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Introduction: Endotoxin is a typical pyrogen derived from the outer membrane of Gram-negative bacteria. In fabricating cell-based medicinal products, it is necessary to control endotoxin in the process and the products. In the quality control tests of our clinical study, endotoxin concentration in the culture supernatant of autologous oral mucosal epithelial cell sheets exceeded the criterion value. Therefore, endotoxin measurements were conducted to clarify the cause of the endotoxin contamination. Methods: The reagents used to prepare the culture medium, the unused culture medium, and the culture supernatants were diluted with pure water. Endotoxin concentrations in the diluted samples were measured. Results: Endotoxin was detected in both the unused culture medium and the culture supernatant of the epithelial cell sheets at higher concentrations than the criterion value. Therefore, endotoxin concentrations in the reagents used to prepare the culture medium were measured and were found to be below the criterion value, except for cholera toxin. On the other hand, three lots of cholera toxin products were used for the measurement, and the endotoxin concentrations were higher than the criterion value. The results indicate that the endotoxin contamination is caused by the cholera toxin product. Conclusions: To prevent endotoxin contamination in cell-based medicinal products, endotoxin concentrations in reagents used for the fabrication should be measured in the facility conducting clinical research or confirmed by an adequate certificate of analysis from the manufacturers of the reagents.

Abstract Background Pulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection. Objective In this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface. Results One month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet’s angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host. Conclusions DFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

Abstract Background Peripheral ossifying fibroma is a nonneoplastic inflammatory hyperplasia that originates in the periodontal ligament or periosteum in response to chronic mechanical irritation. Peripheral ossifying fibroma develops more commonly in young females as a solitary, slow-growing, exophytic nodular mass of the gingiva, no more than 2 cm in diameter. While various synonyms have been used to refer to peripheral ossifying fibroma, very similar names have also been applied to neoplastic diseases that are pathologically distinct from peripheral ossifying fibroma, causing considerable nomenclatural confusion. Herein, we report our experience with an unusual giant peripheral ossifying fibroma with a differential diagnostic challenge in distinguishing it from a malignancy. Case presentation A 68-year-old Japanese male was referred to our department with a suspected gingival malignancy presenting with an elastic hard, pedunculated, exophytic mass 60 mm in diameter in the right maxillary gingiva. In addition to computed tomography showing extensive bone destruction in the right maxillary alveolus, positron emission tomography with computed tomography revealed fluorodeoxyglucose hyperaccumulation in the gingival lesion. Although these clinical findings were highly suggestive of malignancy, repeated preoperative biopsies showed no evidence of malignancy. Since even intraoperative frozen histological examination revealed no malignancy, surgical resection was performed in the form of partial maxillectomy for benign disease, followed by thorough curettage of the surrounding granulation tissue and alveolar bone. Histologically, the excised mass consisted primarily of a fibrous component with sparse proliferation of atypical fibroblast-like cells, partly comprising ossification, leading to a final diagnosis of peripheral ossifying fibroma. No relapse was observed at the 10-month follow-up. Conclusions The clinical presentation of giant peripheral ossifying fibromas can make the differential diagnosis from malignancy difficult. Proper diagnosis relies on recognition of the characteristic histopathology and identification of the underlying chronic mechanical stimuli, while successful treatment mandates complete excision of the lesion and optimization of oral hygiene. Complicated terminological issues associated with peripheral ossifying fibroma require appropriate interpretation and sufficient awareness of the disease names to avoid diagnostic confusion and provide optimal management.

Introduction: The treatment of nasal septum perforation solely by surgical intervention presents significant challenges. This study evaluated the effect of basic fibroblast growth factor (bFGF) in combination with collagen/gelatin on wound healing of nasal septum perforation in a rabbit animal model. Methods: A nasal septum perforation rabbit model was created. bFGF was added to a collagen/gelatin fixture and placed adjacent to the perforation, which is a complete defect. The rabbits were divided into three groups: the sham group that underwent the surgical procedure only, bFGF (−) group that received collagen/gelatin fixture without bFGF, and bFGF(+) group that received collagen/gelatin fixture with bFGF. The dimensions of the perforations were measured after 4 weeks, and the septum was subjected to histological examination. Results: All perforations remained open in the sham group (closure rate: 20.4%–83.1%). The closure rates of the bFGF(−) and bFGF(+) groups were 49.4%–68.8% and 72.7%–100%, respectively. No significant difference was noted in the closure rates between the sham and bFGF(−) groups; however, significant differences were observed between the sham and bFGF(+) groups, and the bFGF(−) and bFGF(+) groups (p

Abstract Background The assessment of a wide range of cognitive functions using video teleconference (VTC) systems cannot be applied in practice yet. We aimed to determine the feasibility and reliability of previously unvalidated remote cognitive function tests in Japan using common information and communication technology (ICT) devices, software, and VTC systems compared with face-to-face (FTF) assessment. Methods The sample consisted of 26 participants from senior citizens clubs and an employment service centre in Sapporo Japan, including 11 females and 15 males (age averaged 78.6 ± 6.8 years). Tests included the RCPM, Story recall, 10/36 spatial recall, selective reminding test, SDMT, PASAT, FAB, TMT-A, TMT-B, visual cancellation task, digit span, tapping span. The experimental design was a counterbalanced crossover randomised controlled trial. Intraclass correlations (ICCs), paired-samples t-tests, Cohen’s Kappa (κ) coefficients, and Wilcoxon signed-rank test were calculated to compare the scores between VTC and FTF assessments. Results All ICCs were significant and ranged from 0.47 (RCPM time) to 0.92 (RCPM score and PASAT), with a mean ICC of 0.75. Digit span using Cohen’s Kappa (κ) coefficient was significant, but the tapping span was not. Paired samples t-test showed statistically significant differences in SDMT, RCPM time, and cancellation time. Conclusions The results suggest that remote video conference-based neuropsychological tests even using familiar devices and software may be able to assess a wide range of cognitive functions in the Japanese older population. As for the processing speed tasks, we need to create our own standards for the remote condition. For the tapping span, we should consider increasing the number of trials.

