A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

Cytotoxicity against human peripheral blood mononuclear cells and T cell lines mediated by anti-T cell immunotoxins in the absence of added potentiator.

Several in vitro assays have indicated that anti-T cell immunotoxins (IT), composed of monoclonal antibodies (MoAbs) conjugated to ricin A chain (RTA), are maximally effective against T cells only in the presence of potentiators. It was thought that such IT might not be sufficiently cytotoxic to deplete T cells in vivo upon administration to patients. Therefore, we have re-evaluated the in vitro assays and report herein that even with a short exposure time (2 h), the two anti-T cell IT, H65-RTA (anti-CD5 MoAb coupled to RTA) and 4MRTA (anti-CD7 MoAb coupled to RTA30), were specifically cytotoxic for peripheral blood mononuclear cells (PBMC) in the absence of potentiators. Moreover, as has been reported for IT when tested against T cell lines, prolonging the exposure time of the IT with PBMC from 2 h to as long as 90 h, without added potentiators, enhanced their cytotoxicity from 2- to 40-fold. In contrast, most T cell lines were more sensitive to IT in the presence of potentiator, and IT cytotoxicity was much less enhanced by prolonging the exposure time. Thus, T cell lines may not serve as accurate models to determine the efficacy of IT against PBMC in vitro or in vivo. We conclude that IT-induced cytotoxicity of PBMC can be demonstrated in vitro at pharmacologically achievable concentrations in the absence of added potentiators.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor γ chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor γ chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor γ chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

Transcatheter arterial embolization of abnormal neovessels in a swine model of knee arthritis

BackgroundIn recent years, transcatheter arterial embolization (TAE) using imipenem/cilastatin (IPM/CS) has attracted attention as a treatment for relieving osteoarthritis (OA) pain. However, IPM/CS is not approved by Japanese medical insurance for use as an embolic material. Therefore, it is necessary to develop new embolic materials for TAE to relieve OA pain. The purpose of this study was to develop a swine model of knee arthritis and embolize abnormal neovessels (ANs) using two different embolic materials. We compared the embolic effects and tissue damage in knees.MethodsKnee arthritis was induced by intra-articular injection of papain into 12 knees in six female swine. The swine were divided into two groups of three swine each (six knees per group) for embolization of ANs in the knees with either IPM/CS or soluble gelatin sponge particles (SGSs). Three days after embolization, we compared the embolic effects using angiography and the tissue damage histopathologically.ResultsANs were observed in all 12 knees at 42 days after papain injection. The ANs disappeared and the patent arteries were recanalized 3 days after TAE in all 12 knees. Histopathological evaluation revealed synovitis changes, such as synovial thickening and inflammatory cell infiltration, in all 12 knees. There was no evidence of skin or muscle necrosis in either group. The appearance of ANs, recanalization of the parent arteries, and histopathological outcomes were not significantly different between the two groups.ConclusionSGSs were as safe as IPM/CS for TAE of ANs in this swine model of knee arthritis.     

基于通用中间件接口服务器的远程医疗信息系统

在远程医疗信息系统共享平台的设计中,提出一种构建通用中间件接口服务器实现多家医院信息系统HIS与PACS的无缝连接方案。着重介绍了DICOM中间件、HL7消息中间件、生态识别和PKI技术在系统集成中的应用。     

Congenital heart defects in CHARGE: The molecular role of CHD7 and effects on cardiac phenotype and clinical outcomes

CHARGE syndrome is characterized by a pattern of congenital anomalies (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth, Genital abnormalities, and Ear abnormalities). De novo mutations of chromodomain helicase DNA binding protein 7 (CHD7) are the primary cause of CHARGE syndrome. The clinical phenotype is highly variable including a wide spectrum of congenital heart defects. Here, we review the range of congenital heart defects and the molecular effects of CHD7 on cardiovascular development that lead to an over‐representation of atrioventricular septal, conotruncal, and aortic arch defects in CHARGE syndrome. Further, we review the overlap of cardiovascular and noncardiovascular comorbidities present in CHARGE and their impact on the peri‐operative morbidity and mortality in individuals with CHARGE syndrome.     

Adhesion dependent release of hepatocyte growth factor and interleukin-1 receptor antagonist from human blood granulocytes and monocytes: Evidence for the involvement of plasma IgG,complement C3 and β2 integrin

Objective:Evolving evidence of anti-inflammatory effects is observed in patients with rheumatoid arthritis or ulcerative colitis following periodic adsorptive granulocyte and monocyte (GM) apheresis with a column containing cellulose acetate (CA) beads as apheresis carriers. This study was undertaken to obtain insights into mechanisms of anti-inflammatory actions of adsorptive GM apheresis with CA beads. Methods:In a series of in-vitro experiments, we investigated the effects of plasma proteins and the leucocytes 2 integrin (CD18) on granulocyte adsorption to CA beads. Results:Granulocyte adsorption to CA beads required plasma IgG, the complement C3 and was inhibited by an antibody to leucocytes CD18. Further, hepatocyte growth factor (HGF) and interleukin-1 receptor antagonist (IL-1ra) which have strong anti-inflammatory actions were released by granulocytes that adhered to CA beads. Conclusions:Plasma IgG, C3 derived complement activation fragments and leucocytes CD18 are involved in granulocyte adhesion to CA beads and hence the release of HGF and IL-1ra.Received 27 October 2003; returned for revision 16 December 2003; accepted by M. J. Parnham 8 January 2004     

Isolation of DNA from the centromere of human chromosome 7 by microdissection

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a micro-dissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3–4 cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.This revised version was published online in November 2005 with corrections to the Cover Date.     

A B7-1-transfected human melanoma line stimulates proliferation and cytotoxicity of autologous and allogeneic lymphocytes

B7 co-stimulation is necessary to activate resting T cells upon antigen recognition by the T cell receptor. To see whether expression of B7 may render human melanoma cells able to stimulate T cells, a cloned melanoma line (Me1B6), which did not express B7-1, was transfected with the human B7-1 gene. In proliferation assays, B7-1 transfected cells (Me1B6/B7) showed greater stimulatory activity of allogeneic and autologous peripheral blood lymphocytes (PBL) compared to parental, non-transfected tumor cells. This effect was also seen when allogeneic CD8⁺ and CD4⁺ subpopulations were used as effectors. In these studies, activation of lymphocytes was B7-1-dependent and HLA classes I and II mediated. The higher proliferation correlated with an increased lytic activity by PBL stimulated with B7-1⁺ tumor cells against the untransfected Me1B6. Furthermore, PBL from a metastatic melanoma patient stimulated by Me1B6/B7 developed an higher lytic activity not only against Me1B6 but also against their autologous, B7-1^? tumor. Finally, after Me1B6/B7 stimulation, PBL released interleukin (IL)-2 and interferon-γ, but not IL-4, suggesting a Th1-mediated response. These data support the use of B7-1 transfected melanoma cells in the therapeutic vaccination of melanoma patients.     

Jak3 activation in human lymphocyte precursor cells

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4⁻CD8⁻) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) β gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.