Characterisation of a transgenic mouse expressing R122H human cationic trypsinogen

BACKGROUND: The R122H mutation of the cationic trypsinogen was found in patients with hereditary pancreatitis. A transgenic animal carrying this mutation could be useful as a genetic model system of pancreatitis. METHODS: Mice transgenic for the human R122H cationic trypsinogen were generated using the -205 fragment of the rat elastase promoter. The presence of the transgene was assayed in the DNA, in pancreatic mRNA and in zymogen granule lysates. Serum levels of amylase, lipase and cytokines (MCP-1, IL-6) were monitored and the histological appearance of the tissue was investigated. Pancreatitis was induced by 7 hourly injections of 50 μg/kg cerulein. The procedure was repeated twice weekly for 10 consecutive weeks. The animals were sacrificed 24 (n = 8) and 48 hours (n = 8) after the first injection and at the end of the whole treatment (n = 7). RESULTS: The transgene was detected at the genomic level and in pancreatic mRNA. The corresponding protein was found in low amounts in zymogen granule lysates. R122H mice showed elevated pancreatic lipase, but there was no spontaneous development of pancreatitis within 18 months. After induction of pancreatitis, levels of lipase (after 24 hours) and amylase (after 48 hours) were higher in R122H mice compared to controls. Repeated treatment with cerulein resulted in a slightly more severe pancreatitis in R122H animals. Amylase, lipase, and the cytokine levels were similar to controls. CONCLUSION: The R122H transgenic mouse failed to develop a spontaneous pancreatitis but a repeatedly provoked cerulein-induced pancreatitis led to a slightly more severe pancreatitis. The rather small difference in comparison to controls could be due to the low expression of the transgene in the mouse pancreas

Culturing of Melanocytes from the Equine Hair Follicle Outer Root Sheath

Hair follicles harbor a heterogeneous regenerative cell pool and represent a putative low-to-non-invasively available source of stem cells. We previously reported a technology for culturing human melanocytes from the hair follicle outer root sheath (ORS) for autologous pigmentation of tissue engineered skin equivalents. This study translated the ORS technology to horses. We de-veloped a culture of equine melanocytes from the ORS (eMORS) from equine forelock hair follicles cultured by means of an analogue human hair follicle-based in vitro methodology. The procedure was adjusted to equine physiology by addition of equine serum to the culture medium. The hair follicles were isolated by macerating forelock skin rests, enzymatically digested and subjected to air-medium-interface cultivation method. The procedure resulted in differentiated equine melanocytes, which exhibited typical morphology, presence of melanosomes, expression of cytoskeleton proteins vimentin, α-SMA, Sox2, S100ß and tyrosinase as well as tyrosinase activity followed by production of melanin. According to all assessed parameters, eMORS could be ranked as partially melanotic melanocytes. The results of the study offer an experimental base for further insight into hair follicle biology in equine and for comparative studies of hair follicles across different species

The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells

Molecular Subtypes of Oral Squamous Cell Carcinoma Based on Immunosuppression Genes Using a Deep Learning Approach

Background: The mechanisms through which immunosuppressed patients bear increased risk and worse survival in oral squamous cell carcinoma (OSCC) are unclear. Here, we used deep learning to investigate the genetic mechanisms underlying immunosuppression in the survival of OSCC patients, especially from the aspect of various survival-related subtypes. Materials and methods: OSCC samples data were obtained from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and OSCCrelated genetic datasets with survival data in the National Center for Biotechnology Information (NCBI). Immunosuppression genes (ISGs) were obtained from the HisgAtlas and DisGeNET databases. Survival analyses were performed to identify the ISGs with significant prognostic values in OSCC. A deep learning (DL)-based model was established for robustly differentiating the survival subpopulations of OSCC samples. In order to understand the characteristics of the different survival-risk subtypes of OSCC samples, differential expression analysis and functional enrichment analysis were performed. Results: A total of 317 OSCC samples were divided into one inferring cohort (TCGA) and four confirmation cohorts (ICGC set, GSE41613, GSE42743, and GSE75538). Eleven ISGs (i.e., BGLAP, CALCA, CTLA4, CXCL8, FGFR3, HPRT1, IL22, ORMDL3, TLR3, SPHK1, and INHBB) showed prognostic value in OSCC. The DL-based model provided two optimal subgroups of TCGA-OSCC samples with significant differences (p = 4.91E-22) and good model fitness [concordance index (C-index) = 0.77]. The DL model was validated by using four external confirmation cohorts: ICGC cohort (n = 40, C-index = 0.39), GSE41613 dataset (n = 97, C-index = 0.86), GSE42743 dataset (n = 71, C-index = 0.87), and GSE75538 dataset (n = 14, C-index = 0.48). Importantly, subtype Sub1 demonstrated a lower probability of survival and thus a more aggressive nature compared with subtype Sub2. ISGs in subtype Sub1 were enriched in the tumorinfiltrating immune cells-related pathways and cancer progression-related pathways, while those in subtype Sub2 were enriched in the metabolism-related pathways. Conclusion: The two survival subtypes of OSCC identified by deep learning can benefit clinical practitioners to divide immunocompromised patients with oral cancer into two subpopulations and give them target drugs and thus might be helpful for improving the survival of these patients and providing novel therapeutic strategies in the precision medicine area

