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Adeno-associated virus (AAV) vectors have become an important tool for delivering therapeutic genes for a wide range of neurological diseases. AAV serotypes possess differential cellular tropism in the central nervous system. Although several AAV serotypes or mutants have been reported to transduce the brain efficiently, conflicting data occur across studies with the use of various rodent strains from different genetic backgrounds. Herein, we performed a systematic comparison of the brain transduction properties among five AAV serotypes (AAV2, 5, 7, 8, and 9) in two common rodent strains (C57BL/6J and FVB/N), following local intrastriatal injection of AAV vectors encoding enhanced green fluorescent protein (EGFP) driven by the CBh promoter. Important differences were found regarding overall cellular tropism and transduction efficiency, including contralateral transduction among the AAV serotypes and between the mouse strains. We have further found loss of NeuN-immunoreactivity and microglial activation from AAV transduction in the different mouse strains. The important strain-specific differences from our study suggest that the genetic background of the mouse may affect AAV serotype transduction properties in the brain. These data can provide valuable information about how to choose an effective AAV vector for clinical application and interpret the data obtained from preclinical studies and clinical trials.

Small extracellular vesicles (sEVs), or exosomes, play important roles in physiological and pathological cellular communication. sEVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. Studying the intricacies of sEV function and RNA-based communication requires tools capable of labeling sEV RNA. Here we developed a novel genetically encodable reporter system for tracking sEV RNAs comprising an sEV-loading RNA sequence, termed the EXO-Code, fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of RNA puncta and colocalization with markers of multivesicular bodies; imaging RNA puncta within sEVs; and quantification of sEVs. This technology represents a useful and versatile tool to interrogate the role of sEVs in cellular communication via RNA trafficking to sEVs, cellular sorting decisions, and sEV RNA cargo transfer to recipient cells.

Proper reporting of metadata is essential to reproduce microscopy experiments, interpret results and share images. Experimental scientists can report details about sample preparation and imaging conditions while imaging scientists have the expertise required to collect and report the image acquisition, hardware and software metadata information. MethodsJ2 is an ImageJ/Fiji based software tool that gathers metadata and automatically generates text for the methods section of publications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes., Micro-Meta App is an intuitive, highly interoperable, open-source software tool designed to facilitate the extraction and collection of relevant microscopy metadata as specified by recent community guidelines.

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we presentMicro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, includingBioImagingNorthAmerica (BINA) andQUAlity Assessment andREProducibility inLightMicroscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates withMethodsJ2andOMERO.mde, two complementary tools described in parallel manuscripts.

Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science. Comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities are reported with the goal of improving microscopy reporting, rigor and reproducibility.

Recombinant adeno-associated virus (rAAV) vectors have been widely used as favored delivery vehicles for the treatment of inherited diseases in clinical trials, including neurological diseases. However, the noninvasive systemic delivery of rAAV to the central nervous system is severely hampered by the blood-brain barrier (BBB). Several approaches have been exploited to enhance AAV vector brain transduction after systemic administration, including genetic modification of AAV capsids and physical methods. However, these approaches are not always predictive of desirable outcomes in humans and induce complications. It is imperative to explore novel strategies to increase the ability of AAV9 to cross the BBB for enhanced brain transduction. Herein, we have conducted a combinatorial in vivo/in vitro phage display library screening in mouse brains and purified AAV9 virions to identify a customized BBB shuttle peptide, designated as PB5-3. The PB5-3 peptide specifically bound to AAV9 virions and enhanced widespread transduction of AAV9 in mouse brains, especially in neuronal cells, after systemic administration. Further study demonstrated that systemic administration of AAV9 vectors encoding IDUA complexed with PB5-3 increased the phenotypic correction in the brains of MPS I mice. Mechanistic studies revealed that the PB5-3 peptide effectively increased AAV9 trafficking and transcytosis efficiency in the human BBB model hCMEC/D3 cell line but did not interfere with AAV9 binding to the receptor terminal N-linked galactosylated glycans. Additionally, the PB5-3 peptide slowed the clearance of AAV9 from blood without hepatic toxicity. This study highlights, for the first time, the potential of this combinatorial approach for the isolation of peptides that interact with specific AAV vectors for enhanced and targeted AAV transduction. This promising approach will open new combined therapeutic avenues and shed light on the potential applications of peptides for the treatment of human diseases in future clinical trials with AAV vector-mediated gene delivery.

