Simple method for selective amplification of cDNA from a defined promoter.

A simplified technique for the detection of transcripts from a defined promoter is described. After reverse transcription, a PCR target sequence is selectively added to the 3' end of cDNA strands by DNA polymerase extension directed by an oligonucleotide template. Those cDNA molecules that do not have ends within a few nucleotides of the promoter start site are not extended and thus are excluded from subsequent amplification. Even when amplified products are visualized by ethidium bromide staining of agarose gels, this method requires only 1% of the RNA usually needed for detection of mRNA by standard RNase protection utilizing radiolabeled probes. In contrast to direct detection of cDNA by PCR, this procedure restricts amplification to a narrow subset of transcripts even when other overlapping colinear transcripts are present. We call this detection procedure specific amplification of cDNA ends (SPACE).

Rapid epidemiologic characterization of cytomegalovirus strains from pediatric bone marrow transplant patients.

BACKGROUND: DNA amplification by the polymerase chain reaction (PCR) of human cytomegalovirus (CMV) nucleotide sequences recently has been reported for differentiation of CMV strains. DESIGN: Retrospective study. OBJECTIVE AND PATIENTS: Evaluate the strain patterns of 15 CMV-positive buffy coat specimens from five pediatric bone marrow transplant patients. SETTING: Pediatric bone marrow transplant unit. METHODS: We perform PCR using primers corresponding to two distinct regions of the CMV genome, the major immediate-early (MIE) region and the a-sequence region, with subsequent restriction enzyme analysis of the amplified products. RESULTS: Restriction enzyme analysis with Hae III and Hinf I of products amplified with nested PCR for the MIE region revealed distinguishable digestion patterns between patients but similar patterns for samples from each patient. All were distinct from the CMV Towne laboratory control strain. In contrast to these results, amplification of specimens with a-sequence primers, followed by restriction enzyme analysis, did not allow differentiation between all patients. CONCLUSION: Our results indicate that nested amplification directly from buffy coat specimens using primers for the CMV MIE gene allows rapid CMV strain characterization that is useful for laboratory quality control and epidemiological studies. Distinct CMV strains were found in each patient, suggesting horizontal transmission was not responsible for acquisition of CMV infection in these patients.

Restriction enzyme fragment length polymorphisms of amplified herpes simplex virus type-1 DNA provide epidemiologic information.

Human herpes simplex type 1 (HSV-1) DNA of isolates from patients in a large teaching hospital was amplified by the polymerase chain reaction (PCR). The PCR products targeted approximately 2100 nt regions of relatively low G + C content. Comparison of restriction enzyme digests of amplified DNA showed variation useful for strain differentiation. Twelve nonrelated HSV-1 were differentiated from one another. In contrast, specimens epidemiologically related in an outbreak were indistinguishable from each other. Restriction endonuclease analysis of amplified HSV-1 sequences appears to be useful for molecular epidemiology and laboratory quality control to detect possible contamination by PCR products.

Detection of human cytomegalovirus in peripheral blood leukocytes by the polymerase chain reaction and a nonradioactive probe.

This study evaluated the effectiveness of polymerase chain reaction (PCR) combined with a nonradioactive probe for the early detection of cytomegalovirus (CMV) in buffy-coat specimens of immunocompromised patients. Dot-blot hybridization with a digoxigenin-labeled probe was used to detect a 262-bp PCR amplified fragment of the major immediate-early gene of CMV DNA. The results were compared with tissue cultures isolation of CMV. The study included 172 buffy-coat specimens from 72 immunocompromised patients. All 28 buffy-coat specimens positive by culture were also positive by PCR. The remaining 144 specimens were negative by culture; however, 47 of these were positive by PCR. Consequently, PCR was in agreement with culture results in 72% of the samples. Of the 47 PCR-positive-culture-negative specimens, 23 were from patients who had positive buffy-coat cultures at other times during their treatment. Chart review showed that an additional 16 of the PCR-positive-culture-negative samples were from patients with clinical evidence of active CMV disease. The eight remaining specimens were from five patients without signs of active disease. Specimens from 11 healthy volunteers were negative by PCR. In this study PCR was shown to be more sensitive than culture because it allowed earlier detection of viremia and demonstrated CMV in buffy-coat specimens that were negative by culture.

Is use of hormone replacement therapy associated with increased detection of human papillomavirus and potential risk of HPV-related genital cancers?

