Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells.

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.

Loss of critical palindromic transgene promoter sequence in chemically induced Tg.AC mouse skin papillomas expressing transgene-derived mRNA.

The Tg.AC transgenic mouse carries a v-Ha-ras transgene. Skin papillomas develop in Tg.AC mice upon repeated dermal application of tumor promoters and carcinogens. The transgene is inserted at a single site on chromosome 11 in a multiple-copy array. Although most of the >or= 40 copies are arranged in a direct-repeat orientation, two copies of the transgene are inserted in a palindromic, inverted-repeat orientation. Deletion of the palindromic transgene promoter sequence is associated strongly with and diagnostic of loss of phenotypic responsiveness to Tg.AC papillomagens, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, a loss of palindromic transgene sequence, in the absence of an observable reduction in copy number of the direct-repeat-oriented transgene sequence, is seen in DNA from papillomas when compared to genomic DNA from tail clips or skin samples away from the application site. Transgene-derived transcripts were detectable in all Tg.AC papillomas sampled. The transgene locus was hypomethylated in papillomas but not in samples from tail clips from the same animal or from skin samples away from the application site in responder Tg.AC mice, as shown by loss of resistance to digestion by HpaII. A cell line derived from a Tg.AC squamous cell carcinoma showed complete loss of the palindromic transgene sequence, hypomethylation of the transgene locus, and strong expression of v-Ha-ras mRNA. These data indicate that the palindromic transgene sequence, which appears to be necessary for initial responsiveness to tumorigens, may be susceptible to deletion during rapid cellular proliferation and is not required for transgene expression in later phases of papilloma growth.

Loss of palindromic symmetry in Tg.AC mice with a nonresponder phenotype.

The Tg.AC transgenic mouse carries the v-Ha-ras oncogene under the control of the zeta-globin promoter and is currently being used in a short-term carcinogenesis assay for safety testing of pharmaceuticals. A subset of hemizygous Tg.AC mice was found to be nonresponsive to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, which characteristically induces skin papillomas in these mice with repeated dermal applications. We previously showed that responder and nonresponder hemizygous Tg.AC mice carry about 40 copies of transgene but that the nonresponders had lost a 2-kb BamHI fragment containing the zeta-globin promoter sequence. The present restriction enzyme and S1 nuclease digestion experiments strongly suggested that the 2-kb BamHI fragment resulted from the orientation of two transgenes in an inverted repeat formation. Two subsets of nonresponder Tg.AC mice were identified. Restriction enzyme and S1 nuclease digestion experiments suggested that one nonresponder genotype was produced by a large deletion of one or more near complete copies of transgene sequence and the other genotype was produced by a small deletion near the apex of the "head-to-head" juncture of the inverted repeat. Polymerase chain reaction amplification, cloning, and sequencing results confirmed the palindromic orientation of transgene in Tg.AC mice. Our results indicated that, despite the presence of multiple copies of transgene in a direct repeat orientation, loss of symmetry in the palindromic array of transgene sequence results in the loss of the responder phenotype in Tg.AC mice. Mol. Carcinog. 30:99-110, 2001. Published 2001 Wiley-Liss, Inc.

Evaluation of in vitro reporter gene induction assays for use in a rapid prescreen for compound selection to test specificity in the Tg.AC mouse short-term carcinogenicity assay.

Under ICH guidelines, short-term carcinogenicity assays such as the Tg.AC assay are allowed alternatives for one species in the 2-year rodent bioassay. The Tg.AC transgenic mouse, which carries the v-Ha-ras oncogene under control of the zeta-globin promoter, develops skin papillomas in response to dermal application of carcinogens and tumor promoters. The appropriate specificity of the Tg.AC model for testing pharmaceuticals has not been systematically evaluated. The selection of candidate test compounds among noncarcinogenic pharmaceuticals would be aided by a high-throughput in vitro prescreen correlative of activity in the in vivo Tg.AC assay. Here we describe the development of a prescreen based on correct response to 24 compounds tested previously in Tg.AC mice. The in vitro prescreens, chosen to reflect molecular pathways possibly involved in Tg.AC papilloma formation, consisted of a zeta-globin promoter-luciferase construct stably expressed in K562 cells (Zeta-Luc) and three of the stress-response element-chloramphenicol acetyltransferase (CAT) fusion constructs stably expressed in HepG2 cells that are part of the CAT-Tox (L)iver assay. The stress response elements chosen were the c-fos promoter, the gadd153 promoter, and p53 response element repeats. Of the four assays, the gadd153-CAT assay showed the strongest concordance with activity in the Tg.AC assay, correctly classifying 78% of Tg.AC positive and 83% of Tg.AC negative compounds. The correlation was further improved by adding the Zeta-Luc assay as a second-stage screen. These cell-based assays will be used in a novel approach to selecting candidate compounds that challenge the specificity of the Tg.AC assay toward pharmaceuticals.

