J. ter Schegget

A case-control study of betapapillomavirus infection and cutaneous squamous cell carcinoma in organ transplant recipients

We examined the association between betapapillomavirus (betaPV) infection and cutaneous squamous cell carcinoma (SCC) in organ transplant recipients. A total of 210 organ transplant recipients with previous SCC and 394 controls without skin cancer were included. The presence of 25 betaPV types in plucked eyebrow hairs was determined using a human papillomavirus (HPV) DNA genotyping assay, and antibodies for the 15 most prevalent betaPV types were detected using multiplex serology. We used multivariate logistic regression models to estimate associations between various measures of betaPV infection and SCC. BetaPV DNA was highly prevalent (>94%) with multiple types frequently detected in both groups. We found a significant association between SCC and the concordant detection of both antibodies and DNA for at least one betaPV type (adjusted OR 1.6; 95% CI 1.1;2.5). A borderline‐significant association with SCC was found for HPV36 (adjusted OR 2.4; CI 1.0;5.4), with similar associations for HPV5, HPV9 and HPV24. These data provide further evidence of an association between betaPV infection and SCC in organ transplant recipients. Confirmation of a betaPV profile predictive of risk for SCC may pave the way for clinically relevant pretransplant HPV screening and the development of preventive and therapeutic HPV vaccination.

Detection of epidermodysplasia verruciformis-like human papillomavirus types in malignant and premalignant skin lesions of renal transplant recipients

To evaluate the putative role of human papillomaviruses (HPV) in the development of skin cancer in renal transplant recipients, a series of skin biopsies from premalignant and malignant skin lesions was analysed using the polymerase chain reaction. Four different consensus primer pairs were used. HPV DNA was detected in five of 24 cases of squamous cell carcinoma, in one of three cases of Bowens disease, in none of four basal cell carcinomas, in two of seven cases of actinic keratosis and in one of five cases of keratoacanthoma. Typing by direct sequencing of the amplified HPV DNA was possible in seven of nine cases, and revealed epidermodysplasia verruciformis (EV)‐associated HPV types, or HPV types related to EV‐associated types. Hence, HPV DNA could be detected in a significant proportion of (pre)malignant skin tumours in renal transplant recipients. The finding that some of the detected HPV types were as yet uncharacterized EV‐related types, suggests that HPV DNA could be present in a higher percentage of lesions, and might be detected with refinement of the techniques.

Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

The presence of antibodies against virus‐like particles of epidermodysplasia verruciformis‐associated humanpapillomavirus type 8 in patients with actinic keratoses

Epidermodysplasia verruciformis‐associated human papillomaviruses (EV‐HPVs) are possibly involved in the development of actinic keratoses and may play a part in the pathogenesis of squamous cell carcinoma (SCC) of the skin, as the DNA of these viruses is frequently detected in biopsies of such lesions. Properly designed epidemiological studies, using serological tests to investigate the role of infection with EV‐HPVs in cutaneous oncogenesis, are still rare. An IgG‐specific enzyme‐linked immunosorbent assay using virus‐like particles composed of the major capsid protein L1 of the EV‐specific HPV 8 (HPV 8 VLPs) was developed and used to test the seroprevalence of HPV 8 in 114 inhabitants of a tropical island, of whom 13 had developed SCC, and 19 had developed basal cell carcinoma. Gender, age, eye and hair colour, sun exposure and number of actinic keratoses were recorded for all individuals. The presence of antibodies against HPV 8 VLPs was associated with the development of large numbers of actinic keratoses. After adjusting for gender, age, eye and hair colour, and sun exposure, the odds ratio to develop 37 (the median in this dataset) or more actinic keratoses in the presence of antibodies against HPV 8 VLPs was 2·3 (95% confidence interval: 1·0; 5·3). Similarly, after adjustment for the same factors, the presence of these antibodies was associated with SCC with an odds ratio of 3·1 (0·74; 13·3), but the small number of individuals with SCC does not permit any definite conclusions. The presence of these antibodies did not appear to be associated with basal cell carcinoma as, after adjustment for the same factors, the odds ratio was 0·73 (0·23; 2·4). This study provides serological evidence that infection with EV‐HPVs may play a part in the pathogenesis of actinic keratoses. The role of EV‐HPVs in the development of SCC, however, remains to be elucidated.

Uniformity of the Splicing Pattern of the E6/E7 Transcripts in Human Papillomavirus Type 16-transformed Human Fibroblasts, Human Cervical Premalignant Lesions and Carcinomas

We utilized the RNA polymerase chain reaction (PCR) to analyse the transcripts of the E6/E7 open reading frames of human papillomavirus type 16 (HPV-16). Total RNA was isolated from 14 cervical squamous carcinomas, nine cervical intraepithelial neoplasias and from human fibroblasts transformed with different HPV-16 constructs. In all specimens two spliced transcripts were detected. Sequence analysis of the cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in one transcript and from nt 226 to 526 in the other. The major transcript present in all RNA specimens had the smallest intron in E6. The RNA PCR described here is the method of choice for analysing splice and donor sites in tissue specimens where a limited amount of RNA is available. Results obtained with transformed cells revealed no difference in splicing whether HPV-16 was controlled by its homologous promoter or by a heterologous promoter, the Rous sarcoma virus long terminal repeat.

