A new perfusion culture method with a self-organized capillary network

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow

Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells

BACKGROUND: Metastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2(+ )patients were mainly enrolled in the study in Western countries. However, HLA-A24(+ )melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. METHODS: Nine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner. RESULTS: The mean percentage of DCs rated as lin(-)HLA-DR(+ )in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c(+)HLA-DR(+)), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined. CONCLUSIONS: These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan

トクシマ ダイガク ビョウイン ノウソッチュウ センター デノ ナイケイドウミャク キュウセイ ヘイソク ニ タイスル チリョウ センリャク

Objective : Strokes related to acute internal carotid artery （ICA） occlusion are associated with extremely poor prognosis. Recently, some studies have reported that the prognosis has been getting better by treating with intra-arterial endovascular therapy. We sought to evaluate clinical outcomes in patients with acute ischemic stroke attributable to ICA occlusion treated with intraarterial endovascular therapy or otherwise. Methods : We reviewed ４４ patients who underwent treatment with intra-arterial endovascular therapy or otherwise in our stroke care unit （SCU） from January, ２０１１ to May, ２０１４. We compared the prognosis of patients in the ２ treatment groups （endovascular group, n＝２３ vs nonendovascular group, n＝２１）. Results : The rate of good prognosis （modified Rankin Score of ≦２） was significantly higher in the endovascular group than non-endovascular group （１７．４％ vs０％，p＝０．０４５）. The rate of patients who had extreme improvements of National Institutes of Health Stroke Scale （NIHSS） ≧８ was higher in the endovascular group than non-endovascular group （３０．４％ vs１４．３％）. Concerning the improvement of NIHSS ≧８, recombinant tissue-type plasminogen activator （rt-PA） or the left side ICA occlusion were associated significantly （p＝０．０１９, p＝０．０４２, respectively）. Although the hypothesis that endovascular therapy could contribute to extreme improvements of NIHSS wasn’t proved （p＝０．２０２）, there was one case that NIHSS was dramatically improved from ２２ to ０ by endovascular therapy. Conclusions : Intra-arterial endovascular therapy of ICA occlusion might result in improved clinical outcomes for indicated cases

Tracing of Afferent Connections in the Zebrafish Cerebellum Using Recombinant Rabies Virus

The cerebellum is involved in some forms of motor coordination and learning, and in cognitive and emotional functions. To elucidate the functions of the cerebellum, it is important to unravel the detailed connections of the cerebellar neurons. Although the cerebellar neural circuit structure is generally conserved among vertebrates, it is not clear whether the cerebellum receives and processes the same or similar information in different vertebrate species. Here, we performed monosynaptic retrograde tracing with recombinant rabies viruses (RV) to identify the afferent connections of the zebrafish cerebellar neurons. We used a G-deleted RV that expressed GFP. The virus was also pseudotyped with EnvA, an envelope protein of avian sarcoma and leucosis virus (ALSV-A). For the specific infection of cerebellar neurons, we expressed the RV glycoprotein (G) gene and the envelope protein TVA, which is the receptor for EnvA, in Purkinje cells (PCs) or granule cells (GCs), using the promoter for aldolase Ca (aldoca) or cerebellin 12 (cbln12), respectively. When the virus infected PCs in the aldoca line, GFP was detected in the PCs’ presynaptic neurons, including GCs and neurons in the inferior olivary nuclei (IOs), which send climbing fibers (CFs). These observations validated the RV tracing method in zebrafish. When the virus infected GCs in the cbln12 line, GFP was again detected in their presynaptic neurons, including neurons in the pretectal nuclei, the nucleus lateralis valvulae (NLV), the central gray (CG), the medial octavolateralis nucleus (MON), and the descending octaval nucleus (DON). GFP was not observed in these neurons when the virus infected PCs in the aldoca line. These precerebellar neurons generally agree with those reported for other teleost species and are at least partly conserved with those in mammals. Our results demonstrate that the RV system can be used for connectome analyses in zebrafish, and provide fundamental information about the cerebellar neural circuits, which will be valuable for elucidating the functions of cerebellar neural circuits in zebrafish

Development Conditions for Tropical Storms over the Western North Pacific Stratified by Large-Scale Flow Patterns