Summary: Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion. : Takacs et al. show that Rab1b, a major regulator of transport from the endoplasmic reticulum to the Golgi, differentially controls the secretion of lipoprotein components ApoE and ApoB100 and infectious hepatitis C virus particles. Keywords: GTPase, hepatitis, lipoprotein, Rab, secretion, HCV, hepatocyte, transport, albumin, apolipoprotein

Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.

To construct a new calibration method that combines usability and accuracy for estimating herbage mass from rising-plate meter readings, we derived four models differing in the way their parameters are related to sampling date and compared their estimation accuracies using cross-validation. The parameters of the linear regression for each sampling date showed seasonal variations, which had a steep decrease from early April to early June and a gradual increase thereafter. The pooled models were less accurate for estimating herbage mass than a separate model, which had specific parameters for each sampling date (S model). Among the pooled models, however, those in which the parameters were assumed to be linear functions (PL model) or combined functions (PC model) of the sampling date showed substantively improved estimation accuracy compared with the traditional pooled model, in which the parameters were assumed to be fixed throughout the year (PF model). Moreover, at the beginning of the season, the models derived from previous years' data were suggested to be applicable as a practical method. Thus, it can be concluded that these types of pooled calibration could be used as ‘compromise methods’ that combine both accuracy and usability.

Dendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts.

DC-SIGN, a Ca2+-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Linescan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DCSIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/ or by protecting against rapid microdomain endocytosis. © 2011 by the Biophysical Society.

邦文要旨:最近の2年間に当院を肛門症状を主訴として来院した症例484例で,主訴や検査内容,診断名,治療などを調査検討した.痔疾患と診断されたものは462症例で,内痔核が85.3%を占めていた.根治手術は89例(19.3%)で行われ,約8割の症例では保存的治療が行われた.大腸ポリープは63例(13.0%)で,55例は痔疾患を,2例は痔疾患と癌を合併しており,63例中29例(46,0%)が内視鏡的処置を受けていた.大腸癌は11例(2.3%)で発見され,主訴では出血と便通異常が多かった.性別では男性5例,女性6例で,部位では直腸7例(63.7%),S状結腸4例(36.3%)であり,下行結腸より口側にはなかった.根治手術ができたのは7例(63.6%)であった.以上から,肛門症状を主訴とした症例では,主訴から症例を絞り込み,S状結腸内視鏡検査を行うことが,癌の見落としを予防するために重要と考えられた.

大腸ポリープの分類不能とされるものの中で,共通の特徴がみられたpolypに対してcolonic mucosubmucosal elongated polyp(CMSEP)という概念が近年提唱されている.われわれは,本概念に相当すると思われる症例を経験したので報告する.症例は,58歳の女性で,血便を主訴とし来院した.注腸検査で上行結腸に細長いポリープを認め,大腸内視鏡的ポリープ切除術を行ったところ,切除されたポリープは65×10×10mmであった.病理学的検討で,表面は正常な大腸粘膜に覆われ,粘膜下に拡張したリンパ管と血管の増殖からなるポリープで,CMSEPの特徴に一致した.現在までに自験例を含めて,27例,29個のCMSEPが報告されており,これらを検討し報告した.CMSEPという新しい概念は,大腸ポリープに対するひとつの分類として確立されるべきと考える.

Chemical treatment with alkaline solution is used to extract enamel from teeth for retrospective ESR (EPR) dosimetry. It was confirmed in the present study that our condition of chemical treatment does not affect retrospective doses of cow teeth. The method was applied to 10 teeth collected in the South Ural region where the soil is contaminated with Sr 90 . As a result, no noticeable doses were detected so far. Significant variations of sensitivity of the radiation-induced ESR signal to radiation were found for different cow teeth, indicating that individual calibration is necessary.

