Genomic progression for local invasion of cutaneous squamous cell carcinoma from the superficial to the deep portion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. While many treatments exist, our understanding of its genomic progression, especially from the epidermis to the deep dermis, remains limited. This study aims to identify genetic mutations associated with the progression of cSCC into the deep dermis, providing insights into its aggressive behavior and high-risk features. We performed high-depth whole-exome sequencing on 12 cSCC tissues, along with paired normal tissues from six patients, using microdissection techniques. The mutational analysis focused on identifying alterations enriched during cSCC progression. Gene Ontology enrichment analysis, immunohistochemical assays, and external single-cell RNA data were utilized for validation. A total of 8863 non-synonymous somatic mutations were identified in 4092 genes across the superficial and deep portions of cSCCs. Analysis of deep portion mutations revealed a significant correlation with gene ontology biological processes, particularly cell junction organization, and cell-cell adhesion. Clonal mutations in these processes were more prevalent in the deep portions, indicating their impact on the cSCC mutation landscape. Genetic evolution analysis identified 29 causal genes associated with dermal invasion in cSCC. We highlight somatic mutations in cSCC, revealing heterogeneity between superficial and deep regions. Altered genes in cell junction organization and cell-cell adhesion emerged as pivotal in dermal invasion. We identified 29 causal genes primarily in deep tumor regions. Our findings emphasize analyzing multiple tumor regions to capture varied mutational landscapes. These insights advance our understanding of cSCC progression, emphasizing genetic and cellular changes during tumor evolution.

NDRG1 and FOXO1 regulate endothelial cell proliferation in infantile haemangioma.

The etiopathogenesis of infantile haemangioma has not been well understood, and it is accepted that angiogenic mediator dysregulation is the main contributor to the abnormal haemangioma capillary formation. The role of NDRG1, a hypoxia-inducible protein; FOXOs, which are tumor suppressor proteins; and the mTOR complex 2 pathway in infantile haemangioma have not been studied yet. The purpose of this study was to investigate NDRG1 and FOXO1 expression in the infantile haemangioma and the correlation of these proteins with proliferation and involution. Primary endothelial cells were obtained, with parental agreement, from 12 infantile haemangioma patients during surgery; 6 patients had proliferating infantile haemangiomas and 6 had involuting IHs. We compared the infantile haemangioma tissues and primary endothelial cells with human vein endothelial cells using microarrays, real-time PCR, Western blotting and immunohistochemical staining. Our data indicated that FOXO1 expression was downregulated in proliferating infantile haemangioma tissue. We found that the expression of NDRG1, a molecule upstream of the FOXO1 pathway, increased during haemangioma proliferation. NDRG1 knockdown decreased haemangioma endothelial cell proliferation and downregulated c-MYC oncoprotein levels. Our findings suggest that NDRG1 positively regulates haemangioma proliferation. FOXO1 dysregulation plays an important role in infantile haemangiomas pathogenesis.

Enhanced tissue remodelling efficacy of adipose-derived mesenchymal stem cells using injectable matrices in radiation-damaged salivary gland model.

The present study was conducted to introduce the use of a delivery carrier for local transplantation of human adipose tissue-derived mesenchymal stem cells (AdMSCs) into the salivary gland (SG) and analyse its ability to enhance radioprotection of AdMSCs against irradiation (IR)-induced damage. An injectable porcine small intestinal submucosa (SIS) matrix was used as a cell delivery carrier, and human AdMSCs were contained within SIS hydrogel (AdMSC/SIS). After local injection into SGs of mice following local IR, morphological and functional changes were evaluated in the sham, vehicle [phosphate-buffered saline (PBS)], SIS, AdMSC and AdMSC/SIS groups. Local transplantation of AdMSC resulted in less fibrosis, regardless of the use of a carrier, but the AdMSC/SIS group showed more mucin-producing acini relative to those in the PBS group. Functional restoration of salivation capacity and salivary protein synthesis was achieved in AdMSC and AdMSC/SIS groups, with a greater tendency being observed in the AdMSC/SIS group. AdMSC treatment resulted in tissue remodelling with a greater number of salivary epithelial cells (AQP-5), SG progenitor cells (c-Kit), endothelial cells (CD31) and myoepithelial cells (α-SMA), among which endothelial and myoepithelial cells significantly increased in the AdMSC/SIS group relative to the AdMSC group. AdMSC treatment alleviated IR-induced cell death, and the anti-apoptotic and anti-oxidative effects of AdMSC were enhanced in the AdMSC/SIS group relative to the AdMSC group. These results suggest local transplantation of AdMSC improves tissue remodelling following radiation damage in SG tissue, and that use of a carrier enhances the protective effects of AdMSC-mediated cellular protection against IR via paracrine secretion. Copyright © 2016 John Wiley & Sons, Ltd.

