The efficacy and safety of Skinner herpes simplex vaccine towards modulation of herpes genitalis; report of a prospective double-blind placebo-controlled trial.

A randomised, placebo-controlled, multi-centre trial of intracellular subunit herpes simplex virus (HSV) type 1 vaccine NFU.Ac.HSV-1(S-)MRC (Skinner vaccine) was conducted at three medical centres in the United States. Subjects with documented herpes genitalis of at least 1-year duration and a history of six or more genital HSV recurrences in the 12 months prior to study entry were randomised to receive vaccine or placebo at 0, 1 and 2 months. Vaccination induced significant neutralising, enzyme-linked immunosorbent assay and lymphocyte transformation response to HSV-1 antigen. The frequency of recurrences was reduced in the vaccinated female patients at both 3 and 6 months following vaccination with an overall reduction in patients of both sexes which did not reach statistical significance. Recurrence severity was reduced as measured by decreased number of lesions and associated symptoms per recurrence (P = 0.04). The data suggest that clinical manifestations of latent HSV genital infection may be modified by therapeutic immunisation.

All-trans-retinoic acid inhibits the proliferation of cell lines derived from human cervical neoplasia.

The purpose of this study was to determine whether all-trans-retinoic acid (RA) can inhibit the growth of cervical neoplastic cells by inducing differentiation or by increasing the secretion of transforming growth factor-beta (TGF-beta). Normal and HPV DNA-positive cervical cells (2 cell lines derived from cervical intraepithelial neoplasia (CIN), 2 HPV DNA-transfected cell lines, and 6 cervical carcinoma cell lines) were treated with RA (1 to 1000 nM), and both total viable cell count and [3H]thymidine incorporation were used to evaluate proliferation. In vitro differentiation was evaluated in organotypic (collagen gel raft) cultures with hematoxylin/eosin staining, and using specific immunostaining for fillagrin and cytokeratin 10. TGF-beta 1 and TGF-beta 2 secretion were measured with specific SELISAs. One-way analysis of variance and t tests were performed. RA causes a dose-dependent (P<0.05) growth arrest of comparable magnitude in normal ectocervical cells, in HPV DNA-transfected cell lines, in CIN-derived cell lines, and in four of six carcinoma cell lines. Endocervical cells and two carcinoma cell lines are unaffected. In vitro differentiation is decreased in CIN cells and is unchanged in carcinomas treated with RA as compared to control. Secretion of either TGF-beta 1 or TGF-beta 2 is significantly increased (P<0.05) in response to RA, both in RA-sensitive and in RA-resistant cells. RA induces growth inhibition in cervical neoplastic cell lines, including cervical carcinoma cells. This does not appear to be the result of increased differentiation or of increased TGF-beta secretion.

Interferon-alpha and all-trans-retinoic acid reversibly inhibit the in vitro proliferation of cell lines derived from cervical cancers.

Interferon and retinoic acid are active agents for the treatment of cervical cancer, but their mechanisms of action are unclear. Results of [3H]thymidine uptake assays showed that exposure to pharmacologic concentrations of interferon-alpha (IFN-alpha) and all-trans-retinoic acid (RA) for 72 hr inhibited growth of the cervical cancer cell lines ME-180, 283, SiHa, C33-A, 621, CaSki, HeLa, and B132, CaSki and SiHa cells continuously exposed to IFN-alpha or RA or both for 9 days developed resistance to growth inhibition, and growth resumed at a rate comparable to control after removal of agents. Similar assays showed no significant difference in effects of RA and its cis isomer. Assays for lactate dehydrogenase release revealed no significant lysis of any cell line following exposure to IFN-alpha, RA, or their combination. In organotypic culture, cells grew in a pattern histologically similar to carcinoma in situ, and exposure to IFN-alpha and RA for 14 days yielded no change in this pattern. Immunohistochemical analysis showed no change in cytokeratin expression by cells in organotypic or monolayer culture. The major in vitro effect of IFN-alpha and RA on cervical cancer cell lines appears to be reversible inhibition of proliferation.

