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•Twenty-one strains of NDM-producing enterobacteraleswere detected in five hospitals in Tokyo over a 7-year period.•The introduction of global multidrug-resistant clones and subsequent nosocomial transmission were suggested as the main mechanisms of spread.•Spread of IncX3 plasmid carrying blaNDM-5across multiple bacterial species was observed.

Respiratory syncytial virus (RSV) is a leading cause of acute respiratory illness, with severe outcomes in older adults. Information on the prevalence, hospitalization rate, and impact on the health-related quality of life (HRQoL) of RSV in older adults with acute respiratory infections (ARI) in outpatient settings in Japan is limited.

We investigated patient survival after palliative radiotherapy for bone metastases while comparing the prognostic accuracies of the 3-variable number of risk factors (NRF) model and the new Katagiri scoring system (Katagiri score). Overall, 485 patients who received radiotherapy for bone metastases were grouped as per the NRF model (groups I, II and III) and Katagiri score (low-, intermediate- and high-risk). Survival was compared using the log-rank or log-rank trend test. Independent prognostic factors were identified using multivariate Cox regression analyses (MCRA). MCRA and receiver operating characteristic (ROC) curves were used to compare both models’ accuracy. For the 376 evaluable patients, the overall survival (OS) rates decreased significantly in the higher-tier groups of both models (P < 0.001). All evaluated factors except ‘previous chemotherapy status’ differed significantly between groups. Both models exhibited independent predictive power (P < 0.001). Per NRF model, hazard ratios (HRs) were 1.44 (P = 0.099) and 2.944 (P < 0.001), respectively, for groups II and III, relative to group I. Per Katagiri score, HRs for intermediate- and high-risk groups were 4.02 (P < 0.001) and 7.09 (P < 0.001), respectively, relative to the low-risk group. Areas under the curve (AUC) for predicting 6-, 18- and 24-month mortality were significantly higher when using the Katagiri score (P = 0.036, 0.039 and 0.022). Both models predict survival. Prognostic accuracy of the Katagiri score is superior, especially in patients with long-term survival potential; however, in patients with short prognosis, no difference occurred between both models; simplicity and patient burden should also be considered.

Tumor metastasis is a primary source of morbidity and mortality in cancer. Adrenomedullin (AM) is a multifunctional peptide regulated by receptor activity-modifying proteins (RAMPs). We previously reported that the AM-RAMP2 system is involved in tumor angiogenesis, but the function of the AM-RAMP3 system remains largely unknown. Here, we investigated the actions of the AM-RAMP2 and 3 systems in the tumor microenvironment and their impact on metastasis. PAN02 pancreatic cancer cells were injected into the spleens of mice, leading to spontaneous liver metastasis. Tumor metastasis was enhanced in vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2−/−). By contrast, metastasis was suppressed in RAMP3−/− mice, where the number of podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) was reduced in the periphery of tumors at metastatic sites. Because PDPN-positive CAFs are a hallmark of tumor malignancy, we assessed the regulation of PDPN and found that Src/Cas/PDPN signaling is mediated by RAMP3. In fact, RAMP3 deficiency CAFs suppressed migration, proliferation, and metastasis in co-cultures with tumor cells in vitro and in vivo. Moreover, the activation of RAMP2 in RAMP3−/− mice suppressed both tumor growth and metastasis. Based on these results, we suggest that the upregulation of PDPN in DI-E-RAMP2−/− mice increases malignancy, while the downregulation of PDPN in RAMP3−/− mice reduces it. Selective activation of RAMP2 and inhibition of RAMP3 would therefore be expected to suppress tumor metastasis. This study provides the first evidence that understanding and targeting to AM-RAMP systems could contribute to the development of novel therapeutics against metastasis.

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene–related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (−/−) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP−/−mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)–positive lymphatic capillaries were decreased and lymphatic capillary formation–related factors were down-regulated in CGRP−/−mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP−/−mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP−/−mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema.