Identifying crosstalk genetic biomarkers linking a neurodegenerative disease, Parkinson’s disease, and periodontitis using integrated bioinformatics analyses

ObjectiveTo identify the genetic linkage mechanisms underlying Parkinson’s disease (PD) and periodontitis, and explore the role of immunology in the crosstalk between both these diseases.MethodsThe gene expression omnibus (GEO) datasets associated with whole blood tissue of PD patients and gingival tissue of periodontitis patients were obtained. Then, differential expression analysis was performed to identify the differentially expressed genes (DEGs) deregulated in both diseases, which were defined as crosstalk genes. Inflammatory response-related genes (IRRGs) were downloaded from the MSigDB database and used for dividing case samples of both diseases into different clusters using k-means cluster analysis. Feature selection was performed using the LASSO model. Thus, the hub crosstalk genes were identified. Next, the crosstalk IRRGs were selected and Pearson correlation coefficient analysis was applied to investigate the correlation between hub crosstalk genes and hub IRRGs. Additionally, immune infiltration analysis was performed to examine the enrichment of immune cells in both diseases. The correlation between hub crosstalk genes and highly enriched immune cells was also investigated.ResultsOverall, 37 crosstalk genes were found to be overlapping between the PD-associated DEGs and periodontitis-associated DEGs. Using clustering analysis, the most optimal clustering effects were obtained for periodontitis and PD when k = 2 and k = 3, respectively. Using the LASSO feature selection, five hub crosstalk genes, namely, FMNL1, MANSC1, PLAUR, RNASE6, and TCIRG1, were identified. In periodontitis, MANSC1 was negatively correlated and the other four hub crosstalk genes (FMNL1, PLAUR, RNASE6, and TCIRG1) were positively correlated with five hub IRRGs, namely, AQP9, C5AR1, CD14, CSF3R, and PLAUR. In PD, all five hub crosstalk genes were positively correlated with all five hub IRRGs. Additionally, RNASE6 was highly correlated with myeloid-derived suppressor cells (MDSCs) in periodontitis, and MANSC1 was highly correlated with plasmacytoid dendritic cells in PD.ConclusionFive genes (i.e., FMNL1, MANSC1, PLAUR, RNASE6, and TCIRG1) were identified as crosstalk biomarkers linking PD and periodontitis. The significant correlation between these crosstalk genes and immune cells strongly suggests the involvement of immunology in linking both diseases

The Angiogenic Potential of Mesenchymal Stem Cells from the Hair Follicle Outer Root Sheath

Neovascularization is regarded as a pre-requisite in successful tissue grafting of both hard and soft tissues alike. This study considers mesenchymal stem cells from hair follicle outer root sheath (MSCORS) as powerful tools with a neat angiogenic potential that could in the future have wide scopes of neo-angiogenesis and tissue engineering. Autologous MSCORS were obtained ex vivo by non-invasive plucking of hair and they were differentiated in vitro into both endothelial cells and vascular smooth muscle cells (SMCs), two crucial cellular components of vascular grafts. Assessment was carried out by immunostaining, confocal laser-scanning microscopy, gene expression analysis (qRT-PCR), quantitative analysis of anastomotic network parameters, and cumulative length quantification of immunostained α-smooth muscle actin-containing stress fibers (α -SMA). In comparison to adipose mesenchymal stem cells, MSCORS exhibited a significantly higher differentiation efficiency according to key quantitative criteria and their endothelial derivatives demonstrated a higher angiogenic potential. Furthermore, the cells were capable of depositing their own extracellular matrix in vitro in the form of a membrane-cell sheet, serving as a base for viable co-culture of endothelial cells and SMCs integrated with their autologous matrix. Differentiated MSCORS hereby provided a complex autologous cell-matrix construct that demonstrates vascularization capacity and can serve as a base for personalized repair grafting applications

Culturing of Melanocytes from the Equine Hair Follicle Outer Root Sheath

Hair follicles harbor a heterogeneous regenerative cell pool and represent a putative low-to-non-invasively available source of stem cells. We previously reported a technology for culturing human melanocytes from the hair follicle outer root sheath (ORS) for autologous pigmentation of tissue engineered skin equivalents. This study translated the ORS technology to horses. We de-veloped a culture of equine melanocytes from the ORS (eMORS) from equine forelock hair follicles cultured by means of an analogue human hair follicle-based in vitro methodology. The procedure was adjusted to equine physiology by addition of equine serum to the culture medium. The hair follicles were isolated by macerating forelock skin rests, enzymatically digested and subjected to air-medium-interface cultivation method. The procedure resulted in differentiated equine melanocytes, which exhibited typical morphology, presence of melanosomes, expression of cytoskeleton proteins vimentin, α-SMA, Sox2, S100ß and tyrosinase as well as tyrosinase activity followed by production of melanin. According to all assessed parameters, eMORS could be ranked as partially melanotic melanocytes. The results of the study offer an experimental base for further insight into hair follicle biology in equine and for comparative studies of hair follicles across different species