Non-clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti-glypican-3 chimeric antigen receptor-expressing natural killer/innate lymphoid cells.

The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 106 iPSC within 7 weeks was 1.8-4.0 × 109 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

The Importance of 11α-OH, 15-oxo, and 16-en Moieties of 11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Its Inhibitory Activity on Melanogenesis.

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis.

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

Salt-inducible kinase 3 deficiency exacerbates lipopolysaccharide-induced endotoxin shock accompanied by increased levels of pro-inflammatory molecules in mice.

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll-like receptor 4 on macrophages, several signalling cascades co-operatively up-regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt-inducible kinase [SIK, a member of the AMP-activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up-regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over-expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin-6 (IL-6), nitric oxide (NO) and IL-12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up-regulated in SIK3-deficient thioglycollate-elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3-deficient TEPM suppressed the levels of the above-mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3-knockout (KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1-KO (n = 8), SIK2-KO (n = 9) and wild-type (n = 8 or 9) groups. In addition, SIK3-KO bone marrow transplantation increased LPS sensitivity of the recipient wild-type mice, which was accompanied by an increased level of circulating IL-6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS-stimulated macrophages.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

Direct comparison of bacterial communities in soils contaminated with different levels of radioactive cesium from the first Fukushima nuclear power plant accident.

The Great East Japan Earthquake caused a serious accident at the first Fukushima nuclear power plant (NPP), which in turn released a large amount of radionuclides. Little attention has been paid to in-situ soil microorganisms exposed to radioactive contamination by the actual NPP accident. We herein investigated bacterial communities in the radioactive cesium (Cs)-contaminated and non-contaminated soils by high-throughput sequencing. The uppermost and ectorhizosphere soil samples were collected from the base of mugwort grown in the same soil type with the same soil-use history in order to compare the bacterial communities at geographically separated areas. The concentrations of radioactive Cs in the soils ranged from 10 to 563,000 Bq 137Cs/kg dry soil, with the highest concentration being detected at 1 km from the NPP. Alpha-diversity indices, i.e., Chao1, Shannon and Simpson reciprocal, of the sequence data showed the lower bacterial diversity in the most highly Cs-contaminated soil. Principal coordinate analysis with principle components 1 and 3 based on unweighted UniFrac distances indicated the significant difference in bacterial communities of the most contaminated area from those of the other areas. Operational taxonomic unit-based assay revealed higher abundance of the radio-resistant Geodermatophilus bullaregiensis relative in the most contaminated soil. Thus, it was strongly suggested that the radioactive accident facilitated the growth and/or survival of radio-resistant bacteria in the Cs-contaminated soils. The results of this study show that information on the soil type, vegetation and soil-use history enhances the direct comparison of geographically distant soil bacterial communities exposed to different levels of radioactive contamination.

Dilemma of physician-mothers faced with an increased home burden and clinical duties in the hospital during the COVID-19 pandemic.

PURPOSE: Since December 2019, coronavirus disease 2019 (COVID-19) has spread rapidly across the world. During the pandemic, physicians in our hospital have had to respond both to the issue of treating the patients and the increasing domestic burden associated with social disruption. The purpose of this study was to assess how much the burden on our doctors, especially female doctors, was increasing. MATERIAL AND METHODS: The Physicians' Career Support Committee in Sapporo Medical University conducted a questionnaire survey. The questionnaire inquired about a wide range of subjects with regard to working style and family life during the first and second waves of the COVID-19 pandemic, and was sent to all medical/dental physicians working in Sapporo Medical University. RESULTS: A total of 266 (42.7%) physicians in our hospital responded to our questionnaire and the data for 264 data were analyzed. The total numbers of males, females, and others, including those who did not want to specify, were 178 (67.4%), 82 (31.0%), and 4 (1.5%), respectively. Among them, 62 (23.5%) and 23 (8.7%) answered that their domestic burden was slightly or markedly increased. The increase in the domestic burden showed a significant difference between genders (p = 0.04). Even after correction for background differences using multivariate analysis, being female (p<0.001), having child dependents (p<0.001), and treating COVID-19 patients (p = 0.03) were significantly related to an increased domestic burden. Regarding family style, 58.1% of the physician-fathers were from two-income families (i.e., families with both parents in employment), and they answered that their partner mainly cared for the children. In contrast, 97.3% of physician-mothers were from two-income families, and 94.6% of the physician-mothers had to take care of children by themselves. CONCLUSION: Physician-mothers are caught in a dilemma between an increased home burden and clinical duties in the hospital, with a significantly higher ratio than physician-fathers during the pandemic. As we showed, female doctors could have not continued their careers and take responsible positions in the same way as male doctors. This is a social risk in the timing of a crisis, such as a pandemic.

Simaomicin α, a polycyclic xanthone, induces G1 arrest with suppression of retinoblastoma protein phosphorylation.