Central retinal vein occlusion (CRVO) is an intractable disease that causes visual acuity loss with retinal ischemia, hemorrhage, and edema. In this study, we developed an experimental CRVO model in mice and evaluated the therapeutic potential of the pleiotropic peptide adrenomedullin (ADM) and its receptor activity–modifying protein 2 (RAMP2). The CRVO model, which had phenotypes resembling those seen in the clinic, was produced by combining i.p. injection of Rose bengal, a photoactivator dye enhancing thrombus formation, with laser photocoagulation. Retinal vascular area, analyzed using fluorescein angiography and fluorescein isothiocyanate–perfused retinal flat mounts, was decreased after induction of CRVO but gradually recovered from day 1 to 7. Measurements of retinal thickness using optical coherence tomography and histology revealed prominent edema early after CRVO, followed by gradual atrophy. Reperfusion after CRVO was diminished in Admand Ramp2knockout (KO) mice but was increased by exogenous ADM administration. CRVO also increased expression of a coagulation factor, oxidative stress markers, and a leukocyte adhesion molecule in both wild-type and AdmKO mice, and the effect was more pronounced in AdmKO mice. Using retinal capillary endothelial cells, ADM was found to directly suppress retinal endothelial injury. The retinoprotective effects of the Adm-Ramp2system make it a novel therapeutic target for the treatment of CRVO.

The fcc lattice of porous Cu prepared by dealloying Al2Cu with HCl aqueous solution exhibits a high density of twinning defects with an average domain size of about 3 nm along the ⟨111⟩ directions. The high density of twinning was verified by X-ray diffraction and qualitatively interpreted by a structural model showing the 5% probability of twinning defect formation. Most of the twinning defects disappeared after annealing at 873 K for 24 h. Twinned Cu reveals much faster oxidation rate in comparison to that without (or with much fewer) twinning defects, as shown by X-ray diffraction and hydrogen differential scanning calorimetry. Using ab initio DFT calculations, we demonstrate that twinning defects in porous Cu are able to form nucleation centers for the growth of Cu2O. The geometry of the V-shaped edges on the twinned {211} surfaces is favorable for formation of the basic structural elements of Cu2O. The fast oxidation of porous Cu prepared by dealloying can thus be explained by the fast formation of the Cu2O nucleation centers and their high density.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

Calcitonin gene-related peptide (CGRP) is a bioactive peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP is largely distributed in the cardiovascular and nervous systems, where it acts as a regulatory factor. CGRP is also expressed in organs and tissues involved in metabolic regulation, including white adipose tissue (WAT), where its function is largely unknown. In this study, we examined the effects of endogenous CGRP on metabolic function. When we administered a high-fat diet to CGRP-specific knockout (CGRP-/-) and wild-type (WT) mice for 10 weeks, we observed that food intake did not differ between the two groups, but body weight and visceral fat weight were significantly lower in CGRP-/- mice. Fatty liver changes were less severe in CGRP-/- mice, which also showed lower serum insulin and leptin levels. Glucose tolerance and insulin sensitivity were better in CGRP-/- than WT mice, and expired gas analysis revealed greater oxygen consumption by CGRP-/- mice. Adipocyte hypertrophy was suppressed in CGRP-/- mice, while expression of β-3-adrenergic receptor, hormone-sensitive lipase and adiponectin was enhanced. Isoproterenol-induced glycerol release from WAT was higher in CGRP-/- than WT mice, and CGRP-/- mice showed elevated sympathetic nervous activity. β-receptor-blockade canceled the beneficial effects of CGRP deletion on obesity. These results suggest that, in addition to its actions in the cardiovascular system, endogenous CGRP is a key regulator of metabolism and energy homeostasis in vivo.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after coronary intervention. We previously described the essential angiogenic function of the adrenomedullin (AM)-receptor activity-modifying protein (RAMP) 2 system. In the present study, we assessed the vasoprotective actions of the endogenous AM-RAMP2 system using a wire-induced vascular injury model. We found that neointima formation and vascular smooth muscle cell proliferation were enhanced in RAMP2+/- male mice. The injured vessels from RAMP2+/- mice showed greater macrophage infiltration, inflammatory cytokine expression, and oxidative stress than vessels from wild-type mice and less re-endothelialization. After endothelial cell-specific RAMP2 deletion in drug-inducible endothelial cell-specific RAMP2-/- (DI-E-RAMP2-/-) male mice, we observed markedly greater neointima formation than in control mice. In addition, neointima formation after vessel injury was enhanced in mice receiving bone marrow transplants from RAMP2+/- or DI-E-RAMP2-/- mice, indicating that bone marrow-derived cells contributed to the enhanced neointima formation. Finally, we found that the AM-RAMP2 system augmented proliferation and migration of endothelial progenitor cells. These results demonstrate that the AM-RAMP2 system exerts crucial vasoprotective effects after vascular injury and could be a therapeutic target for the treatment of vascular diseases.