Abstract Background Melanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. Method We differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. Result We successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. Conclusion The robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders.

Abstract TRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling‐induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5‐azacytidine as a myogenic differentiation inducer in murine bone marrow‐derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD‐positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura‐2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation‐promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Abstract Background We performed the first autologous oral mucosa-derived epithelial cell sheet transplantation therapy in a patient with refractory postoperative anastomotic stricture in congenital esophageal atresia (CEA) and confirmed its safety. In this study, patients with CEA and congenital esophageal stenosis were newly added as subjects to further evaluate the safety and efficacy of cell sheet transplantation therapy. Methods Epithelial cell sheets were prepared from the oral mucosa of the subjects and transplanted into esophageal tears created by endoscopic balloon dilatation (EBD). The safety of the cell sheets was confirmed by quality control testing, and the safety of the transplantation treatment was confirmed by 48-week follow-up examinations. Results Subject 1 had a stenosis resected because the frequency of EBD did not decrease after the second transplantation. Histopathological examination of the resected stenosis revealed marked thickening of the submucosal layer. Subjects 2 and 3 did not require EBD for 48 weeks after transplantation, during which time they were able to maintain a normal diet by mouth. Conclusions Subjects 2 and 3 were free of EBD for a long period of time after transplantation, confirming that cell sheet transplantation therapy is clearly effective in some cases. In the future, it is necessary to study more cases; develop new technologies such as an objective index to evaluate the efficacy of cell sheet transplantation therapy and a device to achieve more accurate transplantation; identify cases in which the current therapy is effective; and find the optimal timing of transplantation; and clarify the mechanism by which the current therapy improves stenosis. Trial registration: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

Abstract This study aimed to determine effects of cooling on contraction‐induced peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) and vascular endothelial growth factor (VEGF) gene expression, phosphorylations of its related protein kinases, and metabolic responses. Male rats were separated into two groups; room temperature (RT) or ice‐treated (COLD) on the right tibialis anterior (TA). The TA was contracted isometrically using nerve electrical stimulation (1‐s stimulation × 30 contractions, with 1‐s intervals, for 10 sets with 1‐min intervals). The TA was treated before the contraction and during 1‐min intervals with an ice pack for the COLD group and a water pack at RT for the RT group. The muscle temperature of the COLD group decreased to 19.42 ± 0.44°C (p

Loud acoustic noise from the scanner during functional magnetic resonance imaging (fMRI) can affect functional connectivity (FC) observed in the resting state, but the exact effect of the MRI acoustic noise on resting state FC is not well understood. Functional ultrasound (fUS) is a neuroimaging method that visualizes brain activity based on relative cerebral blood volume (rCBV), a similar neurovascular coupling response to that measured by fMRI, but without the audible acoustic noise. In this study, we investigated the effects of different acoustic noise levels (silent, 80 dB, and 110 dB) on FC by measuring resting state fUS (rsfUS) in awake mice in an environment similar to fMRI measurement. Then, we compared the results to those of resting state fMRI (rsfMRI) conducted using an 11.7 Tesla scanner. RsfUS experiments revealed a significant reduction in FC between the retrosplenial dysgranular and auditory cortexes (0.56 ± 0.07 at silence vs 0.05 ± 0.05 at 110 dB, p=.01) and a significant increase in FC anticorrelation between the infralimbic and motor cortexes (−0.21 ± 0.08 at silence vs −0.47 ± 0.04 at 110 dB, p=.017) as acoustic noise increased from silence to 80 dB and 110 dB, with increased consistency of FC patterns between rsfUS and rsfMRI being found with the louder noise conditions. Event-related auditory stimulation experiments using fUS showed strong positive rCBV changes (16.5% ± 2.9% at 110 dB) in the auditory cortex, and negative rCBV changes (−6.7% ± 0.8% at 110 dB) in the motor cortex, both being constituents of the brain network that was altered by the presence of acoustic noise in the resting state experiments. Anticorrelation between constituent brain regions of the default mode network (such as the infralimbic cortex) and those of task-positive sensorimotor networks (such as the motor cortex) is known to be an important feature of brain network antagonism, and has been studied as a biological marker of brain disfunction and disease. This study suggests that attention should be paid to the acoustic noise level when using rsfMRI to evaluate the anticorrelation between the default mode network and task-positive sensorimotor network.

