Comparative study on deletions of the dystrophin gene in three Asian populations

The frequency and distribution of deletions of 19 deletion-prone exons clustered in two hot spots in the proximal and central regions of the dystrophin gene were compared in three populations from Singaporean, Japan, and Vietnam. DNA samples obtained from 105 Singaporean, 86 Japanese, and 34 Vietnamese Duchenne muscular dystrophy patients were examined by polymerase chain reaction amplification. Deletions of the examined exons were found in 51.2% of Japanese patients but in 40.0% or less of the Singaporeans and Vietnamese. About two thirds of the deletions were localized in the central region and the remaining deletions were clustered at the proximal region. The most commonly deleted exons at the central deletion hot spot were exon 50 in the Singaporean, exons 49 and 50 in the Japanese, and exon 51 in the Vietnamese population. At the proximal deletion hot spot, the most commonly deleted exons were exons 6 and 8 in the Singaporeans, exons 12 and 17 in the Japanese, and exons 8 and 12 in the Vietnamese. Two cases each from Singapore and Japan had large-scale gross mutations spanning both deletion hot spots. Our results suggest that, although the presence and frequency of the two deletion hot spots may be similar in the three Asian populations analyzed, the distribution and frequency of deletions among the different exons can vary as a result of population-specific intronic sequences that predispose individuals to preferential deletion breakpoints. Received: May 20, 2002 / Accepted: July 1, 2002     

SARS病毒S1蛋白重组C端片段免疫效果的实验研究

为获得纯化的重组SARS病毒S1蛋白C端 ,研究其刺激机体产生针对SARS病毒免疫应答的规律和机制 ,将编码SARS病毒S1蛋白C端 311个氨基酸残基的基因克隆 ,并在原核表达系统中表达 ,纯化获得了的重组蛋白。利用SARS患者恢复期的血清 ,对纯化的重组S1蛋白进行血清学分析。结果表明 ,本研究中克隆表达的重组蛋白序列与公布的SARS病毒S1蛋白C端的序列相同 ,其编码的重组蛋白相对分子质量约为 5 90 0 0Mr。SARS患者恢复期的血清均与重组蛋白反应 ,在5 9 0 0 0Mr处形成特异性的反应条带 ,而来自SARS流行前的正常人对照血清则不能与重组蛋白反应。在本研究中获得的重组蛋白可以为研究SARS病毒识别宿主细胞受体的过程及其机制提供条件。     

Selective cytotoxic lesions of the retrohippocampal region produce a mild deficit in social recognition memory

Although a number of studies have implicated the hippocampal formation in social recognition memory in the rat, a recent study in this laboratory has demonstrated that selective cytotoxic lesions, confined to the hippocampus proper (encompassing the four CA subfields and the dentate gyrus), are without effect on this behaviour. This finding suggests that the hippocampus proper does not subserve social recognition memory in the rat, but does not preclude the possibility that other areas of the hippocampal formation, such as the entorhinal cortex or subiculum, could support this form of learning. The present study addressed this issue by examining the effects of selective cytotoxic retrohippocampal (RHR) lesions (including both the entorhinal cortex and subiculum) on social recognition memory in the rat. RHR lesions produced a mild social recognition memory impairment, although lesioned animals still displayed a reduction in investigation time between the first and second exposure to the juvenile. This result is consistent with other studies which have implicated the retrohippocampal or parahippocampal area in olfactory recognition memory processes. It also suggests, however, that other areas, out with the retrohippocampal region, are also likely to play an important role in social recognition memory.     

Evaluation of the lymphocyte blastogenic response by rapid flow analysis.

The data presented show that the blastogenic response of human peripheral lymphocytes to mitogens and specific antigens can be easily evaluated with rapid flow analysis of mithramycin stained cells. The precision, reproducibility and rapidity of the method make it highly suitable to study lymphocyte response to antigens in patient populations.     

