Evaluation of linear array human papillomavirus genotyping using automatic optical imaging software.

Variations in biological behavior suggest that each carcinogenic human papillomavirus (HPV) type should be considered individually in etiologic studies. HPV genotyping assays might have clinical applications if they are approved for use by the FDA. A widely used genotyping assay is the Roche Linear Array HPV genotyping test (LA). We used LA to genotype the HPV isolates from cervical specimens from women with the full spectrum of cervical disease: cervical cancer, cervical intraepithelial neoplasia (CIN), and HPV infections. To explore the feasibility and value of the automated reading of the LA results, we custom-designed novel, optical imaging software that provides optical density measurements of LA bands. We compared unmagnified visual examination with the automated measurements. The two measurements were highly associated. By either method, the threshold between a negative and a positive result was fairly sharp, with a clear bimodal distribution. Visually, most positive results were judged to be strong or medium, with fewer equivocal results categorized as weak (9.5% of positive samples), very weak (6.5% of positive samples), or extremely weak (7.7% of positive samples). The automated measurements of the intensities were significantly associated with the strength of the visual categories (P < 0.001). At the extremes of the automated signal intensities (< or = 20 units or > or = 120 units), the bands were almost always categorized visually as negative and positive, respectively. In the equivocal zone (20 to 119 units), specimens were more increasingly likely to be judged to be visually positive as the number of other, definite infections on the same strip increased (P for trend < 0.001). Multiple, concurrent infections comprise > or = 25% of HPV infections; thus, any systematic visual tendency that influences their evaluation when the result is equivocal should be minimized. Therefore, automated reading is probably worth development if easy-to-calibrate hardware and software can be optimized.

Targeting hepatocytes with liposomal interferon-alpha: effect on metallothionein gene induction.

Interferon-alpha (INF-alpha) is the only effective drug for the treatment of chronic hepatitis B. However it can produce severe side effects during treatment. Encapsulation of INF-alpha in liposomes may reduce the side effects and enhance its therapeutic activity. We evaluated the activity of free (nonencapsulated) and liposome-encapsulated INF-alpha on in vitro cultured Chang liver cells by measuring the metallothionein gene (MT-IIA). INF-alpha was encapsulated in different liposomal formulations, Dimyristoylphosphatidylcholine (DMPC), Dioleyl-phosphatidyl-ethanolamine/Dimyristoylphosphatidylcholine (DOPE/DMPC) and DOPE/Dimyristoylphosphatidylglycerol (DOPE/DMPG). Chang liver cells were incubated for 10 hours with 100 units/ml of free or one of the aforementioned liposomal INF-alpha formulations. We also evaluated the extended-time effects of DMPC liposomal formulations of INF-alpha and the non-encapsulated (free) INF-alpha on Chang liver cells after 12, 24 and 36 h of incubation. Total RNA was extracted and signals on Northern blots were densitometrically compared following hybridization with MT-IIA and beta actin probes. All INF-alpha formulations (free and liposomal) induced higher MT-IIA gene levels compared to non-treated control cells. Levels of MT-IIA mRNA expression were 80.9, 73.6, 43.9, and 35.3% over the control for the free, DOPE/DMPG, DMPC and DOPE/DMPC liposomal INF-alpha, respectively. The ratios of MT-IIA mRNA amounts expressed after the Chang liver cells were incubated with INF-alpha encapsulated in DMPC liposomes and the MT-IIA mRNA expressed after incubation with nonencapsulated INF-alpha are 0.7, 0.52 and 0.82 at 12, 24, and 36 hours, respectively. The results indicate that the MT-IIA mRNA level depends on the liposomal formulation of INF-alpha, and the sustained-time effect of the INF-alpha encapsulated in DMPC liposomes is parallel to that of nonencapsulated INF-alpha over a period of 36 hours.

HPV DNA testing of the residual sample of liquid-based Pap test: utility as a quality assurance monitor.

