Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Effect of Soil Organic Matters in Dredged Soils to Utilization of their Mixtures Made with a Steel Slag.

Dredged soils have been used as construction materials by alkaline activation with steel slag (steel slag-dredged soil mixtures) at harbors. Such mixtures develop strength chiefly by calcium silicate hydrate (C-S-H) formation by the pozzolanic reaction. However, the strength of such mixtures is unpredictable, and in some cases, mixtures have been too soft for the intended engineering application. An identification of strength development indicators would accelerate evaluation processes for strength development to facilitate and promote the utilization of such materials. This paper focuses on the relationship between the characteristics of soil organic matters in dredged soils and the strength development of the mixtures by a comparison of eight dredged soils collected from eight different Japanese harbors. The characteristics of the soil organic matters were identified to determine as indicators of mixtures with weak strength development, i.e., enriched sulfur content in extracted soil organic matter (humic acid) fraction, and the N/C ratio of humic acid similar to land humic acid standards. Increases in the validated fraction of dredged soils and steel slag by replacing fractions disadvantageous to construction resources would contribute to reduce waste production, which would lower the environmental impact of the use, aiming to achieve sustainable utilization of such materials.

Macrophages fine-tune pupil shape during development.

Tissue macrophages, which are ubiquitously present innate immune cells, play versatile roles in development and organogenesis. During development, macrophages prune transient or unnecessary synapses in neuronal development, and prune blood vessels in vascular development, facilitating appropriate tissue remodeling. In the present study, we identified that macrophages contributed to the development of pupillary morphology. Csf1op/op mutant mice, in which ocular macrophages are nearly absent, exhibited abnormal pupillary edges, with abnormal protrusions of excess iris tissue into the pupillary space. Macrophages located near the pupillary edge engulfed pigmented debris, which likely consisted of unnecessary iris protrusions that emerge during smoothening of the pupillary edge. Indeed, pupillary edge macrophages phenotypically possessed some features of M2 macrophages, consistent with robust tissue engulfment and remodeling activities. Interestingly, protruding irises in Csf1op/op mice were only detected in gaps between regressing blood vessels. Taken together, our findings uncovered a new role for ocular macrophages, demonstrating that this cell population is important for iris pruning during development.

Development of a Unique T Cell Receptor Gene-Transferred Tax-Redirected T Cell Immunotherapy for Adult T Cell Leukemia.

Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by infection with human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains extremely poor. Tax, the most important regulatory protein for HTLV-1, is associated with the aggressive proliferation of host cells and is also a major target antigen for CD8+ cytotoxic T cells (CTLs). Based on our previous findings that Tax-specific CTLs with a T cell receptor (TCR) containing a unique amino-acid sequence motif exhibit strong HLA-A*24:02-restricted, Tax301-309-specific activity against HTLV-1, we aimed to develop a Tax-redirected T cell immunotherapy for ATL. TCR-ɑ/ß genes were cloned from a previously established CTL clone and transduced into peripheral blood mononuclear cells (PBMCs) of healthy volunteers using a retroviral siTCR vector. Then the cytotoxic efficacy against HTLV-1-infected T cells or primary ATL cells was assessed both in vitro and in vivo. The redirected CTLs (Tax-siCTLs) produced a large amount of cytokines and showed strong killing activity against ATL/HTLV-1-infected T cells in vitro, although they did not have universal activity against ATL cells. Next, in a xenograft mouse model using an HTLV-1-infected T cell line (MT-2), in all mice treated with Tax-siCTLs, the tumor rapidly diminished and finally disappeared without normal tissue damage, although all mice that were untreated or treated with non-gene-modified PBMCs died because of tumor progression. Our findings confirm that Tax-siCTLs can exert strong anti-ATL/HTLV-1 effects without a significant reaction against normal cells and have the potential to be a novel immunotherapy for ATL patients.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

M1 macrophage infiltration exacerbate muscle/bone atrophy after peripheral nerve injury.

