YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells.

Fibroblastic reticular cells (FRCs) are immunologically specialized myofibroblasts of lymphoid organ, and FRC maturation is essential for structural and functional properties of lymph nodes (LNs). Here we show that YAP and TAZ (YAP/TAZ), the final effectors of Hippo signaling, regulate FRC commitment and maturation. Selective depletion of YAP/TAZ in FRCs impairs FRC growth and differentiation and compromises the structural organization of LNs, whereas hyperactivation of YAP/TAZ enhances myofibroblastic characteristics of FRCs and aggravates LN fibrosis. Mechanistically, the interaction between YAP/TAZ and p52 promotes chemokine expression that is required for commitment of FRC lineage prior to lymphotoxin-ß receptor (LTßR) engagement, whereas LTßR activation suppresses YAP/TAZ activity for FRC maturation. Our findings thus present YAP/TAZ as critical regulators of commitment and maturation of FRCs, and hold promise for better understanding of FRC-mediated pathophysiologic processes.

Endoscope-assisted transoral accessory parotid mass excision: Multicenter prospective observational study.

OBJECTIVE: Transoral surgery is gaining favor because it has the advantage of leaving no scar after surgery. The aim of this study was to evaluate the technical feasibility and safety of endoscope-assisted transoral accessory parotid mass excision. STUDY DESIGN: Multicenter, prospective, observational study. METHODS: This study was designed as a 7-year, prospective, multicenter evaluation of endoscope-assisted transoral accessory parotid mass excision. Clinical outcomes and complications related to the procedures were evaluated in patients. RESULTS: Twenty patients underwent endoscope-assisted transoral accessory parotid mass excisions, and 22 patients underwent conventional parotidectomy approach excisions. There was no significant difference with respect to overall demographic characteristics between the groups. However, the operation times were shorter in the transoral approach group (P = 0.001), and cosmetic satisfaction was much better in the transoral group (P < 0.001). CONCLUSION: Endoscope-assisted transoral accessory parotid mass excision is a potentially safe and effective procedure with excellent outcomes. LEVEL OF EVIDENCE: 2 Laryngoscope, 130:1218-1226, 2020.

Effect of photobiomodulation therapy on neuronal injuries by ouabain: the regulation of Na, K-ATPase; Src; and mitogen-activated protein kinase signaling pathway.

BACKGROUND: To determine whether photobiomodulation (PBM) rescued the disruption of Na+/Ca2+ homeostasis and mitochondrial membrane potential by ouabain; the Na, K-ATPase inhibitor. For PBM in this study, a 660 nm LED array was used at energy densities of 0.78, 1.56, 3.12, 6.24, and 9.36 J/cm2. RESULTS: HCN-2 neuronal cells treated with ouabain showed loss of cell polarity, disrupted cell morphology, and decreased cell viability, which were improved after PBM treatment. We found that ouabain-induced Na, K-ATPase inhibition promoted activation of downstream signaling through Src, Ras, and mitogen-activated protein kinase (MAPK), which were suppressed after PBM treatment. This provided evidence of Na, K-ATPase α-subunit inactivation and intracellular Ca2+ increase. In response to ouabain, we observed activation of Src and MAPK by Na, K-ATPase, decreased mitochondrial membrane potential, and Na+-dependent Ca2+ increases, which were restored by PBM treatment. CONCLUSIONS: This study demonstrated that Na+/K+ imbalance could be regulated by PBM treatment in neuronal cells, and we suggest that PBM is a potential therapeutic tool for Na, K-ATPase targeted neuronal diseases.

A case of interdigitating dendritic cell sarcoma studied by whole-exome sequencing.

