Generation of monkey iPS cell-derived cartilage lacking MHC class I molecules on the cell surface

Multiple immune reactions when transplanting cartilage into monkeys. 京都大学プレスリリース. 2021-07-07.Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of MHC class I molecules in order to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey (Macaca fascicularis)) iPS cells (cyiPSCs) to obtain B2M⁻/⁻ cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M⁻/⁻ cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M⁻/⁻ cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M⁻/⁻ cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M⁺/⁺ and B2M⁻/⁻ cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M⁺/⁺ and B2M⁻/⁻ cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M⁻/⁻ chondrocytes isolated from B2M⁻/⁻ cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M⁺/⁺ nor B2M⁻/⁻ cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the other hand, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer (NK) cells around B2M⁻/⁻ cyiPSC-derived cartilage than B2M⁺/⁺ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo

卵巣明細胞癌と類内膜癌の鑑別に関するMRIについての知見

BACKGROUND: Common cancerous histological types associated with endometriosis are clear cell carcinoma (CCC) and endometrioid carcinoma (EC). CCC is regarded as an aggressive, chemoresistant histological subtype. Magnetic resonance imaging (MRI) offers some potential advantages to diagnose ovarian tumors compared with ultrasonography or computed tomography. This study aimed to identify MRI features that can be used to differentiate between CCC and EC. METHODS: We searched medical records of patients with ovarian cancers who underwent surgical treatment at Nara Medical University Hospital between January 2008 and September 2018; we identified 98 patients with CCC or EC who had undergone preoperative MRI. Contrasted MRI scans were performed less than 2 months before surgery. Patients were excluded from the study if they had no pathology, other pathological subtype of epithelial ovarian cancer, and/or salvage treatment for recurrence and metastatic ovarian cancer at the time of study initiation. Clinically relevant variables that were statistically significant by univariate analysis were selected for subsequent multivariate regression analysis to identify independent factors to distinguish CCC from EC. RESULTS: MRI of CCC and EC showed a large cystic heterogeneous mixed mass with mural nodules protruding into the cystic space. Univariate logistic regression analysis revealed that the growth pattern (broad-based nodular structures [multifocal/concentric sign] or polypoid structures [focal/eccentric sign]), surface irregularity (a smooth/regular surface or a rough/irregular/lobulated surface), "Width" of mural nodule, "Height-to-Width" ratio (HWR), and presence of preoperative ascites were factors that significantly differed between CCC and EC. In the multivariate logistic regression analysis, the growth pattern of the mural nodule (odds ratio [OR] = 0.69, 95% confidence interval [CI]: 0.013-0.273, p = 0.0004) and the HWR (OR = 3.71, 95% CI: 1.128-13.438, p = 0.036) were independent predictors to distinguish CCC from EC. CONCLUSIONS: In conclusion, MRI data showed that the growth pattern of mural nodules and the HWR were independent factors that could allow differentiation between CCC and EC. This finding may be helpful to predict patient prognosis before operation.博士（医学）・乙第1433号・令和元年9月27日© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

Transcranial Direct Current Stimulation of the Temporoparietal Junction and Inferior Frontal Cortex Improves Imitation-Inhibition and Perspective-Taking with no Effect on the Autism-Spectrum Quotient Score

Lesions to brain regions such as the temporoparietal junction (TPJ) and inferior frontal cortex (IFC) are thought to cause autism-spectrum disorder (ASD). Previous studies indicated that transcranial direct current stimulation (tDCS) of the right TPJ improves social cognitive functions such as imitation-inhibition and perspective-taking. Although previous work shows that tDCS of the right IFC improves imitation-inhibition, its effects on perspective-taking have yet to be determined. In addition, the role of the TPJ and IFC in determining the Autism-Spectrum Quotient (AQ), which is a measure of autism spectrum traits, is still unclear. Thus, the current study performed tDCS on the right TPJ and the right IFC of healthy adults, and examined its effects on imitation-inhibition, perspective-taking and AQ scores. Based on previous studies, we hypothesized that anodal tDCS of the right IFC and right TPJ would improve imitation-inhibition, perspective-taking and the AQ score. Anodal tDCS of the right TPJ or IFC significantly decreased the interference effect in an imitation-inhibition task and the cost of perspective-taking in a perspective-taking task, in comparison to the sham stimulation control. These findings indicated that both the TPJ and the IFC play a role in imitation-inhibition and perspective-taking, i.e., control of self and other representations. However, anodal stimulation of the right TPJ and the right IFC did not alter participants’ AQ. This finding conflicts with results from previous brain imaging studies, which could be attributed to methodological differences such as variation in sex, age and ASD. Therefore, further research is necessary to determine the relationship between the TPJ and IFC, and the AQ

Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface

Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of major histocompatibility complex (MHC) class I molecules to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey [Macaca fascicularis]) iPS cells (cyiPSCs) to obtain B2M−/− cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M−/− cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M−/− cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M−/− cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M+/+ and B2M−/− cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M+/+ and B2M−/− cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M−/− chondrocytes isolated from B2M−/− cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M+/+ nor B2M−/− cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the contrary, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer cells around B2M−/− cyiPSC-derived cartilage than B2M+/+ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo.Okutani Y., Abe K., Yamashita A., et al. Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface. Tissue Engineering - Part A 28, 94 (2022); https://doi.org/10.1089/ten.tea.2021.0053

A novel form of transcutaneous electrical nerve stimulation for the reduction of dysesthesias caused by spinal nerve dysfunction: A case series

Background: Current therapeutic interventions for dysesthesias caused by spinal cord dysfunctions are ineffective.We propose a novel intervention using transcutaneous electrical nerve stimulation (TENS) for dysesthesias, and we present an in-depth case series. Patients and methods: Conventional high-frequency TENS and the novel dysesthesia-matched TENS (DM-TENS) were applied to 16 hands of nine patients with spinal cord dysfunction. The dysesthesia-matched TENS’ stimulus intensity and frequency matched the intensity and somatosensory profile of the patients’ dysesthesias. The Short-Form McGill Pain Questionnaire version-2 (SF-MPQ2) and quantitative sensory testing (QST) were applied during electrical stimulation/no stimulation. We determined intraclass correlation coefficients (ICCs) to evaluate the reliability of the setting and the effects on the dysesthesias and the change in subjective dysesthesia between each patient’s baseline without TENS and DM-TENS.Results: We were able to apply electrical stimulation matching the patients’ subjective dysesthesia for 14 hands (eight patients). TENS could not be applied for the remaining patient due to severe sensory deficits. Compared to the patients’ baseline and high-frequency TENS, the DM-TENS provided significant decreases in tingling/pinsand-needles and numbness on the SF-MPQ2, and it significantly improved the dynamic and static mechanical detection on QST. Regarding the reliability of the dysesthesia-matched TENS settings, the ICCs (1,5) were intensity, 0.95; frequency, 1.00; and effect on dysesthesia, 0.98.Conclusion: DM-TENS improved the dysesthesias and mechanical hypoesthesia caused by spinal cord dysfunction. The effectiveness of DM-TENS particularly for tingling and numbness was clearly higher and was reliable within the patients. These results may suggest an effective treatment of dysesthesias in patients with spinal cord dysfunction

Changes in intentional binding effect during a novel perceptual-motor task

Perceptual-motor learning describes the process of improving the smoothness and accuracy of movements. Intentional binding (IB) is a phenomenon whereby the length of time between performing a voluntary action and the production of a sensory outcome during perceptual-motor control is perceived as being shorter than the reality. How IB may change over the course of perceptual-motor learning, however, has not been explicitly investigated. Here, we developed a set of IB tasks during perceptual-motor learning. Participants were instructed to stop a circular moving object by key press when it reached the center of a target circle on the display screen. The distance between the center of the target circle and the center of the moving object was measured, and the error was used to approximate the perceptual-motor performance index. This task also included an additional exercise that was unrelated to the perceptual-motor task: after pressing the key, a sound was presented after a randomly chosen delay of 200, 500, or 700 ms and the participant had to estimate the delay interval. The difference between the estimated and actual delay was used as the IB value. A cluster analysis was then performed using the error values from the first and last task to group the participants based on their perceptual-motor performance. Participants showing a very small change in error value, and thus demonstrating a small effect of perceptual-motor learning, were classified into cluster 1. Those who exhibited a large decrease in error value from the first to the last set, and thus demonstrated a strong improvement in perceptual-motor performance, were classified into cluster 2. Those who exhibited perceptual-motor learning also showed improvements in the IB value. Our data suggest that IB is elevated when perceptual-motor learning occurs

