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PubMed1997

Rapid detection of different RNA respiratory virus species by multiplex RT-PCR: application to clinical specimens.

BACKGROUND: The polymerase chain reaction (PCR) applied in diagnostic and epidemiologic investigations is very useful for sensitivity, specificity and time saving. OBJECTIVE: We have developed a method for the detection of genomic RNA of two different species of virus, the influenza A virus (IA) and the respiratory syncytial virus (RS), which are responsible for clinical similarities. We applied this multiplex RT-PCR protocol on clinical specimens. STUDY DESIGN: We describe a method which allows rapid diagnosis by performing a single retro-transcriptase (RT) reaction associated with the PCR (multiplex RT-PCR) on different genomes in a single sample. We have evaluated the sensitivity and the specificity of the multiplex test on positive controls, then, on RNA extracted from clinical specimens harvested from 15 children with respiratory symptoms during the spring-winter season 1997. RESULTS AND CONCLUSIONS: The multiplex RT-PCR protocol, applied to respiratory specimens, allows the investigation of RNA IA virus and RS virus in a single sample at the same time. The detection of the etiologic viral agent is rapid and it is possible to evaluate incidental simultaneous infections.

Animals↗
PubMed1986

Both subunits of rat liver ferritin are regulated at a translational level by iron induction.

A few hours after administering iron to rats, liver ferritin synthesis increases several fold. However, Northern blot analysis with cDNA probes for ferritin light (L) and heavy (H) subunit mRNAs failed to show an increase in total population of either messenger. Cytoplasmic distribution of ferritin messages was therefore investigated in control and iron administered rats killed at 3.5 hours. The liver post-mitochondrial supernatant was fractionated on a sucrose gradient to separate polyribosomes, monosomes, ribosomal subunits and cell sap. RNA extracted from each fraction and analyzed using Northern blotting showed that 65% of the total mRNA population for each subunit was present in the cell sap of control rats, presumably as mRNP particles since ribosomal RNA was absent from this fraction. After iron administration, these reserves of free mRNA were recruited onto the polysomes, reducing the free mRNA pool to 15% of the total. We interpret this to be due to activation of blocked ferritin messages on entry of iron into the cell.

Animals↗
PubMed1993

SnR31, snR32, and snR33: three novel, non-essential snRNAs from Saccharomyces cerevisiae.

Genes for three novel yeast snRNAs have been identified and tested for essentiality. Partial sequence information was developed for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database. The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively. Each RNA is derived from a single copy gene. The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein eIF-4E. The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two open reading frames with no known function. Genetic disruption analyses showed that none of the three snRNAs is required for growth. The new RNAs bring the number of non-spliceosomal snRNAs characterized thus far in S. cerevisiae to 14, of which only three are essential.

Base Sequence↗
PubMed1976

Translation of the mRNA for rabbit uteroglobin in cell-free systems. Evidence for a precursor protein.

Uteroglobin, an hormonally induced protein composed of two similar subunits, represents around 50% of the proteins synthesized and secreted into the uterine lumen of rabbits treated sequentially with estradiol and progesterone. The endometrium of these animals was used as a source for the isolation of the mRNA for uteroglobin. Poly(A)-rich RNA, extracted from purified polysomes with phenol chloroform and isolated on oligo(dT)-cellulose columns, contains one fourth of the total protein coding activity of the endometrium. Between 20--25% of the polypeptides synthesized by this RNA in cell-free systems derived from Krebs II ascites cells or wheat germs react with a monospecific antiserum prepared in guinea pigs against uteroglobin. The material bound to the antibody was identified as a precursor of uteroglobin according to the following criteria. 1. The product synthesized in vitro can be displaced from the complex with the specific immunoglobulin by purified uteroglobin. 2. Analysis of the immunoprecipitate on polyacrylamide gels containing urea and dodecylsulfate demonstrate the existence of a single labelled polypeptide with an apparent molecular weight larger than the uteroglobin subunits. 3. The tryptic digest of this polypeptide, labelled in vitro with [3H]lysine, shares seven peptides with mature uteroglobin labelled with the same amino acid in perfused uteri, and exhibits and additional peptide not present in uteroglobin. 4. Injection of the same mRNA preparation into Xenopus oocytes results in the production of uteroglobin. The endometrium of intact animals treated with estradiol alone also contains the same mRNA bound to polysomes but in a smaller proportion, indicating that the progesterone-induced synthesis of uteroglobin is accompanied by an accumulation of the specific mRNA in the polysomes.