Oral contraceptives (OC) are a risk factor for female genital cancers and in vivo studies have shown that progestins stimulate human papillomavirus (HPV) gene expression. A similar role for hormone replacement therapy (HRT) has received little evaluation. Cervical/vaginal specimens were obtained to detect HPV from postmenopausal women (n = 429) seeking annual gynaecologic care. HPV was detected in 14% of women and 4.4% had high-risk, oncogenic types. HPV prevalence was similar across current, past and never HRT users. After adjustment for HPV-related risk factors, current and past user status showed no increased viral detection compared with never users. HRT duration also did not elevate risk among current users. However, longer duration (adj. OR 1.5/year, 95% CI 1.0-2.3) and longer latency (adj. OR 1.2/year, 95% CI 0.9-1.7) among past users of oestrogen/progestin regimens were associated with greater risk. Overall use of HRTs was not associated with HPV detection or disease. However, past users of combination HRTs had significantly greater risk of HPV detection with longer HRT duration and latency, similar to OC-HPV findings. The recommendation that postmenopausal women continue HRTs long term may lead to an increased development of HPV-related diseases, of particular concern among those who discontinue HRTs and subsequent gynaecologic care for early cancer detection.

Human papillomavirus and risk of oral cancer.

Although human papillomavirus (HPV), a sexually transmitted virus, is established as a necessary cause for more than 95% of cervical carcinomas, the association with oral squamous cell carcinoma is less well delineated. The purpose of this study was to determine the frequency and types of HPV in squamous cells of a group of patients with newly diagnosed oral or pharyngeal cancer (n = 93) compared with an age- and gender-frequency-matched control group of patients with no history of oral cancer (n = 205). HPV was evaluated from a mouth rinse collection of cells in the oral cavity and tested by 32P-labeled HPV generic probes and DNA sequencing for HPV types. HPV was identified in 15% of the oral cancer cases but in fewer than 5% of the controls (P < .05). The risk of cancer associated with HPV infection was independent of tobacco and alcohol use (adjusted odds ratio [OR] = 3.70; 95% confidence interval [CI]: 1.47-9.32; P < .05). HPV types included similar and other types not identified previously in the genital tract. There was no statistically significant increased risk of cancer among former tobacco users (former vs. never users: adjusted OR = 0.67, 95% CI: 0.31-1.44, P < .05), but the risk was significantly increased for current users (current vs. never: adjusted OR = 2.63; 95% CI: 1.22-5.71; P < .05). Likewise, former alcohol users were not at increased risk of disease (former vs. never: adjusted OR = 1.78; 95% CI: 0.87-3.67), whereas current alcohol users were (current vs. never: adjusted OR = 2.57; 95% CI: 1.22-5.42; P < .05). HPV-related genital lesions (14.3% vs. 10.6%), oral-genital sexual behavior (42.4% vs. 45.2%), and number (11 or more) of sexual partners (23% v. 17%) were not significantly different between cases and controls. These data suggest that in addition to tobacco and alcohol, HPV plays a role in the development of oral cancer.

Human papillomavirus infection in papillomas and nondiseased respiratory sites of patients with recurrent respiratory papillomatosis using the polymerase chain reaction.

We examined human papillomavirus (HPV) infection in biopsy specimens and cellular scrapes that were taken from respiratory papillomas and six nondiseased sites from the respiratory tract of seven patients. Human papillomavirus was detected by polymerase chain reaction amplification, followed by DNA hybridization with probes for specific HPV types. All papillomas (100.0%, n = 5) were positive only for HPV type 6 or 11. In the nondiseased site specimens, 61.3% (19/31) of the specimens were positive, again only for HPV type 6 or 11. Among the nondiseased site specimens from the cervical trachea, intrathoracic trachea, and bronchus, 80% to 100% were HPV positive compared with only 25% to 50% of HPV infection detected in the nasopharynx, posterior tonsillar pillar, and aryepiglottic fold. These results support the tenet that HPV infection is present in clinically normal respiratory tract tissue and that the reservoir site of reinfection is more commonly in the lower airway. However, patients with upper-airway involvement were more likely to be diagnosed as having severe disease.

Human papillomavirus and risk of laryngeal cancer.

