Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Biogeochemical processes controlling the dynamics of dissolved organic matter in streams in the Shirakami Mountains, Japan.

The Shirakami Mountain range, including the largest primeval beech forest in East-Asia, is undergoing ecological change. Dissolved organic matter (DOM) plays an important role in nutrient and material cycling in forest ecosystems. Because the quality of DOM varies based on its origin and diagenetic and runoff processes, changes in the environment surrounding DOM can be rapidly detected by monitoring its quality. Herein, concentrations and fluorescence composition of DOM at 14 sites in 13 streams in the Shirakami Mountain range were monitored monthly for over 2 years, excluding winter (December-March), to gain insight into the catchment hydrological and soil characteristics affecting DOM concentrations and composition in stream water. Based on the pattern of temporal changes in fluorescent component composition, monitoring sites were categorized into four groups (streams with small catchments, large catchments, catchments facing the Sea of Japan, and open waters in the catchment) with similar catchment characteristics affecting DOM dynamics. Multiple linear regression analysis showed that DOM concentrations in each group could be attributed to rainfall on the survey date, short-term (1-2 days) rainfall, midterm (~1 month) accumulated rainfall, midterm (7-11 days) accumulated temperature, and catchment characteristics as explanatory variables. The degree of influence of these variables differed among the four groups. The results of this study show that grouping streams according to catchment hydrological characteristics can help identify the impact of climate and environmental change on DOM dynamics in stream water.

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.

Discovery of 1,8-naphthyridin-2-one derivative as a potent and selective sphingomyelin synthase 2 inhibitor.

Sphingomyelin synthase 2 (SMS2) has attracted attention as a drug target for the treatment of various cardiovascular and metabolic diseases. The modification of a high throughput screening hit, 2-quinolone 10, enhanced SMS2 inhibition at nanomolar concentrations with good selectivity against SMS1. To improve the pharmaceutical properties such as passive membrane permeability and aqueous solubility, adjustment of lipophilicity was attempted and 1,8-naphthyridin-2-one 37 was identified as a potent and selective SMS2 inhibitor. A significant reduction in hepatic sphingomyelin levels following repeated treatment in mice suggested that compound 37 could be an effective in vivo tool for clarifying the role of SMS2 enzyme and developing the treatment for SMS2-related diseases.

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.

Histopathological significance of microRNA-210 expression in acute peripheral ischemia in a murine femoral artery ligation model.

Under hypoxic conditions, microRNA-210 is upregulated and plays multiple physiological roles including in cell growth arrest, stem cell survival, repression of mitochondrial respiration, angiogenesis, and arrest of DNA repair. In this study, we investigated the histopathological expression of microRNA-210 under hypoxic conditions using a femoral artery ligation model established in C57BL/6J mice to determine the pathological significance of microRNA-210. Following femoral artery ligation, ischemia was represented by decreased blood flow compared to the control, in which a sham operation was performed. On histopathology, degeneration/necrosis of the muscle fibers, inflammatory cell infiltration, and regeneration of the muscle fibers were sequentially observed from 3 h to 3 d after ligation of the artery. The degree of these effects was more severe in the area in which type I muscular fibers are dominant. The histological expression of hypoxia-inducible factor 1α, a well-known biomarker of hypoxia, and microRNA-210 was observed in a few necrotic muscle fibers, macrophages, and myoblasts, a distribution consistent with the histopathological lesions, and their signal increased over time. The expression of microRNA-210 in macrophages and myoblasts under ischemia might be indicative of a significant role in the recovery from ischemic lesions. In addition, the in situ hybridization of microRNA-210 could potentially be used for the detection of hypoxia as a histological marker in addition to the immunohistochemistry of hypoxia-inducible factor 1α.

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.

Clinical significance of discrepant mid-wall late gadolinium enhancement in patients with nonischemic dilated cardiomyopathy.