HPV DNA testing of the residual sample volume of liquid-based Pap tests has been recommended as a way to determine the appropriate follow-up for women who have equivocal results in routine clinical screening. A major aspect of quality assurance in the cytopathology laboratory consists of correlation of smear interpretation with biopsy or conization results as mandated by CLIA '88. However, the use of histology as the gold standard suffers from similar problems of subjectivity and sampling as the Pap smear. In this study we explore the potential use of HPV DNA testing of the residual volume from the ThinPrep Pap Test (Cytyc Corporation, Boxborough, Massachusetts) as a substitute gold standard in quality assurance monitoring of a cervical cytology screening program. The residual samples from 397 ThinPrep Pap cases were retrospectively analyzed for high-risk HPV DNA using the Hybrid Capture II technique. Sensitivity (71.8%), specificity (86.5%), predictive value of positive (77.1%) and negative (82.9%) ThinPrep Pap interpretations were calculated on the basis of HPV DNA results for 266 cases classed as either squamous intraepithelial lesion (SIL) or negative. Overall, there was agreement between the two tests in 80.8% of cases (Cohen's kappa =.59). The percentage of HPV DNA-positive cases interpreted as atypical squamous cells of uncertain significance (ASCUS) was 43.7%, and the percentage of negative cases was 17.1%. We believe that this approach is an objective adjunct to the traditional quality assurance protocol, with the added benefit that it includes cases interpreted as negative, as well as abnormal cases that do not come to biopsy.

Angiotensin-converting enzyme gene polymorphism in a cohort of coronary angiography patients.

An association between a polymorphism of the angiotensin-converting enzyme (ACE) gene and myocardial infarction (MI) in men has been previously reported. The present study examines the association between ACE genotype, atherosclerosis, MI, hypertension and other cardiovascular risk factors in Caucasian men (n=576) and women (n=124) who have undergone coronary angiography. Gene frequencies are also reported for African-American men (n=56). Genotype determination was based on the presence (allele I) or absence (allele D) of a 287 nucleotide Alu sequence in intron 16 of the ACE gene. Genotype frequencies for DD, ID and II were: 30.9, 47.7, 21.4% for Caucasian men; 28.2, 48.4, 23.4% for Caucasian women; and 30.4, 46.4, 23.2% for African-American men. There were no statistically significant associations between ACE genotype and number of plaques (> or =10% obstruction), lipid variables, or body mass index (BMI) for Caucasian men. Caucasian women with the DD genotype had on average fewer plaques, but this was accounted for by their younger ages. In Caucasian males, the DD genotype independently contributed to the presence of hypertension (odds ratio=1.8, 95% CI 1.1-2.9) after adjusting for age and BMI. In Caucasian males with total cholesterol levels less than 200 mg/dl (n=237), the DD (odds ratio=2.5, 95% CI 1.2-5.4) and ID genotypes (odds ratio=2.2, 95% CI 1.1-4.4) were associated with a history of MI.

Expression of p27kip1 and p53 in medulloblastoma: relationship with cell proliferation and survival.

p27kip1 and p21cip1 are cyclin-dependent kinase (cdk) inhibitors which along with p53 play critical roles in the control of cell cycle progression. Accumulation of p27kip1 in post-mitotic neurons is a major event of neurogenesis. We hypothesized that a dysregulation of the expression of p53 and these cdk inhibitors underlies cellular proliferation in medulloblastomas, and tested this hypothesis by investigating p27kip1, p21cip1, Bcl2 and p53 immunoreactivity in 14 medulloblastoma tumors. We noted an inverse relationship between p27kip1 expression and cellular proliferation (MIB1). Focal islands of neuroblastic or glial differentiation expressed high levels of p27kip1, while the undifferentiated, highly-proliferative population of tumor cells showed no detectable p27kip1 expression, thus suggesting a role for p27kip1 in cell cycle control in medulloblastoma. In addition, there was no detectable p21cip1 expression in any of the medulloblastomas studied. The low level of apoptosis displayed by these tumors was not associated with the expression of Bcl-2. A significant relationship was found between detection of p53 protein and poor survival. Since, p21cip1 and p27kip1 are often co-expressed with other INK4 family of cdk inhibitors during the induction of cellular differentiation and are synergistic in their effect, a deregulation of their coordinate expression may underlie the lack of complete differentiation in medulloblastoma.