POSSUM, a Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity, is a scoring system which assesses perioperative surgical risks (Copeland GP et al.: Br J Surg, 1991, Vol 78, 356-360). The POSSUM scoring system consists of two categories of assessment to assess the risk of surgery. A 12-factor (age, cardiac status, pulse rate, systolic blood pressure, respiratory status, Glasgow Coma Score, serum concentration of urea, potassium and sodium, hemoglobin concentration, white cell count and findings on electrocardiography) and 4-grade physiological score (PS) were developed. This was combined with a 6-factor (type of surgical procedure, number of procedures, blood loss, peritoneal soiling, presence of malignancy and mode of surgery) and 4-grade operative severity score (OSS). The present paper attempts to validate it retrospectively. Postoperative hospitalization period and duration of antibiotics administration were both significantly correlated with OSS, but not with PS. These results suggest that the POSSUM scoring system is useful for predicting the postoperative clinical course.

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (∼ 50 nm) pathogen, dengue virus, leading to infection of host cells.

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Recently, a new approach for super-resolution microscopy has emerged which is based on the successive localization of single molecules. The majority of molecules are prepared to reside in a nonfluorescent dark state, leaving only a few single molecules fluorescing. The single molecules can subsequently be localized on the camera image. Successive localization of all molecules allows reconstruction of a super-resolved image of the labeled structure. A variety of ways for limiting the number of locatable molecules have been developed recently which expand this current field of imaging. Here we describe a super-resolution microscopy method that employs the use of reversible, generic dark states, for example radical ion states. This method requires only a single laser source and can be carried out with many fluorescent dyes, in some cases, even in living cells. We provide a step-by-step procedure for this method, which we have called Blink Microscopy.

Recently, a new approach for super-resolution microscopy has emerged which is based on the successive localization of single molecules. The majority of molecules are prepared to reside in a nonfluorescent dark state, leaving only a few single molecules fluorescing. The single molecules can subsequently be localized on the camera image. Successive localization of all molecules allows reconstruction of a super-resolved image of the labeled structure. A variety of ways for limiting the number of locatable molecules have been developed recently which expand this current field of imaging. Here we describe a super-resolution microscopy method that employs the use of reversible, generic dark states, for example radical ion states. This method requires only a single laser source and can be carried out with many fluorescent dyes, in some cases, even in living cells. We provide a step-by-step procedure for this method, which we have called Blink Microscopy.

Dendritic cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN) is a Ca(2+) -dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC-SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in the removal of the N-linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD), as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and fluorescence recovery after photobleaching measurements showed that the cytoplasmic domain and the N-linked glycosylation site do not affect the ability of DC-SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC-SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains.

Evidence in support of the classical lipid raft hypothesis has remained elusive. Data suggests that transmembrane proteins and the actin-containing cortical cytoskeleton can organize lipids into short-lived nanoscale assemblies that can be assembled into larger domains under certain conditions. This supports an evolving view in which interactions between lipids, cholesterol, and proteins create and maintain lateral heterogeneity in the cell membrane.

Hemagglutinin (HA), from influenza virus, and DC-SIGN, a dendritic cell C-type lectin that binds many pathogens including viruses, bacteria, and fungi, form microdomains on the plasma membrane. We investigated the dynamics of these proteins using Scanning FCS (S-FCS), defined valency quantum dot-based Single Particle Tracking (SPT) and Fluorescence Recovery After Photobleaching (FRAP). Using FRAP we verified that HA has a large mobile fraction (∼80%) that is characterized by a diffusion coefficient of ∼0.09 um2/sec and that it exchanges with the surround (Ellens et al. (1990) Biochemistry,29(41): 9697-9707). By contrast, neither DC-SIGN or Δ35-DC-SIGN, a DC-SIGN mutant missing the cytoplasmic tail, recovered after photobleaching, even after many minutes, indicating that these molecules do not exchange significantly with the surround. In order to determine the dynamics of molecules within the domains, we utilized a confocal line S-FCS method that permits the autocorrelation function between the same pixel in successive linescans (acquired in the form of a carpet or kymograph) to be calculated on the ms timescale. HA lateral motion within its microdomain is characterized by a diffusion coefficient of ∼0.10 um2/sec, similar to that measured outside the domain by FRAP. On the other hand, DC-SIGN and Δ35-DC-SIGN do not diffuse within the domain. We are currently using quantum dots that have been conjugated to a single streptavidin in order to examine lateral dynamics within these domains. Such nanoparticles will obviate a consistent interpretative limitation for gold and quantum dot SPT; namely, that particle valency cannot be specified with absolute certainty for the particle whose motion is tracked. Further characterization of these surprisingly stable DC-SIGN domains that are important for pathogen entry into dendritic cells is in progress.Supported by NIH GM 41402 (KJ &NLT)