Animals↗
PubMed2005

Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E.

The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction mixture (5 microl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 10(3) to 10(10) viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 x 10(9) per reaction mixture can be detected, which corresponds to 10(4) to 10(11) viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.

Child↗
PubMed1983

Identification of four complementary RNA species in Akabane virus-infected cells.

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 X 10(6), 1.5 X 10(6), and 0.48 X 10(6) for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind to oligodeoxythymidylate-cellulose. Kinetic analysis of cRNA synthesis in infected cells at various times postinfection suggested that cRNA synthesis could be detected as early as 2 h postinfection and that maximal synthesis occurred at 4 to 6 h postinfection. The RNAs synthesized in infected cells could be partially resolved by sucrose density gradient centrifugation. The RNA fraction that cosedimented with the S segment of viral genomic RNA yielded two duplex RNA species when hybridized with viral genomic RNA, suggesting the presence of two small cRNA species. Specific hybridization with individual viral genomic RNAs confirmed that two species of cRNA are coded by the S RNA segment. Analysis of cRNA synthesis in the presence of the protein synthesis inhibitors cycloheximide and puromycin indicated that cycloheximide completely inhibited virus-specific RNA synthesis early and late in infection, whereas a very low level of synthesis occurred in the presence of puromycin. The inhibitory effects of these drugs were found to be reversible when the drugs were washed from the cells. It is concluded that continued protein synthesis is required for cRNA synthesis to proceed in Akabane virus-infected cells.

Animals↗
PubMed1996

Localization and regulation of corticotropin receptor expression in the midgestation human fetal adrenal cortex: implications for in utero homeostasis.

Developmental changes in the responsiveness of the fetal adrenals to corticotropin (ACTH) play an important role in the regulation of the fetal hypothalamic-pituitary-adrenal axis. Responsiveness of adrenal cortical cells to ACTH is dependent on the extent of ACTH receptor expression. Therefore, we examined the localization and regulation of ACTH receptor expression in the midgestation (16-24 weeks) human fetal adrenal cortex. In situ hybridization analysis was used to localize messenger RNA (mRNA) encoding the ACTH receptor in sections of human fetal adrenal glands. Messenger RNA encoding the ACTH receptor was localized in cells from all cortical zones; abundance was higher in definitive zone than in fetal zone cells and was least abundant in the more central portions of the cortex. Regulation of ACTH receptor expression was studied using Northern blot analysis of total RNA extracted from primary cultures of fetal and definitive zone cells. Two major (1.5 and 3.5 kilobases) and, upon stimulation with ACTH, 3 minor (4.0, 6.0 and 10.0 kb) ACTH receptor mRNA transcripts were detected in RNA from fetal and definitive zone cells. In both cell types, ACTH-(1-24) increased the abundance of mRNA encoding the ACTH receptor 10- to 20-fold compared with untreated cells. The effects of ACTH-(1-24) on ACTH receptor expression in fetal zone cells were time- and dose-dependent. The ED50 for the stimulation of ACTH receptor expression by ACTH-(1-24) was 1-10 pM, and maximal response to 0.1 nm ACTH-(1-24) was detected after 12-16 h. Eight-bromoadenosine cAMP and forskolin also stimulated ACTH receptor expression in fetal zone cells and closely mimicked the effects of ACTH-(1-24). In contrast, stimulation of protein kinase C with 12-O-tetradecanoyl phorbol 13-acetate had no effect on ACTH receptor expression. Changes in ACTH receptor expression in response to ACTH-(1-24), cAMP and forskolin were paralleled by changes in expression of the P450 cholesterol side chain cleavage (P450scc) enzyme. These data demonstrate that expression of the ACTH receptor by the human fetal adrenal cortex is up-regulated by its own ligand and that this effect is mediated by a cAMP-dependent mechanism. In addition, the coordinate stimulation of ACTH receptor and P450scc expression by ACTH indicates that the gene for the ACTH receptor is one of a specific cohort of genes regulated by ACTH that are required to facilitate fetal adrenal cortical response to ACTH. ACTH regulation of its own receptor may represent a mechanism by which fetal adrenal responsiveness to ACTH is maintained and possibly enhanced during fetal development.

