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Biomedical subjects

E R Unger

Publications and source records attributed to E R Unger.

At least 19 recordsLinked to original sources
PubMed2007

Glucocorticoid receptor polymorphisms and haplotypes associated with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.

5' Flanking Region↗
PubMed2001

Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

Animals↗
PubMed2001

Human papillomaviruses: into the new millennium.

Human papillomaviruses (HPV) are newsworthy in this new millennium. Numerous articles have appeared in the lay press ranging in style and quality from informative essays to sensationalized exposes. Women, sensitized by confusing information, are asking obstetricians hard questions about HPV transmission and prevention, partner notification, the need for HPV testing, and methods of treatment. These questions are difficult because none of the answers are clear cut. This article provides the practicing gynecologist and obstetrician a concise and accurate summary of clinically important issues surrounding HPV. Current knowledge about HPV virology, epidemiology, testing, and the prospects for vaccination and other prevention measures is summarized.

Adult↗
PubMed2001

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays.

Computer Systems↗
PubMed2001

Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18.

BACKGROUND: Quantitation of human papillomavirus (HPV) DNA in clinical samples may yield important clinical information. METHODS AND RESULTS: We developed a 5' exonuclease fluorescent probe assay for HPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures. CONCLUSION: The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.

DNA-Binding Proteins↗
PubMed2001

Improved detection of viral RNA isolated from liquid-based cytology samples.

BACKGROUND: Molecular diagnosis requires the ability to obtain high-quality nucleic acids that are representative of the disease state. We evaluated the recovery and detection of limiting amounts of viral oncogenic RNA from cells fixed in liquid-based cytology media. METHODS AND RESULTS: Serial dilutions of a human papillomavirus (HPV)-positive cell line fixed in a liquid media was used as a model system. Total nucleic acid (TNA) extraction produced RNA with clearly visible ribosomal bands even after one year of storage. These TNA extracts, treated with DNase-I, were used in an RT-PCR assay for HPV-16 E6-E7 oncogenic transcripts. With chemiluminscent Southern blot detection, samples with one HPV-positive cell in 30,000 were consistently detected. CONCLUSION: PreservCyt-fixed cells can yield RNA suitable for molecular assays even after one year of storage.

Blotting, Southern↗
PubMed2001

Interphase cytogenetic analysis of 1q12 satellite III DNA in melanocytic lesions: increased aneuploidy with malignant histology.

To examine the relationship of chromosome 1 copy number to melanocytic tumorigenesis, interphase cytogenetic analysis of 1q12 satellite III DNA was performed on the spectrum of melanocytic lesions comprising Clark's tumor progression model. Results showed increased copy number in a "step off" pattern between melanoma in-situ and the intraepidermal component of invasive melanoma rather than a progression between each lesional group. These findings support Clark's concept of independent clonal expansion of a cell population giving rise to the vertical growth phase and further demonstrates increased chromosome 1 copy number as a late event in melanoma tumor progression.

Analysis of Variance↗
PubMed2000

Reproducibility of alternative probe synthesis approaches for gene expression profiling with arrays.

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 microg of purified poly(A)+ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 microg total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 microg total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.

DNA Probes↗
PubMed2000

Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture.

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (P = 0.001) and to detect an HPV type (P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.

Biotinylation↗
PubMed2000

In situ diagnosis of human papillomaviruses.

In situ hybridization (ISH) is the demonstration of specific genetic information within a morphologic context. For HPV, colorimetric ISH has the advantage that it can be applied to routine formalin-fixed paraffin embedded tissues. This conserves patient material and permits histologic selection of optimal material for testing. ISH allows for precise spatial localization of viral sequences within tissues. ISH also allows the integration status of HPV to be determined. The major limitations of the method are the potential for error in HPV typing because of probe cross-hybridization and relatively low sensitivity if the method is not fully optimized.

DNA, Viral↗
PubMed1999

An unusual cervical carcinoma showing exception to epitheliotropism of human papillomavirus.

Human papillomaviruses (HPV) infect epithelial tissues but have not been previously detected within mesenchymal cells. During a systematic investigation of FIGO stage Ib cervical cancers with colorimetric in situ hybridization, we detected HPV 16 DNA within the stromal compartment of an unusual undifferentiated carcinoma. The mesenchymal nature of the HPV-containing cells was confirmed by immunohistochemistry and electron microscopy. No viral particles were identified. Sequencing the majority of the HPV 16 genome identified few changes from the revised reference clone; all previously reported in other HPV 16 variants. These viral changes are unlikely to explain the exceptional mesenchymal localization of the HPV 16 DNA in this case.

Adult↗
PubMed1999

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type.

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.

Adolescent↗
PubMed1999

Association of human papillomavirus with HIV and CD4 cell count in women with high or low numbers of sex partners.

