Biomedical subjects

L Gissmann

Publications and source records attributed to L Gissmann.

At least 37 records · Page 2Linked to original sources

PubMed1998

Serologic response to the E4, E6, and E7 proteins of human papillomavirus type 16 in pregnant women.

OBJECTIVE: In a seroepidemiologic study the effects of pregnancy and other factors on humoral response to human papillomavirus type 16 infection were examined. STUDY DESIGN: Multiple serum samples were taken at 3-month intervals for 15 months from 77 pregnant and 85 nonpregnant women. Serologic response to human papillomavirus type 16 proteins was analyzed with a peptide-based enzyme-linked immunosorbent assay. RESULTS: Seroreactivity was higher in nonpregnant women than in pregnant women, suggesting a reduced humoral immune response against human papillomavirus infections during pregnancy. Among the pregnant women a twofold to threefold decrease in mean reactivity in the E4 protein-based assay was detected between early gestation and delivery. The presence of human papillomavirus type 16 or 18 deoxyribonucleic acid was significantly associated with reactivity to the E6 protein (p = 0.0005) and the E4 protein (p = 0.06). Reactivity to the E4 protein also correlated with an abnormal Papanicolaou smear. CONCLUSIONS: The observation of changes in humoral response to genital human papillomavirus infections during pregnancy warrants further investigation with highly seroreactive assays.

Amino Acid Sequence↗

PubMed1998

Association of rare human papillomavirus types with genital premalignant and malignant lesions.

Due to the limited number of reports concerning their association with particular dysplastic and neoplastic lesions, the oncogenic potential of so-called rare or novel human papillomavirus (HPV) types is still unclear. Cytologic smears or biopsy specimens from 538 patients were analyzed for dysplastic or neoplastic lesions and HPV infection. The HPV detection and typing system utilized allowed identification of all mucosal HPVs amplifiable by L1 polymerase chain reaction. Considering only patients infected with a single HPV type (n = 329), rare or novel HPVs (HPV-59, HPV-61, HPV-62, HPV-66, HPV-70, HPV-73, MM4, MM7, MM8, CP6108, and CP8304) were detected in 28% of normal specimens (n = 46), none of condylomatous lesions (n = 44), 12% of low-grade squamous intraepithelial lesions (SILs) (n = 42), 8% of high-grade SILs (n = 142), and 4% of cervical cancers (n = 54). Prevalence and oncogenic potential of distinct rare HPV types seems to be higher than previously assumed.

Condylomata Acuminata↗

PubMed1998

Calcium is required in reassembly of bovine papillomavirus in vitro.

Papillomaviruses are small DNA viruses which infect and induce benign warts and sometimes malignant tumours in the epithelium of the skin or mucosa. The viruses do not replicate in conventional tissue culture systems and little is known about the requirements for virus assembly. We investigated the effect of ethylene glycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) and dithiothreitol (DTT) treatment on the stability of bovine papillomavirus type 1 (BPV-1) particles in vitro. Removal of calcium ions by 11 mM EGTA at pH 8.0 together with reduction of disulfide bonds by 15 mM DTT destabilized BPV particles. Electron microscopy examination of treated particles showed that the BPV particles had been disrupted to capsomeres. Addition of exogenous calcium ions to the disruption buffer prevented virus destabilization. Adding calcium to the disrupted BPV particles resulted in the reassembly of disrupted particles. The reassembled particles were morphologically similar to intact BPV virions. We further quantified the efficiency of reassembly by focus formation assay. We recorded 500-fold less infectivity for reassembled BPV and 4-fold less haemagglutination activity compared to untreated BPV, pointing towards a decrease in the amount of reassembled particles recovered.

Animals↗

PubMed1998

Antibodies against early proteins of human papillomaviruses as diagnostic markers for invasive cervical cancer.

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.

Adult↗

PubMed1997

Chimeric papillomavirus-like particles.

