Judit Czeglédy

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas.

Aim: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). Methods: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). Results: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. Conclusions: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.

HPV 16 DNA and mRNA in cervical brush samples quantified by PCR and microwell hybridization

To quantitate HPV 16 DNA and mRNA, biotinylated amplicons from PCR and reverse transcription PCR were captured on streptavidin-coated microtitre plates. The amount of amplicon was determined by colorimetric detection after hybridization with an alkaline phosphatase-labelled probe. Dynamic ranges of between 4 and 6 log10, sufficient to cover the amounts of viral DNA and mRNA prepared from cervical samples were achieved. The reproducibility of the colorimetric detection step was reflected in coefficients of variation (C.V.) below 8%, considerably better than that of chemiluminescence detection. In a series of 89 HPV 16 DNA positive cervical samples, as compared with a CIN I/normal diagnosis subgroup, the number of HPV 16 genome copies per assay was significantly greater in a CIN II subgroup (P = 0.014), and a high-grade neoplasia subgroup (P = 0.040), and the content of HPV 16 mRNA significantly greater in the high-grade neoplasia subgroup (P = 0.0021). The number of mRNA equivalents per copy of viral DNA was higher for E5 than for the other three mRNA species analyzed (P < 0.001), and the concentration of E6*I mRNA was higher than those of the E6 full-length (P < 0.001) and E6*II (P < 0.001) transcripts. Despite these differences, no correlation was found between histological/cytological diagnosis and the amount of viral mRNA relative to the viral load.

High-risk human papillomavirus types in cytologically normal cervical scrapes from Kenya

Seventy-seven women with normal cervical cytology on routine visit to a family planning clinic in Nairobi, Kenya, were analysed for genital human papillomavirus (HPV) types by polymerase chain reaction (PCR). We applied a general primer pair (GP60/GP124) recognising sequences conserved among HPV types 6, 11, 16, 18, 31 and 33. Of the 77 specimens tested 15 (19.5%) proved to be positive for genital HPV. Amplification products were examined for the presence of high-risk HPV types by Slot-blot hybridization. Out of the 15 PCR-positive samples, 4 were positive for HPV 16,3 for HPV 18, while 1 contained both HPV 16 and 33. HPV DNA prevalence in this group of women from a “high-risk” area is similar to that in “low-risk” Swedish women but much lower than in cervical cancer samples from the same region.

Follow-up of human papillomavirus (HPV) DNA and local anti-HPV antibodies in cytologically normal pregnant women

Abstract The high level of progesterone during pregnancy may enhance the transcription and replication of genital human papillomaviruses (HPV) through the glucocorticoid/progesterone response element found in the long control region of the viral genome. In this study, cytologically and colposcopically healthy pregnant women were subjected to a follow-up examination. Samples from the uterine cervix were collected during early pregnancy (n = 39), in the third trimester (n = 31), and a few weeks after birth (n = 30). The presence of HPV DNA was detected by polymerase chain reaction (PCR), while local secretory anti-viral IgA antibodies were demonstrated by enzyme-linked immunosorbent assay using synthetic peptide antigens. Follow-up examination by PCR revealed HPV DNA persistence in 5 women. In 5 other cases, HPV positivity changed from negative to positive during the follow-up. There was 1 case which changed from positive to negative and 1 in which the HPV type changed during the study. Altogether, 12 of 39 women (31%) were shown to harbor HPV DNA at some time during follow-up. HPV DNA positivity increased from 18% during early pregnancy to 27% after birth (difference not significant). On the other hand, there was a significant rise in the level of local antibodies against HPV antigens (E 2, E 7, and L 2) between samples collected in early pregnancy and those collected after birth (P<0.0001). This may indicate the reactivation of genital HPV infections during late pregnancy.

Detection of human papillomavirus deoxyribonucleic acid in the female genital tract

A total of 336 biopsies, scrapes and exfoliated cells from the cervix and from the lower genital tract were screened for human papilloma (HP) viral sequences of types 6, 11, 16 and 18 by Southern blot, dot blot and filter in situ (FISH) hybridizations with cloned 32P-radiolabeled HPV DNA probes. The specimens included cervical intraepithelial neoplasias (CIN I–III), carcinoma in situ and invasive carcinoma of the cervix and vagina, adenocarcinomas, vulvar and vaginal condylomata acuminata and healthy epithelial samples. The oncogenic HPV 16 was found in 46% of the cervical carcinomas. Most of the type 16 occurences (75%) represented the third stage of inooperable cases. Similarly, HPV 18 was also most frequently present in this stage as well as in carcinoma in situ and in CIN III (25%, 18%). At the same time, in condylomata acuminata, types 6 and 11 were detectable in 88.7% of cares. In all, 13.5% of the normal samples harboured HPV DNA.

