摘要:

BackgroundThe detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample.MethodsIn this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures.ResultsThe biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample.ConclusionsThe developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

ObjectiveProlonged intestinal replication of polioviruses has not previously been studied in Greek AIDS patients. The objective of our study was to estimate the prevalence of enteroviral infections in this population.MethodsNineteen stool samples were investigated from 19 different patients. Collection took place at the Hellenic Red Cross Hospital, Athens, Greece, between August and October 2002. Samples were processed as follows: virus isolation was attempted by cell culture using three different cell lines (human epidermoid carcinoma [Hep]-2, rabdomyosarcoma [RD], and mouse cells genetically modified in order to express the polio virus receptor in their cell surface [L20B]). An enterovirus-specific reverse transcription (RT)-PCR was then applied. Finally, seroneutralization tests were performed on 11 blood samples taken from a number of the patients who had supplied stool samples.ResultsSamples were negative for enterovirus detection of any serotype on all cell lines. No cytopathic effect was observed. Enterovirus-specific RT-PCR assays were also negative for the detection of enteroviral RNA. Seroneutralization revealed relatively high antibody titers against poliovirus 1 and 2 in three of the eleven blood samples.ConclusionsGreek AIDS patients are not vulnerable to enteroviral infections and do not constitute a potential reservoir of poliovirus-prolonged excretion in Greece.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

IntroductionAntimuscarinic drugs have frequently been used for the treatment for patients with an overactive bladder (OAB) and there have been many studies on the distribution of muscarinic receptor subtypes in the bladder. However, the distribution of muscarinic receptor subtypes in OAB patients has not been well investigated. In this study we investigated the distribution of muscarinic receptor subtypes with mRNA and protein expressions in patients with and without OAB, and investigated both the dome and trigone area.MethodsSamples of bladder smooth muscle were obtained from 10 individuals, five patients with OAB and a non-OAB group consisting of five patients who received radical cystectomy.ResultsThe M2 receptor was predominant, but there was no significant difference in the level of M2 expression between the groups in the dome area. M5 expression in the dome area was significantly higher in the OAB group than in the non-OAB group. In the trigone area, the level of M2 mRNA expression was the highest in the non-OAB group, and was significantly lower in the OAB group. The levels of M1 and M5 mRNA expression were also observed in samples obtained from the trigone area.ConclusionThe multiformity of the muscarinic receptor subtypes in human bladder smooth muscle was confirmed, and our results suggest that the efficacy of a given pharmacologic therapy differs from patient to patient.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundThe routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis.AimThis study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women.MethodsPlasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of theRHDgene using conventional and real-time PCR. The results were then compared with theRHDfetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth.ResultsAfter exclusion of 42 samples from women exhibiting a nonfunctional or rearrangedRHDgene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results.ConclusionFetalRHDgenotyping from maternal plasma DNA in different clinical situations may be used with confidence.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundBiospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies is of interest in clinical genetics. However, few data are available on its use in hereditary diseases caused by genetic mutations.AimThe primary aim of this study was the refinement of BIA technology for use in identifying the β°39 mutation of the β-globin gene, a mutation which causes a common type of β°thalassemia.MethodsTarget-biotinylated PCR products were immobilized on streptavidin-coated sensor chips and diagnosed using SPR-based BIA performed by injecting specific oligonucleotide probes into the sensor chip.ResultsWe demonstrated that the β°39 mutation can be easily and reproducibly identified during the association phase.ConclusionsThis should be considered a pilot study demonstrating the ability of SPR-based BIA to detect point mutations in the β-globin gene by real-time monitoring of hybridization between oligonucleotide probes and target-biotinylated PCR products generated from genomic DNA from normal, heterozygous individuals and homozygous β° thalassemia patients.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundThe different clinical presentations of latent autoimmune diabetes in adults (LADA) and type 1 diabetes mellitus may be the result of susceptibility genes in determining the mode of onset. We analyzed the 5′ polymorphisms of the insulin mini-satellite region (INS), a variable number of tandem repeats (VNTR) [repeat units; RU]. We evaluated the association of the different INS-VNTR alleles in patient susceptibility to LADA autoimmune diabetes. To our knowledge, this constitutes the first study of this kind performed in a Caucasian population.MethodsFrom an group of 160 Argentinean patients previously characterized as having LADA, we selected 44 patients who presented with humoral autoimmunity for genotyping and compared them to 88 patients with type 1 diabetes and 138 healthy individuals. The INS-VNTR allele classes were determined by Southern blotting (class I: 21–44RU; class III: 138–159RU). Subjects with class I alleles were further studied using PCR amplification to determine the exact length of the alleles (short 1S: 22–37RU; medium 1M: 38–41RU; large 1L: 42–43RU). Allelic and genotype frequencies were estimated by χ2tests for independence with 2 × 2 contingency tables and the relative risks (RR) were determined using GraphPad InStat software.ResultsWe observed differential associations among the class I alleles when comparing patients with LADA (80.6%) and type 1 diabetes (81.3%) with the controls (70%; p < 0.005). This increase was largely due to the high frequency of the 1S/S genotype (63.6% LADA vs 37% controls, with a p-value of 0.0019 [p1]; 53.4% type 1 diabetes vs 37% controls, with a p-value of 0.0149 [p2]). Remarkably, all LADA patients genotyped as class I homozygous had the shorter (S) class I allele (100%). Differences in the overall 1S distribution were observed: in LADA the 94.4% of the alleles were equal to or smaller than 35RU, while in patients with type 1 diabetes it was 78.3% and in controls 74.1%. Moreover, the relative risks associated with the 1S/S genotype for patients with LADA showed a substantial increase with respect to those with type 1 diabetes (52%) when we compare them to the controls (1S/S LADA/control, 2.282 [RR1] vs type 1 diabetes/control, 1.497 [RR2]).ConclusionThe presence of the 1S allele could be considered a risk factor in LADA patients, as previously reported for type 1 diabetes. The class I INS-VNTR allele in LADA increases genetic susceptibility to disease development.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