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization–associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-β2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration–related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

Calcitonin gene–related peptide (CGRP; official name CALCA) has a variety of functions and exhibits both angiogenic and anti-inflammatory properties. We previously reported the angiogenic effects of the CGRP family peptide adrenomedullin in oxygen-induced retinopathy; however, the effects of CGRP on ocular angiogenesis remain unknown. Herein, we used CGRP knockout (CGRP−/−) mice to investigate the roles of CGRP in ocular vascular disease. Observation of pathological retinal angiogenesis in the oxygen-induced retinopathy model revealed no difference between CGRP−/−and wild-type mice. However, much higher levels of the CGRP receptor were present in the choroid than the retina. Laser-induced choroidal neovascularization (CNV), a model of exudative age-related macular degeneration, revealed more severe CNV lesions in CGRP−/−than wild-type mice, and fluorescein angiography showed greater leakage from CNV in CGRP−/−. In addition, macrophage infiltration and tumor necrosis factor (TNF)-α production were enhanced within the CNV lesions in CGRP−/−mice, and the TNF-α, in turn, suppressed the barrier formation of retinal pigment epithelial cells. In vivo, CGRP administration suppressed CNV formation, and CGRP also dose dependently suppressed TNF-α production by isolated macrophages.From these data, we conclude that CGRP suppresses the development of leaky CNV through negative regulation of inflammation. CGRP may thus be a promising therapeutic agent for the treatment of ocular vascular diseases associated with inflammation.

Adrenomedullin (ADM), originally identified as a vasodilating peptide, is now recognized to be a pleiotropic molecule involved in both the pathogenesis of cardiovascular diseases and circulatory homeostasis. Homozygotes of ADM knockout mice (ADM-/-) were lethal at mid-gestation with abnormalities of vascular development and this finding clarified the angiogenic potency of ADM. Calcitonin gene-related peptide (CGRP), which has a structure and function similar to that of ADM, has been identified as a family peptide of ADM. Unlike ADM-/-, CGRP-/- were apparently normal. Therefore, the study of knockout mice first clarified the distinctly different physiological roles between ADM and CGRP. In contrast, heterozygotes of ADM knockout mice (ADM/-) were alive but showed blood pressure elevation, reduced neovascularization, and enhanced neointimal formation by arterial injury. Based on these observations, there was hope ADM would have a therapeutic use. However, ADM has a short half-life in the blood stream and its application in chronic disease has limitations. Therefore, we focused on the ADM receptor system. The calcitonin-receptor-like receptor (CLR), which is the ADM receptor, associates with one of the accessory proteins, called receptor activity-modifying proteins (RAMPs). By interacting with RAMP1, CLR exhibits a high affinity for CGRP, whereas by interacting with either RAMP2 or -3, CLR exhibits a high affinity for ADM. We generated RAMP knockout mice and found that vascular phenotypes similar to ADM-/- were reproduced only in RAMP2-/-. This shows that RAMP2 is the key determinant of the vascular functions of ADM. RAMP2 could be an attractive therapeutic target in cardiovascular diseases.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM−/−) or its receptor regulating protein, RAMP2 (RAMP2−/−), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM+/−or RAMP2+/−, respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2−/−). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM+/−retinas compared with wild-type mice. ADM+/−retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2−/−showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly.

