Rubella virus carrier cultures derived from congenitally infected infants.

Spontaneous rubella carrier cultures derived from tissues of infants with congenital rubella were studied in an attempt to elucidate a possible mechanism for viral persistence observed in these infants. Chronically infected cells were found to have a reduced growth rate and the cultures appeared to have a shortened life span. The rubella carrier state was not dependent on serum inhibitors or rubella antibodies. Virtually every cell in the carrier population was found to be producing virus. The carrier cultures could not be cured by rubella antibodies. The rubella-infected cells were resistant to superinfection with vesicular stomatitis virus and herpes simplex virus but were susceptible to infection with echovirus 11. The replication of vesicular stomatitis virus was apparently blocked at an intracellular site, for the virus readily adsorbed to the chronically infected cells and entered into an eclipse phase; however no infectious virus developed. No evidence of interferon production by these cells could be obtained. It is postulated that clones of rubella-infected cells in vivo, with properties similar to those in carrier cultures developed in vitro from tissues of in utero infected infants, might explain the observed viral persistence noted in congenital rubella.

Abnormalities of in vitro lymphocyte responses during rubella virus infections.

In vitro rubella virus infections of lymphocytes from normal adult humans impaired their responsiveness to phytohemagglutinin (PHA) stimulations; a situation which seemed analogous to the PHA unresponsiveness of peripheral lymphocytes from babies with the congenital rubella syndrome. Such in vitro viral infection of normal cells also decreased the synthesis of normal nucleic acids and structural proteins, and abrogated the enhanced DNA synthesis induced by pokeweed and specific antigen stimulations. Furthermore, it was shown that live rubella virus, but not ultraviolet-irradiated virus, was necessary for the impaired mitogenic responses of normal leukocytes. These observations are interpreted to favor the view that the virus achieves its inhibitory effect on the action of mitogens by interference either directly or indirectly at an intracellular site. Such an action could reduce the functional potential of lymphocytes and impair their effectiveness as immunologically competent cells or as effectors in immunologic reactions.

Herpesvirus type 2: association with carcinoma of the cervix.

By utilizing the kinetics of neutralization, it was found that an antibody response to type 1 (oral) and type 2 (genital) herpesvirus infection could be independently measured and correlated with type of virus isolated from the patient. The presence of antibodies to type 1 and type 2 herpesvirus was examined in patients with carcinoma of the cervix, in matched controls, random controls, and patients with other types of malignancy. Antibodies to type 2 were found with greater frequency in patients with carcinoma of the cervix (83 percent) than in the other groups tested (0 to 20 percent). The data suggest an association of genital herpesvirus and carcinoma of the cervix.

Effect of amantadine hydrochloride on the response of human lymphocytes to phytohemagglutinin.

Amantadine hydrochloride (Symmetrel), which is an antiviral agent marketed for the prevention of infection by influenza virus, inhibits the mitogenic response of human lymphocytes stimulated with phytohemagglutinin. The concentrations which effectively inhibited the response to phytohemagglutinin were similar to those which maximally inhibit virus replication. The drug inhibited the mitogenic response without affecting the ability of phytohemagglutinin to agglutinate leukocytes. The data suggest that phytohemagglutinin, amantadine, and certain lipid-containing RNA viruses take part in cell-membrane interactions of common nature.

Antibodies to herpesvirus nonvirion antigens in squamous carcinomas.

Serums from tumor-bearing patients, cured patients, and normal subjects were examined for antibodies to the separated complement-fixing reactive components of nonvirion antigens of herpesvirus type 1 and type 2. The occurrence of antibodies to the antigens was similar in serums from tumor-bearing patients and cured patients. Antibodies to the antigens were observed among 21 of 24 (87 percent) cervical cancer cases, 44 of 49 (90 percent) laryngeal cancer cases, 15 of 24 (62 percent) cases of squamous cell carcinomas of the head and neck excluding the larynx, 2 of 24 (8 percent) nonsquamous cell cancer cases, and 3 of 51 (6 percent) normal subjects. By contrast, no differences were found in the titers of neutralizing antibodies to the virus in serums from laryngeal cancer patients and controls. The observations support an etiologic role of herpesviruses in cervical cancer and in laryngeal cancer, and possibly other squamous cell cancers of the head and neck.

Viral inhibition of lymphocyte response to phytohemagglutinin.