BACKGROUND: Peripheral nerve injury causes limb muscle/bone atrophy, leading to chronic pain. However, the mechanisms underlying muscle/bone atrophy after peripheral nerve injury remain unknown. It was recently reported that M1 macrophages are the main factors responsible for neurogenic inflammation after peripheral nerve injury. We hypothesized that M1 macrophages are important in muscle/bone atrophy after nerve injury. Therefore, we investigated the influence of M1 macrophages on muscle/bone atrophy after nerve injury in mice to prevent muscle/bone atrophy by suppressing M1 macrophages. METHODS: Hindlimb muscle weight and total bone density were measured in a chronic constriction injury (CCI) mouse model. Immunohistochemical analysis and intravital microscopy were performed to visualize hindlimb muscles/bones, and cells were quantified using flow cytometry. We compared M1 macrophage infiltration into muscles/bones and muscle/bone atrophy between macrophage depletion and untreated groups. We also investigated muscle/bone atrophy using administration models for anti-inflammatory and neuropathic pain drugs. RESULTS: Peripheral nerve injury caused significant reduction in muscle weight and total bone density at 1 and 3 weeks after CCI, respectively, compared with that in controls. Osteoclast numbers were significantly higher at 1 week after CCI in the CCI group than in the control group. M1 macrophage infiltration into muscles was observed from 2 h after CCI via intravital microscopy and 1 week after CCI, and it was significantly higher 1 week after CCI than in the control group. In the macrophage depletion group, dexamethasone, pregabalin, and loxoprofen groups, M1 macrophage infiltration into muscles/bones was significantly lower and muscle weight and total bone density were significantly higher than in the untreated group. CONCLUSIONS: M1 macrophage infiltration exacerbates muscle/bone atrophy after peripheral nerve injury. By suppressing M1 macrophages at the neural injury local site, muscle/bone atrophy could be avoided.

Macrophage hypoxia signaling regulates cardiac fibrosis via Oncostatin M.

The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which transforming growth factor-1 (TGF-ß1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-ß1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6Chi monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-ß1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.

Comparison between a rotatable sphincterotome and a conventional sphincterotome for selective bile duct cannulation.

BACKGROUND: Selective biliary cannulation (SBC) is the first challenge of endoscopic retrograde cholangiopancreatography (ERCP), especially for trainees, and a rotatable sphincterotome may be useful to guide the directional axis of the scope and SBC. METHODS: We performed a prospective randomized single-center trial, enrolling 200 patients with a native papilla who required therapeutic biliary ERCP. Patients were randomly assigned to the rotatable sphincterotome group (n = 100) or the conventional sphincterotome group (n = 100). The primary endpoint was successful SBC by the trainees within 10 minutes. RESULTS: The early and late cannulation success rates did not differ significantly between the groups (P = 0.46 and P > 0.99, respectively). For the patients in whom trainees failed to achieve SBC, the rotatable sphincterotome was used as a rescue cannulation technique in four patients from the conventional group; in no patients in the rotatable group was the conventional sphincterotome used for SBC. Post-ERCP pancreatitis (PEP) occurred in 11 patients (5.5 %; 6 mild, 5 moderate); the incidence did not differ significantly between the two groups (rotatable group 3 %, conventional group 8 %; P = 0.21). The two groups were thus combined for evaluation of the factors relating to cannulation difficulty for trainees, which revealed that orientation of the papilla was a significant factor (P < 0.001). CONCLUSIONS: The type of sphincterotome used did not affect the success of SBC by trainees. However, orientation of the papilla was revealed to be a significant factor relating to cannulation difficulty for trainees overall.

Von Willebrand Factor Aggravates Hepatic Ischemia-Reperfusion Injury by Promoting Neutrophil Recruitment in Mice.