Adrenal Cortex↗
PubMed1975

Adoptive transfer of cell-mediated immunity to tuberculin using RNA from tuberculin-sensitive subjects.

Adoptive transfer of delayed hypersensitivity skin test response to tuberculin with 'immune' RNA extracted from the sensitized lymphocytes of a healthy subject or a patient with Hodgkin's disease was successfully demonstrated in previously non-sensitive patients with neoplastic diseases including Hodgkin's disease. 'Non-immune' RNA obtained from non-sensitive man, on the other hand, failed to transfer PPD skin reactivity in non-sensitive recipients. 'Immune' RNA-mediated PPD skin test response remained positive for a considerable period of time, indicating that the effect of 'immune' RNA is systemically active. 'Immune' RNA was found to be RNase-sensitive but DNase-resistant. In vitro adoptive transfer of delayed hypersensitivity with 'immune' RNA, as assayed by lymphocyte transformation test, was unsuccessful.

Deoxyribonucleases↗
PubMed1993

Soluble factors modulate changes in collagen gene expression in abdominal aortic aneurysms.

BACKGROUND: Although increased procollagen gene expression and synthesis have been implicated in the progression of abdominal aortic aneurysms (AAA), factors modulating this change have not been identified. Furthermore, it is not known whether the increase in AAA procollagen expression is specific to this disease or also occurs in tissue affected by atherosclerotic occlusive disease (AOD). If paracrine rather than autocrine factors are responsible for increased gene expression in AAA, this effect should be transferable to target smooth muscle cells through conditioned media. Our objectives were to determine 1 alpha (I) procollagen messenger RNA levels in AOD tissue compared with normal and AAA and to determine whether differences noted in tissue procollagen gene expression could be transferred through conditioned media from normal, AOD, and AAA tissues to target smooth muscle cells in primary culture. METHODS: Normal, AOD, and AAA tissue was used for tissue RNA extraction or was minced and washed with serum-free media (4 degrees C) x 30 minutes and the media applied to human aortic smooth muscle cells (SMC) in primary culture for 36 hours. Total RNA from tissue and SMC exposed to conditioned media was analyzed by Northern and dot blot analysis for 1 alpha (I) procollagen. RESULTS: Relative tissue 1 alpha (I) procollagen levels were not increased in AOD (0.23 +/- 0.05) as compared with normal (0.17 +/- 0.03); both were decreased compared with AAA (0.53 +/- 0.07; p < 0.01). The 1 alpha (I) procollagen levels in SMC exposed to conditioned media from AAA (1.73 +/- 0.15) were increased (p < 0.05) compared with AOD (1.10 +/- 0.12) and normal (1.16 +/- 0.16). CONCLUSIONS: There is no increase in tissue AOD procollagen gene expression. The ability to transfer the same relative patterns of gene expression from tissue to target SMC with conditioned media suggests that paracrine, rather than autocrine, factors modulate procollagen expression in AAA tissues.

Adult↗
PubMed1990

Polyadenylated RNAs as error sources in ribosomal RNA turnover analyses.

An approach to ribosomal RNA turnover studies in which cytoplasmic RNA was extracted and subsequently fractionated to isolate ribosomal RNA is reported. The presumption that the pool of 28S and 18S RNAs represented ribosomal RNA, exclusively, proved false and led to erroneous results of ribosomal RNA turnover. Polyadenylated RNAs exhibited a heterogeneous size distribution and, although constituting only 3% (w/w) of the cytoplasmic RNA extract, accounted for fully 10% of radioactivity of the presumptive ribosomal RNA pool. Profiles from the radioactivity data suggested that the discrepant results were due to these polyadenylated RNAs. An additional analytical procedure, an oligo (dT) cellulose column chromatography of the RNA extract prior to the sucrose density gradient fractionation step, performed as described in this paper, proved an effective remedy for this error.

Animals↗
PubMed1992

Estrogen suppresses hepatitis B virus expression in male athymic mice transplanted with HBV transfected Hep G-2 cells.

Hormones are known to regulate both viral and cellular genes. It has been shown previously that estrogen has an effect on liver gene transcription and mRNA stability. Sex hormones might have a role in the chronic persistence of hepatitis B virus (HBV) infection. In fact, there is a male preponderance in the incidence of chronic HBV infection, and HBsAg expression was reported to be much higher in male transgenic mice than in the females. We investigated the effect of estrogen on HBV gene expression and regulation in athymic mice bearing 2.2.15 cells, a human hepatoblastoma cell line derived from Hep G-2 transfected with HBV sequences. Both male and female mice were treated with estradiol after tumors could be observed. Episomal DNA was extracted from the tumors and hybridized with 32P-labelled HBV DNA. Southern blot and slot blot analyses demonstrated that male mice had higher expression of HBV DNA. Estrogen treatment suppressed HBV DNA expression in males, but had only a minor effect on females. HBeAg production in male mice was also inhibited by estrogen treatment. HBV RNA extracted from 2.2.15 cells showed 2-3-fold reduction following beta-estradiol treatment. Moreover, inhibition of HBV transcription by estrogen was demonstrated by an RNA pulse-labelling experiment. These data indicate that estrogen inhibits HBV expression in the in vivo model presented in this study. These results might contribute to a better understanding of the effect of sex hormones on the pathogenesis of HBV-induced liver disease.

Animals↗
PubMed2025

Assessment of genomic prediction capabilities of transcriptome data in a barley multi-parent RIL population.

Low-cost and high-throughput RNA sequencing data for barley RILs achieved GP performance comparable to or better than traditional SNP array datasets when combined with parental whole-genome sequencing SNP data. The field of genomic selection (GS) is advancing rapidly on many fronts including the utilization of multi-omics datasets with the goal of increasing prediction ability and becoming an integral part of an increasing number of breeding programs ensuring future food security. In this study, we used RNA sequencing (RNA-Seq) data to perform genomic prediction (GP) on three related barley RIL populations. We investigated the potential of increasing prediction ability by combining genomic and transcriptomic datasets, adding whole-genome sequencing (WGS) SNP data, functional annotation-based filtering, and empirical quality filtering. Our RNA-Seq data were generated cost-efficiently using small-footprint plant cultivation, high-throughput RNA extraction, and Library preparation miniaturization. We also examined sequencing depth reduction as an additional cost-saving measure. We used fivefold cross-validation to evaluate the prediction ability of the gene expression dataset, the RNA-Seq SNP dataset, and the consensus SNP dataset between the RNA-Seq and parental WGS data, resulting in prediction abilities between 0.73 and 0.78. The consensus SNP dataset performed best, with five out of eight traits performing significantly better compared to a 50K SNP array, which served as a benchmark. The advantage of the consensus SNP dataset was most prominent in the inter-population predictions, in which the training and validation sets originated from different RIL sub-populations. We were therefore able to not only show that RNA-Seq data alone are able to predict various complex traits in barley using RILs, but also that the performance can be further increased with WGS data for which the public availability will steadily increase.

Hordeum↗
PubMed1983

Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169.

We used cloned subgenomic DNA fragments of human cytomegalovirus strain AD169 as hybridization reagents to analyze the sites of transcription and abundance of viral RNA in permissively infected human embryonic lung cells. RNA extracted from immediate early, early, middle, and late times in the infection was attached to filters and hybridized with excess cloned subgenomic fragments. Each hybridization was performed with an internal standard to allow quantitation of the RNA concentration and standardization for the variation in probe complexity and specific activity. We found immediate early transcription in the AD169 strain occurring primarily from three regions in the long unique segment at 0.061-0.110, 0.117-0.142, and 0.588-0.616 map units. A low level of transcription was also detected from the long unique segment at 0.230-0.372, 0.419-0.437 and 0.703-0.707 map units and the short unique segment and terminal repeats at 0.00-0.046, 0.776-0.854, and 0.892-1.00 map units. At 8 hr after infection in the presence of de novo protein synthesis, the transcription pattern was changed. Two of the major immediate early sites at 0.061-0.110 and 0.117-0.142 map units were represented less abundantly in the absence of cycloheximide while the site at 0.588-0.616 map units was represented at the same level. The most abundant early RNA was synthesized from the terminal repeat sequences and part of the short unique segment at 0.00-0.046, 0.776-0.822, and 0.892-1.00 map units. Steady-state RNA from the midpoint of the infection hybridized with most regions of the genome. Abundantly transcribed regions included the abundant early sites mentioned above (0.00-0.046, 0.588-0.616, 0.776-0.822, and 0.892-1.00 map units), several new sites in the long unique segment at 0.104-0.110, 0.576-0.588, 0.653-0.664, 0.703-0.707, and 0.766-0.776 map units and the L-S junction fragment at 0.804-0.854 map unit. RNA from late in the infection hybridized to all subgenomic fragments. The sites of de novo RNA synthesis along the genome were determined by hybridizing pulse-labeled RNA to individual cloned subgenomic fragments attached to filters. Transcription from the terminal repeat sequences and the long unique segment at 0.576-0.588 map unit accounted for 40% of the total viral RNA synthesized from 22 to 28 hr after infection. Of the total de novo RNA synthesis from 71 to 77 hr after infection, 4.3% was virus specific and 47% of the total de novo synthesized viral RNA hybridized to the terminal repeat sequences.

Cell Line↗
PubMed2004

Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus.

Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak.

Animals↗
PubMed1994

Expression of preproenkephalin A gene and presence of Met-enkephalin in dorsal root ganglia of the adult rat.

The expression of the preproenkephalin A gene was investigated in adult rat dorsal root ganglia (DRG). A radioimmunoassayable Met-enkephalin (ME)-like material was detected in 0.1 M HCl extracts of rat DRG, representing approximately 60 pg of ME equivalents/mg of protein. Chromatographic analyses indicated that the major component of the ME-like material coeluted with authentic ME. In northern blot experiments on total RNA extracted from DRG, a cDNA probe corresponding to the entire coding region of rat preproenkephalin A mRNA yielded a single band of the expected size for this mRNA, i.e., 1.5 kb. Polymerase chain reaction (PCR) experiments were carried out with DRG, striatum, and liver cDNAs using two primers flanking the 1,371-1,771 base region of the preproenkephalin A gene. Thirty PCR cycles performed on both striatum and DRG cDNAs generated a single band of 400 bp, as expected, whereas only trace amounts of this product were detectable using liver cDNAs. Nucleotide sequencing of the PCR product obtained with DRG cDNAs revealed a 100% homology with the 1,371-1,771 sequence of the preproenkephalin A gene. In situ hybridization with a cRNA probe showed that about 3.5% of DRG cells expressed the preproenkephalin A transcript. However, most of these cells probably did not process proenkephalin to enkephalins, as thorough immunohistochemical investigations with anti-ME antibodies allowed the detection of only one in approximately 6,000 cells (in 30 sections of DRG) that exhibited ME-like immunoreactivity. Cells expressing preproenkephalin A mRNA were intermediate-sized neurons, suggesting that primary afferent ME-containing fibers belong to the A category and may participate in a local (spinal) inhibitory control of nociception.

Amino Acid Sequence↗
PubMed1983

Cloning of cDNA for pyruvate, Pi dikinase from maize leaves.

To obtain molecular probes for studies of gene regulation in photosynthetic tissues of maize, we have cloned DNA complementary to poly(A)+RNA extracted from green leaves by insertion into plasmid pBR322 and transformation of E. coli, strain RR1. Colonies were screened by sequential hybridization with 32P-labeled single stranded cDNA synthesized from pooled aliquots of poly(A)+RNA fractionated by sucrose density centrifugation. Among the clones bearing cDNA homologous to high molecular weight poly(A)+RNA, we identified one with an insert of 440 base pairs homologous to mRNA for pyruvate, Pi dikinase, a C-4 carbon cycle protein localized in mesophyll cells of the leaf. Our work indicates that the dikinase subunits are synthesized in the cytoplasm as precursors approximately 13,000 daltons larger than the mature peptide subunits. Leaves of seedlings illuminated during growth have higher levels of pyruvate, Pi dikinase mRNA than leaves of dark-grown plants.

Base Sequence↗
PubMed1996

Studies on the response of carrot cells to a Sclerotinia sclerotiorum elicitor: induction of the expression of an extracellular glycoprotein mRNA.

A heat-released elicitor (HRE) obtained from Sclerotinia sclerotiorum mycelium induced a transient increase in the activity of phenylalanine-ammonia lyase (PAL) in a carrot cell culture. Differential display reactions carried out on total RNA extracted from cells exposed for 7 h to the HRE revealed a complex cell response involving the induction and repression of several mRNAs. One of these elicitor-induced mRNAs encodes a 42.6-kDa protein. Northern (RNA) blot analysis and reverse-transcription polymerase chain reaction studies showed that noninduced cells have a basal expression of the 42.6-kDa protein mRNA that was stimulated five- to 10-fold by treatment with the elicitor.

Amino Acid Sequence↗
PubMed2005

Human papillomavirus (HPV) transcripts in malignant inverted papilloma are from integrated HPV DNA.

OBJECTIVES: The objectives of the study were to detect human papillomavirus (HPV) sequences in nasal inverted papilloma (IP) lesions and to determine whether HPV is involved in the progression of IP to sinonasal squamous cell carcinoma (SCC). STUDY DESIGN: A retrospective study was performed on 14 patients diagnosed with IP within the last 12 years. Three of these 14 patients developed SCC. METHODS: Eighteen formalin-fixed, paraffin-embedded tissue blocks were obtained for these 14 patients. After DNA extraction, polymerase chain reaction (PCR) was performed, followed by hybridization using HPV 6, 11, 16, 18, 31, 33, 35, 45, and 52 specific DNA probes, in an attempt to identify HPV type in each specimen. After RNA extraction, the integration status of the HPV genome was evaluated based on the relative abundance of E7 and E5 viral transcripts, assessed by quantitative real-time PCR. RESULTS: HPV sequences were detected in samples from 3 of the 14 patients with IP. Of the three patients with SCC, HPV sequences were detected in two patients, whereas one patient was negative for the oligoprobes tested. Of the 11 patients diagnosed only with IP, 1 patient was positive for HPV DNA (HPV type 11). This difference in HPV positivity between IP and SCC was not statistically significant (P = .09, Fisher's Exact test, two tailed). Viral transcripts were detected in both patients with SSC who were HPV positive. Because HPV early transcripts are polycistronic, loss of 3' transcript sequences (E5) and retention of 5' sequences (E7) indicates integration. One of the SSC containing HPV 18 sequences showed a E7/E5 ratio of 776:1. The other SSC showed E7 transcripts and an absence of E5 transcripts CONCLUSION: HPV transcripts were present in SCC positive for HPV, and the relative level of E7 to E5 transcripts indicates integration of the viral genome. These findings are suggestive of HPV having an active role in the lesion. More extensive studies are needed to determine the exact role of HPV in IP and progression to SCC.

Carcinoma, Squamous Cell↗