Functional ultrasound (fUS) imaging is a method for visualizing deep brain activity based on cerebral blood volume changes coupled with neural activity, while functional MRI (fMRI) relies on the blood-oxygenation-level-dependent signal coupled with neural activity. Low-frequency fluctuations (LFF) of fMRI signals during resting-state can be measured by resting-state fMRI (rsfMRI), which allows functional imaging of the whole brain, and the distributions of resting-state network (RSN) can then be estimated from these fluctuations using independent component analysis (ICA). This procedure provides an important method for studying cognitive and psychophysiological diseases affecting specific brain networks. The distributions of RSNs in the brain-wide area has been reported primarily by rsfMRI. RSNs using rsfMRI are generally computed from the time-course of fMRI signals for more than 5 min. However, a recent dynamic functional connectivity study revealed that RSNs are still not perfectly stable even after 10 min. Importantly, fUS has a higher temporal resolution and stronger correlation with neural activity compared with fMRI. Therefore, we hypothesized that fUS applied during the resting-state for a shorter than 5 min would provide similar RSNs compared to fMRI. High temporal resolution rsfUS data were acquired at 10 Hz in awake mice. The quality of the default mode network (DMN), a well-known RSN, was evaluated using signal-noise separation (SNS) applied to different measurement durations of rsfUS. The results showed that the SNS did not change when the measurement duration was increased to more than 210 s. Next, we measured short-duration rsfUS multi-slice measurements in the brain-wide area. The results showed that rsfUS with the short duration succeeded in detecting RSNs distributed in the brain-wide area consistent with RSNs detected by 11.7-T MRI under awake conditions (medial prefrontal cortex and cingulate cortex in the anterior DMN, retrosplenial cortex and visual cortex in the posterior DMN, somatosensory and motor cortexes in the lateral cortical network, thalamus, dorsal hippocampus, and medial cerebellum), confirming the reliability of the RSNs detected by rsfUS. However, bilateral RSNs located in the secondary somatosensory cortex, ventral hippocampus, auditory cortex, and lateral cerebellum extracted from rsfUS were different from the unilateral RSNs extracted from rsfMRI. These findings indicate the potential of rsfUS as a method for analyzing functional brain networks and should encourage future research to elucidate functional brain networks and their relationships with disease model mice.

Background/Aims The usefulness of ultrasonography (US) in diseases of the gastrointestinal tract has been reported recently. This prospective study aimed to determine the features of US findings in immune-mediated colitis (IMC), an adverse event induced by immune checkpoint inhibitor, and examine the correlation between US findings, colonoscopy (CS) findings, and severity of colitis. Methods We studied patients examined using CS and US upon suspicion of IMC in Hokkaido University Hospital between April 2018 and February 2021. Endoscopic findings of IMC were assessed using the Ulcerative Colitis Endoscopic Index of Severity (UCEIS). The severity of US findings in IMC was evaluated using US grade, which is the ultrasonographic grading scale in ulcerative colitis. Bowel wall thickness and the intensity of the color Doppler signal were also analyzed. Severity of colitis was evaluated using Common Terminology Criteria for Adverse Events (CTCAE) grade version 5. Results Fourteen patients with IMC were enrolled. The US findings were bowel wall thickening, loss of stratification, ulceration and increased blood flow signal. The US grade was moderately correlated with the UCEIS (r=0.687, P=0.009) and CTCAE grade (r=0.628, P=0.035). Bowel wall thickness and UCEIS (r=0.628, P=0.020), as well as color Doppler signal grade and CTCAE grade (r=0.724, P=0.008), were significantly correlated. Conclusions US findings in IMC were mainly similar to those of ulcerative colitis, but there were some findings that were characteristic only of IMC. Significant correlation was found between US findings, CS findings, and severity of colitis. Hence, US could be useful for the evaluation of IMC.

Introduction: Duchenne muscular dystrophy (DMD) is a progressive disease that leads to damage of muscle and myocardium due to genetic abnormalities in the dystrophin gene. In utero cell transplantation that might facilitate allogenic transplantation is worth considering to treat this disease. Methods: We performed allogeneic in utero transplantation of GFP-positive myoblasts and adipose-derived mesenchymal stem cells into murine DMD model animals. The transplantation route in this study was fetal intraperitoneal transplantation and transplacental transplantation. Transplanted animals were examined at 4-weeks old by immunofluorescence staining and RT-qPCR. Results: No GFP-positive cells were found by immunofluorescence staining of skeletal muscle and no GFP mRNA was detected by RT-qPCR in any animal, transplantation method and cell type. Compared with previous reports, myoblast transplantation exhibited an equivalent mortality rate, but adipose-derived stem cell (ASC) transplantation produced a higher mortality rate. Conclusions: In utero transplantation of myoblasts or ASCs to murine models of DMD does not lead to engraftment and, in ASC transplantation primarily, frequently results in fetal death.

Introduction: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2β1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2β1 to laminin-221. Methods: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis. Results: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts. Conclusion: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2β1 to laminin-221.

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Purpose High-intensity focused ultrasound (HIFU) treatment requires prior evaluation of the HIFU transducer output. A method using micro-capsulated thermochromic liquid crystal (MTLC) to evaluate the temperature distribution in the media during HIFU exposure has been previously developed. However, the color-coded temperature range of commercial MTLC is approximately 10 °C, which is insufficient for temperature measurement for HIFU exposure. We created two layers of tissue-mimicking phantoms with different color-coded temperature ranges, and a new visualization method was developed by utilizing the axisymmetric pressure distribution of a HIFU focus. Methods A two-layer phantom with two sensitivity ranges was created. The HIFU transducer was set to align the focal point to the boundary between the two layers. Images of the upper and lower layers were flipped along the boundary between the two layers such that they overlapped with each other, assuming the pressure distribution of HIFU to be axisymmetric. Results The experimental and simulation results were compared to evaluate the accuracy of the phantom temperature measurement. The experimental time profile of the temperature and spatial distribution around the HIFU focus matched well with that of the simulation. However, there is room for improvement in the accuracy in the axial direction of HIFU focus. Conclusion Users can apply our proposed method in clinical practice to promptly assess the output of the HIFU transducer before treatment.