A drawback of visual assessment for late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) is the subjectivity and reproducibility of the results. The aim of this study was to investigate the relationship between left ventricular (LV) reverse remodeling in response to optimal pharmacotherapies and the definite or discrepant mid-wall LGE with visual assessment in patients with dilated cardiomyopathy (DCM). A total of 65 patients who had been hospitalized with newly diagnosed DCM and had undergone CMR, were enrolled. When the visual assessment of the presence or absence of mid-wall LGE was confirmed by the two observers, patients were classified into either the positive- (n = 20) or negative-LGE (n = 29) groups. If there was discordance between the diagnoses of the two observers, patients were classified into the discrepant-LGE (n = 16) group. LV reverse remodeling was defined as an increase in LV ejection fraction by at least 10% concomitant with a decrease in the LV end-diastolic dimension by at least 10%. Among the three groups, the frequency of early LV reverse remodeling within a 1-year follow-up was significantly different (p = 0.0068). The frequency of LV reverse remodeling within a 1-year follow-up was 59, 31, and 15%, and over 2 years was 83, 62, and 40%, in patients with negative-, discrepant-LGE, and positive-LGE, respectively. The survival rate for composite end-points of cardiovascular mortality, sustained ventricular tachycardia, appropriate cardioverter-defibrillator discharge, or rehospitalization for decompensated heart failure was lower in positive-LGEs than in negative-LGEs (p =0.0011), whereas, there were no significant differences between both negative- and discrepant-LGEs, and discrepant- and positive-LGEs. This study showed that the discordance for LGE visual assessment occupied an intermediate position between positive and negative for LGE in LV reverse remodeling in patients with DCM.

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

Coronary plaque rupture with subsequent thrombosis typifies the culprit lesion of non-ST-segment-elevation myocardial infarction, not unstable angina: non-ST-segment-elevation acute coronary syndrome study.

Recently, unstable angina pectoris (UAP) and non-ST-segment-elevation myocardial infarction (NSTEMI) have been considered together because they exhibit indistinguishable clinical and electrocardiogram features, and constitute non-ST-segment-elevation acute coronary syndrome (NSTE-ACS). However, no optical coherence tomography (OCT) studies have reported the association between vulnerable plaque morphology and clinical characteristics in NSTE-ACS patients based on assessment of clinical symptoms and myocardial necrosis. The aim of this study was to investigate the differences in clinical characteristics and plaque morphology assessed by OCT between patients with UAP and NSTEMI. Preinterventional OCT images of 84 NSTE-ACS patients were studied, 19 with NSTEMI and 65 with UAP, according to levels of high-sensitivity troponin T. The frequency of plaque rupture and thrombus in patients with NSTEMI was higher than in UAP patients with either class I or II + III (rupture: NSTEMI, 68 %; UAP classes II + III, 30 %; UAP class I, 19 %, thrombus: NSTEMI, 73 %; UAP classes II + III, 22 %; UAP class I, 14 %). In NSTEMI patients, the frequency of occurrence of both thrombus and rupture was the highest. Conversely, patients with UAP class I or those with UAP classes II + III most frequently had no thrombus and rupture, and the frequencies of the presence of thrombus were only 14 and 22 %, respectively. Multivariate analysis revealed that thrombus and plaque rupture were independently associated with NSTEMI. This study demonstrates that the morphological features of culprit lesions could be related to clinical severity in NSTE-ACS patients.

[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.

NLRP3 regulates neutrophil functions and contributes to hepatic ischemia-reperfusion injury independently of inflammasomes.

Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.

Four cases of investigational therapy with interleukin-11 against acute myocardial infarction.

We describe four cases of the patients with ST-elevation myocardial infarction (STEMI) that were treated with interleukin-11 (IL-11), a cardioprotective cytokine. Recombinant human IL-11 (rhIL-11), was intravenously administered to two cases at low dose (6 µg/kg) and to two at high dose (25 µg/kg). The cytokine administration started just after the coronary occlusion was confirmed by coronary angiography (CAG), taking 3 h. Following CAG, percutaneous coronary intervention (PCI) was performed as a standard therapy. No serious adverse drug reactions were observed. All the cases left the hospital without the symptom of heart failure. We discuss the possibility of the clinical use of rhIL-11 as an adjunct therapy to PCI for the STEMI patients.

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.

ASC in renal collecting duct epithelial cells contributes to inflammation and injury after unilateral ureteral obstruction.

Inflammation plays a crucial role in the pathophysiological characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a multiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X7-potassium efflux and reactive oxygen species-dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.

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.

Inhibition of Stabilin-2 elevates circulating hyaluronic acid levels and prevents tumor metastasis.

Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.

[Microscopic study and microanalysis of platelets in vivo].

Under physiological and pathological conditions, complex cellular interplays take place in living animals. However, the conventional microscope using two-dimensional analysis is not sufficient for analyzing cell dynamics and functions in vivo. Thus, we improved the in vivo imaging technique based on multi-photon microscopy, and we then identified single platelet behaviors in the developing thrombus. We utilized XYZT high-speed imaging, which visualized platelet fate in vivo. This novel technique is anticipated to provide new insights into physiological and pathological conditions involving platelets.