The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.

ヒト用に開発された心拍メモリー装置を放牧牛に応用し,24時間以上の長期間にわたり心拍数を記録してきた.本報告は,ホルスタイン育成牛あるいは成牛を用いてこの装置の性能と放牧時の測定精度を調査したものである.心拍メモりー装置の心電図誘導法として,QRS群の電位が大きく,しかも波形が安定しているA-B誘導法が最適であった.毎分50拍程度から160拍まで測定できた.A-B誘導法による測定で、草地に昼夜放牧中の育成牛の24時間総心拍数(DHB)は約14万拍で,そのうちの0.2～0.45%がミスカウントで,過大に測定された.このミスカウントの大部分は食草時に見られた.以上の結果から長期間にわたり心拍数を自動記録できる心拍メモリー装置は牛においても測定精度が高く,放牧牛の心拍数記録装置として広く利用できることが再確認された.

症例は,64歳の男性.主訴は,便秘と下血.大腸内視鏡検査にて,S状結腸に直径約1cmのIIc+IIa型の病変が発見され,陥凹型大腸早期癌と診断された.生検標本で高分化腺癌と診断され,S状結腸部分切除術(R2)を施行した.肉眼診断では,13mm×10mmのIIc+IIa,深達度SM,S0,N0,H0,P0でstage Iと診断された.組織学的検討では,高分化腺癌,ly0,v0,n1,中心の陥凹部で深達度smのIIa+IIc型大腸早期癌と診断され,腺腫成分は認められず, de novo癌と考えられた.術後経過は良好で,現在外来にて経過観察中である.近年,本症例のような陥凹型もしくは扁平隆起型の大腸早期癌が発見されるようになってきている.これらの症例の集積によって大腸早期癌の病理学的背景や大腸癌の組織発生そのものの解明に役立つものと期待される.

Pulmonary metastasis is frequently seen in patients with advanced hepatocellular carcinoma. However, information is limited concerning life-threatening complications and effective treatment of pulmonary metastasis because of the poor prognosis of patients with advanced hepatocellular carcinoma. Recent remarkable progress in detection and treatment of hepatocellular carcinoma has improved prognosis, making management of pulmonary metastasis an important clinical issue. We describe a 68-year-old man with pulmonary metastasis of hepatocellular carcinoma and sudden onset of hemoptysis from bronchial invasion. Transcatheter embolization was performed successfully via the bronchial artery with disappearance of bloody sputum. Peribronchial pulmonary metastasis of hepatocellular carcinoma can cause life-threatening hemoptysis. Transcatheter arterial embolization may be one of therapeutics for hemoptysis from invasive pulmonary metastasis of hepatocellular carcinoma.

Contrast-enhanced ultrasonography (arterial infusion) has been clinically established as a qualitative diagnosis imaging tool for hepatocellular carcinoma (HCC). Contrast-enhanced ultrasonography (CEUS) was performed after of Albunex (sonicated serum albumin) or Carbon Dioxide (CO2) microbubble by hand, into the hepatic artery as a diagnostic modality for the early HCC. Here, we discussed the diagnosis of the early HCC by CEUS using Albunex as a contrast medium. Briefly, a diagnosis of the early HCC was made CEUS examination of the hemodynamics of the arteries showed a hypovascular pattern. And tumor size was under 20 mm in diameter, the histopathologic examination was essential to reach a final diagnosis, well-differentiated HCC.