This study investigated the characteristics and environmental conditions of tropical cyclones (TCs) over the western North Pacific from 2009 to 2017 that dissipated before reaching tropical storm strength (TDs) under unfavorable environmental conditions; we compared TDs with TCs that reached tropical storm strength (TSs) in terms of modulations of relevant large-scale flow patterns. The flow patterns were categorized based on five factors: shear line, confluence region, monsoon gyre, easterly waves, and Rossby wave energy dispersion from a preexisting cyclone. Among 476 cases, 263 TDs were detected using best-track data and early stage Dvorak analysis. The TCs in the environments associated with the confluence region or Rossby wave energy dispersion (easterly waves) tended to reach tropical storm strength (remain weak) compared with the other factors. The average locations of TDs at the time of cyclogenesis in the confluence region, monsoon gyre, and easterly waves (Rossby wave energy dispersion) in the summer and autumn were farther to the west (east and north) than those of TSs that exhibited the same factors. The environments around TDs were less favorable for development than those around TSs, as there were significant differences in atmospheric (oceanic) environmental parameters between TDs and TSs in the factors of confluence region, easterly waves, and Rossby wave energy dispersion (shear line, monsoon gyre, and Rossby wave energy dispersion). The environmental conditions for reaching tropical storm strength over their developing stage, using five factors, can be summarized as follows: higher tropical cyclone heat potential in the shear line and monsoon gyre, weak vertical shear in the confluence region, wet conditions in the easterly waves, and higher sea surface temperatures and an intense preexisting cyclone in Rossby wave energy dispersion from a preexisting cyclone.</p

Calaxin is required for cilia-driven determination of vertebrate laterality

Sasaki, K., Shiba, K., Nakamura, A. et al. Calaxin is required for cilia-driven determination of vertebrate laterality. Commun Biol 2, 226 (2019). https://doi.org/10.1038/s42003-019-0462-

Decadal–centennial-scale solar-linked climate variations and millennial-scale internal oscillations during the Early Cretaceous

Understanding climate variability and stability under extremely warm ‘greenhouse’ conditions in the past is essential for future climate predictions. However, information on millennial-scale (and shorter) climate variability during such periods is scarce, owing to a lack of suitable high-resolution, deep-time archives. Here we present a continuous record of decadal- to orbital-scale continental climate variability from annually laminated lacustrine deposits formed during the late Early Cretaceous (123–120 Ma: late Barremian–early Aptian) in southeastern Mongolia. Inter-annual changes in lake algal productivity for a 1091-year interval reveal a pronounced solar influence on decadal- to centennial-scale climatic variations (including the ~ 11-year Schwabe cycle). Decadally-resolved Ca/Ti ratios (proxy for evaporation/precipitation changes) for a ~ 355-kyr long interval further indicate millennial-scale (~ 1000–2000-yr) extreme drought events in inner-continental areas of mid-latitude palaeo-Asia during the Cretaceous. Millennial-scale oscillations in Ca/Ti ratio show distinct amplitude modulation (AM) induced by the precession, obliquity and short eccentricity cycles. Similar millennial-scale AM by Milankovitch cycle band was also previously observed in the abrupt climatic oscillations (known as Dansgaard–Oeschger events) in the ‘intermediate glacial’ state of the late Pleistocene, and in their potential analogues in the Jurassic ‘greenhouse’. Our findings indicate that external solar activity forcing was effective on decadal–centennial timescales, whilst the millennial-scale variations were likely amplified by internal process such as changes in deep-water formation strength, even during the Cretaceous ‘greenhouse’ period

Cutaneous T-cell-attracting chemokine as a novel biomarker for predicting prognosis of idiopathic pulmonary fibrosis: a prospective observational study

[Background] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease that leads to respiratory failure and death. Although there is a greater understanding of the etiology of this disease, accurately predicting the disease course in individual patients is still not possible. This study aimed to evaluate serum cytokines/chemokines as potential biomarkers that can predict outcomes in IPF patients. [Methods] A multi-institutional prospective two-stage discovery and validation design using two independent cohorts was adopted. For the discovery analysis, serum samples from 100 IPF patients and 32 healthy controls were examined using an unbiased, multiplex immunoassay of 48 cytokines/chemokines. The serum cytokine/chemokine values were compared between IPF patients and controls; the association between multiplex measurements and survival time was evaluated in IPF patients. In the validation analysis, the cytokines/chemokines identified in the discovery analysis were examined in serum samples from another 81 IPF patients to verify the ability of these cytokines/chemokines to predict survival. Immunohistochemical assessment of IPF-derived lung samples was also performed to determine where this novel biomarker is expressed. [Results] In the discovery cohort, 18 cytokines/chemokines were significantly elevated in sera from IPF patients compared with those from controls. Interleukin-1 receptor alpha (IL-1Rα), interleukin-8 (IL-8), macrophage inflammatory protein 1 alpha (MIP-1α), and cutaneous T-cell-attracting chemokine (CTACK) were associated with survival: IL-1Rα, hazard ratio (HR) = 1.04 per 10 units, 95% confidence interval (95% CI) 1.01–1.07; IL-8, HR = 1.04, 95% CI 1.01–1.08; MIP-1α, HR = 1.19, 95% CI 1.00–1.36; and CTACK, HR = 1.12 per 100 units, 95% CI 1.02–1.21. A replication analysis was performed only for CTACK because others were previously reported to be potential biomarkers of interstitial lung diseases. In the validation cohort, CTACK was associated with survival: HR = 1.14 per 100 units, 95% CI 1.01–1.28. Immunohistochemistry revealed the expression of CTACK and CC chemokine receptor 10 (a ligand of CTACK) in airway and type II alveolar epithelial cells of IPF patients but not in those of controls. [Conclusions] CTACK is a novel prognostic biomarker of IPF