Abstract Background Congenital esophageal atresia postoperative anastomotic stricture occurs in 30–50% of cases. Patients with severe dysphagia are treated with endoscopic balloon dilatation (EBD) and/or local injection of steroids, but many patients continue to experience frequent stricture. In this study, we investigated the transplantation of autologous oral mucosa-derived cell sheets (epithelial cell sheets) as a prophylactic treatment for congenital esophageal atresia postoperative anastomotic stricture. Methods Epithelial cell sheets were fabricated from a patient’s oral epithelial tissue, and their safety was confirmed by quality control tests. The epithelial cell sheets were transported under controlled conditions from the fabrication facility to the transplantation facility and successfully transplanted onto the lacerations caused by EBD using a newly developed transplantation device for pediatric patients. The safety of the transplantation was confirmed by follow-up examinations over 48 weeks. Results The dates that EBD was performed were recorded for one year before and after epithelial cell sheet transplantation, and the intervals (in days) were evaluated. For about 6 months after transplantation, the intervals between EBDs were longer than in the year before transplantation. The patients were also aware of a reduction in dysphagia after transplantation. Conclusions These results suggest that cell sheet transplantation may be effective in preventing anastomotic stricture after surgery for congenital esophageal atresia, but the effect was temporary and limited in this case. Although we chose a very severe case for the first human clinical study, it may be possible to obtain a more definitive effect if the transplantation is performed before the disease becomes so severe. Future studies are needed to identify cases in which cell sheet transplantation is most effective and to determine the appropriate timeframes for transplantation. Trial registration: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

Introduction: Keratinocytes derived from pluripotent stem cells have a short proliferative lifespan under conventional culture conditions that are optimized for keratinocytes. Recently, a Rho kinase inhibitor, Y-27632, had been used as a standard supplement for culture medium in which the proliferative lifespan of postnatal keratinocytes was markedly expanded. In addition, recombinant human laminin-511 was demonstrated to be an adhesive ligand for promoting proliferation of cultured epidermal keratinocytes. Based on this knowledge, efficacies of Y-27632 and a laminin511-derived recombinant fragment, known as laminin-511 E8 fragment (LN-511-E8), were evaluated for establishing cultivation methods of keratinocyte differentiated from human embryonic stem cells (hESC). Methods: Differentiated cells from hESCs, which were established with clinical grade in previous study, were seeded onto culture dishes coated with LN-511-E8 and co-cultured with a mouse feeder layer in serum-free medium supplemented with Y-27632. Before serial cultivation, hESC-derived keratinocytes were separated from other differentiated cells by trypsinization. The isolated hESC-derived keratinocytes were used for evaluating clonogenicity, gene expression analysis for keratinocyte markers, potency of terminal differentiation by air-lifting culture, and long-term proliferation activity by serial cultivation. Moreover, efficacies of Y-27632, LN-511-E8, and mouse feeder layer were evaluated on proliferation of hESC-derived keratinocytes. Results: hESC-derived keratinocytes with activity of clonal growth were successfully isolated by trypsinization and exhibited potency of differentiation to form stratified epidermal equivalents with expressions of progenitor and differentiation markers of epidermal keratinocyte. Y-27632 and LN-511-E8 were required for maintaining the proliferative activity of the hESC-derived keratinocytes in serially cultivation using mouse feeder layer with stable doubling time during logarithmic growth phase. Conclusions: These results indicate the utility of Y-27632 and LN-511-E8 for serial cultivation of hESC-derived keratinocytes, which have a potential for fabricating allogeneic cellular products in clinical situations for regeneration of stratified epithelial tissues.

BackgroundThe first-line chemotherapy for patients with RAS and BRAF wild-type metastatic colorectal cancer (mCRC) commonly involves cytotoxic regimens, such as FOLFOX and FOLFIRI, combined with epidermal growth factor receptor (EGFR) antibodies. When progression occurs following anti-EGFR antibody-combined chemotherapy, anti-angiogenic inhibitors can be used as second-line treatment. Although randomized controlled trials have shown that anti-angiogenic inhibitors [bevacizumab, ramucirumab, and aflibercept (AFL)] carry survival benefit when combined with FOLFIRI as second-line chemotherapy, such trials did not provide data on patients with mCRC refractory to anti-EGFR antibody-combined chemotherapy. Therefore, our group planned a multicenter, nonrandomized, single-arm, prospective, phase II study to investigate the safety and efficacy of FOLFIRI plus AFL as a second-line chemotherapy for patients with mCRC refractory to oxaliplatin-based chemotherapy combined with anti-EGFR antibodies.MethodsFOLFIRI (irinotecan 180 mg/m2, l-leucovorin 200 mg/m2, bolus 5-FU 400 mg/m2, and infusional 5-FU 2400 mg/m2/46 h) and AFL (4 mg/kg) will be administered every 2 weeks until progression or unacceptable toxicities occur. The primary endpoint will be the 6-month progression-free survival (PFS) rate, whereas the secondary endpoints will include overall survival, PFS, response rate, disease control rate, adverse events, and relative dose intensity for each drug. A sample size of 41 participants will be required. This study will be sponsored by the Non-Profit Organization Hokkaido Gastrointestinal Cancer Study Group and will be supported by a grant from Sanofi.DiscussionThere is only an observational study reporting data on FOLFIRI plus AFL for patients with mCRC who previously received anti-EGFR antibodies; therefore, a prospective clinical trial is needed. This study will prospectively evaluate the efficacy and safety of FOLFIRI plus AFL in patients with mCRC who are resistant to anti-EGFR antibodies and have limited data. Moreover, this study will reveal predictive biomarkers for AFL-based chemotherapy.Clinical trial registrationJapan Registry of Clinical Trials, jRCTs011190006. Registered 19 November, 2019, https://jrct.niph.go.jp/latest-detail/jRCTs011190006.