Histological,immunohistochemical, and ultrastructural features of a rat medullary thyroid carcinoma transfected with a corticotropin-releasing hormone cDNA expression vector

Clonal cell lines producing corticotropin-releasing hormone (CRH) have been generated by transfection of the W2 rat medullary thyroid carcinoma (MTC) cell with a CRH-encoding CMV/ SV40 expression vector. Here, we report the morphological, immunohistochemical, and ultrastructural features of rat tumors derived by implantation of CRH-producing W2CRH-7 cells and compare them with non-CRH-producing W2 MTCs. Both types of tumors grew rapidly and consisted of sheets and nests of pleomorphic cells infiltrating adjacent adipose tissue. Immunohistochemistry revealed CRH in only W2CRH-7 tumors; scattered cells in these tumors also were immunoreactive for chromogranin and for vasoactive intestinal peptide. Otherwise, the two tumor types exhibited similar profiles of various neuroendocrine markers, including neuron-specific enolase, synaptophysin, calcitonin, and somatostatin. Ultrastructurally, the tumors contained abundant dilated rough endoplasmic reticulum, a prominent Golgi apparatus, and numerous lysosomes. Very few secretory granules were noted in the W2 tumors; by contrast, secretory granules, although still not numerous in the majority of W2CRH-7 cells, were more abundant in scattered cells of those tumors. The positive immunostaining for CRH is consistent with the observations of increased plasma CRH and pituitary-adrenal activation induced by these transplanted tumors. This system provides a valuable model for CRH excess mimicking tumoral CRH-dependent Cushing’s syndrome in human patients.     

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.     

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. RelB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which RelB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4+ T cells induced by the DCs transfer antigen-specific "infectious" tolerance to primed recipients in an interleukin-10-dependent fashion. Thus CD40, regulated by RelB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.     

Functional evidence of decreased tumorigenicity associated with monochromosome transfer of chromosome 14 in esophageal cancer and the mapping of tumor-suppressive regions to 14q32

Despite the abundant evidence of high allelic loss of chromosome arm 14q in human cancers, tumor-suppressor genes mapped to this chromosome have yet to be identified. To narrow the search for candidate genes, we performed monochromosome transfer of chromosome 14 into an esophageal carcinoma cell line, SLMT-1 S1. Statistically significant suppression of the tumorigenic potential of microcell hybrids containing the transferred chromosome 14 provided functional evidence that tumor-suppressive regions of chromosome 14 are essential for esophageal cancer. Tumor segregants emerging in nude mice during the tumorigenicity assay were analyzed by detailed PCR-microsatellite typing to identify critical nonrandomly eliminated regions (CRs). A 680-kb CR mapped to 14q32.13 and an approximately 2.2-Mb CR mapped to 14q32.33 were delineated. Dual-color BAC FISH analysis of microcell hybrids and tumor segregants verified the selective loss of the 14q32.13 region. In contrast, similar transfers of an intact chromosome 11 into SLMT-1 S1 did not significantly suppress tumor formation. These functional complementation studies showing the correlation of tumorigenic potential with critical regions of chromosome 14 validated the importance of the 14q32 region in tumor suppression in esophageal cancer. The present study also paved the path for further identification of novel tumor-suppressor genes that are relevant to the molecular pathogenesis of esophageal cancer.     

Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies

BACKGROUND: Idiopathic autonomic neuropathy is a severe, subacute disorder with a presumed autoimmune basis. It is indistinguishable from the subacute autonomic neuropathy that may accompany lung cancer or other tumors. Autoantibodies specific for nicotinic acetylcholine receptors in the autonomic ganglia are potentially pathogenic and may serve as serologic markers of various forms of autoimmune autonomic neuropathy. METHODS: We tested serum from 157 patients with a variety of types of dysautonomia. Immunoprecipitation assays with iodine-125-labeled epibatidine and solubilized human neuroblastoma acetylcholine receptors were used to detect autoantibodies that bound to or blocked ganglionic receptors. RESULTS: Ganglionic-receptor-binding antibodies were found in 19 of 46 patients with idiopathic or paraneoplastic autonomic neuropathy (41 percent), in 6 of 67 patients with postural tachycardia syndrome, idiopathic gastrointestinal dysmotility, or diabetic autonomic neuropathy (9 percent), and in none of 44 patients with other autonomic disorders. High levels of the binding antibodies correlated with more severe autonomic dysfunction (including the presence of tonic pupils). Levels of these antibodies decreased in patients who had clinical improvement. All seven patients with ganglionic-receptor-blocking antibodies had ganglionic-receptor-binding antibodies and had idiopathic or paraneoplastic autonomic neuropathy. CONCLUSIONS: Seropositivity for antibodies that bind to or block ganglionic acetylcholine receptors identifies patients with various forms of autoimmune autonomic neuropathy and distinguishes these disorders from other types of dysautonomia. The positive correlation between high levels of ganglionic-receptor antibodies and the severity of autonomic dysfunction suggests that the antibodies have a pathogenic role in these types of neuropathy.