AimHepatitis B virus (HBV) e antigen (HBeAg)-negative variants are reported to harbor 1896 precore mutants, and predict a worse clinical outcome. The aim of this study was to estimate the incidence of a precore mutation (1896) in both patients with chronic hepatitis B (CH-B) infection and blood donors in a tertiary care hospital in south India.MethodsOne hundred and twenty-two consecutive HBV DNA-positive CH-B patients (group I) and 102 HBsAg-positive ‘healthy’ blood donors (group II) were recruited. Samples found to be positive for HBV DNA were further studied. A nested PCR was used for the detection of HBV DNA. The 1896 precore mutation was detected using PCR-restriction fragment length polymorphism (RFLP). Nucleotide sequencing was performed on representative samples to confirm PCR-RFLP findings. The study population was stratified comprising: group IA: 17 HBeAg-positive CH-B patients; group IB: 105 HBeAg-negative CH-B patients; group IIA: 12 HBeAg-positive blood donors; and group IIB: 55 HBeAg-negative blood donors.ResultsThere was no significant difference in the HBeAg-positive status between groups I and II. Significantly higher levels of alanine transaminase (ALT) were seen in groups IA and IB than in groups IIA and IIB, respectively (p = 0.033; p = 0.004). A significantly higher proportion of CH-B patients (32.7%) were positive for anti-HBc IgM compared with the blood donor groups (10.4%; p = 0.0006). Among the HBeAg-negative subjects, 69% of the CH-B patients and 65% of the blood donors showed evidence of 1896 precore mutant. This infection included the 1896 mutant exclusively or mixed infection involving the 1896 mutant and 1896 wild-type.DiscussionThe absence of detectable HBeAg in most of the viremic blood donors and patients emphasizes the need for HBV DNA testing irrespective of HBeAg status. Mixed infection was detected in a higher proportion (42.6%) of CH-B patients than in blood donors (26.8%; p = 0.031). Among those with mixed infection, a significant proportion (44.2%) of CH-B patients, had ALT levels greater than the upper limit of normal (ULN), as compared with the blood donor groups (16.6%; p = 0.036).ConclusionsThe majority of CH-B patients and blood donors were negative for HBeAg despite their positive HIV DNA status. About two-thirds of the HBsAg-positive blood donors were viremic. Mixed infection was detected more frequently in CH-B patients and appears to be associated with more pronounced liver damage, as indicated by increased ALT levels.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundThe viral transforming genes E6 and E7 of human papillomavirus (HPV) 16 cause the degradation of tumor suppressor proteins. Expression of these oncoproteins increases following the integration of viral DNA into the host cell, resulting in the disruption of the E2 open reading frame (ORF).AimTo detect and correlate HPV-16 oncogene transcripts and HPV-16 E2 DNA in cervical biopsies obtained from women (n = 68) with cervical neoplasia.MethodsHPV-16 E6/E7 transcript and HPV-16 E2 DNA detection was performed on the cervical biopsies of 42 women positive for HPV-16 (36 with invasive cervical carcinoma and 6 with cervical intraepithelial neoplasia [CIN]). PCR was used to detect HPV DNA in cervical biopsies then restriction fragment length polymorphism (RFLP) was used to type the HPV DNA. Reverse-transcription (RT)-PCR for HPV-16 E6/E7 oncogene mRNA transcripts and a PCR to detect the HPV-16 E2 DNA was performed on HPV-16-positive samples.ResultsHPV-16 E6/E7 mRNA transcripts were not detected in any of the CIN I or II biopsies, but were detected in all cases of CIN III and invasive cancer in different combinations (E6 alone, E6*I, E6*I/E6*II, E6/E6*I/E6*II) except for one patient with stage IIB cancer treated with radiotherapy. The incidence of episomal E2 DNA was high in this study with 52.4% of the samples positive for episomal E2. It was even detected in patients with advanced stage cancer with 50%, 42%, and 66.6% of samples positive in stages IIB, IIIB, and IV, respectively.DiscussionHPV-16 E6/E7 mRNA oncogene transcripts, in various combinations, were uniformly detectable in the majority of the high-grade cervical lesions examined. Intact episomal E2 DNA was seen in a high proportion of samples, even from advanced cervical lesions. Conservation of the E2 gene with concomitant expression of viral oncogenes in advanced cervical lesions may point to alternate mechanisms, other than integration, bringing about the enhanced expression of E6/E7 mRNA.ConclusionsThis study suggests that the detection of the HPV-16 oncogene transcripts could serve as an indicator for assessing the prognosis of patients on radiotherapy. The majority of HPV-16-positive cervical neoplastic lesions are transcriptionally active and express the oncogene transcripts. The increased occurrence of intact HPV-16 episomal E2 DNA in advanced lesions further substantiates the fact that the disruption of E2 ORF is not mandatory for increased oncogene expression. Thus, this study underscores the significance of investigating alternative mechanisms of oncogene expression in HPV-16.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS