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L Gissmann

Publications and source records attributed to L Gissmann.

At least 73 records · Page 4Linked to original sources
PubMed1992

Definition of immunogenic determinants of the human papillomavirus type 16 nucleoprotein E7.

Specific T lymphocyte lines and T cell clones were established from peripheral blood mononuclear cells of asymptomatic seropositive individuals employing synthetic peptides which correspond to the sequence of the human papillomavirus (HPV) type 16 transforming protein E7. Specificity analysis of T cells as determined by means of [3H] thymidine incorporation after stimulation with individual peptides revealed three immunogenic determinants of E7 that are recognised in association with at least two different HLA haplotypes. One N-terminal region (aminoacids 5-18) was recognised by one T cell line. T cell clones and the corresponding T cell line established from another donor responded to a different N-terminal (17-38) and to a C-terminal region (69-86). The N-terminal sequence 5-18 and the C-terminal determinant contain a periodicity of hydrophilic and hydrophobic residues that have been found in many T cell epitopes. Phenotypic characterisation of T cell clones by indirect immunofluorescence revealed that the T cell clones expressed the CD4 surface glycoprotein suggesting that the specific E7 determinants were recognised in association with major histocompatibility complex (MHC) class II molecules. With regard to functional properties, at least three T cell clones exhibited specific cytotoxic activity towards autologous B lymphocytes transformed by Epstein-Barr virus in the presence of the relevant HPV16 E7 peptides. The implications of these results regarding the development of vaccination strategies and host-virus interaction are discussed.

Amino Acid Sequence↗
PubMed1992

Geographical dependence of sequence variation in the E7 gene of human papillomavirus type 16.

We have determined nucleotide sequences of the E7 open reading frame (ORF) of human papillomavirus type 16 (HPV-16) isolates obtained from 32 genital tumours and two HPV-16-transformed human keratinocyte cell lines. In comparison to the prototype HPV-16 isolated from a German cervical cancer biopsy, no sequence variations were noticed in either the two cell lines or the 10 biopsies that were obtained from German patients. In contrast only three of 22 (13.6%) of Tanzanian isolates showed the prototype sequence. In 18 of these biopsies two alterations (T to C and T to G) not affecting the amino acid sequence were found within the HPV-16 E7 ORF (nucleotide positions 789 and 795) but eight of these isolates contained an additional change (nucleotide position 647) coding for serine instead of asparagine (amino acid position 29). One tumour harbours HPV-16 DNA with a mutation (C to T) at nucleotide 790 changing the E7 amino acid sequence (arginine to cysteine) at position 76. Our findings suggest that clustering of E7 sequence variants may occur in different geographical regions of the world.

Amino Acid Sequence↗
PubMed1992

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Female↗
PubMed1992

Subclinical Epstein-Barr virus infection of both the male and female genital tract--indication for sexual transmission.

Epstein-Barr Virus (EBV) can infect B lymphocytes as well as epithelial cells of the oral cavity. Recently, infection of epithelial cells of the inflamed uterine cervix has been demonstrated, and EBV-DNA has been detected in urethral discharge of men suffering from genital infection. We investigated whether EBV can be found in the genital tract of both sexes independently from inflammatory disease states. Genital specimens of men and women of a sexually transmitted diseases outpatient clinic after excluding sexually transmitted diseases and clinically apparent signs of inflammation were investigated using the polymerase chain reaction to screen for EBV-DNA. In 13 of 47 samples (27.7%) swabbed from the uterine cervix, EBV-DNA could be detected. Similarly, 6 of 45 samples (13.3%) scraped from the sulcus coronarius contained EBV-DNA. Our study shows that the female genital tract and likewise the male genital tract can subclinically harbor EBV. These findings suggest i) that in addition to the oral cavity, the female and the male genital tract may be a reservoir for EBV and ii) that sexual transmission of this virus associated with an epidemiology different from that of oral infection may be possible.

Base Sequence↗
PubMed1992

General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions.

We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV types X [HPV X]) in cervical smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n = 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types.

Amino Acid Sequence↗
PubMed1992

Repeated evaluation of human papillomavirus 16 status in cervical swabs of young women with a history of normal Papanicolaou smears.

This study was designed to compare single-point prevalence estimates with a cumulative prevalence estimate of human papillomavirus (HPV) type 16 in cervical smears. The influence of the menstrual cycle and the long-term effect of HPV 16 positivity on the development of cervical intraepithelial neoplasia (CIN) were monitored. We examined 21 women (median age 23.6 years) every 5 weeks for 1 year. All women had a history of negative Papanicolaou smears for at least 5 years before enrollment. Cervical swabs were collected at each visit for Papanicolaou smears and HPV 16 detection by the polymerase chain reaction. Five years after completion of the last visit, self-reported information regarding cervical neoplasia was obtained. Human papillomavirus type 16 point-prevalence estimates per visit varied between 14.3-33.3%. The cumulative prevalence was 66.7%; 14 women were positive at least once and seven women were continuously negative for HPV 16. Detection of HPV 16 was significantly higher in the luteal phase. Repeated positivity for HPV 16 by Southern blot and polymerase chain reaction preceded and accompanied CIN in one patient, whereas in the remaining patients, positivity for HPV 16 by polymerase chain reaction alone was not associated with CIN during a 5-year follow-up. Single-point measurements of HPV 16 in cervical smears by polymerase chain reaction are of limited value for assessment of an individual's HPV status. This should be kept in mind when HPV testing for screening programs is considered.

Adolescent↗
PubMed1992

Human papillomaviruses and genital cancer.

Human papillomaviruses (HPVs) are epitheliotropic agents which preferentially infect either mucosal surfaces or the skin. A subset of the mucosotropic HPVs, in particular HPV types 16 and 18, are strongly linked with various forms of genital carcinomas. The great majority of these tumours carry integrated HPV DNA sequences and constitutively express two early viral genes, E6 and E7. DNA transfection studies show that these same genes can co-operate to immortalise human epithelial cells in vitro and that immortalised cells subsequently acquire a malignant phenotype through additional cellular genetic changes. Cell lines established from HPV-positive tumours also express E6 and E7, upon which continued tumour cell proliferation appears to depend. The development of molecular and serological markers specific for 'high risk' HPV types should allow the natural history of infection with these viruses, and their role in tumour development, to be better understood.

Cell Transformation, Neoplastic↗
PubMed1991

Comparison of ELISA and western blotting for human papillomavirus type 16 E7 antibody determination.

A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35.2%) than in the other individuals (six of 106, 5.7%). Ten children's (1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.

Adult↗
PubMed1991

Detection of Epstein-Barr virus-DNA in tongue epithelium of human immunodeficiency virus-infected patients.

Oral hairy leukoplakia is a lesion on the lateral part of the tongue that contains replicating Epstein-Barr virus (EBV) and presages progression from human immunodeficiency virus (HIV) infection to AIDS. To clarify the role of EBV in the development of the lesions, we used filter in situ DNA hybridization to determine the prevalence of EBV and of human papillomavirus (HPV) in epithelial cells obtained on swabs from the tongue of HIV-infected patients who had hairy leukoplakia, HIV-infected patients who did not have hairy leukoplakia, and healthy uninfected control persons. In samples collected from the 35 uninfected control persons, EBV DNA could not be detected except at low concentrations in three people. In contrast, all but one of the samples from 11 HIV-infected patients who had hairy leukoplakia contained EBV DNA. Of greatest interest, in 19 of 32 HIV-infected patients who had no signs of hairy leukoplakia, EBV DNA was also detected on the epithelium of the tongue. DNA filter in situ hybridization for the detection of HPV serotypes 6, 11, 16, and 18 in all cases yielded negative results. Statistical analysis showed that the presence of EBV DNA was significantly correlated with the clinical status of the HIV-infected persons, as determined by Walter Reed staging classification, whereas hairy leukoplakia was not. It is concluded that detection of EBV DNA in oral epithelium may be an earlier and more powerful predictor of progression to AIDS than is hairy leukoplakia.

DNA, Viral↗
PubMed1991

Human papillomavirus type 18 E6*, E6, and E7 protein synthesis in cell-free translation systems and comparison of E6 and E7 in vitro translation products to proteins immunoprecipitated from human epithelial cells.