The presence of DNA molecules with a displacement loop in standard mitochondrial DNA preparations.

Abstract In electron micrographs of standard closed circular duplex mtDNA preparations from chick liver, spread by the protein monolayer technique from solutions containing 40 or 76% formamide, up to 30% of the molecules contained a singled-stranded displacement loop, as depicted in Fig. 1. The average lengths of the loops and of the doublestranded sections between the attachment points were about the same and equivalent to 3.5% of the contour length of the mtDNA circle.

Antibody responses to 26 skin human papillomavirus types in the Netherlands, Italy and Australia

Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infections with skin human papillomavirus (HPV) types have also been linked to the development of SCC. Little is known about the natural history of these infections and whether the seroprevalence of skin HPV types is affected by ambient or individual levels of sun exposure. This study investigated this by analysing sera for antibodies to 26 skin HPV types from five phylogenetic genera obtained from 807 healthy individuals from the Netherlands, Italy and Australia, countries with strong differences in sunlight intensity. Overall HPV seroprevalence was similar across the three countries (50-57 % for beta-HPV types, 40-48 % for gamma-HPV types), and the most frequent beta-HPV and gamma-HPV types were the same in all countries. The highest seroprevalences for 24 of the 26 skin HPV types were observed in Italy (14 types) and Australia (ten types). Seroprevalence among men was generally higher than among women, and the male sex was significantly associated with both beta-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64-4.82] and gamma-HPV (OR 2.42, 95 % CI 1.40-4.18) antibodies in Australia. The only measure of sun sensitivity or UV exposure significantly associated with skin HPV seroprevalence was found for weekend sun exposure in Australia and beta-HPV antibodies. It was concluded that type spectra and HPV seroprevalence are similar in countries with different sunlight intensity, and that levels of UV exposure do not play a strong role in the development of skin HPV antibodies in this study population.

The synthesis and utilization of dCDP-diglyceride by a mitochondrial fraction from rat liver

SUMMARY I. Mitochondrial preparations from rat liver incorporate added dCTP into a compound that is readily extracted with chloroform. This compound was identified as dCDP-diglyceride by co-chromatography with authentic dCDP-diglyceride on silica gels with three different solvent systems and by the absence of an effect of borate on its RF value. 2. The apparent K, for dCTP in dCDP-diglyceride synthesis by intact mitochondria was 4.10-5 M; the vmex was 1.1 .10-z nmoles/min per mg protein. Synthesis was inhibited by rCTP, whereas the incorporation of the rCDP moiety of rCTP into rCDP-diglyceride was inhibited by dCTP. 3. dCDP-Diglyceride substituted for rCDP-diglyceride in the synthesis of phosphatidylglycerol from sgz-glycero-3-phosphate by these mitochondrial preparations. The apparent Km’s were z .10-s M for dCDP-diglyceride and 7.10-~ M for rCDP-diglyceride. The vUmax was 0.42.10-z nmole/min per mg protein with dCDPdiglyceride and 2.5. IO-~ nmole/min per mg protein with rCDP-diglyceride as substrate.

Localization of human papillomavirus type 16 DNA using the polymerase chain reaction in the cervix uteri of women with cervical intraepithelial neoplasia

The localization of human papillomavirus type 16 (HPV-16) DNA throughout the cervix uteri of women with cervical intraepithelial neoplasia (CIN) was studied by utilizing the polymerase chain reaction technique directly on histologically defined sections of paraffin-embedded cervical tissue obtained by conizations. HPV-16 DNA was detected only in the sections that contained CIN lesions and/or koilocytes. No HPV-16 DNA was detected in sections that contained only normal epithelium. This is in accordance with HPV-16 playing a role in the development of CIN lesions.

DNA synthesis by isolated mitochondria: I. Effect of inhibitors and characterization of the product

Abstract 1. 1. Chick-liver mitochondria incorporate deoxyribonucleosides and dNTPs into endogenous mitochondrial DNA. The maximal rate of dATP incorporation was 0.5 pmole/mg protein per 10 min. This corresponds to about 0.1 % net synthesis. The apparent Km for dATP in the presence of 15 μM of the other dNTPs was 1.5 · 10−6 M. 2. 2. dATP incorporation into acid-insoluble material by isolated chick-liver mitochondria was not inhibited by phenethyl alcohol and only to a limited extent by high concentrations of nalidixic acid. Half-maximal inhibition by ethidium bromide was observed at about 1 μM and 12.5 μM was sufficient to completely block the incorporation of dATP in high-molecular-weight DNA. 3. 3. Up to 70 % of the purified product DNA co-sedimented with closed circular duplex mitochondrial DNA in sucrose gradients and CsCl gradients containing ethidium. Treatment of this I∗ with deoxyribonuclease converted it into material with the sedimentation properties of open circular mitochondrial DNA (II∗).