The process of receptor-mediated endocytosis is common to a variety of species and cell types. One of the best characterized receptor-ligand systems is the hepatocyte receptor for asialoglycoproteins. We investigated the morphological features of the uptake and intracellular transport of gold-conjugated asialofetuin in isolated rat hepatocyte couplets. We assessed the effects of colchicine, lumicolchicine, cytochalasin B, and chloroquine on the uptake and intracellular transport of asialoglycoproteins. Isolated rat hepatocyte couplets were incubated with gold-conjugated asialofetuin, and transmission electron micrographs of these cells were analyzed to determine the density and distribution of gold particles in the peripheral and pericanalicular areas. Results were analyzed morphometrically. Colchicine significantly inhibited the uptake and intracellular transport of asialoglycoproteins, but did not affect membrane fusion of endocytic compartments in the peripheral area. Lumicolchicine and cytochalasin B had minimal effects on these processes. Chloroquine inhibited the uptake of asialoglycoproteins, but did not affect the intracellular transport of asialoglycoproteins. Results suggest that the microtubule is essential for intracellular movement of endocytosed asialoglycoproteins and receptor recycling, and that endocytic structures in the peripheral regions can fuse in the absence of intact microtubules. We also found that uptake and intracellular transport of asialoglycoproteins were independent of the microfilaments, and the pH gradient in endocytic compartments was important in receptor-mediated endocytosis of asialoglycoproteins.

The subjects were 41 cases with implantable reservoir systems for unresectable hepatic tumors. We evaluated the difference in drug distribution between intra-hepatic arterial continuous infusion and rapid infusion using hepatic perfusion scintigraphy by 99mTc-macroaggregated albumin via reservoirs. The 41 cases were divided into 2 groups (A and B) according to the imaging of the perfusion scintigraphy. There were 17 cases in group A with equal distribution and 24 cases in group B with a different distribution between the continuous infusion and the rapid infusion. The 24 cases in group B were subdivided into groups according to the distribution of 99mTc-MAA in and around liver. In some cases the distribution pattern after rapid infusion improved more than with continuous infusion. These results suggested that the infusion method must be selected for every case.

Various treatments were performed in 14 patients with liver abscess, and their clinical effects were compared. The treatments were intra-arterial antibiotic infusion therapy (Group A, 4 patients), intra-arterial infusion therapy plus percutaneous drainage of the abscess (Group B, 4 patients), and intravenous antibiotics alone (Group C, 6 patients). The period from the beginning of treatment to negative conversion of CRP* (period until cure) was compared among the groups. Groups A and B had received intravenous antibiotics for a mean of 10 days and 16 days, respectively, but the treatment was ineffective. The mean period until cure from the beginning of intra-arterial infusion therapy was 36 days for Group A and 18 days for Group B. In Group C, the mean period until cure was 28 days. All patients recovered, and recurrence was observed in only one patient. Although liver abscess can be treated successfully in many patients by intravenous antibiotics alone, intra-arterial therapy is effective in those in whom intravenous therapy is not effective.

The subjects were 20 cases with implantable reservoir systems for unresectable hepatic tumors. We evaluated the correlation between a complication of the gastro-duodenum after intra-hepatic arterial chemotherapy and a gastro-duodenal distribution of anti-cancer drugs using the hepatic perfusion scintigram by 99mTc-macroaggregated albumin via reservoirs. The 20 cases were divided into 3 groups (A, B, C) according to the level of gastro-duodenal accumulation of 99mTc-MAA. In group A with the strongest accumulation in the gastro-duodenum, we frequently found a gastro-duodenal ulcer after intra-hepatic arterial chemotherapy. There was a significant difference between group A and group C, the latter without a gastro-duodenal accumulation. These results suggested that the hepatic perfusion scintigram via reservoir was a useful examination to expect and prevent the complication of gastro-duodenum on intra-hepatic arterial infusion (especially continuous infusion) chemotherapy.