Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-α.

Lupeol and stigmasterol, major phytosterols in various herbal plants, possess anti-inflammatory activities and have been proposed as candidates for anti-cancer agents, but their molecular mechanisms are still unclear. Here, we investigated the effects of lupeol and stigmasterol on tumor and endothelial cells in vitro and their anti-cancer activities in vivo. Our results demonstrated that lupeol and stigmasterol suppressed cell viability, migration, and morphogenesis of human umbilical vein endothelial cells (HUVECs) but not cholangiocarcinoma (CCA) cells. Expression analyses showed that the treatment of both compounds significantly reduced the transcript level of tumor necrosis factor-α (TNF-α), and Western blot analyses further revealed a decrease in downstream effector levels of VEGFR-2 signaling, including phosphorylated forms of Src, Akt, PCL, and FAK, which were rescued by TNF-α treatment. In vivo, lupeol and stigmasterol disrupted tumor angiogenesis and reduced the growth of CCA tumor xenografts. Immunohistochemical analyses confirmed a decrease in CD31-positive vessel content and macrophage recruitment upon treatment. These findings indicate that lupeol and stigmasterol effectively target tumor endothelial cells and suppress CCA tumor growth by their anti-inflammatory activities and are attractive candidates for anti-cancer treatment of CCA tumors.

Development and application of single-tube multiplex real-time PCR for lineage classification of Mycobacterium tuberculosis based on large sequence polymorphism in Northeast Thailand.

An appreciation of the genetic diversity of Mycobacterium tuberculosis (Mtb) is needed for effective planning of strategies in tuberculosis (TB) control. Large sequence polymorphisms (LSPs) are the molecular epidemiological and evolutionary markers for classification of Mtb into East Asian (EA) or Beijing, Indo-Oceanic (IO), Euro-American (EuA) and East African-Indian (EAI) lineages. We aimed to develop a single-tube multiplex real-time PCR assay using melting curve analysis for lineage classification of Mtb based on LSPs. The technique was optimized and tested with well-characterized strains (n = 89). The developed technique was then applied to classify Mtb isolates from TB patients (n = 256) randomly recruited from 19 provinces covering Northeast Thailand in 2013-2014. The technique demonstrated 100% sensitivity and specificity based on well-characterized strains compared to conventional techniques. The detection limit of the technique is 0.05 ng of genomic DNA of Mtb. The 256 Mtb isolates represented IO (n = 178, 70%), Beijing (n = 60, 23%) and EuA (n = 18, 7%) lineages. Significant associations of the Beijing lineage with drug resistance (p < 0.001) and younger average age of TB patients (p < 0.001) compared to other lineages were shown. The single-tube multiplex real-time PCR technique provides a simple, rapid and high performance tool for characterizing Mtb based on LSPs.

Development of single-tube multiplex PCR for classification of Mycobacterium tuberculosis lineages based on large sequence polymorphisms.

Large sequence polymorphisms (LSPs) or regions of differences (RDs) are molecular epidemiological and evolutionary markers used to classify Mycobacterium tuberculosis (MTB) into East Asian (Beijing), Indo-Oceanic (IO), Euro-American (EuA) and East African Indian (EAI) lineages. The most used method is separate PCR and sequencing for each RD. We developed a single-tube multiplex PCR using four primer pairs specific to the four MTB lineages and a primer pair for species-specific RD9 with genomic DNA extracted from isolated colonies. The single-tube multiplex PCR produced lineage-specific amplicon patterns capable of differentiating the four MTB lineages. Sensitivity and specificity of the assay were 100% when differentiating MTB lineages from other species and strains of bacteria. The limit of detection of genomic MTB DNA was 12.5 ng. This single-tube multiplex PCR method offers a simple, rapid and reliable method for classification of MTB lineages based on LSPs.