Enhancing the utility of a prM/E-expressing chimeric vaccine for Japanese encephalitis by addition of the JEV NS1 gene

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.     

Antidiuretic effect of desmopressin chimera agglomerates by nasal administration in rats

In the present study, a nasal powder of the antidiuretic peptide desmopressin (DDAVP) formulated as chimera agglomerates was studied to improve drug bioavailability and provide a flexible drug product. Firstly, DDAVP was spray-dried along with mannitol and lecithin to produce primary microparticles capable of instantaneous dissolution in water. The chimera agglomerates were spontaneously formed by mechanically vibrating the microparticles on two stacked sieves. Agglomerate formation and strength were favored by the presence of lecithin. Drug content and dissolution rate remained unmodified after agglomeration. However, owing to the agglomerate larger size, powder flowability was greatly improved in comparison with the original microparticles, allowing accurate powder dosing into the nasal delivery device. DDAVP in vitro permeation across excised rabbit nasal mucosa from the agglomerates was significantly higher than that obtained from a commercial liquid nasal spray.     

应用荧光原位杂交技术检测卵巢早衰患者X染色体嵌合型

目的　探讨卵巢早衰与低百分率 45 ,X/ 46,XX嵌合核型的关系 ,了解荧光原位杂交(FISH)技术检测染色体嵌合核型的敏感性和特异性。方法　取 18例卵巢早衰患者和 9例正常妇女(对照 )的外周血进行培养 ,行常规G显带和用X染色体记数探针行FISH ,对每例计数 60 0～ 70 0个细胞的荧光信号。结果　 18例卵巢早衰患者的常规染色体分析核型为 46,XX。卵巢早衰患者的 45 ,X/46,XX染色体嵌合型比率为 7.6% ,显著高于对照者的 2 .2 % ,两者比较 ,差异有极显著性 (P <0 .0 1)。结论　某些卵巢早衰患者的致病机理可能与 45 ,X/ 46,XX嵌合核型比例增高 ,X染色体数量不足有关。对此类低百分率染色体嵌合核型 ,FISH技术在敏感性和特异性上优于常规核型检查     

A true hermaphrodite dispermic chimera with 46, XX and 46, XY karyotypes

A 16-year-old male with hypospadias and gynaecomastia had a rudimentary uterus with a right Fallopian tube and ovary; the left gonad was a functioning testis. Cytogenetic studies showed cells with 46 , XX and 46 , XY sex chromosomes in cultured blood, skin and gonadal tissues. Cells with the 46 , XX constitution predominated in all tissues. Extensive investigations failed to demonstrate blood cell and serum chimerism, but there was little genetic variation of these characters between family members. Cytogenetic studies demonstrated that the father had contributed different marker chromosomes to the 46 , XY and 46 , XX cell lines of the propositus, whereas the mother had contributed the same two informative markers to both cell lines. The patient was a chimera with two diploid cell lines of different sex that had developed from the products of two separate acts of syn-gamy. Dispermy was demonstrated, and, whereas there was no evidence of different maternal contributions to the chimeric cell lines, uncertainty remains that these were identical.     

B7反义肽预处理脾细胞诱导异基因嵌合体形成及延长移植物存活

目的　尝试以B7 CD2 8/CTLA4为靶点 ,用B7反义肽 (B7AP)预处理供者的脾细胞 ,免疫受者后结合输注供者骨髓细胞 ,诱导受体形成异基因嵌合体。方法　每只用 5× 10 6/ 10 0 μlB7AP预处理的供者小鼠 (C5 7BL/ 6 )脾细胞免疫受者小鼠 (BALB/c) ,诱导特异性同种低免疫应答反应 ;在此基础上每只受者小鼠输注供者骨髓细胞 2× 10 7/ 10 0 μl,诱导受者形成异基因嵌合体 ,以流式细胞术 (FACS)连续动态监测嵌合状态的维持情况并用小鼠心肌移植模型研究移植物存活的影响因素。结果　在受者体内成功诱导出了特异性同种低免疫应答反应 ,应答水平只有正常未免疫组的 4 3%(n =6 ,P <0 0 0 1) ;嵌合状态可以维持到 10 0d以上 (n =6 ) ,15 0d时嵌合率仍有 3.4 5 % ;嵌合小鼠进行同种异体小鼠心肌移植存活超过 10 0d(n =6 ) ,而对照组阿霉素组只能延长至 16d(n =6 )。结论　B7AP预处理小鼠脾细胞可诱导形成异基因嵌合体 ,并显著延长同种小鼠心肌移植存活的时间 ,为移植免疫的研究提供了一个简单易行的嵌合体平台技术 ,也为用B7AP在移植免疫中的应用进行了有益的尝试。     