Neuromuscular dysfunction in the jejunum and colon of human leukocyte antigen B27 transgenic rats.

HLA-B27 transgenic rats are a model of spontaneous gastrointestinal inflammation associated with expression of human leukocyte antigen (HLA) B27 and beta(2)-microglobulin. Our goal was to investigate in vitro enteric nerve regulation and contractile activity in isolated longitudinal muscles from the jejunum and colon of HLA-B27 rats. Nontransgenic age-matched Fisher 344 rats were used as controls. Intestinal inflammation and tissue injury, quantified histologically and through tissue myeloperoxidase activity, were evident in both the jejunum and colon of HLA-B27 rats. Although resting tension and spontaneous activity of the jejunal and colonic muscles from HLA-B27 rats did not differ significantly from controls, responses to both enteric nerve stimulation or direct muscle activation were significantly inhibited. In muscles from HLA-B27 rats, electrical field stimulation (0.5 ms, 0.5-20 Hz) induced low-amplitude contractions (maximal reduction 60-65%) compared with respective controls. In the presence of atropine and guanethidine, nonadrenergic and noncholinergic contractile responses to higher frequencies of stimulation (8-20 Hz) were also of lower amplitude. These changes were accompanied by a shift in neurally mediated contractions from predominantly cholinergic in the jejunum and colon of Fisher 344 rats to predominantly nonadrenergic and noncholinergic in HLA-B27 rats. Furthermore, maximal contractions to carbachol or KCl depolarization were reduced (up to 2.7-fold) compared with respective controls. In the jejunum of HLA-B27 rats the EC(50) level for carbachol was decreased. The data indicate that gastrointestinal inflammation induced by expression of HLA-B27 is associated with hypocontractility and inhibition of enteric cholinergic control of the longitudinal muscle in both the small and large intestine.

Evaluation of role of factor V Leiden mutation in fatal pulmonary thromboembolism.

Association of a mutation in the coagulation factor V gene (FV Leiden) with deep vein thrombosis and pulmonary thromboembolism has been well documented in the literature, but no study has specifically screened cases of fatal pulmonary thromboembolism for the mutation. This study sought to determine whether FV Leiden plays a role in the pathogenesis of fatal pulmonary thromboembolism. We isolated DNA from archived paraffin-embedded tissues derived from 46 necropsy cases in which pulmonary thromboembolism was listed as the cause of death (n = 27) or was secondarily associated with death (n = 19). FV Leiden genotypes were determined by using polymerase chain reaction and MnlI digestion of amplified products. The Leiden mutation occurred in the heterozygous state in one (2.1%) of the necropsy specimens. The prevalence of the mutation was higher (8.7%) in gender- and ethnic-matched blood donor controls. The FV Leiden mutation is not independently associated with fatal pulmonary thromboembolism in the group of patients analyzed. The results suggest different etiologies for nonfatal, chronic deep vein thrombosis/pulmonary thromboembolism and fatal, acute pulmonary thromboembolism.

Role of factor V Leiden mutation in patients with angiographically demonstrated coronary artery disease.

The study sought to determine whether coagulation factor V Leiden (FV Leiden) plays a role in the pathogenesis of coronary artery disease and/or myocardial infarction. Association of FV Leiden with venous thromboembolism is well established in the literature, but the role of the mutation in arterial thrombotic events is controversial. Some studies have documented an association between the mutation and myocardial infarction and stroke in juveniles. Few studies have explored its possible contribution to coronary atherosclerosis. We screened FV genotype in 850 predominantly white coronary angiography patients. Coronary artery disease risk factors and history of myocardial infarction were then analyzed by genotype. The FV Leiden mutation occurred in 54 (6.4%) patients. There was one homozygote; a 37-year-old, white male smoker with a history of myocardial infarction. Gene frequencies for white males and females were similar: 0.965 for the normal allele and 0.035 for FV Leiden. Gene frequencies for both genders were in Hardy-Weinberg equilibrium. FV Leiden was not a useful predictor (p=0.23) of the presence of clinically defined atherosclerosis (> or = 50% stenosis) in a logistic regression model adjusting for age, lipoprotein (a), total cholesterol, triglycerides, high density lipoprotein cholesterol, and fibrinogen. In addition, there was no difference in frequency of FV Leiden among those with and without medical histories of myocardial infarction (p=0.51). Allelic frequencies of FV Leiden in this patient group do not differ significantly from those reported for white populations. The FV Leiden mutation in its heterozygous state is not independently associated with coronary artery disease or myocardial infarction.