The response to phytohemagglutinin by lymphocytes from eight of fourteen patients with congenital rubella was inhibited, whereas that of lymphocytes from patients with other diseases was not. The response of normal lymphocytes infected in vitro was also inhibited. The results suggest that early association of lymphocytes with virus inhibits the function of the cell and contributes to persistent carrying of virus in congenital rubella. This phenomenon may be a means of detecting viruses not now recognizable by routine methods of tissue culture.

Antibody to herpes simplex virus type 2-induced nonstructural proteins in women with cervical cancer and in control groups.

Sera obtained from 15 patients with cervical cancer, 10 patients with breast cancer, and 15 control women, individually matched with the cervical cancer patients, were examined for antibodies to early proteins synthesized in herpes simplex virus type 2 (HSV-2)-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. The relative reactivity to a major early nonstructural protein (VP134) was used to compare these selected sera. The results obtained suggest that cervical cancer patients possess sera with a higher reactivity to VP134 than breast cancer patients or matched healthy women,and that serum reactivity is independent of the level of neutralizing antibodies to HSV-2.

Invasive cervical cancer and smoking in Latin America.

A case-control study of 667 patients with invasive squamous cell carcinoma of the cervix and 1,430 controls from four Latin American countries showed an age-adjusted relative risk (RR) of 1.2 [95% confidence interval (CI) = 1.0-1.4] for women who had ever smoked, with risk rising to 1.7 (95% CI, 0.8-3.6) for women who smoked greater than or equal to 30 cigarettes per day. The associations were practically eliminated after adjustment for the number of sexual partners and alcohol consumption, probably a surrogate for an unidentified life-style risk factor. Some excess risk persisted among women who smoked for extended periods (RR = 1.5 for greater than or equal to 40 yr), as well as those who began smoking at older ages (RR = 1.7 for greater than 30 yr), which suggests a late-stage effect. In addition, among women who tested positive for human papillomavirus (HPV) type 16 or 18 by filter in situ hybridization, there was an increased risk for women who had ever smoked and a dose-response relationship with the number of cigarettes smoked (adjusted RRs compared with HPV-negative nonsmokers = 5.0 for HPV-positive nonsmokers, 5.5 for less than 10 cigarettes/day, and 8.4 for greater than or equal to 10 cigarettes/day). In contrast, HPV-negative women had no increased risk associated with smoking. These results, from a high-incidence area where intensive smoking among women is still relatively rare, suggest that smoking has a limited effect on cervical cancer risk, possibly only among women with specific types of HPV.

Factors affecting the expression of carcinoembryonic antigen at the surface of cultured human colon carcinoma cells.

Factors affecting the expression of carcinoembryonic antigen (CEA) at the surface of in vitro human colon carcinoma cells were determined using 125I-labeled antibodies. Binding of specific anti-CEA antibodies resulted in polar redistribution of CEA, followed by endocytosis of most of the CEA-anti-CEA complexes. These processes were temperature and energy dependent. CEA removed from the tumor cell surface by antibody was totally replaced within 6 hr at 37 degrees, and the reexpression of CEA required protein synthesis. Examination of clonally derived subpopulations of various strains of human colon cancer cells indicated that control over the level of cell surface CEA expressed was genetically stable in vitro. CEA expression was enhanced in a low-CEA-producing strain by incorporating theophylline in the culture medium, and the inclusions of bromodeoxyuridine enhanced the expression of a high-CEA-producing strain. The kinetics of enhancement of CEA expression by these two drugs differed. These findings suggest that CEA expression may be controlled by more than a single gene function.

Reaction patterns of herpes simplex virus type 1 and type 2 proteins with sera of patients with uterine cervical carcinoma and matched controls.

Serum from 105 individuals with diagnosed uterine cervical cancer and 231 matched controls were examined for their ability to react with a large number of herpes simplex virus type 1 or type 2 (HSV-1, HSV-2) proteins. Radiolabeled HSV-1 or HSV-2 proteins were mixed with test serum and immune complexes were isolated with staphylococcal protein A. Viral proteins in the immune complexes were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. When the frequency of precipitation for cancer and control serum was calculated for each HSV-1 and HSV-2 protein, the results demonstrated that four HSV-1 and 11 HSV-2 proteins were precipitated more frequently by cases than by controls (p less than or equal to 0.05). However, since these results could be influenced by the presence or absence of HSV-2 specific antibodies as well as social, economic, and sexual history, the data were grouped and analyzed according to these parameters. This enabled all significant differences between case and control sera in the precipitation of HSV-1 or HSV-2 proteins to be abolished except for two HSV-2 proteins with molecular weights of 38,000 and 118,000. These two proteins appear to be tumor associated and not merely covariables of past infection or risk factors alone.