We have constructed chimeric papillomavirus-like particles (CVLPs) by replacing the 34-carboxy-terminal amino acids of the HPV 16 L1 protein with various parts of the HPV 16 E7 protein. Chimeric proteins were expressed by recombinant baculoviruses and analyzed by electron microscopy for their ability to assemble into virus capsids. We were able to produce CVLPs in high efficiencies with inserts of up to 60 amino acids. CVLPs are able to induce a neutralizing antibody response, assayed by inhibition of hemagglutination of mouse erythrocytes. CVLPs are interacting with the putative receptor for papillomaviruses as they were shown to hemagglutinate mouse red blood cells and bind to and penetrate cells in vitro. As CVLPs follow a similar intracellular pathway as observed earlier for BPV VLPs, we speculate that CVLPs can be used to deliver peptides into mammalian cells in vitro and in vivo, possibly reaching the pathway for MHC class I presentation.

Amino Acid Sequence↗

PubMed1997

Sequence close to the N-terminus of L2 protein is displayed on the surface of bovine papillomavirus type 1 virions.

The bovine papillomavirus type 1 (BPV1) L2 protein purified from Escherichia coli was used as an antigen to produce monoclonal antibodies (MAbs). A total of 26 individual clones which recognized the BPV1 L2 protein were obtained. Using infectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles. Binding of the MAbs to BPV1 was confirmed by immunoelectron microscopy. A set of 92 13-mer peptides overlapping by 8 amino acids spanning the entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies. Seventeen linear epitopes were identified. Our results revealed that a sequence toward the N-terminus of the L2 protein (aa 61-123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid. Although the polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity for the 3 L2-specific monoclonal antibodies which bound to the BPV1 particles. This suggests that extra binding sites may be needed for neutralization. This study prompted us to propose a model about how L1 and L2 proteins may interact during infectious papillomavirus assembly.

Amino Acid Sequence↗

PubMed1997

Interferon-gamma enhances susceptibility of cervical cancer cells to lysis by tumor-specific cytotoxic T cells.

Recently we have demonstrated that tumor-specific cytotoxic T lymphocytes (CTLs) can be activated by cervical carcinoma cells expressing the costimulatory molecule CD80, which may be used as a therapeutic vaccine for patients with cervical cancer. For activated CTLs to be effective, appropriate amounts of MHC class I expression are required on target tumor cells. In this study, we found that some cervical carcinoma cells expressed only low levels of MHC class I and adhesion molecules such as CD54. We further demonstrated that tumor cells (CaSki and SiHa) expressing low levels of MHC class I were more resistant to lysis by specific CTLs than tumor cells (HeLa) expressing high levels of MHC class I. Treatment of CaSki or SiHa cells with interferon-gamma resulted in an increased expression of MHC class I, MHC class II, and CD54. Expression of CD58 and CD80 was not up-regulated or induced. Treatment of the tumor cells with interferon-gamma significantly enhanced the lysis of the tumor cells by specific CTLs which had been activated by the respective CD80-expressing tumor cells. The enhancement of cytolysis could be blocked by monoclonal antibodies to MHC class I and CD54, but not by that to MHC class II. Furthermore, we found that interferon-gamma induced apoptosis in cervical carcinoma cells but not in tumor-specific CTLs.

B7-1 Antigen↗

PubMed1997

Regulation of human papillomavirus type 18 in vivo: effects of estrogen and progesterone in transgenic mice.

We examined the in vivo effect of estrogen, progesterone, RU 486, and pregnancy on the upstream regulatory region (URR) of human papillomavirus (HPV) 18 transgenic mice. The mice contain the bacterial reporter beta-galactosidase gene under control of the HPV 18 URR. Pregnant transgenic mice were sacrificed on various days of gestation and the level of URR activation was determined. Another group of female transgenic mice was ovariectomized at 4 to 6 weeks of age. Pellets of estradiol, progesterone, progesterone + RU 486, or placebo were implanted 1 to 2 weeks after ovariectomy. Mice were sacrificed after pellet implantation to examine acute and chronic effects. Marked increases in URR activation during pregnancy were observed. Progesterone was found to activate the URR acutely. Significantly higher activation was demonstrated at 24 hr in the progesterone group compared to placebo (P < 0.01). Activation with progesterone at 24 hr was significantly higher than at any other time point (P < 0.001). A trend toward decreasing activation over time was demonstrated in the progesterone group (r = -0.87, P = 0.0001). RU 486 does not block the activation of progesterone in our model. Estradiol activates the URR acutely compared to placebo (P = 0.034). This in vivo model demonstrates activation of the URR in response to exogenous estrogen, progesterone, and pregnancy. These data may have clinical implications for women who harbor high-risk HPV.

