Collagen patches releasing phosphatidylserine liposomes guide M1-to-M2 macrophage polarization and accelerate simultaneous bone and muscle healing.

Bilateral communication between bones and muscles is essential for healing composite bone-muscle injuries from orthopedic surgeries and trauma. However, these injuries are often characterized by exaggerated inflammation, which can disrupt bone-muscle crosstalk, thereby seriously delaying the healing of either tissue. Existing approaches are largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging, with little research conducted to date. Here we introduce collagen patches that overcome this overlooked issue by harnessing the plasticity of macrophage phenotypes. Phosphatidylserine liposomes (PSLs) capable of shifting the macrophage phenotype from inflammatory M1 into anti-inflammatory/prohealing M2 were coated on collagen patches via a layer-by-layer method. Original collagen patches failed to improve tissue healing under inflammatory conditions coordinated by M1 macrophages. In contrast, PSL-coated collagen patches succeeded in accelerating bone and muscle healing by inducing a microenvironment dominated by M2 macrophages. In cell experiments, differentiation of preosteoblasts and myoblasts was completely inhibited by secretions of M1 macrophages but unaffected by those of M2 macrophages. RNA-seq analysis revealed that type I interferon and interleukin-6 signaling pathways were commonly upregulated in preosteoblasts and myoblasts upon stimulation with M1 macrophage secretions, thereby compromising their differentiation. This study demonstrates the benefit of PSL-mediated M1-to-M2 macrophage polarization for simultaneous bone and muscle healing, offering a potential strategy toward simultaneous regeneration of multiple tissues. STATEMENT OF SIGNIFICANCE: Existing approaches for tissue regeneration, which primarily utilize growth factors, have been largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging and has been little studied. Here we demonstrate that collagen patches releasing phosphatidylserine liposomes (PSLs) promote M1-to-M2 macrophage polarization and are effective for simultaneous healing of bone and muscle. Transcriptome analysis using next-generation sequencing reveals that differentiation of preosteoblasts and myoblasts is inhibited by the secretions of M1 macrophages but promoted by those of M2 macrophages, highlighting the importance of timely regulation of M1-to-M2 polarization in tissue regeneration. These findings provide new insight to tissue healing of multiple tissues.

Opioid prescription status around surgery, bone metastasis, or death events among patients with breast cancer in Japan: an analysis of the Japanese public health insurance comprehensive claims database (the National Database).

OBJECTIVE: To investigate the opioid prescription status around clinical events among patients with breast cancer in Japan using a comprehensive claims database. METHODS: This was a retrospective cohort study using the National Database (April 2009-March 2020). The target patients had a first breast cancer diagnosis in April 2010 or later. The percentages of patients prescribed opioids before and after surgery, before and after bone metastasis, and before death with a breast cancer diagnosis in the same month were analyzed by month and by clinical facility characteristics and location. RESULTS: We identified 1 085 388 target patients, including 216 503, 72 645, and 70 832 patients with data for the events of surgery, bone metastasis, and death, respectively. The percentage of patients prescribed opioids in the month of surgery was the highest of the entire study period at ≥70%. The percentage of patients prescribed opioids increased before bone metastasis, peaked 1 month later, and decreased thereafter while remaining higher than that before the event. The percentage of patients who were prescribed opioids before death increased over time, peaking at 33.4% 1 month before death. Prescriptions differed by facility characteristics and facility location around surgery; no differences by facility characteristics, including location, were noted around the other events. The percentage of patients prescribed opioids was consistently lower than that reported in other countries for all events. CONCLUSIONS: We showed the opioid prescription status around clinical events, including some distinct patterns depending on facility characteristics for the period around surgery, among patients with breast cancer in Japan.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells.

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Size control of induced pluripotent stem cells colonies in two-dimensional culture for differentiation into functional monocyte-like cells.

