Prospective study on the relationship between cervical neoplasia and herpes simplex type-2 virus. II. Herpes simplex type-2 antibody presence in sera taken at enrollment.

Sera obtained at enrollment in the study from patients suffering from moderate to sever dysplasia (cervical intraepithelial neoplasia grade II), carcinoma in situ (cervical intraepithelial neoplasia grade III) and invasive carcinoma, or developing any of these conditions in the course of the prospective study, and from control subjects, were examined for herpes simplex type-2 (HSV-2) antibody presence. The controls were matched with the patients by age, age at first intercourse, number of sexual partners, smoking habits and history of diathermoelectrocoagulation of the ectopic epithelium and transformation zone of cervix. Only those subjects were selected as controls who remained free of pathological colposcopical and cytological findings throughout the observation period, i.e. for at least 4 years after their serum sample was obtained. The microneutralization test (MNT) and type-2-specific solid-phase radioimmunoassay (SPRIA) were used as serological tests. No difference in the prevalence of HSV-2 antibody between the patients and controls was revealed by either test. Various combinations of the results from the two tests also failed to show any difference between patients and controls. Moreover, no significant differences were observed in the prevalence of HSV-2 antibody between patients suffering from the various pathological conditions and those diagnosed at enrollment and later in the course of the study. These results do not provide any support for the hypothesis of the involvement of HSV-2 in cervical neoplasia.

Presence of Epstein-Barr virus DNA in tonsillar tissues.

Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.

Production and some properties of neutralizing antigens of herpes simplex virus.

Human diploid cells (LEP) infected with herpes simplex virus (HSV) type 1 were extracted by treatment with Nonidet P-40. The content of antigens reactive with neutralizing antibody in the clarified extract was measured by the blocking test. Influence of various factors on the production of the blocking antigens (BA) was tested. Highest BA titres in LEP cells were achieved at a multiplicity of infection (m.o.i.) of 0.5 TCD50/cell, medium 199 and 22 to 29 hours incubation at 37 degrees C. The use of resting cultures or the presence of cytosine arabinoside in medium reduced the BA production. The activity of BA was reduced by repeated freezing and thawing. The antigen was stable at -20 and -70 degrees C. One hour heating at 60 degrees C resulted in a marked decrease of the antigen titre. BA was resistant to formalin; formalin treatment did not influence its thermosensitivity.

Biochemical characteristics of collagen produced by long term cultivated amniotic fluid cells.

The synthesis of the most common type I of collagen was proven in human amniotic fluid cell culture with fibroblast morphology in the cells as well as in the culture medium. Mesenchymal origin of long term cultivated amniotic fluid cells is indicated and the possibility of prenatal investigation of hereditary disorders of connective tissue is pointed out.

Thymidine-kinase in cytomegalovirus infected cells.

In human diploid fibroblast LEP cells infected with AD169 strain of human cytomegalovirus (CMV) a sharp increase of cytosol thymidine kinase activity was observed. The properties of the cytosol enzymes from infected and non-infected cells were compared. No significant differences between the enzymes from infected and control cells were observed in substrate specificity, pH dependence, thermostability and relative electrophoretic mobility. Human sera containing high titres of CMV complement-fixing antibodies did not neutralize the enzyme from infected cells. It is concluded from these results that the increase of cytosol thymidinekinase activity in CMV-infected cells was due to an enhancement of cellular thymidine kinase.

Creatinephosphokinase in human diploid cell lines.

The activity of CPK was proved in cultivated cells from amniotic fluid cells, thymus, spleen, pericardium, umbilical cord, skin and muscle. The activity in these strains did not significantly differ. The average value is 224 plus or minus 68 i.u. CPK/mg N. No differences of activity were disclosed between embryonal, foetal and children cultures. The activity of CPK in post mortem cultures was not altered. The relationship between collagen synthesis and CPK activity is suggested. The evidence of CPK activity in amniotic fluid cells corresponding to the activity in other foetal cultures and the whole sample under investigation opens the way to the prenatal investigation of pathophysiology and inborn errors of the metabolism of this enzyme.

Biosynthesis of collagen during the life cycle of human diploid cell lines.

In the course of the active phase of growth collagen synthesis appears relatively stable and decreases when the culture passes into the degenerative phase III. The increase of cell nitrogen indicates the increasing accumulation of cell proteins with their aging in vitro. The ratio of soluble to insoluble collagen is relatively constant in fibroblast cultures from different tissues, periods of development during the active phase of growth. The increase of insoluble collagen in long term cultivated Marfan syndrome fibroblasts indicates the possible relationship between increasing stability of collagen and ageing of fibroblasts in vitro. Further studies confirmed the significant differences between control fibroblast and fibroblast derived from tissues of patients affected by Marfan syndrome. No differences were found in collagen synthesis and in the ratio of soluble to insoluble collagen in fibroblast strains from embryos, foetuses, children and young adults. The collagen synthesis and the ratio of soluble to insoluble collagen does not differ in fibroblast cultures derived from or post-natal period of life. The collagen synthesis in amniotic fluid cells was confirmed by analysis of cell proteins and proteins produced into the cultivation medium. The ultrastructural analysis of these cells reveals formation of intracellular fibrills and their growth in the extracellular space. The possibility of prenatal investigation of inborn errors of collagen metabolism is pointed out.

The nature of adenovirus persistence in human adenoid vegetations.

Ninety-seven adenoid vegetations (AV) originating from children aged 2 to 11 years were examined for the presence of adenoviruses. No infectious virus was detected in cell-free homogenates. However, adenovirus was recovered in 30 instances from either fragment or trypsinized cell cultures, or both, of the same tissues. The viruses belonged to types 1, 2, 5 and 6. It was determined by the infectious center assay that the frequency of virus-producing cells in different AV varied between 1 of every 10(5) cells to 1 of every 10(7) cells. Cells reactive with hamster sera containing antibody against the adenovirus early ("T") antigen and with rabbit sera containing antibody against the virus structural antigens were detected in cell smears from trypsinized virus-positive AV. The frequency of positive cells was very low. Nearly 80 per cent of extracts from virus-positive AV contained substances neutralizing the homotypic viruses. Adenovirus-neutralizing substances were only rarely detected in extracts from virus-negative AV. The neutralizing substances could be removed from the extracts by absorbtion with concentrates of the homotypic viruses. Most of the subjects from whom AV containing neutralizing substances were obtained, possessed high levels of homotypic serum antibodies.