Serological and epidemiological considerations of the role of herpes simplex virus type 2 in cervical cancer.

To assess the possible biological significance of the observations that women with cervical cancer tend to be younger at first intercourse than control women, data from 1823 women were analyzed for the relationship between age at 1st intercourse and number of sex partners. Women who were younger at first intercourse had more sex partners than did women who were older at first intercourse. The interdependence of age at first intercourse and number of sex partners does not exlude the possibility that intercourse at an early age represents a biologically significant event in which the neoplastic process is initiated. However, it is equally possible that younger women at first intercourse may have multiple sex partners and be at greater risk of coming in contact with a putative oncogenic agent later in life. In addition, sera from patients with herpesvirus infections were assayed for cross-reacting and type-specific antibodies. Approximately 80% of the total antibody activity was to the cross-reacting antigen and only 20% was to the type-specific antigens in the sera of patients infected with either type 1 or type 2 virus. Among patients infected with both types of virus, less antibody activity to the type-specific antigens and more antibody activity to the cross-reacting antigens were found. These observations are discussed with respect to case-control seroepidemiological studies.

Variants of a human colon adenocarcinoma cell line which differ in morphology and carcinoembryonic antigen production.

A variant, HCT-8R, of the colon adenocarcinoma cell line HCT-8 was located and cloned. The variant and parent cell line were characterized with respect to morphology, growth characteristics, karyotype, production of Cea, and ability to form tumors in nude mice. The variant cells differed from the parent cells in morphology, marker chromosomes, and ability to form colonies in soft agar and produced more carcinoembryonic antigen. The two cell strains were equally oncogenic in nude mice, although HCT-8R cells produced poorly differentiated tumors while HCT-8 cells produced tumors with both differentiated and poorly differentiated areas. Thus, in nude mice, no correlation was observed between carcinoembryonic antigen production by cells and their oncogenicity.

Regional distribution of human papillomavirus DNA and other risk factors for invasive cervical cancer in Panama.

A population-based national cancer registry has documented strikingly different regional incidence rates of cervical cancer in the Republic of Panama. Such regional differences in disease rates could represent regional differences in the occurrence of risk factors, in particular, human genital papillomaviruses (HPV). This study enrolled newly diagnosed invasive cancer patients in the Republic of Panama over an 18-mo period. Behavioral risk factors were measured by interviewing cases and matched controls. In addition, DNA extracted from biopsies of the cancers was tested for HPV sequences. Early age at first coitus, multiple pregnancies, and nonparticipation in Pap smear screening programs were significant risk factors for cervical cancer in this population. These factors and low levels of education occurred more frequently among women residing in regions with higher cancer rates than women residing in the region with lower cancer rates. HPV DNA was detected most frequently (70%) among cases from the region with the lowest cancer rate (30 of 100,000) and least frequent (54%) among cases where the cancer rate was the highest (51 of 100,000). The observations suggest that risk factors other than HPV contribute to the differences in cervical cancer rates among women residing in various regions of Panama.

Occurrence of IgA and IgG antibodies to select peptides representing human papillomavirus type 16 among cervical cancer cases and controls.

We used an enzyme-linked immunosorbent assay to test sera from 186 cases of invasive cervical cancer and 172 age-matched controls for IgG and IgA antibodies to an human papillomavirus 16 E7 peptide and to peptide 245, representing an epitope in E2. Cases had significantly higher mean absorbance values than controls for both immunoglobulin isotypes to E7 and elevated mean values for IgG to peptide 245. Since absorbances were not normally distributed we analyzed cervical cancer risk for seropositive and seronegative women. Of the traditional cervical cancer risk factors, cigarette smoking, educational level, number of pregnancies, time interval since last Papanicolaou smear, and age at first intercourse influenced the distribution of seropositivity to some of the viral antigens. Adjusting for these variables, the odds ratios of cervical cancer associated with IgG to E7 was 5.28 [95% confidence (95% CI) = 2.4-11.6] and that with IgA to E7 was 2.67 (95% CI = 1.3-5.3). IgG to peptide 245 was less strongly associated, odds ratio 1.68 (95% CI = 1.2-3.3), and IgA to peptide 245 was not significantly associated with disease. These findings suggest that antibodies to E7 are markers for invasive cervical cancer. However, seropositivity correlated poorly with clinical state, survival, or the presence of human papillomavirus DNA in the cancer tissue.