BACKGROUND AIMS: Monocytes, derived from hematopoietic stem cells (HSCs), play a pivotal role in the immune response to cancer. Although they are an attractive source of cell therapy for cancer, a method for ex vivo expansion has not yet been established. Monocytes differentiated from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs), can be an alternative source of HSC-derived monocytes because of their self-renewal and pluripotency. To develop a standardized method for the generation of iPSC-derived monocytes for future clinical applications, we aim to control the size of the iPSC colony. METHODS: To this end, we developed a plate with multiple dots containing a chemical substrate for the iPSC scaffold. iPSCs placed in the plate expanded only on the dots and created colonies of the same size. The cells were then differentiated into monocytes by adding cytokines to the colonies. RESULTS: The dot plate substantially reduced variability in monocyte-like cell generation when compared with cultivating cells on a plate with the substrate covering the entire surface area. Furthermore, more monocyte-like cells were obtained by adjusting the dot size and the distance between the dots. The iPSC-derived monocyte-like cells phagocytosed cancer cells and secreted proinflammatory cytokines. The cells also expressed Fc receptors and exerted immunoglobulin G-mediated killing of cancer cells with the corresponding antibodies. CONCLUSIONS: The dot plate enabled the control of iPSC colony size in two-dimensional culture, which resulted in a reduction in the generation-variation of functional monocyte-like cells. This standardized method for generating iPSC-derived monocyte-like cells using the dot plate could also facilitate the development of an automated closed system on a large scale for clinical applications.

Improved Production of Induced Pluripotent Stem Cells Using Dot Pattern Culture Plates.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

Opioid prescriptions at the point of surgery, bone metastasis, or death among patients with breast cancer in Japanese acute care hospitals: a claims-based, retrospective, longitudinal study.

PURPOSE: Breast cancer is the most common cancer among Japanese women and often yields a better prognosis than other cancers. However, few studies have been conducted on pain control using opioids in Japan. In this study, we aimed to examine actual opioid use among breast cancer patients. METHODS: Breast cancer patients were defined as female patients with a first breast cancer diagnosis during the observational period in an acute care hospital database (April 2008 - February 2020). We examined the percentage of patients prescribed opioids, the opioid amount per patient, and the opioid dosage per day around surgery, bone metastasis diagnosis, or death. RESULTS: Overall, 217,722 breast cancer patients were identified. The percentage of patients prescribed opioids and the average amount of opioids per patient were highest in the month of surgery, 78% and 27 morphine milligram equivalents (MMEs), respectively. The average opioid dosage increased with time after surgery from 19 to 28 MMEs. Around bone metastasis, the percentage of patients prescribed opioids and the average opioid amount per patient peaked one month after the diagnosis, 31% and 371 MMEs, respectively. The average opioid dosage gradually increased from 22 to 35 MMEs in succeeding days after a bone metastasis diagnosis. The percentage of patients prescribed opioids and the average opioid amount per patient increased as the month of death approached. CONCLUSION: We investigated opioid prescription trends around clinical events in breast cancer patients on a large scale in Japan. These results may be useful to control cancer pain among breast cancer patients.

Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit.

Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.

Diagnosis and treatment of influenza based on health insurance claims between the 2010-2011 and 2019-2020 influenza seasons in Japan.

Medical practices for influenza virus infection vary among countries. In Japan, treatment with anti-influenza drugs is recommended for patients diagnosed with influenza. This health claims database study provides quantitative information aimed at describing the actual medical practices, including diagnostic testing and medication use, for managing influenza in Japan. Most patients diagnosed with influenza underwent diagnostic tests and were prescribed anti-influenza drugs. Meanwhile, the majority of patients prescribed anti-influenza drugs had undergone diagnostic testing. However, an increase in the percentage of anti-influenza prescriptions without diagnostic testing was observed during the 2019-2020 influenza season, which may be associated with the COVID-19 pandemic.

A chemically-defined plastic scaffold for the xeno-free production of human pluripotent stem cells.