Interdigitating dendritic cell sarcoma (IDCS) is an aggressive neoplasm and is an extremely rare disease, with a challenging diagnosis. Etiology of IDCS is also unknown and most studies with only case reports. In our case, immunohistochemistry showed that the tumor cells were positive for S100, CD45, and CD68, but negative for CD1a and CD21. This study aimed to investigate the causative factors of IDCS by sequencing the protein-coding regions of IDCS. We performed whole-exome sequencing with genomic DNA from blood and sarcoma tissue of the IDCS patient using the Illumina Hiseq 2500 platform. After that, we conducted Sanger sequencing for validation of sarcoma-specific variants and gene ontology analysis using DAVID bioinformatics resources. Through comparing sequencing data of sarcoma with normal blood, we obtained 15 nonsynonymous single nucleotide polymorphisms (SNPs) as sarcoma-specific variants. Although the 15 SNPs were not validated by Sanger sequencing due to tumor heterogeneity and low sensitivity of Sanger sequencing, we examined the function of the genes in which each SNP is located. Based on previous studies and gene ontology database, we found that POLQ encoding DNA polymerase theta enzyme and FNIP1 encoding tumor suppressor folliculin-interacting protein might have contributed to the IDCS. Our study provides potential causative genetic factors of IDCS and plays a role in advancing the understanding of IDCS pathogenesis.

Transoral Dermoid Cyst Excision: A Multicenter Prospective Observational Study.

OBJECTIVE: Transoral surgery is becoming a preferred technique because it does not leave any scar after surgery. However, transoral surgery for a dermoid cyst of the oral cavity is not standardized yet, due to the anatomic complexity of this region. The aim of this study was to evaluate the safety and efficacy of a transoral dermoid cyst excision. STUDY DESIGN: Multicenter prospective observational study. SETTING: University hospital. SUBJECTS AND METHODS: This study was designed as a 4-year prospective multicenter evaluation of dermoid cyst excisions within the floor of mouth. Clinical outcomes and complications related to procedures were evaluated among patients. The primary outcome was the efficacy of the procedure, and the secondary outcome was cosmetic satisfaction of each procedure. RESULTS: Twenty-one patients underwent transoral dermoid cyst excisions, and 22 underwent transcervical excisions. In the transoral surgery group, the mean size of the dermoid cyst was 5.35 cm (95% CI, 4.79-5.91), and in the transcervical surgery group, it was 6.19 cm (95% CI, 5.67-6.71). There was no significant differences with respect to overall demographic characteristics between the groups. However, the duration of the operation was shorter with the transoral group than with the transcervical group ( P = .001), and cosmetic satisfaction was much better in the transoral group ( P < .001). CONCLUSION: Transoral dermoid cyst excision is a potentially safe and effective method that can lead to easy and quick removal of an oral cavity dermoid cyst, with excellent cosmetic outcomes.

mTOR and ROS regulation by anethole on adipogenic differentiation in human mesenchymal stem cells.

BACKGROUND: Adipocyte differentiation of human mesenchymal stem cells (hMSCs) is dependent on mitochondrial metabolism and reactive oxygen species (ROS) to initiate adipocyte differentiation. Although anethole has been known as an anti-oxidant and lipid peroxidation inhibitor, there is little investigated about its role in adipogenic differentiation. METHODS: The effects on cytotoxicity and proliferation of anethole in hMSCs were measured by the MTT assay. The anti-adipogenic effect of anethole on hMSCs was analyzed by Oil Red O staining and western blot analysis. The anti-oxidant activity of anethole on hMSC was assessed by flowcytometry and fluorescence staining using 2',7' -dichlorofluorescin diacetate (DCFDA). The western blotting was used to detect of phospho-Akt, phospho-mTOR, phospho-p70S6K, PPARγ, and phsopho-AMP-activated kinase (AMPK). RESULTS: Anethole suppressed the adipogenic differentiation of hMSCs through down-regulation of Akt-mTOR-p70S6K-PPARγ and up-regulation of AMPK. Anethole affected oxidative conditions through ROS generation. Anethole also rescued AMPK activity and reduced activation of mTOR-p70S6K-PPARγ under oxidative conditions in presence of exogenous hydrogen peroxide. CONCLUSION: ROS and mTOR regulation is a crucial factor in adipogenic differentiation, anethole has an important role in regulating activities of mTOR/PPARγ and ROS control in adipogenic differentiation of hMSCs.

Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca2+ overload and ER stress during hyperglycemia.

