Patient and parental attitudes toward genetic screening and its implications at an adult cystic fibrosis centre

General population screening for cystic fibrosis carrier status in the United Kingdom would detect 72% of at-risk couples. Proper counselling would allow these couples to make informed reproductive choices, including the possibility of prenatal diagnosis and the termination of an affected pregnancy. However, children with cystic fibrosis born in this decade, given optimum treatment, now have an average life expectancy of 40 years, and there is no unanimity of opinion on how, where, when, or even if, screening should be offered. The purpose of this questionnaire-based study was to examine the attitudes of an adult clinic population who have grown up with cystic fibrosis, and of their parents, towards genetic screening programmes and the controversies and ethical dilemmas surrounding such programmes in cystic fibrosis. Both patients and parents supported prenatal screening (88% and 90%) and the option of terminating an affected pregnancy (68% and 84%). Only 22% of patients and 10% of parents felt that screening should be limited to families with a history of cystic fibrosis, and 19% and 6%, respectively, that prenatal diagnosis should be restricted to those with a previous child with cystic fibrosis. Despite the negative aspects of any screening programme and the acknowledged ethical problems peculiar to cystic fibrosis, the conclusion of our patients and parents who have lived intimately with the illness is that there should be the option of utilising information available from genetic screening for cystic fibrosis to guide reproductive choices. Pilot programmes to define the optimum management of such screening should continue.     

ST2作为Th2细胞亚群标志以及其与支气管哮喘的关系的研究

各自主要分泌IFN γ和IL 4的Th1和Th2亚群 ,与临床疾病的关系十分密切。如何从表面标志上加以区分是一项迫切需要解决的问题。ST2是近年来提出的Th2细胞的稳定标志物。本工作在体外成功地诱导人脐带血T细胞向Th1或Th2分化的基础上 ,应用逆转录PCR分析了ST2mRNA的表达特点。证实ST2在人Th2细胞上的选择性表达。为了探索ST2、Th2与支气管哮喘的关系 ,本工作进一步检测了正常人和支气管哮喘患者外周血单个核细胞中 β actin、ST2以及IFN γ和IL 4的mRNA水平。结果显示 :支气管哮喘患者ST2mRNA水平升高 ,IL 4水平也明显升高 ,但IFN γ无变化。这提示ST2作为Th2细胞的标志物 ,有可能成为Th2极化性疾病如哮喘发病机制研究的一个参考性标志 ,至于ST2是否有可能作为治疗的靶分子 ,有待进一步探讨     

Detection of single nucleotide substitution by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip

Recent advances in human genome research have revealed that genetic polymorphisms, such as single nucleotide polymorphisms (SNPs), are closely associated with susceptibility to various common diseases and adverse drug reactions. Also, numerous mutations responsible for a number of genetic diseases have been identified. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We have devised a novel low-tech method for the detection of a single nucleotide substitution using competitive allele-specific short oligonucleotide hybridization with immunochromatographic strip. The gene of interest is PCR-amplified, hybridized to an allele-specific short oligonucleotide probe in the presence of a competitive oligonucleotide, and subjected to chromatography using a DNA test strip at room temperature. The genotype is unambiguously determined by the presence or the absence of visible purple lines on a strip. Feasibility of the method was demonstrated by the detection of a prevalent disease-causing mutations in glycogen storage disease type Ia (G6PC), medium-chain acyl-CoA dehydrogenase deficiency (ACADM), non-ketotic hyperglycinemia (GLDC), and clinically important polymorphisms in the CYP2C19 gene and the aldehyde dehydrogenase 2 gene (ALDH2). The procedure does not demand either technical expertise or expensive instruments and is readily performed in local clinical laboratories. The result is obtained within 10 min after PCR. This rapid and simple method of SNP detection may be used for point-of-care genetic diagnosis with potentially diverse clinical applications. Hum Mutat 22:166-172, 2003.     

同种异体移植免疫耐受的进展

探索组织器官移植后的排斥反应,提高移植物的存活率是移植免疫研究者致力攻克的堡垒。20世纪90年代以来,随着器官移植术的广泛开展,人们对移植排斥反应的认识不断加深,对移植免疫耐受提出了新的研究思路和方法。目前普遍认为,解决异体移植排斥反应的关键在于诱导受体对供体的细胞、组织或器官产生免疫耐受,即受体免疫系统对移植物抗原产生特异性免疫无反应性。本文将对近年来阻断特异性免疫应答、诱导免疫偏离、主动免疫诱导同种移植耐受及建立受者体内嵌合体诱导移植耐受等主要方面的进展综述。     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Real-time automatic extraction of lumen region and boundary from endoscopic images

A new approach to the automatic extraction of the lumen region and its boundary for gastrointestinal (GI) endoscopic images is presented. First, a quasi region of interest, the darker regions of the image, is segmented using a region splitting scheme termed progressive thresholding. The centre of mass of this segmented region acts as a seed for further processing. Then the lumen region is obtained using a region growing technique called the integrated neighbourhood search (INS). A new quad structure based technique is introduced to enhance the INS speed significantly. A back projection algorithm is suggested to optimise the search for pixels belonging to the lumen region and boundary. A boundary-thinning algorithm is also proposed to remove the redundant pixels from the lumen boundary and to generate a connected single pixel width boundary. The proposed approach does not need a priori knowledge about the image characteristics. The experimental results indicate that the proposed technique enhances the speed of conventional INS by 45.5% to 28.6% based on the lumen size varying from 22,709 pixels to 4947 pixels. The main advantage of the proposed technique is its high-speed response that facilitates real-time analysis of endoscopic images.     

Experimental rat model for therapeutic retinal pigment epithelium transplantation—unequivocal microscopic identification of human donor cells by in situ hybridisation of human-specific Alu sequences

Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.     

基于小波变换的小鼠QRS复合波检测

采用二进小波变换与斜率和幅度相结合的方法，对小鼠QRS复合波进行检测。根据小鼠QRS复合波的特点，采用Daubechics小波为母函烽，按照ECG的频谱特点选用尺度因子，对有噪声污染和形态变异的QRS复合波进行了检测。结果表明：小波变换对小鼠QRS复合波的检测是一种有效的方法。     

An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.