An evaluation of the hemizygous transgenic Tg.AC mouse for carcinogenicity testing of pharmaceuticals. I. Evidence for a confounding nonresponder phenotype.

We have completed 2 26-wk studies to evaluate the hemizygous transgenic Tg.AC mouse, which has been proposed as an alternative short term model for testing carcinogenicity. We attempted to evaluate the response to the known rodent carcinogens cyclophosphamide, phenolphthalein, and tamoxifen and to the noncarcinogen chlorpheniramine following topical application. In the first study, a weak response (2/17 animals) was observed to the positive control 12-O-tetradecanoylphorbol 13-acetate (TPA in ethanol, 1.25 micrograms), and no response was observed to cyclophosphamide, phenolphthalein, or chlorpheniramine, despite evidence for skin penetration. The second study compared 1.25 micrograms and 6.25 micrograms of TPA in ethanol and acetone solutions. Tamoxifen was also evaluated in both solvents and orally. No significant response was observed to tamoxifen by skin paint or oral routes. Over 60% of the high dose TPA-treated animals showed no (0 or 1) papilloma response, and 30% of the animals each developed more than 32 papillomas. The heterogenous response to high dose TPA may be related to variability in the responsiveness of hemizygous animals. In light of these findings, further Tg.AC studies should employ homozygous animals, and the underlying cause for heterogeneity in the tumorigenic response of Tg.AC mice should be identified and eliminated.

An evaluation of the hemizygous transgenic Tg.AC mouse for carcinogenicity testing of pharmaceuticals. II. A genotypic marker that predicts tumorigenic responsiveness.

The Tg.AC transgenic mouse skin paint assay is one of the short-term carcinogenesis models that has been proposed as a replacement for 1 species in the conventional 2-yr bioassay required for safety testing of pharmaceuticals. In our initial efforts to evaluate the sensitivity and specificity of this model for human pharmaceuticals, 61% of the hemizygous Tg.AC mice in the positive control groups were refractory to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Tg.AC mice are reported to carry < or = 10 copies of a transgene consisting of a zeta-globin promoter fused to the v-Ha-ras structural gene with a terminal simian virus 40 (SV40) polyadenylation signal. Southern blot hybridization of genomic DNA from 66 tail biopsies using a zeta-globin probe revealed that all of the hemizygous. Tg.AC mice screened contained approximately 40 copies of the transgene and that mice unresponsive to TPA had lost a 2-kb BamHI fragment containing zeta-globin promoter sequence. By systematic screening of Tg.AC breeder mice for this diagnostic marker of phenotypic responsiveness, it should be possible to selectively enrich the Tg.AC mouse colony to consist exclusively of responders and to guard against further genetic instability.

Observations of scanning electron microscopy detected abnormalities of non-lubricated latex condoms.