Adrenomedullin (AM), originally identified as a vasodilating peptide, is now recognized to be a pleiotropic vasoactive molecule involved in both the pathogenesis of cardiovascular diseases and circulatory homeostasis. To elucidate the in vivo roles of AM, we have established and analyzed genetically engineered AM mice and its receptor components. Heterozygotes of AM knockout mice (AM/-) showed blood pressure elevation, severe cardiac hypertrophy, and fibrosis by pressure overload or angiotensin II infusion. On the other hand, vascular-specific AM-overexpressing mice were resistant to neointimal formation by arterial injury. Therefore, endogenous AM exerts protective effects against stress-induced cardiac hypertrophy, fibrosis, and arteriosclerosis, as well as a vasodilating effect. Homozygotes of AM knockout mice (AM-/-) were lethal at mid-gestation with abnormalities in vascular development. This finding first clarified the indispensable roles of AM in vascular development. We also showed that AM possesses novel angiogenic properties not only during development, but also in adults. Based on these observations, there is hope that AM can be used therapeutically. However, AM has a short half-life in the blood stream and its application in chronic disease has limitations. We generated knockout mice of receptor activitymodifying protein 2 (RAMP2), a small membrane protein that associates with the AM receptor. We found that the important vascular phenotypes of AM knockout mice were reproduced in RAMP2 knockout mice. This shows that RAMP2 is the key determinant of the vascular functions of AM. RAMP2 could be an attractive therapeutic target in cardiovascular diseases.

Translational control of specific mRNAs by cytoplasmic polyadenylation has fundamental roles in gametogenesis. The cytoplasmic polyadenylation element binding (CPEB) protein regulates cytoplasmic polyadenylation of mRNAs as a trans factor in oogenesis and spermatogenesis. The CPEB protein contains two RNA recognition motifs and a Zn-finger structure. Proteins (KIAA0940 and KIAA1673) with similar structures are predicted from the genome database, but nothing is known about their expression and function. Here, we report another novel member of the CPEB protein family, CPEB2. Comparison of the amino acid sequences of CPEB family members suggests that the family can be divided structurally and, perhaps, functionally into two groups: the CPEB group, and the CPEB2-KIAA0940-KIAA1673 group. The CPEB2 maps to mouse chromosome distal 5B and is abundantly expressed in testis. However, it was detected by reverse transcription-polymerase chain reaction in all tissues that we examined. It preferentially binds to poly(U) and localizes to the cytoplasm in transfected HeLa cells. The CPEB2 is expressed postmeiotically in mouse spermatogenesis, suggesting a possible role in translational regulation of stored mRNAs in transcriptionally inactive haploid spermatids.

Bis-, tris-, and tetrakis-trimethylgermylpropanoyl esters of p-tert-butylcalix[4]arenes 5, 7, and 8 were synthesized and their conformations in solution were determined by ¹H and ¹³C NMR spectroscopy. The characteristic pair of doublets from the Ar&bond;CH2&bond;Ar methylene protons was observed, indicating that all the prepared calixarenes were in the cone conformation. These germylated calixarenes failed to exhibit any appreciable cation-capturing ability, indicating that further modification is required to make these compounds to be effective ditopic hosts. © 2003 Wiley Periodicals, Inc. Heteroatom Chem 14:365–373, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10164

Bis‐, tris‐, and tetrakis‐trimethylgermylpropanoyl esters of p‐tert‐butylcalix[4]arenes 5, 7, and 8were synthesized and their conformations in solution were determined by 1H and 13C NMR spectroscopy. The characteristic pair of doublets from the ArCH2Ar methylene protons was observed, indicating that all the prepared calixarenes were in the cone conformation. These germylated calixarenes failed to exhibit any appreciable cation‐capturing ability, indicating that further modification is required to make these compounds to be effective ditopic hosts. © 2003 Wiley Periodicals, Inc. Heteroatom Chem 14:365–373, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10164