Radioprotective effects of Keratinocyte Growth Factor-1 against irradiation-induced salivary gland hypofunction.

Irradiation can cause salivary gland hypofunction, with hyposalivation producing discomfort, health risks, and reducing function in daily life. Despite increasing translational research interest in radioprotection, there are no satisfactory treatments available. Keratinocyte growth factor-1 stimulates proliferation of salivary epithelial cells or salivary stem/progenitor cells. However, the exact mechanism of its radioprotection against radiation-induced salivary hypofunction is not fully elucidated. Our results reveal that the radioprotective effects of keratinocyte growth factor-1 involved alleviation of growth inhibition and anti-apoptotic cell death of human parotid epithelial cells. Furthermore, keratinocyte growth factor-1 protected human parotid epithelial cells through the phosphoinositide 3-kinase - protein kinase B (Akt) pathway and inhibition of p53-mediated apoptosis through activation of mouse double minute 2. Local delivery of keratinocyte growth factor-1 into the irradiated salivary glands could protect radiation-induced salivary cell damages, suppress p53-mediated apoptosis and prevent salivary hypofunction in vivo. This suggests that keratinocyte growth factor-1 is a promising candidate to prevent radiation-induced salivary hypofunction and raise rational development keratinocyte growth factor-1 local delivery system.

Radioprotective Effect of Epigallocatechin-3-Gallate on Salivary Gland Dysfunction After Radioiodine Ablation in a Murine Model.

OBJECTIVES: Radioiodine (RI) therapy is known to subject cellular components of salivary glands (SG) to oxidative stress leading to SG dysfunction. However, the protective effects of antioxidants on RI-induced SG damage have not been well investigated. The authors investigated the morphometric and functional effects of epigallocatechin-3-gallate (EGCG) administered prior to RI therapy and compared this with the effects of amifostine (a well-known antioxidant) in a murine model of RI sialadenitis. METHODS: Four-week-old female C57BL/6 mice (n=48) were divided into four groups; a normal control group, a RI-treated group (0.01 mCi/g mouse, orally), an EGCG and RI-treated group, and an amifostine and RI-treated group. Animals in these groups were divided into 3 subgroups and euthanized at 15, 30, and 90 days post-RI treatment. Salivary flow rates and lag times were measured, and morphologic and histologic examinations and TUNEL (terminal deoxynucleotidyl transferase biotin-dUDP nick end labeling) assays were performed. Changes in salivary (99m)Tc pertechnetate uptake and excretion were followed by single-photon emission computed tomography. RESULTS: Salivary flow rates and lag times to salivation in the EGCG or amifostine groups were better than in the RI-treated group. Histologic examinations of SGs in the EGCG or amifostine group showed more mucin-rich parenchyma and less periductal fibrosis than in the RI-treated group. Fewer apoptotic cells were observed in acini, ducts, and among endothelial cells in the EGCG or amifostine group than in the RI group. In addition, patterns of (99m)Tc pertechnetate excretion were quite different in the EGCG or amifostine group than in the RI group. CONCLUSION: EGCG supplementation before RI therapy could protect from RI-induced SG damage in a manner comparable to amifostine, and thus, offers a possible means of preventing SG damage by RI.

Adipose Mesenchymal Stem Cell Secretome Modulated in Hypoxia for Remodeling of Radiation-Induced Salivary Gland Damage.

BACKGROUND AND PURPOSE: This study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model. MATERIALS AND METHODS: Human adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O2 < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 µL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects. RESULTS: The conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia-cultured hAdMSC secretome. CONCLUSION: These results show that the hAdMSC secretome from hypoxic-conditioned medium may provide radioprotection and tissue remodeling via release of paracrine mediators.

Functional regeneration of recurrent laryngeal nerve injury during thyroid surgery using an asymmetrically porous nerve guide conduit in an animal model.