We determined the relationship between human papillomavirus (HPV) infection and the HPV types detected in 44 patients with squamous cell carcinoma, 10 laryngeal leukoplakia patients, and 12 patients evaluated for benign laryngeal conditions (controls). The sources of HPV DNA were from brushings from the upper respiratory tract and lesion (benign or malignant), oral rinses, and biopsies of patient lesions. Polymerase chain reaction (PCR) and DNA sequencing were used to identify and type HPV. We detected HPV in 25.0% (11/44) of patients with laryngeal cancer, in 30.0% (3/10) of patients with laryngeal leukoplakia, and in 16.7% (2/12) of noncancer controls. Patients with cancer were not more likely to be identified with oncogenic HPV types ( 18.2%) than either the leukoplakia group (20%) or the control group (16.7%). An increased risk of disease was associated with current tobacco use and former alcohol drinking in cancer patients versus controls and in leukoplakia patients versus controls (all p < .05). After we controlled for tobacco and alcohol effects on the risk of disease, exposure to oncogenic HPV types was associated with an increased risk of laryngeal cancer (odds ratio = 3.0) and of laryngeal leukoplakia (odds ratio = 6.0) compared to controls, although the results were not statistically significant. This study suggests that although HPV infection and HPV oncogenic types are not found at a higher frequency in laryngeal cancer or laryngeal leukoplakia as compared to controls, infection is associated with an increased risk of disease after controlling for the effects of alcohol and tobacco use.

Persistent HPV infection in postmenopausal age women.

OBJECTIVE: Persistence of human papillomavirus (HPV) is associated with an increased risk of developing cervical SIL and cancer in young women. Because this association in older, postmenopausal age women has received little attention, we evaluated persistence of HPV among women in this age group. METHODS: Women (n=105) ages 45-64 were examined annually for 7 years to evaluate HPV in cervical cytologic specimens. PCR, dot blot hybridization and DNA sequencing were used to detect HPV types. RESULTS: The cumulative prevalence of HPV was 34%, and 24% had HPV high-risk oncogenic types which are associated with genital cancers. The most common oncogenic types were HPV-16 (72%) and HPV-31 (16%). The persistence rate of HPV infection was 16%. No specific risk factors were associated with repeat viral positivity. CONCLUSION: Postmenopausal women are infected with persistent oncogenic HPV at a substantial rate, supporting the need for continued screening in postmenopausal women to detect preneoplastic genital lesions.

p53 polymorphism, human papillomavirus infection in the oral cavity, and oral cancer.

OBJECTIVES: Human papillomavirus (HPV) infection has emerged as a risk factor in oral carcinogenesis. An arginine-coding polymorphism of the tumor suppressor protein p53 at codon 72 is more readily degraded by the HPV oncoprotein E6. Our objective was to evaluate the association between p53 polymorphism at codon 72 and HPV infection in the oral cavity, as well as its association with oral cancer. STUDY DESIGN: Oral squamous cells from 202 patients with oral cancer and 333 age-sex frequency matched controls were evaluated by polymerase chain reaction for the presence and type of HPV and for alleles of codon 72 in p53. Fisher exact test and chi(2) tests were used to evaluate the data. RESULTS: The p53 codon 72 polymorphism is not associated with HPV infection, whether comparing HPV-negative controls with HPV-positive controls or comparing HPV-negative cases with HPV-positive cases. Additionally, we found no association with the codon 72 polymorphism and oral cancer, whether comparing HPV-negative controls with HPV-negative cases or comparing HPV-positive controls with HPV-positive cases. CONCLUSIONS: There is no association between p53 codon 72 polymorphism and HPV infection or between the p53 polymorphism and the risk of oral cancer.

Human papillomavirus in the oral cavities of children and adolescents.

OBJECTIVE: The purpose of this pilot study was to determine the frequency of human papillomavirus (HPV) in the oral cavities of children and adolescents and to identify potential risk factors for HPV infection. STUDY DESIGN: Sociodemographic information was obtained on 268 healthy infants, children, and adolescents who were < or = 20 years old. Oral squamous cells were collected from swabs with young children and from oral saline solution rinses with older children and adolescents. Extracted DNA was evaluated for HPV by polymerase chain reaction, dot blot hybridization, and DNA sequencing. Factors associated with the presence of HPV were tested by using chi(2), Fisher's exact test, and logistic regression tests. RESULTS: HPV was detected in 6.0% of the participants. HPV frequency among young children (<7 years old) was 8.7% (11/127), and among adolescents (13-20 years old) it was 5.2% (5/97). HPV was not detected in children aged 7 to 12 years old (0/44). Fifty-four percent (6/11) of HPV-positive children were 1 year of age or less; 3 of the HPV-positive children (<7 years old) were delivered by cesarean section. No statistically significant association was found between the detection of HPV in the oral cavity and method of delivery or gender; parent's race, education, HPV-related conditions, smoking history, or number of sex partners; or adolescent's smoking history or history of sexual activity. CONCLUSIONS: This study suggests that HPV is present in the oral cavity primarily in children 2 years old and younger and in adolescents 13 years and older. Cesarean delivery was not protective against oral HPV infection; in fact, half of the HPV-positive infants were born by cesarean delivery.