To describe surface abnormalities found by scanning electron microscopy of non-lubricated latex condoms, thirty samples of 15 condoms were photographed at low (26-94x), medium (240-940x) and high (> 1000x) magnification. Each sample was initially scanned at low magnification to determine the presence and location of surface abnormalities. Photographs taken at medium and high magnification were of the area most representative of the abnormality. Nine samples (30%) were completely normal at all magnifications. Of the total viewed at low power, 18 (60%) were normal, 11 (37%) revealed ridging and 1 (3%) revealed melting. Of the total viewed at medium power, 14 (47%) were normal, 3 (10%) showed cracking, 2 (7%) showed melting or a combination of ridging and cracking, and 11 (37%) showed ridging. At high power, 10 (34%) were normal, 11 (38%) revealed either melting or a combination of two defects, 5 (17%) showed ridging, and 3 (10%) revealed cracking. Fifteen (50%) of the samples revealed a surface abnormality (cracking or melting) other than ridging. The majority of condoms viewed under scanning electron microscopy revealed surface abnormalities. The implication of the detected abnormalities needs further evaluations. While ridging may represent a minor abnormality associated with condom handling, multiple abnormalities or melting may represent a more significant atypicality.

P2 purinergic receptors potentiate parathyroid hormone receptor-mediated increases in intracellular calcium and inositol trisphosphate in UMR-106 rat osteoblasts.

The PTH receptor has been cloned and shown to activate both adenylate cyclase and phospholipase C. Evidence exists that both signaling pathways are important for mediating the net physiological effects of this hormone on bone remodeling. We have shown previously that UMR-106 osteoblastic sarcoma cells express two calcium-signaling P2 purinergic receptors, a P2U and a unique P2T receptor. Neither receptor modulates PTH receptor-mediated activation of adenylate cyclase. We now report that stimulation of either P2 receptor will, however, potentiate the magnitude of the calcium signal observed after subsequent addition of human (h) PTH-(1-34) to fluo-3-loaded UMR-106 cells. Results from experiments with staurosporine and phorbol 12-myristate 13-acetate argue against a role for protein kinase C as a mediator of this potentiating effect of P2 receptor ligands. The P2 receptor-mediated intracellular calcium elevation itself cannot account for the potentiating mechanism, because addition of ionomycin will not replicate the effect of P2 receptor ligands on hPTH-(1-34) signaling. Addition of EGTA after exposure to P2 ligands does not prevent the potentiation of hPTH-(1-34), indicating that P2 ligands potentiate the release of intracellular calcium after PTH receptor stimulation. Inositol trisphosphate production is potentiated in response to hPTH-(1-34) after first priming [3H]inositol-labeled cells with a P2 agonist. We conclude that UMR-106 cells express PTH receptors that are capable of activating adenylate cyclase, but may be unable to activate phospholipase C until cells receive a signal as a consequence of P2 receptor activation. The nature of the signal is unclear, but appears not to be mediated by either calcium or protein kinase C.

Location and concentration of estrogen, progesterone, and androgen receptors in the bladder and urethra of the rabbit.

The objective of this study was to determine location and concentration of estrogen, androgen, and progesterone receptors in the bladder and urethra of the rabbit. Two urethral and two bladder specimens were obtained from four 12-week-old female New Zealand white rabbits. Rat monoclonal antibody (AN1-15) to human androgen receptor and (H222) to human estrogen receptor and mouse monoclonal antibody (PR6) to chicken progesterone receptor were used. Immunocytochemical staining was performed and specimens were evaluated for presence and location of steroid receptors. Androgen receptors were found in the highest concentrations in urethral and bladder epithelium. Low to low/moderate concentration were found in smooth muscle. Estrogen receptors were found in moderate to moderate/high concentrations in urethral epithelium and bladder and urethral smooth muscle. Progesterone receptors were not found in appreciable concentrations from any location, though the animals were not pretreated with estrogen. The rabbit model suggests a mechanism by which estrogen therapy can be effective in treating postmenopausal lower urinary tract symptoms. Progesterone receptors were not found in appreciable concentrations, suggesting progesterone therapy may not diminish the effectiveness of estrogen therapy by acting on urethral progesterone receptors. The effect of androgens on the lower urinary tract needs further investigation to determine if androgen therapy can alleviate lower urinary tract symptoms.

Separate P2T and P2U purinergic receptors with similar second messenger signaling pathways in UMR-106 osteoblasts.