BACKGROUND: Vocal cord paralysis (VCP) caused by recurrent laryngeal nerve (RLN) damage during thyroidectomy commonly results in serious medico-legal problems. The purpose of this study was to evaluate the usefulness of an asymmetrically porous polycaprolactone (PCL)/Pluronic F127 nerve guide conduit (NGC) for functional regeneration in a RLN injury animal model. METHODS: A biodegradable, asymmetrically porous PCL/F127 NGC with selective permeability was fabricated for use in this study. A 10-mm segment of left RLN was resected in 28 New Zealand white rabbits, and then an asymmetrically porous NGC or a nonporous silicone tube was interposed between both stumps and securely fixed. Vocal cord mobility was endoscopically evaluated at one, four, and eight weeks postoperatively. Nerve growth through NGCs was assessed by toluidine blue staining, and thyroarytenoid (TA) muscle atrophy was evaluated by hematoxylin and eosin staining. Immunohistochemical stainings for acetylcholinesterase (AchE), anti-neurofilament (NF), and anti-S100 protein were also conducted, and transmission electron microscopy (TEM) was used to evaluate functional nerve regeneration. RESULTS: At eight weeks postoperatively, endoscopic evaluations showed significantly better recovery from VCP in the asymmetrically porous PCL/F127 NGC group (6 of 10 rabbits) than in the silicone tube group (1 of 10 rabbits). Continued nerve growth on the damaged nerve endings was observed with time in the asymmetrically porous PCL/F127 NGC-interposed RLNs. TA muscle dimensions and AchE expressions in TA muscle were significantly greater in the asymmetrically porous PCL/F127 NGC group than in the silicone tube group. Furthermore, immunohistochemical staining revealed the expression of NF and S100 protein in the regenerated nerves in the asymmetrically porous PCL/F127 NGC group at eight weeks postoperatively, and at this time, TEM imaging showed myelinated axons in the regenerated RLNs. CONCLUSION: The study shows that asymmetrically porous PCL/F127 NGC provides a favorable environment for RLN regeneration and that it has therapeutic potential for the regeneration of RLN damage.

Systemic transplantation of human adipose tissue-derived mesenchymal stem cells for the regeneration of irradiation-induced salivary gland damage.

OBJECTIVES: Cell-based therapy has been reported to repair or restore damaged salivary gland (SG) tissue after irradiation. This study was aimed at determining whether systemic administration of human adipose-derived mesenchymal stem cells (hAdMSCs) can ameliorate radiation-induced SG damage. METHODS: hAdMSCs (1 × 10(6)) were administered through a tail vein of C3H mice immediately after local irradiation, and then this infusion was repeated once a week for 3 consecutive weeks. At 12 weeks after irradiation, functional evaluations were conducted by measuring salivary flow rates (SFRs) and salivation lag times, and histopathologic and immunofluorescence histochemistry studies were performed to assay microstructural changes, apoptosis, and proliferation indices. The engraftment and in vivo differentiation of infused hAdMSCs were also investigated, and the transdifferentiation of hAdMSCs into amylase-producing SG epithelial cells (SGCs) was observed in vitro using a co-culture system. RESULTS: The systemic administration of hAdMSCs exhibited improved SFRs at 12 weeks after irradiation. hAdMSC-transplanted SGs showed fewer damaged and atrophied acinar cells and higher mucin and amylase production levels than untreated irradiated SGs. Immunofluorescence TUNEL assays revealed fewer apoptotic cells in the hAdMSC group than in the untreated group. Infused hAdMSCs were detected in transplanted SGs at 4 weeks after irradiation and some cells were found to have differentiated into SGCs. In vitro, a low number of co-cultured hAdMSCs (13%-18%) were observed to transdifferentiate into SGCs. CONCLUSION: The findings of this study indicate that hAdMSCs have the potential to protect against irradiation-induced cell loss and to transdifferentiate into SGCs, and suggest that hAdMSC administration should be viewed as a candidate therapy for the treatment of radiation-induced SG damage.

Patterns of temperament and character in patients with posttraumatic stress disorder and their association with symptom severity.

OBJECTIVES: The aim of this study was to identify the patterns of temperament and character of patients with posttraumatic stress disorder (PTSD) and to explore the relationship between the patterns of temperament and character and PTSD symptoms severity. METHODS: Temperament and character features of 130 patients with PTSD (n = 65) and age and sex-matched healthy controls (n = 65) were evaluated using the Temperament and Character Inventory. Severity of PTSD symptoms and general anxiety symptoms was measured with the Impact of Events Scale-Revised (IES-R) and the Hamilton Anxiety Rating Scale (HARS). RESULTS: Patients with PTSD showed significantly higher scores on subscales of harm avoidance and self-transcendence and lower scores on self-directedness and cooperativeness compared to controls. Harm avoidance and self-transcendence scores were significantly associated with PTSD symptom severity as measured by IES-R but not with general anxiety symptom severity as measured by HARS. CONCLUSIONS: Patterns of temperament and character of patients with PTSD were significantly different from those of healthy controls. In addition, these patterns are specifically associated with the PTSD symptom severity.