The overall goal is to study the effect of low-level laser therapy (LLLT) on membrane distribution of major water channel protein aquaporin 5 (AQP5) in salivary gland during hyperglycemia. Par C10 cells treated with high glucose (50 mM) showed a reduced membrane distribution of AQP5. The functional expression of AQP5 was downregulated due to intracellular Ca2+ overload and ER stress. This reduction in AQP5 expression impairs water permeability and therefore results in hypo-salivation. A reduced salivary flow was also observed in streptozotocin (STZ)-induced diabetic mice model and the expression of AQP5 and phospho-AQP5 was downregulated. Low-level laser treatment with 850 nm (30 mW, 10 min = 18 J/cm2) reduced ER stress and recovered AQP5 membrane distribution via serine phosphorylation in the cells. In the STZ-induced diabetic mouse, LLLT with 850 nm (60 J/cm2) increased salivary flow and upregulated of AQP5 and p-AQP5. ER stress was also reduced via downregulation of caspase 12 and CHOP. In silico analysis confirmed that the serine 156 is one of the most favorable phosphorylation sites of AQP5 and may contribute to the stability of the protein. Therefore, this study suggests high glucose inhibits phosphorylation-dependent AQP5 membrane distribution. High glucose induces intracellular Ca2+ overload and ER stress that disrupt AQP5 functional expression. Low-level laser therapy with 850 nm improves salivary function by increasing AQP5 membrane distribution in hyperglycemia-induced hyposalivation.

Effect of Topical Propolis on Wound Healing Process After Tonsillectomy: Randomized Controlled Study.

OBJECTIVES: The post-tonsillectomy pain and post-tonsillectomy hemorrhage are the two main problems after tonsillectomy. The aim of this study was to investigate the beneficial effects of water soluble ethanol extract propolis on post-tonsillectomy patient. METHODS: One hundred and thirty patients who underwent tonsillectomy or adenotonsillectomy were randomly divided into the control and propolis groups, each including 65 patients. The propolis group was applied with propolis orally immediately after surgery and by gargle. The pain scores were assessed on post-tonsillectomy 0, 1st, 2nd, 3rd, and 7th-10th day using a visual analogue scale score. Postoperative wound healing was evaluated by scoring pinkish membrane of tonsillar fossae on postoperative days 3 and 7-10. The incidence of post-tonsillectomy bleeding was examined in each group. RESULTS: Post-tonsillectomy pain was significantly less in propolis group compared to control group on postoperative days 3 and 7-10. Post-tonsillectomy hemorrhage was significantly less in the propolis group compared to the control group (P<0.05). The wound healing was significantly better in the propolis group compared to the control group on postoperative day 7-10 (P=0.002). CONCLUSION: Applying the propolis to post-tonsillectomy wound showed beneficial effect of reducing postoperative pain, preventing hemorrhage, and accelerating of wound healing of tonsillar fossae.

Enhanced survival of ischemic skin flap by combined treatment with bone marrow-derived stem cells and low-level light irradiation.

The aim of this study is to examine the enhanced survival effect of ischemic skin flap by combined treatment with bone marrow-derived stem cells (BMSCs) and low-level light irradiation (LLLI). The neovasculogenic effect of BMSCs induced by LLLI was detected using a wound healing and tube formation assay. ICR mice were divided into four groups: control group, LLLI group, BMSCs group, and combine-treated group. The percentage of skin flap necrosis area was calculated on the seventh post-operative day. Specimens were harvested for histologic analyses. LLLI promoted BMSC migration and tube formation. The flap survival rate of combined treated group was significantly higher than that of the control group. Histologic results demonstrated a significant increase in neovascularization in the combined treatment group. This study demonstrates that combination treatment of BMSCs and LLLI could enhance the survival of ischemic skin flap in a mouse model.

Low-Level Laser Therapy Promoted Aggressive Proliferation and Angiogenesis Through Decreasing of Transforming Growth Factor-ß1 and Increasing of Akt/Hypoxia Inducible Factor-1α in Anaplastic Thyroid Cancer.