UMR-106 rat osteogenic sarcoma cells express two calcium signaling P2 purinergic receptors. One is a P2U receptor with EC50's for adenosine triphosphate (ATP) and uridine triphosphate (UTP) of 2.6 and 2.4 microM, respectively. The other is a novel P2T receptor for adenosine diphosphate (ADP) (EC50 0.4 microM), adenosine 5'-O-(2-thiodiphosphate). (EC50 5 microM), 2-methylthio ATP (EC50 0.2 microM), and 2-methylthio ADP (EC50 0.04 microgram M). Responses to these ligands are desensitized by ADP but not by UTP. Responses to UTP and ATP are desensitized by UTP but not by ADP or adenosine 5'-O-(2-thiodiphosphate). 2-ChloroATP interacts with both receptors at high concentrations but with only the P2T receptor at low concentrations (EC50 0.05 microM). The weak platelet P2T receptor antagonist AMP blocks this P2T and not the P2U receptor. Addition of ATP after UTP desensitization of P2U receptors inhibits subsequent responsiveness to ADP but evidence for rapid conversion of ATP to ADP complicates interpretation of this apparent antagonism of P2T receptors by ATP. A subpassage of UMR-106.P135 cells lose P2U but retain P2T ligand responsiveness. Activation of either P2 receptor increases cellular IP3 concentrations in UMR-106 cells. Neither receptor can activate divalent cation entry as evidenced by their lack of effect on Mn++ quenching of fura-2 fluorescence. Neither receptor can modify parathyroid hormone receptor-mediated elevation of cellular cyclic AMP. This receptor for ADP demonstrates many of the same characteristics of the ADP receptor which we have previously reported as a P2T receptor expressed in K562 and Dami cells.

Cervical pregnancy: case reports and a current literature review.

The increasing number of reports of successful conservative treatment of cervical pregnancy, such as using the folinic acid antagonist methotrexate, constitutes a breakthrough in the management of this unusual but potentially life-threatening complication of pregnancy. We are reporting two cases of cervical pregnancy, both diagnosed in the first trimester of pregnancy. The first one was successfully treated by transvaginal ultrasound-guided administration of 2 mEq KCI intraamniotically in order to stop the fetal heart activity, followed by administration of 84 mg of methotrexate intraaminiotically (1 mg/kg); whereas in the second case, we encountered a technical failure of the above method. Both patients expressed desire to maintain their reproductive capability. These two cases gave us the opportunity to review the recent literature on cervical pregnancy.

Artificial urinary sphincter for recurrent/severe stress incontinence in women. Urogynecologic perspective.

Eight American Medical Systems 800 artificial urinary sphincters were implanted in seven women with severe and/or recurrent stress urinary incontinence. The surgical approach to urethral cuff implantation consisted of both abdominal and combined abdominal and vaginal approaches. Indications, preoperative evaluations, surgical technique and complications are discussed. Postoperative follow-up for 6-24 months showed all the patients to be completely continent subjectively and objectively. The artificial urinary sphincter is a viable option in these difficult cases of urinary incontinence.

Colchicine and post-inflammatory adhesions in a rabbit model: a dose-response study.

OBJECTIVE: To determine the dose-response relationship of colchicine in reducing inflammatory adhesive disease secondary to Neisseria gonorrhoeae in the rabbit. METHODS: Following intrauterine inoculation of a suspension of N gonorrhoeae, the rabbits were divided into five groups of 11 rabbits each. The control group received no medications. The remaining four groups received 0.1, 0.5, 1, and 2 mg, respectively, of colchicine intramuscularly daily for 14 days. The day after the last injection of colchicine, the peritoneal cavity was explored and assessed for the presence, number, and grade of adhesions. RESULTS: With increasing doses of colchicine, the incidence of adhesions decreased. A linear model (log [dose + 0.5]) showed, however, that as the dose of colchicine increased, the response tended to plateau (P < .05). CONCLUSION: In this model, colchicine was effective in preventing inflammatory adhesions in the rabbit. Increasing doses produced a greater effect in reducing adhesion formation. However, there was a plateau of the response at the 1-mg dose.

Vulvar carcinoma in pregnancy: a case report and literature review.