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-β increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-β-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-β-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

Maternally and transiently accumulated SpCas9 (maternal SpCas9) in a zygote derived from a systemically SpCas9-expressing transgenic mouse strain was used to generate single- and multiple-gene-modified mice. Maternal SpCas9-based gene editing allows for high indel and knockin mutation efficiency, low mosaicism, increased pup delivery rate, and simultaneous induction of mutations at multiple loci in contrast to conventional CRISPR/SpCas9-based gene editing.

Turbulence in the solar wind plasma was studied using angular broadening measurements of 10 extragalactic compact radio sources (quasars) with a very long baseline interferometer (VLBI) at 4.99 GHz. Unlike other angular broadening studies, the measured broadening size was corrected for intrinsic source structures which were obtained from a separate VLBI observation. The solar elongations of the sources ranged from 18 RSto 243 RS, and five sources with elongations ≤ 61 RSshowed varying degrees of broadening. The measured angular sizes are considerably less than predicted by the well‐known empirical relationship of Erickson [1964], as well as two other models for strength of scattering as a function of solar elongation. However, the data are in good agreement with a model for the spatial power spectrum of the turbulence proposed by Coles and Harmon [1989]. This model consists of a Kolmogorov spectrum at large scales, but with an enhancement of power near the wavenumber corresponding to the ion inertial length. Two of our sources, 1148–001 and 1253–053 (3C279), show substantial differences in the amount of scattering, even though they are at similar solar elongations (29 versus 35 RS). Data to which we have access indicate that the state of the corona along the lines of sight to these sources may have been quite different. Angular broadening measurements with VLBI interferometers currently under development (primarily the very long baseline array) will allow a global view of plasma turbulence out of the ecliptic plane and thus be complementary to the point in situ measurements with Ulysses.

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity–modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (KO) (AM–/–, RAMP2–/–) is embryonically lethal in mice, whereas RAMP3–/– mice are apparently normal. AM, RAMP2, and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress. Cardiomyocyte-specific RAMP2–/– (C-RAMP2–/–) and RAMP3–/– showed no apparent heart failure at base line. After 1 week of transverse aortic constriction (TAC), however, C-RAMP2–/– exhibited significant cardiac hypertrophy, decreased ejection fraction, and increased fibrosis compared with wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2–/–, indicating reduced ventricular contractility and relaxation. Exposing C-RAMP2–/– cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress compared with wild-type cells. C-RAMP2–/– cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3–/– also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3–/–. These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti–vascular endothelial growth factor drugs are used to treat AMD, but they may induce subretinal fibrosis. We have focused on adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. Herein, the therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-β and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, the effects of transforming growth factor-β and CXCR4 inhibitors were assessed, and they eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.

ABSTRACTThe immunogenicity of pandemic influenza A H1N1 virus (A/H1pdm) vaccine might be modified by prior seasonal trivalent influenza vaccine (sTIV) administration. We conducted a retrospective analysis of immunogenicity of 243 health care workers (number of sTIV-positive [sTIV+] subjects, 216; number of sTIV-subjects, 27) by hemagglutination inhibition. There was no significant difference in the ratios of antibody titers of =40 (41.2% versus 48.1%; P= 0.49) and fold increases in geometric mean titer (3.8 versus 4.5; P= 0.37). sTIV injected 7 to 10 days prior to A/H1pdm vaccine administration did not interfere with the immunogenicity of the latter.

A series of dendritic ligands with an α-glycol core was synthesized through the coupling of Dl-dithiothreitol with poly(arylether) dendron in high yields. The corresponding aluminum dendrimers were applied as initiator to ring-opening polymerization of ε-caprolactone. A dendritic effect on the polydispersity (Mw/Mn) of poly(ε-caprolactone) was observed.

Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.

Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody,confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.