OBJECTIVE: We assessed the cause of increased tumor after low-level laser therapy (LLLT) by histological analysis. BACKGROUND DATA: LLLT is a nonthermal phototherapy used in several medical applications, including wound healing, reduction of pain, and amelioration of oral mucositis. We discovered by accident that LLLT increased tumor size while testing a photodynamic therapy (PDT) model for the treatment of thyroid cancer. Although therapeutic effects of LLLT on cancer or dysplastic cells have been studied, LLLT has been recently reported to stimulate the aggressiveness of the tumor. METHODS: The anaplastic thyroid cancer cell line FRO was injected into thyroid glands of nude mice orthotopically and then laser irradiation was performed with 0, 15, and 30 J/cm(2) (100 mW/cm(2)) on the thyroid after 10 days. The tumor volume was measured for 4 weeks and the thyroid tissues underwent histological analysis. We observed that proliferation of FRO cells and macrophage infiltration was increased with energy delivery to the thyroid glands. We also assessed overproliferated FRO cells using an immunohistochemical staining with hypoxia inducible factor 1α (HIF-1α), p-Akt, vascular endothelial growth factor (VEGF), and transforming growth factor ß1 (TGF-ß1). RESULTS: HIF-1α and p-Akt were elevated after LLLT, which suggested that the phosphorylation of Akt by LLLT led to the activation of HIF-1α. Moreover, TGF-ß1 expression was decreased after LLLT, which led to loss of cell cycle regulation. CONCLUSIONS: In conclusion, LLLT led to a decrease in TGF-ß1 and increase of p-Akt/HIF-1α which resulted to overproliferation and angiogenesis of anaplastic thyroid carcinoma (ATC). Therefore, we suggest that LLLT can influence cancer aggressiveness associated with TGF-ß1 and Akt/HIF-1α cascades in some poorly differentiated head and neck cancers.

Metastatic Lymph Node Ratio of Central Neck Compartment Has Predictive Values for Locoregional Recurrence in Papillary Thyroid Microcarcinoma.

OBJECTIVES: This study aimed to evaluate the significance of metastatic lymph node ratio (the ratio between the metastatic lymph node and the harvested lymph nodes; MLNR) in the central neck for the prediction of locoregional recurrence in patients with papillary thyroid microcarcinoma. METHODS: After reviewing medical records of papillary thyroid microcarcinoma patients who received total thyroidectomy with central neck node dissection, 573 consecutive adult patients were enrolled in this study, with a follow-up period of more than 36 months. Regarding the risk of recurrence, multivariate analyses were performed with the following variables; sex, age, multiplicity of the primary tumor, presence of pathological extrathyroidal extension, the level of postoperative stimulated serum thyroglobulin, the number of harvested lymph nodes, the number of lymph node metastasis and MLNR. RESULTS: The MLNR showed a predictive significance for the locoregional recurrence (P<0.05). Most recurrences were occurred in the lateral neck (n=12, 80%) with a median interval of 20 months. The lowest cutoff value of the MLNR for a meaningful separation of disease recurrence was 0.44 (hazard ratio, 8.86; 95% confidence interval, 1.49 to 52.58; P=0.001). CONCLUSION: When the MLNR is higher than 0.44, there is an increased risk of locoregional recurrence mostly in the lateral neck. Therefore, MLNR of the central neck in a permanent or frozen biopsy may be helpful in decision making in the extent of thyroidectomy and/or the need for contralateral central neck lymph nodes dissection.

Chlorin e6 derivative radachlorin mainly accumulates in mitochondria, lysosome and endoplasmic reticulum and shows high affinity toward tumors in nude mice in photodynamic therapy.

The efficacy of photodynamic therapy (PDT) depends upon the amount of photosensitizer accumulated in the malignant tissues. Radachlorin is a popular photosensitizer used in photodynamic therapy to treat various types of cancer. In this study, we have studied the main organelles responsible for the accumulation of radachlorin in human anaplastic thyroid cancer in vitro and in vivo. The optimal time window for uptake and clearance of radachlorin also was studied. Confocal microscopic images confirmed that the radachlorin is mainly acquired by mitochondria and partially by lysosome and endoplasmic reticulum. Studies also showed that the maximum amount of radachlorin was accumulated within 3-6 h after the treatment. Radachlorin also showed a higher affinity toward malignant tumors compared to the other organs in mice xenograft model. Uptake of radachlorin reached an optimum amount within 6 h and most of the radachlorins were also cleared from the body in next 48 h. Therefore, detailed information regarding exact accumulation sites and a time window in which maximum amount of drug is accumulated and cleared were obtained by this study. Hence, not only the efficacy of the treatment can be increased but the phototoxicity after the treatment also can be controlled.

