Immunoglobulin G responses against human papillomavirus type 16 virus-like particles in a prospective nonintervention cohort study of women with cervical intraepithelial neoplasia.

BACKGROUND: Infection with cancer-linked human papillomavirus (HPV) types such as HPV type 16 (HPV16) is the most important risk factor in the development of cervical cancer. It has been shown that immunoglobulin G (IgG) antibody responses against HPV16 virus-like particles (VLPs) are specifically associated with genital HPV16 infection. PURPOSE: The aim of this study was to determine the temporal relationships between the presence of HPV16 VLP-specific IgGs, HPV16 infection patterns, and the course of premalignant cervical disease. METHODS: Plasma samples from 133 women who had been diagnosed originally with mild to moderate cervical dyskaryosis and enrolled in a prospective non-intervention cohort study conducted in Amsterdam, The Netherlands, from 1991 through 1996 were analyzed for the presence of HPV16 VLP-specific IgGs by use of an enzyme-linked immunosorbent assay. A detailed analysis was performed on 43 women with different HPV16 infection patterns during a follow-up period of 10-34 months. Progression or regression of cervical intraepithelial neoplasia (CIN) lesions was monitored by cytologic and colposcopic testing at intervals of 3-4 months. HPV typing in cervical smears was performed by use of a polymerase chain reaction-based assay. Statistical analysis of the serologic data was performed by use of the Mann-Whitney U test or 2 x 2 table analyses. RESULTS: The presence of HPV16 VLP-specific IgGs in the plasma of the patients was found to be associated with the presence of HPV16 DNA in the cervical smear. Significantly higher proportions of patients with persistent HPV16 infections (i.e., who were polymerase chain reaction positive in three to 11 consecutive tests) than of patients with cleared HPV16 infections were found to be positive for the presence of HPV16 VLP-specific IgGs (18 [69.2%] of 26 versus nine [28.1%] of 32, respectively; P = .003). HPV16 VLP-specific IgGs were consistently detected in all women (n = 11) who were persistently HPV16 DNA positive during follow-up and whose disease ultimately progressed to CIN III (histologically diagnosed severe dysplasia or carcinoma in situ). CONCLUSION: HPV16 VLP-specific IgG responses are present in the plasma of a majority of patients with persistent HPV16 infections and histologically confirmed high-grade lesions but only in a smaller subset of patients with cleared HPV16 infections and either normal cervical histology or low-grade CIN lesions. IMPLICATIONS: These results suggest that HPV16 VLP-specific antibodies are not responsible for the clearance of virally induced CIN lesions but that they might, in patients with persistent HPV16 infections, be indicative of an increased cervical cancer risk.

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.