Abstract Background An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). Methods Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. Results Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. Conclusions Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

Abstract Background Portal hepatic schwannoma is a rare benign tumor and difficult to diagnose preoperatively because of its rarity and imaging manifestations that mimic malignancy. We present a case of portal hepatic schwannoma that showed moderate contrast enhancement on computed tomography (CT), extension along the bile duct on T2-weighted imaging and magnetic resonance cholangiopancreatography (MRCP), and uptake of 18F-fluorodeoxyglucose (FDG) on positron emission tomography. Case presentation Ultrasonography at an annual health checkup identified a hepatic mass in a 38-year-old woman. CT showed a well-defined portal hepatic tumor with mild contrast enhancement. T2-weighted imaging and MRCP showed a clavate tumor extending along the intrahepatic bile ducts but no dilatation of the ducts. The tumor exhibited increased FDG uptake, such as maximum standardized uptake values of 5.0 and 6.5 in the early and late phases, respectively. Neither dilatation of intrahepatic bile ducts nor lymphadenopathy was identified, and the multimodality imaging suggested hepatic portal lymphoma, gastrointestinal tumor, or IgG4-related disease rather than cholangiocarcinoma. A needle biopsy via endoscopic ultrasonography was performed, and immunohistology confirmed the tumor as a schwannoma. Conclusions The diagnosis of a portal hepatic schwannoma requires immunohistological examinations in addition to multimodality imaging studies to reflect fully the pathohistological characteristics of the tumor.

Introduction: Endoscopic sinus surgery is an effective surgical procedure for treating chronic sinusitis; however, extensive exposure of the bone in the nasal cavity can result in permanent disability postoperatively. Particularly, closure of the sinus drainage pathway due to bone hyperplasia associated with bone exposure can trigger the recurrence of sinusitis. It is essential to regenerate the nasal mucosa after surgery to avoid bone hyperplasia. Regenerative medicine, including cell therapy, could be one of the leading options for nasal mucosa regeneration. To date, there is a lack of effective models for evaluating treatments for prevention of bone hyperplasia that occurs after sinus surgery. The purpose of this study was to develop a model of nasal mucosal removal to evaluate cellular therapies. Methods: The model was created in rabbits, a species with a wide nasal structure, and was generated by approaching the maxillary sinus from the nasal bone side and solely removing the maxillary sinus mucosa without destroying the structures in the nasal cavity. Adipose-derived mesenchymal stromal cell sheets prepared in temperature-responsive cell culture dishes were examined for the effect of transplantation in the animal model. Intranasal evaluation was assessed by micro-computed tomography and tissue staining. Results: Significant bone hyperplasia in the maxillary sinus occurred on the side of mucosal removal, and no bone hyperplasia occurred in the control sham side in the same rabbits on postoperative day 28. Bone hyperplasia was observed over a short time period, with the presence of bone hyperplasia in the maxillary sinus on day 14 and calcification of the bone on day 28. The adipose-derived mesenchymal stromal cell (ADSC) sheet was transplantable in a nasal mucosa-removal model. No significant differences in bone hyperplasia were found between the transplantation side and the sham side in terms of the effect of transplantation of the ADSC sheet; however, bone hyperplasia tended to be suppressed on the transplantation side. Conclusions: This animal model is simple, highly reproducible, and does not require special equipment or drugs. In addition, this model can be used for various therapeutic interventions, including cell therapy. The presence or absence of the nasal mucosa affects bone remodeling, which highlights the importance of regeneration of the nasal mucosa. In the nasal mucosal regeneration therapy, the ADSC sheet had an inhibitory effect on bone hyperplasia. The nasal mucosa-removal model allows observation of conditions associated with nasal mucosa removal and evaluation of the effectiveness of cell therapy.

Abstract Lung air leaks (LALs) due to visceral pleura injury during surgery are a difficult-to-avoid complication in thoracic surgery (TS). Reliable LAL closure is an important patient management issue after TS. We demonstrated both safeties of transplantation of a cultured human autologous dermal fibroblast sheet (DFS) to LALs. From May 2016 to March 2018, five patients who underwent thoracoscopic lung resection met all the inclusion criteria. Skin biopsies were acquired from each patient to source autologous dermal cells for DFS fabrication. During the primary culture, fibroblasts migrated from the dermal tissue pieces and proliferated to form cell monolayers. These fibroblasts were subcultured to confluence. Transplantable DFSs were fabricated from these subcultured fibroblasts that were trypsinized and seeded onto temperature-responsive culture dishes. After 10 days of fabrication culture, intact patient-specific DFS were harvested. DFSs were analyzed for fibroblast cell content and tissue contaminants prior to application. For closing intraoperative LAL, mean number of transplanted autologous DFS per patient was 6 ± 2 sheets. Mean chest drainage duration was 5.0 ± 4.8 days. The two patients with major LAL had a drainage duration of more than 7 days. All patients currently have no LAL recurrence after discharge. DFSs effectively maintain LAL closure via remodeling of the deposited extracellular matrix. The use of autologous DFSs to permanently close air leaks using a patient-derived source is expected to reduce surgical complications during high-risk lung resections.