Clinical use of human pluripotent stem cells (hPSCs) is hampered by the technical limitations of their expansion. Here, we developed a chemically synthetic culture substrate for human pluripotent stem cell attachment and maintenance. The substrate comprises a hydrophobic polyvinyl butyral-based polymer (PVB) and a short peptide that enables easy and uniform coating of various types of cell culture ware. The coated ware exhibited thermotolerance, underwater stability and could be stored at room temperature. The substrate supported hPSC expansion in combination with most commercial culture media with an efficiency similar to that of commercial substrates. It supported not only the long-term expansion of examined iPS and ES cell lines with normal karyotypes during their undifferentiated state but also directed differentiation of three germ layers. This substrate resolves major concerns associated with currently used recombinant protein substrates and could be applied in large-scale automated manufacturing; it is suitable for affordable and stable production of clinical-grade hPSCs and hPSC-derived products.

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

Protective and healing effects of apoptotic mimic-induced M2-like macrophage polarization on pressure ulcers in young and middle-aged mice.

Pressure ulcers (PUs) have no cure and are of significant health and economic concern worldwide, owing to the increasing population of elderly individuals at high risk for PU and who have impaired tissue repair. Macrophages play a pivotal role in PU development and healing. Imbalances between M1 (inflammatory) and M2 (anti-inflammatory/reparative) macrophages result in delayed resolution of inflammation and wound healing. We hypothesized that M1-to-M2 macrophage polarization mediated by artificial apoptotic cell mimics, phosphatidylserine-containing liposomes (PSLs), would protect against PU formation and accelerate PU healing in young (2-month-old) and middle-aged (12-month-old) mice. We used a clinically relevant murine model of ischemia-reperfusion-induced PU. Middle-aged mice displayed the delayed wound healing associated with increased inflammation, decreased collagen deposition, reduced angiogenesis, and delayed wound closure relative to their younger counterparts. PSL treatment significantly inhibited PU formation and promoted tissue remodeling in both age groups. These effects were mediated by increased M1-to-M2 macrophage polarization, induced by the PSLs. Thus, this study suggests, for the first time, that PSL-induced M2-like macrophage polarization is a promising strategy to protect against PU formation and promote PU repair in human patients of all ages.

Effect of sodium-glucose cotransporter-2 inhibitors on aldosterone-to-renin ratio in diabetic patients with hypertension: a retrospective observational study.

BACKGROUND: Plasma aldosterone-to-renin ratio (ARR) is popularly used for screening primary aldosteronism (PA). Some medications, including diuretics, are known to have an effect on ARR and cause false-negative and false-positive results in PA screening. Currently, there are no studies on the effects of sodium-glucose cotransporter-2 (SGLT2) inhibitors, which are known to have diuretic effects, on ARR. We aimed to investigate the effects of SGLT2 inhibitors on ARR. METHODS: We employed a retrospective design; the study was conducted from April 2016 to December 2018 and carried out in three hospitals. Forty patients with diabetes and hypertension were administered SGLT2 inhibitors. ARR was evaluated before 2 to 6 months after the administration of SGLT2 inhibitors to determine their effects on ARR. RESULTS: No significant changes in the levels of ARR (90.9 ± 51.6 vs. 81.4 ± 62.9) were found. Body mass index, diastolic blood pressure, heart rate, fasting plasma glucose, and hemoglobin A1c were significantly decreased by SGLT2 inhibitors. Serum creatinine was significantly increased. CONCLUSION: SGLT2 inhibitor administration yielded minimal effects on ARR and did not increase false-negative results in PA screening in patients with diabetes and hypertension more than 2 months after administration.

Sequential peripheral enrichment of H2A.Zac and H3K9me2 during trophoblast differentiation in human embryonic stem cells.

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure, involving covalent modification of histone tails, use of histone variants and gene position changes with respect to the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone modifications for changes in nuclear peripheral enrichment during differentiation of human embryonic stem cells to a trophoblast-like lineage. We found two dynamic modifications at the nuclear periphery, acetylation of histone H2A.Z (H2A.Zac), and dimethylation of histone H3 at lysine 9 (H3K9me2). We demonstrate successive peripheral enrichment of these markers, with H2A.Zac followed by H3K9me2, over the course of 4 days. We find that H3K9me2 increases concomitantly with, but independently of, expression of lamin A, since deletion of lamin A did not affect H3K9me2 enrichment. We further show that inhibition of histone deacetylases causes persistent and increased H2A.Z acetylation at the periphery, delayed H3K9me2 enrichment and failure to differentiate. Our results show a concerted change in the nature of peripheral chromatin occurs upon differentiation into the trophoblast state.