Hepatic ischaemia-reperfusion (I/R) injury is a serious liver damage that critically influences the clinical outcome of liver surgery or transplantation. Since recent studies indicated the critical involvement of von Willebrand factor (VWF) in reperfusion injuries of brain and myocardium, we hypothesized that VWF-dependent thrombotic or inflammatory responses also play a role in hepatic I/R injury. Using a mouse model of hepatic I/R injury, we explored the functional relevance of the VWF-ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) axis in this pathologic condition. Time-course studies during hepatic I/R revealed significantly lower alanine aminotransferase (ALT) values, as well as greater hepatic blood flow, in VWF gene-deleted (KO) mice in comparison with wild-type (WT) mice. Histological analysis revealed a significantly lesser extent of neutrophil infiltration and hepatocellular necrosis in liver tissues of VWF-KO mice. Human recombinant ADAMTS13 significantly improved the impairment in ALT values and hepatic blood flow and decreased neutrophil infiltration within the liver tissue of WT mice. Real-time intravital imaging successfully visualized significantly reduced leukocyte-vessel wall interactions in I/R liver of VWF-KO mice. Taken together, our results indicate that VWF promotes neutrophil recruitment in ischaemic mouse liver, critically aggravating reperfusion injury, and suggest that functional regulation of VWF by ADAMTS13 represents a promising therapeutic option for hepatic I/R injury.

Transplantation of bioengineered rat lungs recellularized with endothelial and adipose-derived stromal cells.

Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient's own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Hemostatic function to regulate perioperative bleeding in patients undergoing spinal surgery: A prospective observational study.

Although bleeding is a common complication of surgery, routine laboratory tests have been demonstrated to have a low ability to predict perioperative bleeding. Better understanding of hemostatic function during surgery would lead to identification of high-risk patients for bleeding. Here, we aimed to elucidate hemostatic mechanisms to determine perioperative bleeding. We prospectively enrolled 104 patients undergoing cervical spinal surgery without bleeding diathesis. Blood sampling was performed just before the operation. Volumes of perioperative blood loss were compared with the results of detailed laboratory tests assessing primary hemostasis, secondary hemostasis, and fibrinolysis. Platelet aggregations induced by several agonists correlated with each other, and only two latent factors determined inter-individual difference. Platelet aggregability independently determined perioperative bleeding. We also identified low levels of plasminogen-activator inhibitor-1 (PAI-1) and α2-plasmin inhibitor to be independent risk factors for intraoperative and postoperative bleeding, respectively. Most important independent factor to determine postoperative bleeding was body weight. Of note, obese patients with low levels of PAI-1 became high-risk patients for bleeding during surgery. Our data suggest that bleeding after surgical procedure may be influenced by inter-individual differences of hemostatic function including platelet function and fibrinolysis, even in the patients without bleeding diathesis.

CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice.

Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.

Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets.

Donor-independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature-dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen-activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC-derived GPIbα+ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP-457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP-457 blocked GPIbα shedding from iPSC platelets at a lower half-maximal inhibitory concentration than panmetalloproteinase inhibitor GM-6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP-457 exhibited improved GPIbα-dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP-457 exerted better hemostatic function in vivo. Our findings suggest that KP-457, unlike GM-6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC-derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720-730.

Electrocapillary Phenomena at Edible Oil/Saline Interfaces.

Interfacial tension between edible oil and saline was measured under applied electric fields to understand the electrocapillary phenomena at the edible oil/saline interfaces. The electric responses of saline droplets in edible oil were also observed microscopically to examine the relationship between the electrocapillary phenomena and interfacial polarization. When sodium oleate (SO) was added to edible oil (SO-oil), the interfacial tension between SO-oil and saline decreased. However, no decrease was observed for additive-free oil or oleic acid (OA)-added oil (OA-oil). Microscopic observations suggested that the magnitude of interfacial polarization increased in the order of additive-free oil < OA-oil < SO-oil. The difference in electrocapillary phenomena between OA- and SO-oils was closely related to the polarization magnitude. In the case of SO-oil, the decrease in interfacial tension was remarkably larger for saline (pH 5.4~5.6) than that for phosphate-buffered saline (PBS, pH 7.2~7.4). However, no difference was observed between the electric responses of PBS and saline droplets in SO-oil. The difference in electrocapillary phenomena for PBS and saline could not be simply explained in terms of polarization magnitude. The ratio of ionized and non-ionized OA at the interfaces changed with the saline pH, possibly leading to the above difference.

[A Case of Laparoscopic Total Gastrectomy for Gastric Cancer with Diffuse Cystic Malformation].