Oral contraceptive use and human papillomavirus infection in women without abnormal cytological results.

Both experimental and epidemiological data support the idea that oral contraceptive (OC) use may have a stimulating effect to a certain point on cervical carcinogenesis. The current investigation tries to answer the question whether OC use might have an influence on early human papillomavirus (HPV) infections. A total of 425 women without abnormal cytological results were examined colposcopically, and filter in situ hybridisation (FISH) was used to determine the presence of human papillomavirus (HPV) types 6, 11, 16 and 18. Eighty-one cervical specimens (19.1%) were found to be positive for one or more of the HPV types in FISH. HPV positivity was found to correlate with age and parity, being the highest among women under 25 and with less than two births. The use of OCs was inversely correlated with the presence of ectopy or dysplasia in this group of women. On the other hand, HPV positivity was not significantly higher among OC users than among non-users in any colposcopic group. Neither the type of pill used, nor the duration of use had any significant effect on HPV positivity. Further investigations are needed to evaluate the effects of OC use on more severe HPV- induced cervical lesions.

Detection of transforming gene regions of human papillomavirus type 16 in cervical dysplasias by the polymerase chain reaction.

In a study of 29 cases of histologically confirmed, characterized colposcopically and cytologically, cervical intraepithelial neoplasias 48.3% (14/ 29) of biopsies were positive for human papillomavirus (HPV) type 16 DNA by polymerase chain reaction. We used two oligonucleotide primer pairs (position 215–514 and 606–805) flanking a 300 and a 199 base pair fragment from the early 6 (E6) and early 7 (E7) genes. The results were concordant both with the E6 and with the E7 regions. Of the amplified products 85.7% (12/14) could be confirmed; these carried 16 specific sequences by Southern blot hybridization. HPV 16 DNA was present in 6.7% (2/30) of the colposcopically directed cytologically normal matched control samples using the same methods.

Detection of high-risk HPV DNA in semen and its association with the quality of semen

vaccination in advanced HIV patients (CD4 lymphocytes o500/mL) and the adverse event profile, this report should caution physicians to balance the risk and questionable benefit of pneumococcal vaccination in advanced HIV patients. Given this serious adverse event, we strongly encourage caregivers to counsel patients, prior to vaccination, in contrast to a previous report. This report and the lack of efficacy data in the literature support the contention that pneumococcal vaccination guidelines in HIV-infected individuals need to be reassessed. It may be more cost-effective to favour strategies that improve adherence to antiretroviral therapy as this has unequivocally been shown to reduce the incidence of pneumococcal disease. Additional safety and efficacy studies should address patients with symptomatic HIV disease as they seem to be at higher risk for adverse events to pneumococcal vaccinogens. Ulrich R Hengge, Ruediger E Scharf, Frank P Kroon and Klaus Pfeffer Department of Dermatology, University of Duesseldorf, Moorenstrasse 5, D-40225 Duesseldorf; Institute of Hemostasis and Transfusion Medicine, University of Duesseldorf, Germany; Department of Infectious Diseases, Leiden University Medical Center, Netherlands; Institute of Virology, University of Duesseldorf, Germany Correspondence to: Ulrich R Hengge Email: ulrich.hengge@uni-duesseldorf.de

Detection of human cytomegalovirus-induced DNA-binding proteins on Western blots

DNA-binding proteins have been isolated from human embryonic fibroblast cells infected with human cytomegalovirus strain AD169 by native and denatured DNA-cellulose affinity chromatography and Polymin P precipitation. The DNA-binding proteins separated by polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose sheet were identified by a modified ELISA reaction. Six DNA-binding proteins were found in infected cells, their molecular weights range from 52K to 18K.

Early Nuclear Antigen as DNA-Binding Protein in Cytomegalovirus-Infected Cells

Early nuclear complement-fixing antigen in cytomegalovirus (CMV) (strain Ad 169)-infected human embryonic fibroblasts is described. The nuclear antigen was solubilized from CMV-infected cells by high salt treatment. DNA-binding properties were studied by DNA-cellulose chromatography. The purified antigen, eluted from double-stranded DNA-cellulose columns, was added to acid-fixed nuclear preparations from human embryonic fibroblasts and then exposed to human sera containing antibodies to CMV. Positive staining was obtained by anti-complement immunofluorescence. These data identify the CMV-determined nuclear antigen as a DNA-binding protein. In this respect, it is similar to the Epstein-Barr virus nuclear antigen.