De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase.

The de novo biosynthesis of RNase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) was studied in a cell-free rabbit reticulocyte translation system using a polyadenylylated fraction of mRNA isolated from bovine pancreas. Analysis of the [35S]methionine-labeled translation products of pancreas mRNA by polyacrylamide gel electrophoresis indicated the presence of several proteins, one of which corresponded to 16,500 daltons, or approximately 2800 daltons greater than native RNase A. This protein was specifically precipitated from the mixture of products by anti-RNase antibodies. Partial sequence determination of the NH2-terminal region of the anti-RNase antibody-precipitable species indicated that it is a precursor form of RNase A with 25 additional amino acids on its NH2 terminus. The precursor nature of the protein was confirmed by demonstration that a mixture of RNase A and a glycosylated form of the enzyme, RNase B, is formed when translation of the mRNA is conducted in the presence of dog pancreas membranes. Assay of the putative precursor form of RNase for catalytic activity with polycytidylic acid as substrate indicated that the protein has a specific enzymatic activity identical to that of native RNase A.

Biosynthesis of bacterial glycogen. The nature of the binding of substrates and effectors to ADP-glucose synthase.

Kinetic studies with ADP-glucose synthase show that 1,6-hexanediol bisphosphate (1,6-hexanediol-P2) is an effective activator that causes the enzyme to have a higher apparent affinity for ATP- and ADP-glucose than when fructose-1,6-P2 is the activator. Furthermore, in the presence of 1,6-hexanediol-P2, substrate saturation curves are hyperbolic shaped rather than sigmoidal shaped. CrATP behaves like a nonreactive analogue of ATP. Kinetic studies show that it is competitive with ATP. CrATP is not a competitive inhibitor of ADP-glucose. However, the combined addition of CrATP and glucose-1-P inhibits the enzyme competitively when ADP-glucose is the substrate. In binding experiments, CrATP, ATP, and fructose-P2 appear to bind to only half of the expected sites in the tetrameric enzyme, while ADP-glucose, the activators, pyridoxal-P and 1,6-hexanediol-P2, and the inhibitor, AMP, bind to four sites/tetrameric enzyme. Fructose-P2 inhibits 1,6-hexanediol-P2 binding, suggesting competition for the same sites. Glucose-1-P does not bind to the enzyme unless MgCl2 and CrATP are present and binds to four sites/tetrameric enzyme. Alternatively, CrATP in the presence of glucose-1-P binds to four sites/tetrameric enzyme. Thus, there are binding sites for the substrates, activators, and inhibitor located on each subunit and the binding sites can interact homotropically and heterotropically. ATP and fructose-P2 binding is synergistic showing heterotropic cooperativity. ATP and fructose-P2 must also be present together to effectively inhibit AMP binding. A mechanism is proposed which explains some of the kinetic and binding properties in terms of an asymmetry in the distribution of the conformational states of the four identical subunits.

Haptoglobin. A novel mode of biosynthesis of a liver secretory glycoprotein.

The de novo biosynthesis of the heterotetrameric (alpha 2 beta 2) serum glycoprotein, haptoglobin, was studied using a rabbit reticulocyte cell-free translation system and mRNA preparations from the livers of turpentine-treated rats. Analysis of the translation mixtures with either antisera specific for the alpha-subunit (anti-alpha-IgG) or the beta-subunit (anti-beta-IgG) or the native haptoglobin tetramer (anti-alpha 2 beta 2-IgG) resulted in each instance in the recovery of a single protein exhibiting a Mr = approximately 38,000. Cleavage of the translation product with cyanogen bromide or trypsin resulted in the generation of small peptide fragments that were specifically immunoadsorbed with either anti-alpha-IgG or anti-beta-IgG. These results indicated that the primary translation product of haptoglobin mRNA is a single polypeptide that contains the elements of both the alpha-subunit and the beta-subunit of the protein. Presumably haptoglobin is synthesized in vitro as a single precursor protein that is proteolytically processed post-translationally to form the dissimilar alpha- and beta-subunits of the native protein.