Effects of calcitonin gene-related peptide on somatostatin and gastrin gene expression in rat antrum.

The ability of exogenous calcitonin gene-related peptide (CGRP) to regulate gastric somatostatin and gastrin messenger RNA was studied in vitro in rat antral mucosal/submucosal tissues. Somatostatin and gastrin mRNA were quantified by Northern and dot blot hybridization and regulatory peptides were measured by radioimmunoassay. Incubation of antral tissues in the presence of CGRP [1 x 10(-7) M] for 60 min resulted in a reciprocal increase in somatostatin and a decrease in gastrin release: 214.7+/-28.5 vs. control of 81.7+/-5.9 pg somatostatin per gram of tissue and 2.2+/-0.3 vs. control of 5.5+/-0.7 ng gastrin per gram of tissue (P < 0.001). CGRP caused parallel changes in somatostatin and gastrin mRNA levels: somatostatin mRNA increased by 212% from 0.40+/-0.02 to 1.25+/-0.09 absorbance units (AU) (P < 0.001) and gastrin mRNA decreased by 73% from 0.55+/-0.08 to 0.15+/-0.02 AU (P < 0.001). Somatostatin monoclonal antibody prevented CGRP-mediated inhibition of both gastrin release and gastrin mRNA levels. In conclusion, CGRP is capable of modulating both the secretion and gene expression of regulatory peptides from antral G and D cells. Somatostatin immunoneutralization studies suggest that the actions of CGRP on gastrin release and gene expression are indirect and mediated through the paracrine influences of somatostatin.

Survey of sinonasal inverted papillomata for Epstein-Barr virus.

BACKGROUND: Several studies have indicated an etiologic role for viruses in the development of sinonasal inverted papillomata (IP). A recent report demonstrates a strong relationship (65%) between Epstein-Barr virus (EBV) and these lesions using polymerase chain reaction (PCR) analysis. METHODS: The present study analyzes a series of paraffin-embedded tissues, comprising 25 surgically resected IPs and four fungiform papillomata (FP) for the presence of EBV using a sensitive in situ hybridization (ISH) assay and PCR. RESULTS: None of the specimens examined showed evidence of EBV infection by ISH, and only two papillomata (one sinonasal IP and one FP) gave positive reactions for EBV using PCR. CONCLUSIONS: These data challenge the previous report and suggest that EBV is not a significant etiopathologic factor to be considered in the development of sinonasal IP.

Flow cytometric analysis of nuclear matrix proteins: method and potential applications.

The nuclear matrix (NM) is an important structural component of the nucleus that participates in the regulation of several diverse metabolic processes. Immunometric assays have shown that alterations in NM-associated functions and morphological characteristics may occur as a result of changes in NM composition. Recent evidence suggests that detection of quantitative or qualitative changes in nuclear matrix protein (NMP) composition may be useful in the diagnosis of cancer and as a reliable indicator of cell death. We have developed an in situ flow cytometric technique for the simultaneous detection of specific NMPs and DNA content in fixed, permeabilized cells. Illustrative results from two different applications of these methods involving two different cell lines (human melanoma and promyelocytic leukemia) are presented, including: 1) measurements of NM breakdown in necrotic and apoptotic cells after treatment with the cytotoxic agents camptothecin, etoposide, or hyperthermia; and 2) detection of changes in NMP content immediately after heat shock. We demonstrate that the technique is useful for the identification of cell-cycle specificity of NM breakdown and allows correlations to be made between the kinetics of DNA fragmentation and NMP solubilization. Furthermore, our studies indicate that flow cytometric detection of changes in NM composition may be useful for identifying different modes and temporal patterns of cell death. We discuss other potential applications of the technique and advantages over standard biochemical assays.