Carcinoma of the vulva has commonly been recognized as a disease of postmenopausal women, although some series have noted up to 15% of cases occur in women under the age of 40 years. The following is a report of a case of invasive vulvar carcinoma diagnosed and treated with radical surgery during pregnancy. Recommendations for treatment of this disease during pregnancy are presented and guidelines for subsequent route of delivery are proposed. A review of previously reported cases is also presented.

Endoscopic management of incidental cystotomy during operative laparoscopy.

Operative laparoscopy is rapidly becoming an important technique used by all surgical specialties. More sophisticated and difficult procedures are continually being performed endoscopically. Complications from these procedures are inevitable and are more frequently being managed with the laparoscope. We describe a case of inadvertent cystotomy during a laparoscopic gynecological operation, which was subsequently repaired endoscopically. The technique and patient followup are presented.

Modified suburethral sling procedure for treatment of recurrent or severe stress urinary incontinence.

The most suitable material used to perform suburethral sling procedures for recurrent or severe stress urinary incontinence remains controversial. A comparison was made between two commonly used materials, synthetic Gore-Tex (expanded reinforced polytetrafluoroethylene) and autologous fascia lata. Both groups showed improved urethral pressure profiles postoperatively, but there was no difference in the magnitude of change between groups. The objective cure rate at six months for the Gore-Tex group was 100.0 versus 87.5 percent for the fascia lata group (p = 0.155). While there was no statistical difference between the incidence of de novo detrusor instability or length of postoperative bladder drainage (p = 0.104 and p = 0.978, respectively), there was a trend toward more postoperative complications of urinary obstruction in the Gore-Tex group.

Prevalence of abnormal urodynamic test results in continent women with severe genitourinary prolapse.

Twenty-two clinically continent women with severe genitourinary prolapse were evaluated urodynamically to determine the prevalence of urodynamic abnormalities that could lead to potential urinary incontinence. Urodynamic testing found an occult incontinence disorder in 13 women (59%), of whom four had urine loss during cough pressure profiles after pessary placement, four had uninhibited detrusor contractions during retrograde medium-fill water cystometry, and five had both stress urinary incontinence and an unstable bladder. Therefore, nine of the 22 patients (41%) had uninhibited detrusor contractions during urodynamic testing. However, uroflowmetry did not reveal voiding dysfunction in this group, although peak flow rates appeared to be lower in the subgroup of women manifesting uninhibited detrusor contractions. Associated symptoms of frequency, nocturia, and urgency occurred in 41% of the women in this study; four of nine (44%) who had normal urodynamic test results, five of 13 (38%) who had abnormal test results, and five of nine (56%) who had an unstable bladder. Therefore, associated symptoms could not be used to determine which women would have abnormal urodynamic test results. These preliminary results suggest that women with genitourinary prolapse may be at risk for an occult incontinence disorder that is masked by the prolapse and that could manifest after corrective surgery for prolapse. Urodynamic testing is suggested for women with genitourinary prolapse who present with or without symptoms of incontinence, so that more data can be obtained to determine the importance of abnormal test results.

Urodynamic evaluation of voiding in women with cystocele.

To determine the effect of cystocele upon voiding, 30 women with various degrees of genitourinary prolapse were studied. The patients were divided into three groups depending on the severity of the cystocele and were evaluated with uroflowmetry, urethrocystoscopy, water cystometry, urethral pressure profilometry and voiding urethrocystometry. The three groups were similar in most parameters except maximum urethral closure pressure (P less than .05). The patients with cystocele did not demonstrate the abnormal voiding patterns characteristic of outflow obstruction.

Temporal separation of cough-induced urethral and bladder pressure spikes in women with urinary incontinence.

The time separation of cough-induced urethral and bladder pressure spikes were studied in 32 female patients: 16 with urinary incontinence due to sphincter incompetence, 9 with urinary incontinence and a competent urethral closure mechanism, and 7 after incontinence surgery. There were no significant differences in time separation between the three groups at different positions in the urethra. Age and menopausal status did not affect time separation of pressure spikes. It is concluded that time separation of pressure spikes during cough cannot be used as a discriminator of the etiology of urinary incontinence. Surgery does not restore the latency seen in normal continent women and may restore continence by a mechanism different from that of normal continent women.