Biosynthesis and processing of rat haptoglobin.

Native rat haptoglobin is an heterotetramer consisting of two alpha-subunits (Mr = approximately 9,500) and two glycosylated beta-subunits (Mr = approximately 38,000) joined by interchain disulfide bonds. We previously reported (Haugen, T. H., Hanley, J. M., and Heath, E. C. (1981) J. Biol. Chem. 256, 1055-1057) that the synthesis of rat haptoglobin is encoded by a single mRNA, and that the primary in vitro translation product is a single polypeptide, preprohaptoglobin (Mr = approximately 40,000), that contains an NH2-terminal signal sequence as well as an alpha-subunit region and a beta-subunit region. We now report that partial sequence analysis of preprohaptoglobin indicates that the protein possesses an NH2-terminal hydrophobic signal peptide of 18 amino acid residues, followed directly by the alpha-subunit region, with the beta-subunit region located in the carboxyl-terminal portion of the protein. The co-translationally processed translation product consists of a core glycosylated polypeptide, prohaptoglobin (Mr = approximately 45,000), that is devoid of the signal sequence and possesses both the alpha-subunit and beta-subunit regions of haptoglobin. Pulse-chase experiments in cultures of isolated hepatocytes, and analysis of haptoglobin biosynthetic intermediates in the various subcellular organelles of in vivo labeled rat liver indicate that: (a) in the endoplasmic reticulum, core glycosylated prohaptoglobin is dimerized and a portion of the protein is processed to form the individual alpha- and beta-subunits; (b) the carbohydrate side chains of prohaptoglobin and of core glycosylated beta-subunit (Mr = approximately 35,000) are converted to complex, sialylated side chains in the Golgi apparatus, resulting in the formation of fully glycosylated prohaptoglobin (Mr = approximately 48,000) and beta-subunit (Mr = approximately 38,000), and these forms of the protein, as well as the alpha-subunit (Mr = approximately 9,500), are secreted; (c) inhibition of glycosylation with tunicamycin does not significantly affect the rate of synthesis, processing, or secretion of the various haptoglobin polypeptides in isolated hepatocytes; (d) similar experiments conducted in the presence of colchicine also had no effect on the rate of synthesis and processing of the intermediates; and (e) the species of haptoglobin secreted in vivo and from isolated hepatocytes consist of approximately 60-70% in the form of the alpha 2 beta 2 tetramer, and the remainder as dimerized prohaptoglobin. Presumably, secreted prohaptoglobin may be processed to the native subunit structure after secretion, as we demonstrated that incubation of prohaptoglobin with either normal rat serum, rat plasma, or with the sera of other animal species results in its conversion to the corresponding alpha- and beta-subunits of the native protein.

Biosynthesis of bacterial glycogen. Characterization of the subunit structure of Escherichia coli B glucose-1-phosphate adenylyltransferase (EC 2.7.7.27).

ADP-glucose pyrophosphorylase has been isolated in homogeneous form from an Escherichia coli B mutant, AC70R1, derepressed in the synthesis of glycogen synthetic enzymes. The enzyme has been found to be identical with the wild type enzyme with respect to kinetic properties, molecular weight, and immunological reactivity. The AC70R1 enzyme is composed of four identical subunits of molecular weight of approximately 50,000. This is based on the findings that: (a) gel electrophoresis under denaturing conditions shows only one component; (b) tryptic mapping shows only enough peptides to account for a single polypeptide subunit; (c) digestion with carboxypeptidase B releases stoichiometric amounts of arginine; and (d) NH2-terminal sequencing shows a single sequence for the first 27 residues.

Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene.

The approximately 1000 nucleotide long upstream regulatory region (URR) of bovine papilloma virus-1 (BPV-1) contains a cis element which responds to trans-activation by a diffusible factor encoded in the viral E2 open reading frame (ORF). A series of URR DNA fragments have been linked to two heterologous genes, bacterial chloramphenicol acetyl transferase (cat) or herpes simplex virus-1 thymidine kinase (tk), and tested in transient transfection assays for transcription initiating at the authentic upstream early viral promoter, P89. Transcriptional activity of the P89 promoter was greatly elevated in the presence of the E2 trans-activator gene product. The E2-responsive cis element (E2R) of P89 has been mapped to sequences -277 to -131 nucleotides upstream from the transcription start site (BPV nucleotide 89). The E2R element functioned as a strong transcriptional enhancer in cis with the SV40 early or the tk promoter in the presence, but not in the absence, of the E2 gene product. However, several heterologous promoters which lack sequences related to the E2R element were also trans-activated in transient cotransfections by a function encoded in the E2 ORF of BPV-1, albeit to a much lesser extent. In addition to activation of early viral gene transcription, the E2 regulatory gene(s) may therefore have the potential to alter cellular gene expression.