Background: The utility of endoscopic transplantation of epithelial cell sheets to ulcer sites after endoscopic submucosal dissection (ESD) has been shown to prevent scar stenosis after ESD of early esophageal cancer. Previously, our group reported use of an endoscopic transplantation device fabricated with a 3-dimensional printer. Cell sheets are transplanted to the esophageal wound site with the following procedure: first, a cell sheet harvested from temperature-responsive culture dishes is placed on the device's deflated balloon surface and transported to the wound site with endoscopic forceps; second, by applying pressure from inflating the balloon locally at the wound site, the cell sheet is successfully transferred and adhered to the wound tissue; third, the balloon is deflated, and the device is removed. By repeating the procedure, several cell sheets can be safely transplanted to a wider ESD area. Nonetheless, possible damage to cell sheets using this procedure has not yet been assessed. Objective: Effects of endoscopic transplantation balloon inflation on cell viability and damage of normal human epidermal keratinocyte sheets resident on the device's balloon surface were evaluated by histology after sheet placement onto lumenal surfaces in the ex vivo porcine submucosal dissection esophagus model. Endoscopic transplantation of these same cell sheets with conventional methods using a polyvinylidene fluoride (PVDF) cell sheet support membrane, balloon device transfer, and also using a novel modified balloon transfer procedure was also examined. Cell sheet transfer results obtained with these three procedures were compared. Method: Normal human epidermal keratinocyte sheets were fabricated on temperature-responsive culture inserts. By temperature reduction to 20 °C, all cells were harvested as a single contiguous cell sheet. Freshly excised porcine esophagi purchased in a slaughter house were turned inside-out, and the exposed lumenal mucosa and submucosal layers were removed by Cooper scissors. This luminal surface was then utilized as a transplantation bed in ex vivo cell sheet experiments. Cell sheets were adhered to the endoscopic transfer device balloon, expanded by balloon inflation and resulting cell viability was evaluated by trypan blue exclusion test after cell sheet trypsinization and dispersion. Cell sheets were transferred onto the esophagus lumen ex vivo using forceps and the balloon device, and also using a modified balloon transfer method. The obtained results were compared with those without balloon expansion, and evaluated for sheet thickness and lumenal histology. Finally, TUNEL staining was performed to examine cell apoptosis. Result: Cell sheets thinned after cell sheet balloon expansion, but no apoptosis was observed after these procedures. Conclusion: Expanding keratinocyte cell sheets on a balloon endoscopic transfer device did not damage the cell sheets. This sheet transplantation method using the endoscopic balloon transfer device may be considered as a future standard cell sheet endoscopic transplantation procedure for repairing the esophagus.

Introduction: Cultured stratified epithelial cell sheets have been clinically utilized as transplantable grafts for the regeneration of epithelial tissues, such as the esophagus, cornea, skin, and intraoral cavity. These cell sheets are expected to gain widespread use as regenerative medicine products and save many patients. For this purpose, establishing and disseminating the stale protocol of fabricating the cell sheet is crucial. The fabrication of cultured stratified epithelial cell sheets consists of many important steps, and since the patients’ epithelial cell conditions vary widely and are sometimes unstable, the qualities of the epithelial cell grafts are likewise potentially unstable. Therefore, in this paper, we report the stable protocol for fabrication of the transplantable cell sheet particularly from patient-derived oral mucosal tissues. Methods: Serum extracted from blood and buccal mucosal tissue were collected in Nagasaki University and transported to Tokyo Women's Medical University. Oral mucosal epithelial cells were collected by minimum trypsin method, and this treatment was studied whether to be a critical procedure. After 14 days cultivation, cultured cells were examined whether to be transplantable as cell sheets. Results: We successfully transported buccal mucosal tissue and serum without damage and contamination. Oral mucosal epithelial cells were collected with high viability by minimum trypsin method. Finally, we succeeded to stably fabricate oral mucosal epithelial cell sheets in all 10 patients. Conclusions: We established a stable protocol for the fabrication of human oral mucosal epithelial cell sheets and their transportation in clinical settings in this study. These methodologies could also be basis for transplantation therapy using cultured cell sheets of various types other than oral mucosal epithelial cell and will contribute largely to the future development of regenerative medicine.