Animals↗

PubMed1997

A general purification protocol for E7 proteins from "high- and low-risk" human papillomavirus types expressed in the yeast Schizosaccharomyces pombe.

A purification protocol was developed to obtain human papillomavirus (HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pombe. Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11.

Animals↗

PubMed1997

Specificity of human cytotoxic T lymphocytes induced by a human papillomavirus type 16 E7-derived peptide.

In order to establish tumour-specific cytotoxic T lymphocyte (CTL) cell lines, T cells from a human papillomavirus (HPV) type 16-positive patient with a cervical carcinoma in situ and from a healthy volunteer were stimulated in vitro with autologous dendritic cells loaded with peptides derived from the viral transforming proteins E6 and E7 and corresponding to potential HLA-A*0201-restricted T cell epitopes. From each donor a small number of low-affinity CTL lines against the peptide E7/86-93 was obtained, which specifically lysed HLA-A*0201-expressing B-lymphocytes (cell line 721) loaded with this peptide. Cytotoxicity was also observed against two HLA-A*0201-E7-positive epithelial cell lines, the cervical carcinoma cell line CaSki and the HPV-16-immortalized foreskin-keratinocyte cell line HPK IA. However, since none of the CTL recognized both cell lines, and E7-expressing 721 transfectants were never lysed, it was concluded that the reactivity against CaSki and HPK IA cells was due to cross-reactivity on allogeneic HLA molecules rather than to E7 recognition, which emphasizes that the specificity of tumour cell lysis by peptide-induced CTL has to be interpreted with caution.

Carcinoma in Situ↗

PubMed1997

Detection of human papillomavirus type 16 DNA and of antibodies to human papillomavirus type 16 proteins in children.

We have measured markers of human papillomavirus type 16 (HPV 16) infection in children (1-10 years of age) who were hospitalized for reasons unrelated to papillomavirus infection. Genital and buccal swabs obtained from 79 children were tested for the presence of HPV 16 DNA by PCR. Low-level positivity was found in 34 donors, twice as often in oral than in genital swabs. There was no sex-specific difference, but there was a trend towards a higher positivity rate with young age. Serum antibodies (IgG) were measured by ELISA based on peptides derived from the HPV 16 early proteins E4 (one peptide), E6 (two peptides) or E7 (two peptides) in 75 children and by ELISA based on virus-like particles in 66 children. Low-positivity rates were found for E6 (5.1%), E7 (2.5%) or capsid proteins (1.5%), but 20.3% of the sera reacted with the E4-specific peptide. There was no correlation between sero-positivity and the detection of HPV 16 DNA. In those instances where HPV DNA positivity in young children represents true infection and not environmental contamination, we speculate that this infection is accompanied by low-level virus replication that does not induce a measurable antibody response. Reactivity to the E4 protein is likely due to cross-reacting antibodies directed either against E4 proteins of other HPV types or against unrelated antigens.

Antibodies, Viral↗

PubMed1996

Carboxyl terminus of bovine papillomavirus type-1 L1 protein is not required for capsid formation.

The papillomavirus major capsid protein L1 can assemble into capsids in vitro. To identify areas within the bovine papillomavirus type-1 L1 (BPV L1) protein that are important for virus assembly, we constructed a set of 24 baculovirus recombinants expressing BPV L1 deletion mutants that span the entire L1 open reading frame. Virus-like particle (VLP) formation of the L1 mutants was examined by electron microscopy. Wild-type (wt) BPV L1 expressed in recombinant baculovirus formed VLPs, while capsomeres and aggregates were seen for most of the mutants screened. However, the C-terminal truncation mutant, lacking the last 24 amino acids (delta C2), was observed to form VLPs (threefold more efficiently than wt BPV L1). This suggests that this C-terminal region of L1 protein is not critical for capsid formation. As capsids assembled from BPV L1 are able to agglutinate mouse red blood cells (RBC) by binding to a membrane protein, we tested the ability of the mutants to hemagglutinate mouse RBCs. Most aberrant capsids or aggregates derived from deletion mutants were unable to agglutinate the RBCs with the exception of deletion mutants delta 11 (aa 231-271), delta 14 (aa 291-331), delta 21 (aa 431-471), and the carboxyl-terminus truncation mutant delta C2.

