophysiology of lung disease in response to oxidative stress. MATERIALS AND METHODS: The human cell lines of epithelial, fibroblast, endothelial, monocyte, eosinophil and neutrophil were incubated with tert-butyl hydroperoxide (tBHP) or cigarette smoke condensate (CSC). Following stimulation, cell viability, total oxidant and antioxidant activity were assessed in both residential and inflammatory cells. Human Oxidative Stress Plus RT2 Profiler PCR array was used to determine 84 gene expression differences in oxidant and antioxidant pathways following oxidant stimulus in all cells. KEY FINDINGS: We showed that various cell types respond differently to oxidative stress inducers, with distinct gene expression and oxidant-antioxidant generation. Most importantly, eosinophils increased the activity of all main antioxidant enzymes in response to both oxidants. Monocytes, on the other hand, showed no change in response to each stimulation, whereas neutrophils only increased their CAT activity in response to both stimuli. The increase in NRF2-regulated genes HSPA1A, HMOX1 and DUSP1 after both tBHP and CSC in epithelial cells and fibroblasts indicates Nfr2 pathway activation. SIGNIFICANCE: This study advances our knowledge of the molecular and cellular mechanisms of cell-specific antioxidant response upon exposure to oxidative stress. Additionally, our observations imply that the eosinophils' distinct biological response may be utilized for endotype-based cell-targeted antioxidant therapy.

House dust mite-derived allergens effect on matrix metalloproteases in airway epithelial cells.

Aim of the Study: Many allergens have protease activities. Although the immunomodulatory effects of these antigens are well known, the effects attributed to their protease activities are not thoroughly investigated. We set out to determine the effects of house dust mite (HDM) allergens with varying protease activities on bronchial epithelial cell functions. Materials and methods: BEAS-2B cells were maintained in ALI-culture and stimulated with Der p1 (cysteine protease), Der p6 (serine protease), and Der p2 (non-protease) with and without specific protease inhibitors or heat denaturation. Cell viability and epithelial permeability were measured with MTT and paracellular flux assay, respectively. The effect of heat denaturation on allergen structure was examined using in silico models. Matrix metalloproteinases (MMPs) were investigated at the transcription (qPCR), protein (ELISA), and functional (zymography) levels. Results: Epithelial permeability increased only after Der p6 but not after Der p1 or Der p2 stimulation. Der p2 increased both MMP-2 and MMP-9 expression, while Der p1 increased only MMP-9 expression. The heat-denatured form of Der p1 unexpectedly increased MMP-9 gene expression, which, through the use of in silico models, was attributed to its ability to change receptor connections by the formation of new electrostatic and hydrogen bonds. IL-8 and GM-CSF production were increased after Der p1 and Der p2 but decreased after Der p6 stimulation. IL-6 decreased after Der p1 but increased following stimulation with Der p6 and heat-denatured Der p2. Conclusion: Allergens in house dust mites are capable of inducing various changes in the epithelial cell functions by virtue of their protease activities.

The Genetic Variants of Interferon Regulatory Factor-3 in Children with Asthma.

Interferon Regulatory Factor-3 (IRF-3) is one of the key players in the inflammatory response mediated by the innate immune system. Although many studies have implicated a role for IRF-3 in the pathogenesis of inflammatory airway diseases, information about the possible association of IRF-3 genetic variants with asthma is scarce. We aimed to investigate the potential effects of IRF-3 polymorphisms in childhood asthma and asthma-related phenotypes. IRF-3 polymorphisms were first determined by sequencing 25 asthmatic and 25 healthy children. For further analysis, 609 asthmatic children and 191 healthy controls were screened for the genetic variants, such as rs2304204, rs2304205, rs320440, rs34739574, and rs7251. In addition, the relationship between these polymorphisms and asthma-related phenotypic features, including forced expiratory volume in one second values, eosinophil counts, and IgE levels was determined. rs7251 was associated with asthma in the codominant (P = 0.049) and G dominant (P = 0.025) model, however this significance was lost after False Discovery Rate analysis. Other investigated single nucleotide polymorphisms (SNPs) showed no significant association with asthma or asthma-related phenotypes. In conclusion, the seven SNPs of IRF-3 gene are not associated with asthma or asthma-related phenotypes in Turkish asthmatic children.

[Relationship Between Alcohol Dependence and Neuropeptide Y (NPY) Gene Promoter Polymorphisms in A Turkish Sample]. / Türkiye Örnekleminde Nöropeptit Y Geni (NPY) Promotor Polimorfizmleri ve Alkol Bagimliligi Arasindaki Iliski.

OBJECTIVE: Neuropeptide Y (NPY) is a protein widely expressed in the central nervous system and involved in diverse physiological processes, such as emotional regulation, nutritional behavior, and stress. In some populations, studies on alcohol dependence (AD) and the NPY gene have found that NPY variations increase alcohol consumption and thus may potentially be associated with AD. In this study, we investigated the relationship between NPY gene promoter polymorphisms and phenotypes related to alcohol use. METHOD: A total of 417 male participants comprising 252 individuals with AD and 165 healthy individuals were included in this study and phenotypic data were collected. Polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) and DNA sequencing METHODS were used for genotyping the rs16147 and rs17149106 polymorphisms in the promoter region of the NPY gene. The data of 384 participants were analysed to evaluate the possible relationship between genotypes and the diagnosis of AD, family history of AD, the severity of AD using the Michigan Alcoholism Screening Test (MAST), the age of onset of problematic alcohol use, the average amount of alcohol consumed per day for the last six months and the lifetime maximum alcohol consumption in one day. RESULTS: A significant difference was found between the AD and control groups concerning rs16147 polymorphism genotype distribution (p=0.025). No association with polymorphisms and alcohol-related phenotypes were demonstrated in the AD group. CONCLUSION: To our knowledge, this study shows for the first time in the literature that alcohol dependence is associated with NPY rs16147 polymorphism in the Turkish population.