A 75-years-old man was diagnosed with a cystic submucosal tumor of the middle body of the stomach and diffuse cystic malformation. Laparoscopic total gastrectomy was performed since a type II c gastric cancer was found at the lower body of the stomach after 1-year follow-up. Histopathological examination revealed mucosal adenocarcinoma with diffuse cystic malformation. Strict observation and appropriate treatment are needed for diffuse cystic malformation because of its significant carcinogenic rate.

A case of neuroendocrine carcinoma in the hepatic hilar lymph nodes concomitant with an adenocarcinoma of the gallbladder.

BACKGROUND: Neuroendocrine tumors (NETs) are rare especially in the gallbladder. They have not been elucidated in the pathogenesis, clinicopathological characteristics, and treatment options. CASE PRESENTATION: We present a 76-year-old woman with a gallbladder tumor and hepatic hilar lymph node swelling. The lymph node biopsy demonstrated neuroendocrine carcinoma (NEC). We performed cholecystectomy, hepatic hilar lymphadenectomy, extrahepatic biliary duct resection, and hepaticojejunostomy prior to chemotherapy. Pathological examination revealed the gallbladder mass was an adenocarcinoma invading to the muscular layer without any NEC components, whereas the hepatic hilar lymph nodes were filled with high-grade NEC cells with negligible area of adenocarcinoma. The patient received general chemotherapy consisting of carboplatin and etoposide, but a recurrence in the para-aortic lymph nodes occurred 4 months after surgery. CONCLUSIONS: We report a rare case of NEC of the hepatic hilar lymph nodes that were concomitant with an adenocarcinoma of the gallbladder. High-grade NEC generally has an aggressive behavior and an optimal treatment strategy should be chosen for each patient.

A rare case of ulcerative colitis exacerbated by VZV infection.

A 16-years old man with severe ulcerative colitis (UC) was admitted to our hospital. After initiating treatment with corticosteroid for UC, chicken pox appeared. At the same time of appearance of chicken pox, the disease activity of UC was exacerbated. After initiating the treatment with acyclovir, both chicken pox and UC improved. Because colonoscopic findings revealed the remaining of moderately active UC, initiating the treatment with infliximab could induce clinical remission of UC without relapse of varicella-zoster virus (VZV) infection. This is a very rare case of UC with concomitant VZV infection. According to our report, the vaccination for VZV prior to immunosuppressive treatments would be necessary for VZV naïve patients with UC.

Successful palliation for an aged patient with primary pericardial mesothelioma.

An 85-year-old Japanese man with a complaint of exertional dyspnea was admitted to our hospital. Sixty-three years prior to admission at our hospital, he handled asbestos for 2 years in a factory. His chest computed tomography showed a massive pericardial effusion leading to cardiac tamponade and right pleural plaque. After a pericardiocentesis was performed, he recovered from cardiac failure caused by the cardiac tamponade. Pathological examination of the pericardial effusion revealed malignant mesothelial cells. Therefore, he was diagnosed with primary pericardial mesothelioma (PPM) related to asbestos exposure. Although his disease slowly progressed over 18 months, he remained active without any adjuvant treatments such as chemotherapy. Long-term palliation in an aged patient with PPM is rarely obtained using supportive care alone because the prognosis of PPM has been consistently reported to be very poor and almost fatal within a year. Clinical oncologists and thoracic surgeons should be aware of this disease because the accumulation of knowledge on PPM may lead to successful treatment even in aged patients.

IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs.

Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture-dependent thrombopoiesis and increased platelet counts. IL-1α-IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs.

[Frontiers in Live Bone Imaging Researches. In vivo vascular imaging of thrombosis and thrombopoiesis].

In vivo imaging technique can elucidate dynamic molecular and cellular mechanisms in various fields. We applied two photon microscopy technique to living animals, and combined with light based cell manipulation system. We applied two photon microscopy technique to living animals, and combined with light based cell manipulation system. We elucidated the detailed cellular processes of thrombosis and thrombopoiesis in vivo. In this review, we introduce our recent findings of in vivo platelet activation, hemostasis and thrombopoiesis from bone marrow megakaryocytes.