Amino Acid Sequence↗

PubMed1996

Expression of CD80 enhances immunogenicity of cervical carcinoma cells in vitro.

Although cervical carcinoma cells may express the human papillomavirus oncoproteins E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. This failure may be due to a lack of expression of costimulatory molecules, such as CD80 (B7.1). To augment the immunogenicity of cervical carcinoma cells, we transfected human papillomavirus (HPV)-transformed cell lines, CaSki and HeLa, with the CD80 expression vector pBJ. Alloantigens on the tumor cells were used for the stimulation of peripheral blood lymphocytes (PBLs). Cocultivation of PBLs and tumor cells resulted in proliferation of CD4+ and CD8+ T lymphocyte subsets. CD80-expressing tumor cells induced proliferation of allogeneic PBLs two-to sixfold compared to control cell lines. Cocultivation of allogeneic PBLs with CD80-positive tumor cells for 3 weeks gave rise to cytotoxic T cells capable of lysing untransfected parental tumor cell lines. Our results demonstrate an immunostimulatory effect of CD80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

B7-1 Antigen↗

PubMed1996

Influence of human papillomavirus type 16 gene expression on in vitro differentiation of the human teratocarcinoma cell line 2102Ep.

Human papillomaviruses (HPVs) are known to infect human keratinocytes and cause alterations in epithelial differentiation. We showed in this study that expression of the HPV-16 genome was able to interfere with the in vitro differentiation of a human simple-epithelial cell type, the 2102Ep teratocarcinoma cell line. Stable HPV-16 genome-expressing 2102Ep cell lines were generated, and subsequent alterations in differentiation were analyzed in comparison with parental 2102Ep cells. We found that in 2102Ep cells phorbol ester-induced differentiation led to changes in the expression of SSEA antigens, whereas in HPV-transfected cell lines only minor changes were observed.

Antigens, Neoplasm↗

PubMed1996

The association of human papillomavirus type 16 E6 and E7 antibodies with stage of cervical cancer.

OBJECTIVE: Previous investigators have reported higher HPV type 16 antibody positivity among cervical cancer patients than among healthy women. The objective of this study was to determine the association of HPV 16 antibody levels with the stage of cervical cancer. METHODS: Pretreatment tumor biopsies and sera were obtained from 137 newly diagnosed cervical cancer patients residing in Mexico. Using peptide ELISA and radioimmunoprecipitation assay (RIPA), HPV 16 E6- and E7-specific antibodies were measured. RESULTS: By ELISA, elevated antibody titers to HPV 16 E6 and E7 were detected in 16.8 and 32.8% of the women, respectively. While sera positivity did not differ by disease stage, the mean absorbance in the E7-positive sera was 0.42, 0.62, 0.91, and 0.81 for stages I to IV, respectively. Using RIPA, anti-E6 and E7 positivity was demonstrated in 46.7 and 38.7% of the females, respectively. Although no difference across disease stage was detected for E6, increasing proportions of positivity to E7 with stage of disease was detected. The rates for increasing disease stage were 0.14, 0.37, 0.40, and 0.67. Sera from the 6-month postradiation follow-up examinations of a small group of patients demonstrated a statistically significant decrease in antibody positivity from pretreatment positivity to HPV 16 E6 (n = 14; P = 0.01) and HPV 16 E7 (n = 20; P = 0.0001) using ELISA. CONCLUSIONS: These data suggest that HPV 16 E7 antibody positivity may be associated with stage of cervical cancer. Such immune parameters may be applicable to disease staging, monitoring of recurrence and, perhaps, diagnosis. Further investigation into the relationship of HPV 16 E6 and E7 antibodies with stage of cervical cancer and response to therapy is warranted.

Adult↗

PubMed1996

Human papillomavirus type 16 (HPV 16) E7 and major histocompatibility complex (MHC) class I and II expression in human keratinocytes in culture.