Background: Recently, the efficacy of cryotherapy and compression therapy to prevent taxane-induced peripheral neuropathy has been reported. We prospectively compared the efficacy of cryotherapy using a frozen glove (FG) and compression therapy using a surgical glove (SG) to prevent nanoparticle albumin-bound paclitaxel (nab-PTX)-induced peripheral neuropathy. Patients and methods: Breast cancer patients who received 260 mg/m2 of nab-PTX were eligible to participate in this trial. Patients wore a FG on one hand (60 min) without changing and two SGs of the same size (i.e., one size smaller than the size that best fit their hand) on the other hand (90 min) during chemotherapy. Peripheral neuropathy was evaluated at each treatment cycle using Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, the Patient Neurotoxicity Questionnaire (PNQ), and the Functional Assessment of Cancer Therapy-Taxane subscale. Temperatures at each fingertip in both groups were measured thermographically. Results: Between August 2017 and March 2019, 43 patients were enrolled and 38 were evaluated. No cases showed discordance of peripheral neuropathy between each gloved group in cases of CTCAE ≥ grade 2. In cases of PNQ ≥ grade D, using the Nam equivalence test, the upper test (P = 0.0329) and lower test (P = 0.0052) both showed negative results in comparisons between each gloved group. Fingertip temperature was significantly lower in the FG group than in the SG group after treatment (P

Abstract It is an open question as to whether cooling‐induced muscle contraction occurs in the in vivo environment. In this investigation, we tested the hypotheses that a rise in intracellular Ca²⁺ concentration ([Ca²⁺]i) and concomitant muscle contraction could be evoked in vivo by reducing muscle temperature and that this phenomenon would be facilitated or inhibited by specific pharmacological interventions designed to impact Ca²⁺‐induced Ca²⁺‐release (CICR). Progressive temperature reductions were imposed on the spinotrapezius muscle of Wistar rats under isoflurane anesthesia by means of cold fluid immersion. The magnitude, location, and temporal profile of [Ca²⁺]i were estimated using fura‐2 loading. Caffeine (1.25–5.0 mM) and procaine (1.6–25.6 mM) loading were applied in separatum to evaluate response plasticity by promoting or inhibiting CICR, respectively. Lowering the temperature of the muscle surface to ~5°C produced active tension and discrete sites with elevated [Ca²⁺]i. This [Ca²⁺]i elevation differed in magnitude from fiber to fiber and also from site to site within a fiber. Caffeine at 1.25 and 5.0 mM reduced the magnitude of cooling necessary to elevate [Ca²⁺]i (i.e., from ~5°C to ~8 and ~16°C, respectively, both p

Abstract Background The aim of this study was to evaluate the efficacy and safety of carbon‐ion radiation therapy for nonsquamous cell carcinomas of the oral cavity in a multicenter study. Methods Retrospective analysis of the clinicopathological features and outcomes of 76 patients with oral nonsquamous cell carcinomas with N0‐1 M0 status and were treated with carbon‐ion radiation therapy at four institutions in Japan between November 2003 and December 2014 was performed. Results Salivary gland carcinoma, mucosal melanoma, and three other carcinomas were found in 46, 27, and 3 patients, respectively. T1‐3, T4a, and T4b disease was diagnosed in 27, 18, and 31 patients, respectively. Median follow‐up period was 31.1 months (range, 3‐118 months). Three‐year local control, progression‐free survival, and overall survival of all patients were 86.8%, 63.1%, and 78.4%, respectively. Multivariate analysis showed T classification (T4) to be a significant independent poor prognostic factor for local control. Acute grade 3 mucositis was observed in 38 patients. Grades 3 and 4 late morbidities were observed in 9 and 4 patients, respectively. No grade 5 late toxicity was observed. Conclusions Oral nonsquamous cell carcinomas could be treated effectively, with acceptable toxicity, by carbon‐ion radiation therapy.

Introduction: Carrier-free autologous mucosal epithelial cell sheets have been clinically utilized as a cell therapy for various epithelial disorders. Fabrication of a transplantable oral mucosal epithelial cell sheet without mouse feeder layers requires a higher seeding density than that of a sheet with mouse feeder layer culture; therefore, a large amount of donor mucosal tissue is needed. However, cell grafts co-cultured with mouse feeder layers are classified by the US Food and Drug Administration (FDA) as xenogeneic products. The goal of this study was to evaluate the utility of oral mucosal epithelial cells expanded by primary explant culture for the fabrication of an adequate number of transplantable epithelial cell sheets without mouse feeder layers. Methods: Small fragments derived from minced oral mucosal tissue were placed into culture dishes for primary explant culture in keratinocyte culture medium. After primary explant culture, the outgrown cells were treated with trypsin-EDTA and were seeded on a temperature-responsive cell culture insert. After subculture, the cultured cells were harvested as a confluent cell sheet from the culture vessel by temperature reduction. Results: Carrier-free human oral mucosal epithelial cell sheets were fabricated in all human cases, and autologous transplantation of the harvested cell sheets showed rapid epithelial regeneration to cover epithelial defects in a rabbit model. The explant culture method, involving the use of small fragments for primary culture, was sufficient for preparing a large number of mucosal epithelial cells without mouse feeder layers. Moreover, oral mucosal epithelial cells derived from the primary explant culture after cryopreservation allowed for the fabrication of cell sheets. Conclusions: This method for fabricating transplantable oral mucosal epithelial cell sheets is an attractive technique for regenerative medicine. It offers a patient-friendly manufacturing method in which a small amount of biopsy material from the patient represents a sufficient epithelial cell source, and a manufacturing plan for preparing cell grafts can be easily tailored. Keywords: Cell therapy, Regenerative medicine, A small amount of biopsy, A sufficient epithelial cell source, Autologous transplantation, Without mouse feeder layers

PEDOT:Tos, a PSS-free PEDOT-based material, is a promising possible organic thermoelectric material for a practical conversion module because the material reportedly has a large power factor. However, since PEDOT:Tos is mainly reported to be a p-type thermoelectric material, the development of PSS-free PEDOT with n-type thermoelectric properties is desirable. Thus, in order to search for PSS-free PEDOT with n-type thermoelectric properties, we investigated the doping concentration of PTSA dependence of the thermoelectric property using the first-principle calculation. The band structure and the density of state indicated that the n-type thermal electromotive force was attributed to the electrons’ large effective mass. Such electrons were produced thanks to the binding of the dopant PTSA to the benzene ring. The contribution of the electron to the Seebeck coefficient increased with increasing PTSA doping concentrations.