21セイキ ノ カンドウミャク インターベンション

Coronary intervention has come to achieve good results with the use of new devices, such as Rotablator (ROTA), new directional coronary atherectomy (DCA), and a special guide wire, even for lesions in which good results were not obtained with plain old balloon angioplasty. In the present study, we evaluated the initial results in patients who underwent ROTA procedures, coronary intervention for chronic total occlusion (CTO), and new DCA procedures in our hospital between January and December 2001. (1) There were 99 patients who underwent ROTA, with an average age of 68±12 years, a lesion length of 15.9±9.9 mm, a reference vessel diameter of 2.7±0.6 mm, and a success rate of 98%. Among these 99 patients, there were 82 patients (83%) with B2 or C type lesion, which is difficult to treat. (2) There were 61 patients with CTO who underwent coronary intervention, with an average age of 63±9 years, an occlusion length of 22.8±13.3 mm, a reference vessel diameter of 2.6±0.7 mm, and a success rate of 82%. (3) There were 5 patients who underwent DCA for ostial lesion of left anterior desending artery and the target lesion was successfully dilated in all these patients. These results indicated that new devices for coronary intervention have made it possible to treat a wider range of lesions, but restenosis still remains to be solved. In Europe and the U.S.A., restenosis is reported to have been drastically reduced by drug eluting stents, which are expected to be introduced in Japan in the future

Narrow safety range of intraoperative rectal irradiation exposure volume for avoiding bleeding after seed implant brachytherapy

<p>Abstract</p> <p>Background & Purpose</p> <p>Rectal toxicity is less common after ¹²⁵I seed implant brachytherapy for prostate cancer, and intraoperative rectal dose-volume constraints (the constraint) is still undetermined in pioneering studies. As our constraint failed to prevent grade 2 or 3 rectal bleeding (bled-pts) in 5.1% of patients, we retrospectively explored another constraint for the prevention of rectal bleeding.</p> <p>Materials and methods</p> <p>The study population consisted of 197 patients treated with the brachytherapy as monotherapy using real-time intraoperative transrectal ultrasound (US)-guided treatment at a prescribed dose of 145 Gy. Post-implant dosimetry was performed on Day 1 and Day 30 after implantation using computed tomography (CT) imaging. Rectal bleeding toxicity was classified by CTC-AE ver. 3.0 during a mean 29-month (range, 12-48 months) period after implantation. The differences in rV100s were compared among intraoperative, Day 1 and Day 30 dosimetry, and between that of patients with grade 2 or 3 rectal bleeding (the bled-pts) and of the others (the spared-pts). All patients were divided into groups based on provisional rV100s that were increased stepwise in 0.1-cc increments from 0 to 1.0 cc. The difference in the ratios of the bled-pts to the spared-pts was tested by chi-square tests, and their odds ratios were calculated (bled-OR). All statistical analyses were performed by <it>t</it>-tests.</p> <p>Results</p> <p>The mean values of rV100us, rV100CT_1, and rV100CT_30 were 0.31 ± 0.43, 0.22 ± 0.36, and 0.59 ± 0.68 cc, respectively. These values temporarily decreased (p = 0.020) on Day 1 and increased (p = 0.000) on Day 30. There was no significant difference in rV100s between the bled-pts and spared-pts at any time of dosimetry. The maximum bled-OR was identified among patients with an rV100us value above 0.1 cc (p = 0.025; OR = 7.8; 95% CI, 1.4-145.8); an rV100CT_1 value above 0.3 cc (p = 0.014; OR = 16.2; 95% CI, 3.9-110.7), and an rV100CT_30 value above 0.5 cc (p = 0.019; OR = 6.3; 95% CI, 1.5-42.3).</p> <p>Conclusion</p> <p>By retrospective analysis exploring rV100 as intraoperative rectal dose-volume thresholds in ¹²⁵I seed implant brachytherapy for prostate cancer, it is proved that rV100 should be less than 0.1 cc for preventing rectal bleeding.</p