RT-PCR microlocalization of mRNAs for calbindin D28k and vitamin D receptor in the murine nephron.

The spatial relationship between vitamin D receptor (VDR) and calbindin D28k [calcium binding protein D28k (CaBP-D28k)] gene expression within the murine kidney was studied by localizing their mRNAs in discrete nephron structures using reverse transcription-polymerase chain reaction (RT-PCR). Primers for beta-actin mRNA were used as a control for the presence of tissue during RT-PCR for CaBP-D28k mRNA. mRNA for CaBP-D28k was found only in distal convoluted tubules (DCTs), connecting tubules (CNTs), and cortical collecting ducts (CCDs). In contrast, VDR mRNA was detected in glomeruli, S2 proximal convoluted tubules, cortical thick ascending limbs of Henle's loop, DCTs, CNTs, and initial CCDs. The presence of both VDR and CaBP-D28k mRNA in DCTs, CNTs, and CCDs is consistent with the hypothesis that cacitriol acts via the VDR to stimulate CaBP-D28k synthesis. Conversely, the presence of VDR mRNA in other parts of the nephron suggests that calcitriol has genomically mediated actions within the kidney in addition to stimulation of CaBP-D28k synthesis.

The influence of antiestrogens on pS2 and cathepsin D mRNA induction in MCF-7 breast cancer cells.

Tamoxifen, although widely used in the treatment of estrogen-dependent tumors, is a partial estrogen agonist producing undesirable effects in breast cancer patients. ICI 182,780 a steroidal antiestrogen displays pure antagonist activity which is due to its ability to prevent dimerization of the estrogen receptor (ER). Our previous studies have shown that 1,1-dichloro-cis-2,3-diaryl cyclopropane (Analog II), a diarylcyclopropyl compound is devoid of estrogenic activity, has a weak binding affinity for the estrogen receptor in the mouse uterine tissue and inhibits the growth of breast cancer cells in culture. These findings suggest that Analog II may not inhibit tumor cell growth at the cellular level by an ER-mediated mechanism of action. Since these three antiestrogens appear to have different mechanisms of antiestrogenic activity, the purpose of this study was to compare the influence of the three antiestrogens on estradiol-induced expression of pS2 and cathepsin D (cath-D). These genes are known to be primarily under the influence of estrogen in ER positive MCF-7 human breast cancer cells. The results of this study demonstrate different mechanisms of regulation of the cath-D and pS2 genes by antiestrogens in MCF-7 cells. This study indicates that ICI 182,780 is a pure antagonist at the levels of gene regulation and cell proliferation. The relative order of inhibitory action was found to be ICI 182,780 > tamoxifen > Analog II.

Cloning, genetic mapping, and expression analysis of a mouse renal sodium-dependent phosphate cotransporter.

Renal tubular reabsorption of phosphate is critical to the maintenance of phosphate homeostasis in mammals, and the brush-border membrane Na-P(i) cotransport systems in proximal tubules play a major role in this process. We have isolated a cDNA encoding a mouse sodium-dependent phosphate transport protein (Npt1), which is expressed primarily in the kidney. This protein is highly similar to its human and rabbit homologues, based on nucleotide and amino acid comparisons. The presence of potential Asn-linked glycosylation and protein kinase C phosphorylation sites that are conserved among all three homologues suggests that these sites may be important in the function and regulation of this protein. The Npt1 gene was mapped to mouse chromosome 13, close to the Tcrg locus. By both in situ hybridization and reverse transcription-polymerase chain reaction, Npt1 mRNA was localized predominantly to the proximal tubule.

Immunohistochemical analysis of proliferation and differentiation in organotypic cultures of cervical tumor cell lines.