OBJECTIVE: To explore whether HIV serostatus (HIV-1, HIV-2, and dual (HIV-D) reactivity) and CD4 cell count affect human papillomavirus (HPV) in two groups of women from Côte d'Ivoire. METHODS: We conducted a cross sectional study of two groups of women. One group had low numbers of lifetime sex partners (maternal women, n = 258) and were enrolled based on HIV serostatus. The other group had high numbers of sex partners (female sex workers, n = 278) and all consenting self identified sex workers were admitted to this study. We collected epidemiological and clinical data, and cervicovaginal lavage for HPV testing. RESULTS: The groups had different distributions of HIV seroreactivity, but the rates of HPV DNA detection were similar. Most of the HPV DNAs detected in both groups were high risk types. A strong association of high risk HPV DNA and HIV-1 seropositivity was found in both maternal women (adjusted odds ratio (OR) 7.5 (95% CI 3.2-17.4)) and in sex workers (OR 5.0 (2.1-12.0)). The maternal group also showed an association of high risk HPV DNA detection with HIV-2 (OR 3.7 (1.6-8.5)) and HIV-D (OR 12.7 (4.3-37.5)) that was not observed in the sex workers. In addition, the association of high risk HPV DNA with HIV-1 in the maternal group was independent of low CD4 cell count, while in the sex workers the association depended on CD4 cell counts < or = 500 x 10(6)/l. CONCLUSIONS: We found that an association between HPV and HIV varied depending on the sexual behaviour and CD4 cell count of the population examined.

AIDS Serodiagnosis↗
PubMed1999

HIV and human papillomavirus as independent risk factors for cervical neoplasia in women with high or low numbers of sex partners.

OBJECTIVE: To explore whether HIV types 1 and 2 and CD4 cell count affect cervical neoplasia independent of human papillomavirus (HPV) in women with high or low numbers of sexual partners residing in Abidjan, Côte d'Ivoire. METHODS: The study population and methods are described in the companion paper. Additional methods include a Papanicolaou smear for cytological diagnosis and statistical analysis. RESULTS: In maternal women, both HIV-1 and high risk HPV were significant independent risk factors for squamous intraepithelial lesions (SIL) (adjusted odds ratio (OR) 11.0 (95% CI 1.1-112) and 5.4 (1.5-18.8), respectively). Only high levels of HPV DNA in the lavage were associated with SIL (OR 13.2 (3.6-47.8)) in the maternal group. In female sex workers, high risk HPV was significantly associated with SIL (OR 23.7 (4.4-126)); HIV seropositivity was not. Any positive level (high or low amounts) of HPV DNA was significantly associated with SIL in sex workers (ORs 15.9 (3.3-76) and 12.7 (3.6-44), respectively). There was no association of SIL with CD4 cell counts < or = 500 x 10(6)/l in HIV seropositive women from either group. CONCLUSION: HPV or HIV-1 infection independently affect cervical neoplasia in women with low numbers of sex partners.

Adolescent↗
PubMed1999

Chemiluminescent analysis of gene expression on high-density filter arrays.

We have optimized conditions for the chemiluminescent analysis of gene expression using high-density filter arrays (HDFAs). High sensitivity and specificity were achieved by optimizing cDNA probe synthesis, hybridization, and detection parameters. The chemiluminescent expression profile reflected expected differences in the transcripts isolated from different sources (placenta and keratinocytes). We estimated the detection limit for low-abundance message to be 1-15 transcripts per cell, a sensitivity rivaling that reported for microarray formats and exceeding that reported for autoradiographic HDFAs. The method allows for short exposure times and reuse of probe. It should be equally applicable to techniques such as differential screening of cDNA libraries and differential display PCR.

DNA, Complementary↗
PubMed1998

Human papillomavirus and disease status following therapy for cervical cancer.

We enrolled 85 patients with invasive cervical cancer and collected cervicovaginal lavage samples at each clinical visit for diagnosis, staging, treatment, and follow-up. Lavage samples were tested by L1 consensus polymerase chain reaction for human papillomavirus (HPV). Results were compared with HPV demonstrated in tumor tissue and the clinical status at time of sample collection. Sensitivity and specificity of the lavage for detection of tumor HPV, determined on the basis of results of tests on lavage samples collected prior to therapy, were found to be 56% and 76.9%, respectively. The proportion of lavage samples detecting tumor HPV decreased significantly with treatment, from 0.54 at diagnosis to 0.03 at complete response (P < .001). Local treatment failure was associated with increased detection of tumor HPV; however, no samples were positive prior to clinically detected treatment failure. These results suggest that cervicovaginal lavage is not an effective sampling method for epidemiological analysis of HPV in cervical tumors.

Adult↗