The low expression of major histocompatibility complex (MHC) class I antigens on human papillomavirus (HPV)-infected cervical carcinoma cells may be responsible for an insufficient cytotoxic T cell response against these cells. To investigate in vitro whether the HPV type 16 early gene product E7 influences cell surface expression of MHC class I and II molecules the HPV negative keratinocyte cell line HaCaT was either stably transfected with the E7 gene or infected with E7-recombinant vaccinia viruses. No difference in MHC class I transcription was detected between E7-transfected and untransfected HaCaT cells. MHC class I cell surface expression as determined by FACS analysis was stronger in some of the transfectants and less intensive in others when compared to untransfected HaCaT cells. In wildtype as well as in E7-recombinant vaccinia virus infected HaCaT cells downregulation of MHC class I molecules on protein and transcriptional level was observed. The alterations in MHC class I expression were independent of the presence and amount of E7-specific transcripts. None of the transfectants or infected HaCaT cells had MHC class II molecules on their cell surface. Hence, our data did not show a correlation between HPV 16 E7 and MHC expression in vitro.

Cell Line↗

PubMed1996

The E7 protein of human papillomavirus (HPV) type 16 expressed by recombinant vaccinia virus can be used for detection of antibodies in sera from cervical cancer patients.

Sera from 128 Mexican cervical cancer patients (age 30-80; mean 53.6) and from 47 healthy women (age 25-69; mean 49.2) were investigated using a newly developed assay for the detection of serum antibodies to the human papillomavirus (HPV) type 16 early protein E7. This test (CIPA), based upon immunoprecipitation followed by Western blot analysis, uses the complete E7 protein expressed in HeLa cells infected with recombinant vaccinia virus. To determine the sensitivity and specificity of this assay, these results were compared with previous results of the same sera tested by enzyme-linked immunosorbent assay (ELISA; using synthetic peptides derived from HPV 16 E7) and radio-immunoprecipitation (RIPA) using in vitro translated HPV 16 E7 protein. CIPA (45% positives) demonstrated a significant increase in detection rate compared to the peptide-ELISA (30% positives; P = 0.014, chi2-test) and only a slight increase compared to RIPA (38% positives; P = 0.204, chi2-test). Based on the testing of sera from patients with HPV 16 DNA positive tumors the specificity and sensitivity of the CIPA were 0.98 and 0.59, respectively.

Adult↗

PubMed1996

The E6 variant proteins E6I-E6IV of human papillomavirus 16: expression in cell free systems and bacteria and study of their interaction with p53.

Several species of alternatively spliced mRNAs are transcribed from the E6 gene region of human papillomavirus (HPV) 16. These have the coding capacity for either the full length E6 of 151 amino acids (aa) or four truncated variants, E6I-E6IV, of 43-64 aa. As the first step to identify the putative E6 variants and their functions, we generated cDNAs corresponding to the various E6 open reading frames (ORF) and examined their expression employing in vitro transcription/translation systems and the bacterial pET system. In wheat germ extract, in vitro translation resulted in the production of all five proteins, E6 and E6I-E6IV. These proteins were also expressed as stable fusion proteins from the pET16b and pET17 x b vectors in Escherichia coli. Mobilites of the E6 variant proteins on SDS-acrylamide gels were consistent with their predicted sizes. The authenticity of the synthesized proteins was confirmed by immunoprecipitation with specific antibodies directed against epitopes in the N-terminal portion of E6 as well as antibodies raised against the individual variant proteins produced in E. coli. In rabbit reticulocyte lysate, however, only the full length E6 and the E6IV variant were synthesized. This could be due to inefficient translation as well as lower stability of the short variants. E6I-III, in reticulocyte lysate (RTL). The ability of the E6 variants to associate with p53 and target its proteolytic degradation in vitro, was examined in coimmunoprecipitation assays, using in vitro synthesized proteins and monoclonal antibodies to p53. Results of these assays indicated that only the full length E6 efficiently binds to and promotes the degradation of p53. The E6 variants E6I-E6IV, although able to associate with p53 at a low efficiency, were unable to target its degradation.

Amino Acid Sequence↗