Recent progress in cancer biology research has shown that abnormal proliferation in tumor cells can be attributed to aberrations in cell cycle regulation, especially in G1 phase. During the course of searching for microbial metabolites that affect cell cycle distribution, we have found that simaomicin α, a polycyclic xanthone antibiotic, arrests the cell cycle at G1 phase. Treatment of T-cell leukemia Jurkat cells with 3 nM simaomicin α induced an increase in the number of cells in G1 and a decrease in those in G2­M phase. Cell cycle aberrations induced by simaomicin α were also detected in colon adenocarcinoma HCT15 cells. Simaomicin α had antiproliferative activities in various tumor cell lines with 50% inhibitory concentration values in the range of 0.3­19 nM. Furthermore, simaomicin α induced an increase in cellular caspase-3 activity and DNA fragmentation, indicating that simaomicin α promotes apoptosis. The retinoblastoma protein phosphorylation status of simaomicin α-treated cell lysate was lower than that of control cells, suggesting that the target molecule of simaomicin α is in a pathway upstream of retinoblastoma protein phosphorylation. In the course of evaluating polycyclic xanthone antibiotics structurally related to simaomicin α, we also found that cervinomycin A1 stimulated accumulation of treated cells in G1 phase. These results indicate that the polycyclic xanthones, including simaomicin α and cervinomycin A1, may be candidate cancer chemotherapeutic agents.

Generation of HIV-Resistant Macrophages from IPSCs by Using Transcriptional Gene Silencing and Promoter-Targeted RNA.

Highly active antiretroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy.

Keratinocytes in atopic dermatitis express abundant ΔNp73 regulating thymic stromal lymphopoietin production via NF-κB.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that often cannot be completely controlled by modern medicine. Since multiple factors are intricately involved in the pathogenesis of AD, wide-ranging research is required for further advancement of AD treatment. Epidermal keratinocytes are the forefront to the external environment and play a pivotal role in the initiation of immune reaction against exogenous invasion. OBJECTIVE: Thymic stromal lymphopoietin (TSLP) is a keratinocyte-derived cytokine that induces differentiation and activation of type 2 helper T cells and innate lymphoid cells, cardinal effectors in pathophysiology of AD. We previously reported that ΔNp63, a p53-related molecule, regulates the expression of TSLP receptors and suggested the entity of a potential TSLP autocrine loop in the AD epidermis. In this study, we further explored the significance of p53 family transcription factors in TSLP production from human keratinocytes. METHOD: Expression profile of p73, a p53-related molecule, was evaluated in human AD tissue by immunohistochemistry. In addition, the function of p73 in producing TSLP was investigated with in vitro cultured keratinocytes via molecular biological analysis. RESULTS: ΔNp73 was abundantly expressed in the AD epidermis and increased the release of TSLP via NF-κB activation. Furthermore, the Toll-like receptor 3 signal enhanced ΔNp73 expression and thereby induced TSLP expression. CONCLUSION: Our results indicate that ΔNp73 is an additional participant in the mechanism of TSLP production. Amending the aberrant state of keratinocytes, represented by overexpression of ΔNp73, can be a novel therapeutic target of AD.

A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency.

Mitochondrial oxidative phosphorylation is a major source of cellular ATP. Its usage as an energy source varies, not only according to the extracellular environment, but also during development and differentiation, as indicated by the reported changes in the flux ratio of glycolysis to oxidative phosphorylation during embryonic stem (ES) cell differentiation. The fluorescent probe JC-1 allows visualization of changes in the mitochondrial membrane potential produced by oxidative phosphorylation. Strong JC-1 signals were localized in the differentiated cells located at the edge of H9 ES colonies that expressed vimentin, an early differentiation maker. The JC-1 signals were further intensified when individual adjacent colonies were in contact with each other. Time-lapse analyses revealed that JC-1-labeled H9 cells under an overconfluent condition were highly differentiated after subculture, suggesting that monitoring oxidative phosphorylation in live cells might facilitate the prediction of induced pluripotent stem cells, as well as ES cells, that are destined to lose their undifferentiated potency.

Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells.

Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Effect of 3-O-octanoyl-(+)-catechin on the responses of GABA(A) receptors and Na+/glucose cotransporters expressed in xenopus oocytes and on the oocyte membrane potential.

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.

Anti-Staphylococcus aureus activity and oxacillin resistance modulating capacity of 3-O-acyl-catechins.

Epicatechin gallate (ECg), a component of green tea with weak activity against Staphylococcus aureus, reduces oxacillin resistance in methicillin-resistant S. aureus at concentrations below the MIC. Because catechins bind to artificial lipid bilayers, we investigated whether the anti-staphylococcal activity of catechins could be improved by increasing their capacity to interact with the cytoplasmic membrane. Substitution of the gallate group of ECg with 3-O-acyl chains of varying lengths (C(4)-C(18)) led to enhanced anti-staphylococcal activity with chain lengths of C(8) (octanoyl) and C(10) (decanoyl). 3-O-octanoyl catechin was bactericidal against MRSA as the result of membrane damage. 3-O-acyl catechins tested at a 1/4 MIC did not reduce the oxacillin MIC greater than two-fold. 3-O-acyl catechins warrant further investigation as anti-staphylococcal agents.