Researchers have previously demonstrated that organotypic cultures of cervical tumor cell lines exhibit morphological characteristics similar to the in vivo biopsies from which they were derived (Rader et al., 1990). Both the in vivo biopsy and organotypic culture appeared undifferentiated. We have extended these studies with immunohistochemical analysis using the proliferation and differentiation markers, proliferating cell nuclear antigen (PCNA) and involucrin, respectively, to evaluate in more detail the ability of cervical tumor cell lines to differentiate in organotypic culture. An HPV-immortalized keratinocyte cell line, PE-4, expressed PCNA in the lower half and involucrin in the upper half of the organotypic culture which is consistent with the characteristics of a preneoplastic lesion in vivo. The CC-1 cell line, derived from an invasive squamous cell carcinoma, appeared undifferentiated, but expressed involucrin in the upper half of the organotypic culture. This is the first observation of expression of a differentiation marker in an organotypic culture of a cervical tumor cell line. The other cervical tumor cell lines, SiHa and HeLa, derived from a squamous cell carcinoma, and an adenocarcinoma of the cervix, respectively, did not express detectable levels of involucrin or mucin. All three cervical tumor cell lines, CC-1, SiHa and HeLa, expressed PCNA throughout their entire thickness. The majority of nuclei in SiHa and HeLa cultures were PCNA-positive, while the CC-1 cell line exhibited a lower growth fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D receptor gene expression in mammalian kidney.

The vitamin D-receptor protein and its mRNA were localized in microscope sections of paraffin-embedded mammalian kidneys by means of immunocytochemistry and in situ hybridization, respectively. A monoclonal antibody against chicken intestinal vitamin D receptor immunostained the nucleus and cytoplasm of cells within the distal convoluted tubule, connecting segment, and initial cortical collecting duct of both rats and pigs. Although fainter, immunostaining also was present over proximal tubular cells. (35S)UTP-labeled cRNA probes were detected over both the proximal and distal portions of the mouse nephron, but silver grain densities were 5.8-fold greater over the latter. In conclusion, localization of both the vitamin D-receptor protein and its mRNA in both the proximal and distal nephron of adult mammals suggests that the gene for this protein is expressed in cells at both of these sites. The intensity of immunostaining and the density of cRNA-associated silver grains suggest that vitamin D-receptor gene expression is greatest in the distal nephron.

Calbindin-D28k gene expression in the developing mouse kidney.

Calbindin-D28k appears in the metanephric kidney during embryogenesis. We studied the temporal appearance and spatial distribution of calbindin-D28k mRNA in the developing kidneys of 12-day fetal through 21-day postnatal mice by in situ hybridization. 35S-UTP-labeled antisense (cRNA) probe to calbindin-D28k mRNA hybridized to the ureteric buds of 12-day embryos, whereas adjacent metanephrogenic tissue was unlabeled. By embryonic day 13, Y-shaped bodies of "advancing" ureteric buds were labeled intensely. In 16-day embryos, ampullae of ureteric buds were located immediately beneath the renal capsule and labeled strongly, in contrast to metanephric tubules and S-shaped bodies. The former were unlabeled and the latter were labeled only at points of contact with the ampullae. Subsequently, the ampullae of the metanephric ureteric buds hybridized with the cRNA probe, and from the 18th embryonic to the 21st postnatal day, this labeling was intense. The cRNA probe did not hybridize with the renal vesicles, proximal tubules, or tubular segments of Henle's loop derived from nephrogenic blastema, but it did label distal nephron segments. By the 21st postnatal day, collecting ducts and ureter no longer were labeled. In conclusion, calbindin-D28k mRNA is present in the developing mouse kidney, and its distribution during nephrogenesis is identical to that of calbindin-D28k per se. Collectively, these findings show that the calbindin-D28k gene is transcribed and its message is translated by the cells of the ureteric bud during the initial stage of renal morphogenesis.

Fourier transform infrared spectrometers: their recent history, current status, and commercial future.

The commercial FT-IR market has grown to a respectable $8,000,000 annual business area. It is of importance to both the users and suppliers alike that this market continues to solidify its gains and embark into new areas and applications. This paper reviews the evolution of commercial FT-IR in the context of the over-all infrared field. The purpose of the review is to discuss the progress to date and to relate the growth of this field to the future. The future uses of the FT-IR are projected as well as the requirements for new instrumentation.