Prompt improvement of a pressure ulcer by the administration of high viscosity semi-solid nutrition via a nasogastric tube in a man with tuberculosis: a case report

INTRODUCTION: Semi-solid nutrition with high viscosity has the advantage of reducing gastroesophageal reflux and diarrhea and shortens the duration of administration compared with liquid nutrition. This is the first report describing the administration of semi-solid nutrition with high viscosity via a nasogastric tube, which achieved a remarkable improvement in the patient's nutritional state. CASE PRESENTATION: A 67-year-old man (mongoloid race, Japanese) with tuberculosis, a pressure ulcer and malnutrition was admitted to our hospital. He also had right hemiplegia, dysphagia and aphasia as sequelae of a cerebral hemorrhage. Before his admission, he had been treated at another hospital with 600 kcal/day of liquid nutrition via a nasogastric tube, which was insufficient and induced severe malnutrition. After he was admitted to our hospital, we increased the quantity of his liquid nutrition without success because of complications, specifically diarrhea and gastroesophageal reflux. As it was difficult to confirm whether or not he would accept gastrostomy feeding, we administered semi-solid nutrition with high viscosity (20,000 mPa x s) via a large-bore nasogastric tube (18 French). Soon after he was started on semi-solid nutrition, his pressure ulcer and malnutrition improved without diarrhea or complications accompanying the large-bore nasogastric tube. CONCLUSION: When patients have problems with liquid nutrition, such as diarrhea or gastroesophageal reflux, semi-solid nutrition via a nasogastric tube is a useful method of achieving improvements in nutritional state in a short period of time

Japanese nationwide questionnaire survey on delayed cerebral infarction due to vasospasm after subarachnoid hemorrhage

Background and purposeVarious prophylactic drugs for cerebral vasospasm and delayed cerebral infarction (DCI) after subarachnoid hemorrhage (SAH) have been used in Japan. To investigate the treatment trends for cerebral vasospasm and frequency of DCI after SAH throughout Japan in 2021.MethodsIn 2021 we conducted an anonymous questionnaire survey on management for preventing cerebral vasospasm after aneurysmal SAH, and the frequency of DCI. The questionnaire was emailed to 955 certified neurosurgeons at 553 hospitals in Japan. Of them, 162 hospitals (29% response rate) responded to the questionnaire. Of these, 158 were included in this study, while four hospitals that responded insufficiently were excluded. The efficacy of treatments for reducing DCI were examined through a logistic regression analysis.ResultsAmong 3,093 patients treated with aneurysmal SAH, 281 patients (9.1%) were diagnosed with DCI related to cerebral vasospasm. Coil embolization had significantly lower DCI frequency (6.9%), compared to microsurgical clipping (11.8%, odds ratio, 0.90; 95% confidential intervals, 0.84–0.96; P, 0.007). In addition, cilostazol administration was associated with significantly lower DCI frequency (0.48; 0.27–0.82; 0.026). The efficacy of cilostazol in reducing DCI remained unchanged after adjustment for covariates. The most effective combination of multiple prophylactic drugs in reducing DCI related to cerebral vasospasm was cilostazol, fasudil, and statin (0.38; 0.22–0.67; 0.005).ConclusionsThis study elucidated the trends in prophylactic drugs to prevent cerebral vasospasm and frequency of DCI after aneurysmal SAH in Japan. Coil embolization and cilostazol administration showed effectiveness in reducing DCI related to cerebral vasospasm in 2021

“I don’t want to live too long!”: Successful ageing and the failure of longevity in Japan

This chapter examines ‘successful aging’ through its impacts on formal care workers in Japan. It is based on one year of fieldwork conducted in urban Japan and examines the affective, ethical, and cultural forces that result at times in resilience, compassion, and intimacy between carers and elderly clients, and at other times, in violence, abuse, and abandonment. I argue that locating the source of this divergence in individuals (i.e., adverse coping strategy) reproduces the same neoliberal model of success for care workers as it does for the elderly. Instead, care and abuse in formal care settings can be seen as symptoms of broader political and economic transformations that have been occurring in Japan since the 1990s