Nose tissue that helps the ear heal Transplanting a sheet of cells from the nose to the middle ear could improve postoperative prognosis for middle ear inflammatory conditions. Kazuhisa Yamamoto of Jikei University School of Medicine and colleagues in Japan developed a novel surgical technique to prevent postoperative recurrence of chronic middle ear conditions. In adhesive otitis media, the eardrum gets sucked into and stuck in the middle ear space. Cholesteatoma involves abnormal skin growth in the middle ear. Recurrence often follows surgical treatment for these conditions due to delays in regeneration of the middle ear’s mucosal epithelium. The new technique involves transplanting cultured cells from the lining of the nasal cavity to the middle ear during surgery. Five patients treated this way had favorable postoperative outcomes with no adverse effects or complications and satisfactory improvement in hearing levels.

Abstract Resistance exercise training induces muscle hypertrophy, and recovery between sessions is one of the major determinants of this effect. However, the effect of the recovery period between sessions on muscle hypertrophy following resistance exercise training remains unclear. To elucidate the effect of recovery period on hypertrophy, in the present study, we investigated changes in protein degradation systems and hypertrophic responses in rat skeletal muscle to resistance training with variable recovery periods. In the conventional recovery group (exercised every 72 h) and a shorter recovery group (exercised every 24 h), 18 bouts of resistance exercise consisting of 50 repetitions of a 3‐sec maximal isometric contraction caused muscle hypertrophy and slight activation of muscle protein degradation systems. By contrast, in an excessively shorter recovery group (exercised every 8 h), 18 bouts of resistance exercise did not cause hypertrophy and markedly activated protein degradation systems, accompanied by inflammatory responses. These observations indicate that excessive shortening of recovery between sessions does not cause skeletal muscle hypertrophy, likely due to the activation of proteolysis induced by inflammatory responses to resistance exercise training.

We have utilized patients' own oral mucosa as a cell source for the fabrication of transplantable epithelial cell sheets to treat limbal stem cell deficiency and mucosal defects after endoscopic submucosal dissection of esophageal cancer. Because there are abundant microbiotas in the human oral cavity, the oral mucosa was sterilized and 40 μg/mL gentamicin and 0.27 μg/mL amphotericin B were added to the culture medium in our protocol. Although an oral surgeon carefully checked each patient's oral cavity and although candidiasis was not observed before taking the biopsy, contamination with Candida albicans (C. albicans) was detected in the conditioned medium during cell sheet fabrication. After adding 1 μg/mL amphotericin B to the transportation medium during transport from Nagasaki University Hospital to Tokyo Women's Medical University, which are 1200 km apart, no proliferation of C. albicans was observed. These results indicated that the supplementation of transportation medium with antimycotics would be useful for preventing contamination with C. albicans derived from the oral mucosa without hampering cell proliferation.

To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA) significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.

A target tissue can be thermally coagulated in high-intensity focused ultrasound (HIFU) treatment noninvasively. HIFU thermal treatments have been clinically applied to various solid tumors. One of the problems in HIFU treatments is a long treatment time. Acoustically driven microbubbles can accelerate the ultrasonic heating, resulting in the significant reduction of the treatment time. In this paper, a method named “trigger HIFU exposure” which employs cavitation microbubbles is introduced and its results are reviewed. A trigger HIFU sequence consists of high-intensity short pulses followed by moderate-intensity long bursts. Cavitation bubbles induced in a multiple focal regions by rapidly scanning the focus of high-intensity pulses enhanced the temperature increase significantly and produced a large coagulation region with high efficiency.

The medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors. The sexually dimorphic nucleus (SDN) of the MPOA has been studied to understand sexual dimorphism, although the anatomy and physiology of the SDN is not fully understood. Here, we characterized SDN neurons that contribute to sexual dimorphism and investigated the mechanisms underlying the emergence of such neurons and their roles in social behaviors. A target-specific neuroanatomical study using transgenic mice expressing Cre recombinase under the control of Calb1, a gene expressed abundantly in the SDN, revealed that SDN neurons are divided into two subpopulations, GABA neurons projecting to the ventral tegmental area (VTA), where they link to the dopamine system (CalbVTA neurons), and GABA neurons that extend axons in the MPOA or project to neighboring regions (CalbnonVTA neurons). CalbVTA neurons were abundant in males, but were scarce or absent in females. There was no difference in the number of CalbnonVTA neurons between sexes. Additionally, we found that emergence of CalbVTA neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. Chemogenetic analyses of CalbVTA neurons indicated a role in modulating sexual motivation in males. Knockdown of Calb1 in the MPOA reduced the intromission required for males to complete copulation. These findings provide strong evidence that a male-specific neural pathway from the MPOA to the VTA is organized by the two-step actions of testicular androgens for the modulation of sexually motivated behavior. [ABSTRACT FROM AUTHOR]