Carboplatin synergistically triggers the efficacy of photodynamic therapy via caspase 3-, 8-, and 12-dependent pathways in human anaplastic thyroid cancer cells.

Anaplastic thyroid cancer is one of the most aggressive forms of malignancies which grow very rapidly. Several conventional methods have been applied for the treatment of anaplastic thyroid cancer, but most of them were not successful in complete recovery of the patients. Therefore, a combination of two or more conventional modalities is being applied nowadays for the treatment of this type of cancer. In this present study, the combination of photodynamic therapy (PDT) and chemotherapy has been studied in anaplastic thyroid cancer. Human anaplastic thyroid cancer cells FRO were treated with a chemotherapy drug, carboplatin (cis-diammine-1,1-cyclobutanedicarboxyl-ateplatinum II (CBDCA)), and radachlorin-mediated PDT individually and in combination. Several parameters like cytotoxicity assay by MTT, apoptosis study by annexin V and propidium iodide, cell cycle analysis by flow cytometry, confocal microscopic study, and Western blot analysis for different apoptosis-related proteins like Bax, cytochrome c, caspases 3, 9, 8, and 12, etc. were studied to check the efficacy of the combination treatment as well as to find out the mechanism of this enhanced efficacy. Results showed that both PDT and CBDCA can induce apoptosis in FRO cells. However, a synergistic efficacy was observed when the cells were treated with CBDCA and PDT in combination. Changes in mitochondrial membrane potential and an increase in reactive oxygen species generation were observed in combination treatments. The enhanced expression of different apoptotic pathway-related proteins like Bax, cytochrome c, caspase 3, caspase 8, caspase 12, etc. also confirmed the higher efficacy of combination treatment. Therefore, with this combination treatment, not only a higher efficacy can be achieved but also the effective dose of the chemotherapy drug can be reduced, and hence, the adverse side effects of the chemotherapy drugs can also be controlled.

Cellular uptake of 9-hydroxypheophorbide-α and its photoactivation to induce ER stress-related apoptosis in human cervical cancer cells.

The 9-hydroxypheophorbide-α (9-HPbD) is a chlorophyll derivative and was found to be very effective for photodynamic therapy of tumor cells. The current study investigates uptake, retention, and intracellular localization of 9-HPbD by HeLa, human cervical cancer cells via fluorescence spectrophotometry and confocal laser scanning microscopy, and its photodynamic effect against human cervical carcinoma cell. HeLa cells exposed to 9-HPbD exhibited a linear uptake of photosensitizer during the first 12 h, and after removal of 9-HPbD, cell fluorescence was observed to decrease gradually over the next 12 h. Cells treated with 9-HPbD and stained with a panel of organelle-specific fluorescence probes (MitoTracker, LysoTracker, and ER-Tracker) revealed an intracellular fluorescence distribution restricted to cytoplasmic compartments with no detectable fluorescence in the nucleus. The 9-HPbD showed cytotoxicity effect against HeLa cells in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Endoplasmic reticulum (ER) disruption and cellular calcium dynamics also showed a photoactivation followed by cell death. The apoptotic effect of 9-HPbD was confirmed by caspase 3 activity study and immunofluorescence study of caspase 12. Morphological observation through the transmission electron microscopy and scanning electron microscopy also confirmed that 9-HPbD can induce apoptosis in HeLa cells. Therefore, it can be concluded that maximum uptake and clearance time of 9-HPbD was 12 h with endoplasmic reticulum as the major organelle site in cellular uptake, and 9-HPbD can induce apoptosis in HeLa cells through ER stress-related pathways via activation of caspase 12.

Extent of local invasion and safe resection in cT1-2 tonsil cancer.