基因工程人-鼠嵌合抗体ch-BDI在荷瘤鼠体内外功能的研究

目的　鉴定抗人膀胱癌人 鼠嵌合抗体ch BDI在荷瘤鼠体内外功能 ,探讨其临床应用价值。　方法　通过超滤浓缩和亲合层析 ,得到较高纯度的ch BDI,用还原法标记99mTc,体外测定了其对人膀胱癌细胞的结合能力 ,应用放射免疫显像观察了其在荷瘤鼠体内的分布。　结果　99mTc标记的ch BDI的免疫活性分数约为 76% ,体外的亲合常数约为 3 5 6× 10 9M-1,在荷瘤鼠体内能够迅速浓缩聚集于肿瘤部位。　结论　抗人膀胱癌人 鼠嵌合抗体ch BDI在体内、体外均具有良好的与人膀胱癌组织的结合活性 ,有较理想的临床应用前景     

47,XXX综合征的研究进展

47,XXX综合征是女性中最常见的性染色体非整倍体疾病,表型多样且症状较轻,缺乏普遍的生理、心理异常特点,有些女性甚至还可以有正常的月经周期和生育能力。一直以来,47,XXX综合征虽然有较高的发病率,诊断率却只有10%左右。近年随着非侵入性产前基因筛查技术的开展与普及,越来越多的性染色体非整倍体疾病被筛查出来。为提高该疾病的诊断率和方便遗传咨询,亟需对47,XXX综合征有一个全面的理解,以便为患者和医者提供临床总结和诊疗指导。本文介绍47,XXX综合征形成机制、表型、行为和认知表现、神经成像特点、遗传学诊断及其研究进展。     

The chimeric<Emphasis Type="Italic"> CYP21P/CYP21</Emphasis> gene and 21-hydroxylase deficiency

The chimeric CYP21P/CYP21 gene is a consequence of a 26- or 32-kb deletion in the C4-CYP21 repeat module of CYP21P, tenascin A (XA), serine/threonine nuclear protein kinase (RP2), and the C4B and CYP21 genes in congenital adrenal hyperplasia (CAH) with steroid 21-hydroxylase deficiency. To date, there have been three distinct chimeras found in CAH patients in ethnic Chinese. Initiation for production of these molecules is proposed to be chi-like sequences and a minisatellite consensus existing in several noncoding regions in CYP21 genes. These molecules have the 5 end of the CYP21P-specific sequence in common but differ in the 3 end of CYP21-specific genes. In addition, there appears to be a 3.2-kb fragment generated by Taq I digestion, which leads to allele dropout in PCR amplification for detecting the aberrant splicing site of the IVS2 –12A/C>G mutation at nucleotide (nt) 655 in the CYP21 gene. Therefore, the chimeric CYP21P/CYP21 cannot be detected by conventional methods. It has been demonstrated that a PCR product amplified with allele-specific primers covering tenascin B (TNXB) to the 5 end of the CYP21 gene combined with Southern analysis by Ase I and Nde I digestion may be used for identifying the chimera in the CYP21 gene.     

Antigen-specific B cells are required for the secondary response of T cells but not for their priming

We studied the potential role of B cells in T cell responses using severe-combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B-17 mice (TG mice). These mice developed both CD4⁺ and CD8⁺ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed-type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)-2, IL-6 and interferon (IFN)-γ and the expression of IL-4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B-17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen-primed B cells, but not by naive B cells, lipopolysaccharide-activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen-primed B cells could induce T cell responses without professional antigen-presenting cells (APC). T and B cells were purified from OVA-immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN-γ in the absence of non-B APC. When anti-CD80 or anti-CD86 was added in the assay, proliferation and IFN-γ production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.     

A semiquantitative PCR technique for detecting chimerism in hamster-to-rat bone marrow xenotransplantation

Although bone marrow transplantation has been used to induce donor-specific tolerance in many allogeneic models, similar effort in xenogeneic transplantation is met with obstacles like more severe graft versus host disease (GVHD). We are currently engaged in developing a GVHD-free hamster-to-rat xenotransplantation model using splenectomy, total body irradiation, and donor bone marrow transplantation. To test donor cell chimerism, particularly in the solid tissues, we developed a semiquantitative polymerase chain reaction (PCR) method using primers specific for hamster beta-actin and mitochondrial cytochrome C oxidase I and II (MCO I and II) genes and rat sex determination region on the Y chromosome (SRY) gene. Using this method, we estimated the level of hamster cells chimerism in rats subjected to splenectomy, total body irradiation (10 Gy), and hamster bone marrow transplantation (3 x 10(8) cell/recipient) and observed high levels of donor cells in all recipient tissues tested.