HPV prevalence and concordance in the cervix and oral cavity of pregnant women.

OBJECTIVES: This investigation examined human papillomavirus (HPV) in pregnant women in order to characterize viral prevalence, types and concordance between infection in the cervix and in the oral cavity. METHODS: A total of 577 pregnant women seeking routine obstetric care were evaluated for HPV infection in their cervix during gestation and immediately before delivery, and in the oral cavity during gestation. Male partners present during the gestational clinic visit also provided a specimen from their oral cavity. HPV assessment was performed by PCR, dot blot hybridization and DNA sequencing. A sexual and health questionnaire was completed by the pregnant women. RESULTS: HPV prevalence in women was 29% in the cervix and 2.4% in the oral cavity. Among those with both gestational and delivery specimens, 35% were infected at least once and 20% had infection at both intervals. At delivery, 68% of infected women had an oncogenic HPV type in the cervix. There was no type-specific HPV concordance between the two cervical specimens, nor cervical and oral results in women, nor with cervical and oral findings between partners. CONCLUSION: The lack of association in HPV positivity and types between the cervix and oral cavity in these women suggests that self-inoculation is uncommon. This source of infection does not appear to be from oral contact with a current male partner, since there also was no concordance between partners. These results suggest either other modes of HPV transmission or differences in susceptibility to HPV infection or its clearance in the oral cavity and genital mucosa.

Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis.

The transcriptional promoter of the candidate E6-E7 transforming gene region of human papillomavirus (HPV)-16 (P97) was active in transiently transfected cervical carcinoma cells when linked to the HSV-1 tk or bacterial cat genes. Sequences 5' to P97 contain a short enhancer element responding to cellular factor(s) in uninfected human foreskin keratinocytes and in cervical carcinoma cells, but not in human or animal fibroblasts. The E2 trans-activator products of HPV-16 or of the related bovine papillomavirus (BPV)-1 further elevated HPV-16-driven transcripts in co-transfections, and required the presence of E2-binding ACC(N)6GGT cores in cis. A 'short E2' C-terminal repressor gene product (sE2) of HPV-16 or the BPV-1 sE2 repressor not only inhibited viral E2 trans-activation, but also suppressed enhancer response to keratinocytic factors. Suppression by the sE2 products was abolished by deletion of the E2-binding cores in cis or by a mutation in the sE2 DNA binding domain. The keratinocyte-dependent enhancer is likely to contribute to the epithelial cell tropism of HPV-16, and may direct persistent E6-E7 gene transcription in response to cellular factors in cervical carcinoma cells in which the viral E2 genes are inactive.

The frequency of human papillomavirus detection in postmenopausal women on hormone replacement therapy.

Postmenopausal women enrolled in the Iowa portion of the postmenopausal estrogen/progestin interventions randomized clinical trial (n = 105) during 1989-1991 were studied for (i) the prevalence of human papillomavirus (HPV) in this older age population (ages 45-64), and (ii) the association between hormone replacement therapies (HRTs) and changes in detection of HPV over a 2-year time period. HPV is causative in most cervical and some other genital cancers and in the presence of steroid hormones has been shown to increase neoplastic transformation by HPV in vitro. Using PCR to detect HPV DNA, the overall frequency of the virus regardless of time period was 50.3% (n = 53) with a baseline (BL) frequency of 38.1% and the second year follow-up (FU) of 22.9%. The oncogenic types HPV-16 (75.5%) and HPV-31 (20.8%) were the most commonly reported. All those with persistently detected infection (10.5%), defined as HPV+ at both BL and FU, were identified with HPV-16 or -18. Between these two time periods there were no significant differences in HPV frequency between the placebo and combined HRT groups (BL-/FU+, 21% vs 18%; BL+/FU-, 71% vs 80%). While the study is based on a small sample, the findings suggest that short-term use of HRTs is not associated with an increased risk of HPV detection, but assessment of effects from long-term use is needed. The data also indicate that the frequency of HPV found in older women is higher than previously suspected but that short-term changes in HPV detected in this age group are unrelated to the development of precancerous cervical lesions.