Unique cellular interaction of macrophage-targeted liposomes potentiates anti-inflammatory activity.

Nanomedicines that suppress macrophage-mediated chronic inflammation are important therapeutics for many inflammatory diseases. The small-sized (<100 nm) apoptotic-cell-mimetic macrophage-targeted liposomes served as a long-lasting immunosuppressive agent through preferential association with CD300a receptor, unlike larger liposomes, enabling the amelioration of hepatic inflammation in mice.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

A novel interleukin-13 receptor alpha 2-targeted hybrid peptide for effective glioblastoma therapy.

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB.

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.

Identification of a wide range of motifs inhibitory to shiga toxin by affinity-driven screening of customized divalent peptides synthesized on a membrane.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli, binds to target cells through a multivalent interaction between its B-subunit pentamer and the cell surface receptor globotriaosylceramide, resulting in a remarkable increase in its binding affinity. This phenomenon is referred to as the "clustering effect." Previously, we developed a multivalent peptide library that can exert the clustering effect and identified Stx neutralizers with tetravalent peptides by screening this library for high-affinity binding to the specific receptor-binding site of the B subunit. However, this technique yielded only a limited number of binding motifs, with some redundancy in amino acid selectivity. In this study, we established a novel technique to synthesize up to 384 divalent peptides whose structures were customized to exert the clustering effect on the B subunit on a single cellulose membrane. By targeting Stx1a, a major Stx subtype, the customized divalent peptides were screened to identify high-affinity binding motifs. The sequences of the peptides were designed based on information obtained from the multivalent peptide library technique. A total of 64 candidate motifs were successfully identified, and 11 of these were selected to synthesize tetravalent forms of the peptides. All of the synthesized tetravalent peptides bound to the B subunit with high affinities and effectively inhibited the cytotoxicity of Stx1a in Vero cells. Thus, the combination of the two techniques results in greatly improved efficiency in identifying biologically active neutralizers of Stx.

Role of a NK receptor, KLRE-1, in bone marrow allograft rejection: analysis with KLRE-1-deficient mice.

Natural killer (NK) cells play a pivotal role in the immune reaction during the bone marrow allograft rejection. Little is known, however, about the molecular mechanisms underlying the NK cell-mediated allograft recognition and rejection. In this report, we assessed the role of a recently identified NK receptor, killer cell lectinlike receptor 1 (KLRE-1), by generating knock-out mice. KLRE-1-deficient mice were born at an expected frequency and showed no aberrant phenotype on growth and lymphoid development. Nevertheless, KLRE-1-deficient cells showed a severely compromised allogeneic cytotoxic activity compared with the wild-type cells. Furthermore, allogeneic bone marrow transfer culminated in colony formation in the spleen of KLRE-1-deficient mice, whereas no colony formation was observed in wild-type recipient mice. These results demonstrate that KLRE-1 is a receptor mediating recognition and rejection of allogeneic target cells in the host immune system.

Expansion of lung V alpha 14 NKT cells by administration of alpha-galactosylceramide-pulsed dendritic cells.

NKT cells, a novel murine lymphoid lineage bearing an invariant T cell receptor encoded by V alpha 14 and J alpha 281 gene segments, recognize a specific ligand glycolipid, alpha-galactosylceramide (alpha-GalCer) in a CD1d-dependent manner. Recent research has revealed that activated V alpha 14 NKT cells have dramatic antitumor effects against a wide variety of tumor cell lines in vivo and in vitro. Here, we demonstrate strong in vivo antitumor effects brought about by treatment with alpha-GalCer-pulsed dendritic cells in comparison with in vitro-activated V alpha 14 NKT cells. Furthermore, we show a significant expansion of endogenous V alpha 14 NKT cells in the lung following the administration of alpha-GalCer-pulsed dendritic cells. The feasibility of immunotherapy with alpha-GalCer-pulsed dendritic cells is discussed.