Oral contraceptive use and risk of invasive cervical cancer.

A case-control study of 759 invasive cervical cancer patients and 1430 controls in Panama, Costa Rica, Colombia and Mexico enabled an evaluation of risk in relation to oral contraceptive use. Overall use was associated with a 21% nonsignificant elevation in risk, with some further increases in risk for more extensive durations of use. Although risks were similar for recent and non-recent users (RRs = 1.3 versus 1.2), recent long-term users were at highest risk (RR for 5+ years use = 1.7, 95% Cl 1.1-2.6). Relationships were similar for women with and without a recent Pap smear, arguing against detection bias. There was little evidence that other risk factors, including smoking and detection of human papillomaviruses (HPV), altered the effects of oral contraceptives. The risk associated with oral contraceptives was significantly increased for adenocarcinomas (RR = 2.2), whereas for squamous cell tumours the effect was minimal (RR = 1.1). These results provide some support for an adverse effect of oral contraceptives on cervical cancer risk, although possibly limited only to a subpopulation of cases.

Canalicular function during herpetic keratoconjunctivitis in rabbits.

Canalicular function during acute herpetic keratoconjunctivitis was investigated in rabbits. Evidence of partial obstruction of the duct was obtained in the infected as compared with the mock-infected eyes. Direct damage of the ductal epithelium by virus could not be demonstrated by histologic and immunofluorescent studies. Our findings suggest that canalicular dysfunction associated with viral infection may result from accompanying inflammatory changes.

Production of hybrid cell lines secreting antibodies to herpes simplex virus type 2.

Following different immunization schedules we have produced 102 myeloma--spleen cell hybrid lines (30.8% of the total) secreting antibodies to HSV-2 infected cells. The highest yield of hybrids was derived from fusion of myeloma cells with spleen cells from mice sensitized by footpad injection of formalin-inactivated virus and boosted intravenously with the same antigen. To date, twelve of the antibody-producing hybrids have been cloned by limiting dilutions to generate over 100 monoclonal lines reacting specifically with HSV-infected but not with mock-infected cells. Several hybrid lines have also been injected into mice to generate tumors and high titers of anti-HSV-specific antibodies have been detected in ascitic fluids. All of the hybrids so far tested have retained the capacity to secrete anti-HSV antibodies after long-term in culture or repeated passages through mice. Antibodies reacting with cytoplasmic or nuclear antigens in infected cells as well as type-specific and type common antibodies have been detected.

Risks associated with blood transfusion in newborn infants.

As with many other effective therapies, there are untoward side effects associated with blood transfusions in newborn infants. Discussed in this article are infectious agents transmitted by blood components, metabolic complications of transfusion, cardiac and vascular complications, hemorrhagic complications, donor erythrocyte injury, and alloimmunization to red cell and white cell antigens. Thus, potential complications must always be borne in mind when transfusing blood products.

Susceptibility to fatal Pichinde virus infection in the Syrian hamster.

The data presented in this paper suggest that the susceptible MHA hamster strain possesses a spleen target cell for Pichinde virus replication which is minimally expressed in the resistant strain. This target cell co-purifies with cells mediating NK activity, raising the possibility that the NK cell itself may be the additional target cell for Pichinde virus replication in the susceptible hamster strain. We hypothesize that early virus replication in the spleens of IP-inoculated hamsters leads to an overwhelming proliferation of virus. In contrast, a footpad inoculation of Pichinde virus retards virus spread into the spleen, and the host's immune response can effectively clear the relatively low amount of virus. In addition, data have been presented that show that a footpad inoculation of Pichinde virus elicits swelling in resistant hamster strains at eight days after infection, but fails to evoke a response in the susceptible MHA hamster strain. The response is controlled by a single autosomal dominant gene, and suggests that the MHA hamster strain has a defective delayed-type hypersensitivity response. The gene responsible for footpad swelling appears to be distinct from the single autosomal dominant gene that controls virus replication in the popliteal lymph nodes of footpad-injected hamsters. The phenotype of survival, then, may be the result of either limited virus replication early in infection, or an effective anti-viral cell-mediated immune response, or both.