BACKGROUND: Although intraoral resection of small-sized tonsil cancer achieves excellent tumor control, the extent of local invasion and adequate safety margin in resection have not been studied. Thus, we aim to determine the extent of local invasion in terms of mucosal spread and deep infiltration in stage T1-2 tonsil cancer. METHODS: We re-analyzed the surgical specimens from 49 cT1-2 tonsil cancers. Microscopic tumor cell extension from the tumor gross boundary of specimens was assessed in representative sections of each tumor. We also tested whether local extension correlates with human papilloma virus (HPV) status of tumors. RESULTS: The extent of microscopic deep invasion from the gross tumor border was 0.52 ± 0.41 mm, which was significantly less than that of mucosal spread (0.83 ± 0.61 mm, P = 0.01) in cT1-2 tonsil cancer. The microscopic deep invasion correlated with tumor size (rho = 0.703, P < 0.001). We found tumor invasion into superior constrictor muscles in 58.1%, no cases of tumor invasion into the deep fascia. In terms of HPV status (genotyping plus p16 staining), there were no differences in microscopic tumor extension. CONCLUSION: Our detailed pathologic analyses confirm that an oropharyngectomy including the superior constrictor muscle is an oncologically safe procedure for stage T1-2 tonsil cancer.

Immunotherapy with dendritic cells in an animal model of early pulmonary metastatic squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Distant metastases is becoming a more frequently recognized pattern of treatment failure in patients with squamous cell carcinoma of the head and neck (SCCHN). In this study, we evaluated the effect of a dendritic cell (DC)-based vaccine in an early pulmonary metastatic murine model with the aim of providing an effective treatment for SCCHN patients presenting with occult pulmonary metastasis. STUDY DESIGN: In vivo animal experiments were conducted in C3H/He immunocompetent mice using the SCCVII syngeneic squamous carcinoma cell line. METHODS: SCCVII cells were injected through the tail vein to establish early pulmonary metastases. Bone marrow-derived DCs were cultured and educated with ultraviolet B-irradiated apoptotic SCCVII cells before adoptive transfer into the inguinal area. Control groups were vaccinated with normal saline, naïve DCs, or apoptotic tumor cells. RESULTS: In the apoptotic SCCVII-pulsed DC group, the number of pulmonary tumor nodules was reduced, extirpated lung weight was less, and survival was longer than in control groups. Differences were statistically significant (P < .0001). Specific antitumor immunity was established only in the pulsed DC group, which was confirmed by an interferon-γ enzyme-linked immunosorbent assay. CONCLUSIONS: In an early pulmonary metastatic SCC murine model, systemic antitumor responses can be elicited by adoptive transfers of DCs, which were primed with apoptotic tumor cells. We hope this study will help improve overall survival of patients with SCCHN, especially when they have early or occult pulmonary metastasis.

The bone regenerative effect of growth hormone on consolidation in mandibular distraction osteogenesis of a dog model.

The purpose of this project was to study the effect of growth hormone on early bony consolidation in distraction osteogenesis of a dog model. Sixteen dogs were used for this study. The vertical osteotomy on the mandibular body was extended downward. An external distraction device was applied to the mandibular body and the mandibular distraction was started 5 days after the operation at a rate of 1 mm/d up to a 10-mm distraction. The experimental group was divided into a control group and growth hormone group. Dogs in the growth hormone group received a daily subcutaneous injection of 100 microg (1 IU) of recombinant human growth hormone per kilogram of body weight. The daily administration of growth hormone was performed from the day of the osteotomy through the whole distraction period to the sacrifice. Normal saline was injected in the control group. Eight dogs were allocated to each group. Two dogs in each group, a total of four dogs, were killed at 2 weeks after completion of distraction, four dogs were killed at 4 weeks, and the other eight dogs were killed at 6 weeks. The level of serum IGF-I in the growth hormone group was elevated and peaked between 8 days and 12 days after systemic administration of growth hormone. Bone mineral density was higher in the growth hormone group and lower in the control group for the whole period. Bone mechanical strength was 300% higher in the growth hormone group than in the control group. However, results were more suggestive than conclusive. On histological examination, the formation of a substantial amount of active woven bone was observed throughout the distracted zone at six weeks in the growth hormone group. In the control group, new bone was generated from the edge to the center of the distracted zone. In addition, most of the central area of the distracted zone was filled with fibrous tissue at six weeks. In conclusion, these findings suggest that growth hormone appears to be effective in early bony consolidation in distraction osteogenesis.