Differential response of cervical intraepithelial and cervical carcinoma cell lines to transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent inhibitor of epithelial cell proliferation. It has been proposed that loss of sensitivity to growth inhibition by TGF-beta 1 may be an important step in the development of cervical carcinoma, but it remains unclear whether this represents an early or a late event. We compared the sensitivity to TGF-beta 1 of nontumorigenic human papillomavirus deoxyribonucleic acid (HPV DNA)-positive cell lines derived from cervical intraepithelial neoplasia (CIN), of newly established cervical carcinoma cell lines, of nontumorigenic HPV DNA-transfected cervical cell lines, and of normal ectocervical cells. There is a dose-dependent inhibition of DNA synthesis by TGF-beta 1 in the CIN cell lines and the HPV DNA-transfected cell lines. The carcinoma cell lines are resistant to the growth inhibitory effects of TGF-beta 1. The CIN cell lines are significantly more sensitive than the carcinoma cell lines (P < 0.001), but significantly less sensitive than normal cervical cells (P < 0.05). A CIN cell line which contains HPV 31b DNA is more sensitive to TGF-beta 1 at early passage than at late passage (P < 0.05). There are no differences in the sensitivity to the growth inhibitory effects of TGF-beta 1 between subclones of this cell line that have different episomal HPV DNA content, population-doubling time, or differentiation characteristics. Both normal and abnormal cervical epithelial cells were able to secrete latent TGF-beta 1 or TGF-beta 2. We conclude that resistance to growth inhibition by TGF-beta 1 is likely to be a late event in the development of cervical carcinoma; it is not the mere consequence of immortalization by HPV genes acquired following transfection in vitro or infection in vivo.

Growth and differentiation of human papillomavirus type 31b positive human cervical cell lines.

Human papillomavirus (HPV) containing cell lines derived from human cervical intraepithelial neoplasia (CIN) can offer valuable insights into the role of HPV in cervical neoplasia and can help in the understanding of the cellular changes that fuel the progression toward malignancy. We describe the growth and differentiation properties of an epithelial cell line established from a CIN I lesion. The cell line, designated as CIN 612, contains predominantly episomal copies of HPV 31b deoxyribonucleic acid (DNA). In vitro differentiation in a collagen raft system, growth characteristics, and episomal HPV DNA content of the CIN 612 cell line and two of its subclones were analyzed. Early passage CIN 612 cells differentiate in a manner which resembles the original low-grade intraepithelial lesion. On further passage, these cells exhibit increasingly poor differentiation in vitro. Two subclones with different growth characteristics and morphology were identified. A more rapidly growing, poorly differentiated subclone contains less episomal copies of viral DNA compared to a slower growing and better-differentiated subclone. Cell populations with similar growth characteristics yet different ploidy were observed. The CIN 612 cell line with its episomal copies of viral DNA shows promise for the development of an in vitro culture system for HPV 31b. The isolation of subclones with consistently different growth and differentiation properties in vitro creates an opportunity to identify the cellular events that lead to the progression from low-grade to high-grade cervical neoplasia.

Amplification of human papillomavirus genomes in vitro is dependent on epithelial differentiation.

Human papillomaviruses (HPVs) infect squamous epithelium and establish their genomes as episomes in proliferating basal cells. As infected cells differentiate, the viral DNA is amplified to high copy number and infectious virus is produced. Viral production has not yet been observed in vitro due to the inability of standard culture methods to duplicate most stages of epithelial differentiation. In this study, we have examined a cell line derived from a low-grade cervical lesion and found that it contained episomal copies of an HPV-31 subtype, HPV-31b, at approximately 50 copies per cell. When allowed to stratify at the air-liquid interface of in vitro raft cultures, this cell line differentiates in a manner which histologically resembles a low-grade cervical lesion in vivo. We have observed the amplification of HPV-31b genomes in distinct foci in the upper portion of the in vitro-stratified epithelium similar to that found in productive HPV infections in vivo. Although transcripts from the late region of HPV-31b were also detected specifically in stratified raft cultures, no capsid protein was found. These studies duplicate one important aspect of a productive HPV infection in vitro, the differentiation-dependent amplification of papillomavirus genomes.

Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix.

The human uterine cervix consists of an endocervical canal lined with a single layer of columnar mucus-secreting cells and an outer ectocervix covered by a stratified squamous epithelium. We report here the culture of human endocervical epithelial cells (HEnE) and human ectocervical epithelial cells (HEcE) in serum-free medium (KGM). Both HEnE and HEcE cultures were composed of keratinocytelike cells which formed desmosomal contacts and stratified in the presence of high concentrations of calcium ions. Cells with a pleomorphic epithelial morphology were observed in HEnE cultures, but not in HEcE cultures. Keratin 18, which is characteristic of endocervix in vivo, was detected by indirect immunofluorescent staining in all HEnE cells but was never detected in cultured HEcE. HEcE expressed keratin 13 which is characteristic of ectocervix in vivo. Although keratin 13 was never detected in primary HEnE cultures, it was expressed in passaged HEnE cultures grown in medium with high concentrations of calcium and in late passage HEnE cultures. HEnE underwent an average of 15.1 population doublings during serial culture. Mean colony-forming efficiency during Passages 2 to 3 was 14.7% and mean population doubling time was 17.8 h. HEcE cultures underwent significantly more population doublings (29.0) than HEnE cultures, whereas colony-forming efficiencies and doubling times were similar to those determined for HEnE. HEnE and HEcE cells may be useful in developing in vitro models of cervical squamous metaplasia and for exploring the interactions between target cell differentiation, carcinogens, and papillomaviruses in the development of cervical neoplasia.