Complete coding region of the new HLA-DQB1*0612 allele, obtained by RT-PCR

A novel HLA-DQB1 allele was detected by oligotyping in the Bubi population of Equatorial Guinea. In order to characterize the new allelic variant, a RT-PCR method which permitted the cloning of its complete coding region was designed. With this method, we have determined the nucleotide sequence of the new DQB1*0612 allele, related to *0604 and *0609 but differing from them at polymorphic codon 70. A proposal for the improvement of the sequencing strategies of HLA class II alleles is made.     

Expression of mucin genes in the human testis and its relationship to spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

Macrophage mannose receptor in chronic sinus disease

BACKGROUND: The role of infectious agents in the onset and maintenance of chronic sinus disease is still not fully understood. Macrophage mannose receptor (MMR), an innate pattern recognizing receptor, capable of phagocytosis of invaders and signal transduction for proinflammatory mechanisms, might be of importance in immune interactions in chronic sinus disease. OBJECTIVE: We examined the MMR in sinonasal airway mucosa to evaluate its possible role in chronic rhinosinusitis (CS) and nasal polyposis (NPs). METHODS: Surgical samples from patients with sinonasal disease were investigated with real-time RT-PCR for quantification of MMR mRNA expression, and the presence and location of MMR-positive cells was analysed by immunohistochemistry. RESULTS: Quantification of MMR mRNA showed a statistically significant higher expression in NPs compared to CS without NP and controls. Immunohistochemistry revealed expression of MMR in all tissue samples; however, in NP we found an enhanced positive cellular staining including cell aggregates. CONCLUSIONS: We could demonstrate for the first time that the expression of MMR is significantly upregulated in NP compared to patients with CS without NP or turbinate tissue of controls. Macrophages expressing MMR, accumulated in cell aggregates in NPs, play a possible key role in pathogen-macrophage interaction in NP disease.     

实时荧光RT-PCR法和NASBA方法在呼吸道多病原检测中的应用评价

目的 评价实时荧光RT-PCR和核酸依赖性序列扩增法（NASBA）两种方法在呼吸道多病原检测中的应用价值。方法 收集北京地区急性呼吸道感染病例标本140份,分别采用实时荧光RT-PCR方法和NASBA方法对样本进行平行检测并对两种方法的病原检出限、重复性、检测一致性及检测时间等指标进行比较。结果 两种检测方法批内重复CV值均<5%,重复性较好;实时荧光RT-PCR方法对脊灰毒株核酸和H1N1病毒核酸最低检出限分别为5.62 CCID50 /0.1 ml（107 倍稀释）和10³倍稀释度,NASBA方法最低检出限分别为56.2 CCID50 /0.1 ml（106 倍稀释）和10²倍稀释。实时荧光RT-PCR灵敏度略优于NASBA方法。针对两种方法共同检测呼吸道病原体种类,实时荧光RT-PCR和NASBA方法检测阳性率分别为89.28%（125/140）、70%（98/140）。两种方法对甲型H1N1病毒、肠道病毒及新型冠状病毒的检测结果一致性达100%,对甲型流感病毒、甲型H3N2、副流感病毒1-4型的检测结果基本一致, Kappa 值范围为0.81~1;对冠状病毒229E/HKU1/OC43/NL63、鼻病毒、呼吸道合胞病毒和乙型流感病毒的检测结果一致性较好, Kappa 值范围为0.61~0.80;对人偏肺病毒的检测结果一致性差, Kappa 值仅为0.55。实时荧光RT-PCR方法检测时间为90 min,NASBA方法仅需40 min即可完成。 结论 实时荧光RT-PCR方法灵敏度较高,适合对大批量临床样本进行筛查;NASBA方法检测时间短,不易污染,适用于时限性强但对灵敏度要求不太高的应用场景,如临床上重症呼吸道感染者的快速检测。建议根据不同需求和应用场景,选择合适的检测方法和产品。     

c-kit在血液病中的表达

目的 :探讨原癌基因c -kit在血液病中表达的情况。方法 :用Ficoll分离液分离 ,收集骨髓单个核细胞。c -kit的表达采用RT -PCR法。结果 :在不同血液病中c -kit的表达情况不一 ,在急性髓细胞性白血病 (AML)为 5 7.8% ,急性淋巴细胞性白血病 (ALL)为 8.3 % ,骨髓增生异常综合征 (MDS)为 40 %。结论 :c -kit主要在AML中表达 ,提示c -kit表达是粒细胞的一个特异性标志。     

人类巨细胞病毒感染对人胚肺细胞HOXB1,HOXB5,HOXB6及HOXB9基因表达的影响

目的 :研究人胚肺 (HEL)细胞HOXB1,HOXB5 ,HOXB6及HOXB9基因的表达状态及人类巨细胞病毒(HCMV)感染对上述基因表达的影响。方法 :采用半定量逆转录 -聚合酶链反应 (RT -PCR)技术。结果 :①HEL细胞表达HOXB5和HOXB6 ,但不表达HOXB1和HOXB9;②HCMV感染后 ,HEL细胞HOXB6基因的表达上调 ,且被HCMV诱导表达HOXB9T ,而HOXB5基因的表达无明显改变 ,HOXB1基因仍旧不表达 ;③全反式维甲酸 (ATRA)能上调HCMV感染后HOXB9基因的表达 ,但对HCMV感染晚期HOXB6基因的表达有抑制作用。结论 :HCMV能诱导HOXB6和HOXB9基因表达异常 ,这可能在先天性HCMV感染导致胚胎发育畸形中起重要作用