Expression of the E6 and E7 transforming genes of human papillomavirus type 18 (HPV18) occurs via structurally bicistronic mRNAs in which the downstream open reading frame (ORF) E7 is preceded either by the full-length ORF E6 or by a spliced ORF, E6*. We have used in vitro transcription and translation of HPV18 cDNAs in order to analyze the synthesis of E6*, E6, and E7 proteins and to compare the E6 and E7 in vitro translation products with the authentic proteins immunoprecipitated from cervical cancer cells. In wheat germ extract, in vitro translation resulted in the production of all three proteins, E6*, E6, and E7. In rabbit reticulocyte lysate, however, only the E6 and E7 proteins were produced. The lack of E6* protein was due neither to template RNA degradation nor to an inhibitory influence of the RNA 5' leader sequences, thus indicating the possibility of either inhibition of synthesis or degradation of E6* protein in reticulocyte lysate. The E7 protein was synthesized from both E6*-E7 and E6-E7 RNAs. In vitro-synthesized and authentic HPV18 E7 proteins revealed identical electrophoretic mobilities in two-dimensional gel electrophoresis, thus indicating similar modifications. By using a monoclonal antibody against the N terminus of HPV18 E6* and E6, an 18-kDa protein was detected not only in HPV18-positive but also in HPV18-negative epithelial cells. The 18-kDa proteins and the in vitro-synthesized HPV18 E6 protein exhibited comparable electrophoretic characteristics in two-dimensional gels. These results suggest the possible existence of a cellular protein related to HPV18 E6.

Cell-Free System↗
PubMed1990

Growth-regulating functions of human papillomavirus early gene products in cervical cancer cells acting dominant over enhanced epidermal growth factor receptor expression.

Squamous cell carcinomas of the human anogenital tract are usually associated with infection of specific types of human papillomaviruses (HPV 16, 18, 31, 33, 35). The intracellular concentration of human papillomavirus early gene products E6 and E7 has been directly linked to the proliferative capacity of cervical cancer cells. Since the expression rate of epidermal growth factor receptor correlates to growth properties in squamous carcinoma cell lines, it has been presumed that human papillomavirus early genes influence cell growth via enhanced epidermal growth factor receptor expression. This hypothesis implies that growth regulation by epidermal growth factor receptor overexpression dominates over a growth-regulatory influence of human papillomavirus early gene products in squamous carcinoma cells. To test this hypothesis epidermal growth factor receptor expression was analyzed in various clones of the C4-1 cervical cancer cell line which, upon dexamethasone treatment, express either increased or decreased levels of human papillomavirus 18 early gene products. In C4-1 clones expressing reduced levels of viral E6/E7 gene products upon glucocorticoid treatment expression of epidermal growth factor receptor was the same as in those clones displaying increased levels of papillomavirus proteins under identical culture conditions. The growth rate of the cells correlated with the level of viral gene products rather than with the expression of epidermal growth factor receptor. These findings suggest that unregulated overexpression of epidermal growth factor receptor is not the dominant mechanism of growth control in papillomavirus-positive carcinoma cells. Other, yet unknown pathways associated with papillomavirus early genes are essentially involved in growth control mechanisms of human cervical cancer cells.

Carcinoma, Squamous Cell↗
PubMed1990

Identification of seroreactive regions of the human papillomavirus type 16 protein E4, E6, E7 and L1.

Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid. Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants. Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and L1 (three regions) open reading frames could be found by this approach. Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids. In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum. Using an approach to predict 'receptor-like' regions within the respective proteins, five of the seven regions were also identified. From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA.

Amino Acid Sequence↗
PubMed1989

Antibodies against the human papillomavirus type 16 early proteins in human sera: correlation of anti-E7 reactivity with cervical cancer.

By Western blot technique, 519 samples of human sera were tested for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E4 and E7 that had been expressed in Escherichia coli as fusion proteins. Sera were obtained from patients attending the University hospitals for reasons unrelated to HPV infections (controls), from patients with HPV-associated lesions, as well as from patients suffering from cervical cancer. Within the control population, 18.1% of them had antibodies that reacted with the E4 protein, and 3.9% of them had antibodies that reacted with the E7 protein. No sex-specific difference in the antibody prevalence was observed. The highest proportion of anti-E4 antibody-positive individuals (40.7%) was observed in the age group between 11 and 20 years. The frequency of anti-E4-positive sera was threefold higher in patients with HPV-associated genital lesions than that in age-matched controls. Antibodies against the HPV16 E7 protein were found 14 times more frequently in patients with cervical cancer, compared with age- and sex-matched controls (P less than .00001). From these data, we concluded that anti-E4 antibodies may be correlated with virus replication and that anti-E7 antibodies may represent a marker for cervical cancer development.

Adolescent↗
PubMed1989

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus.

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.

Animals↗