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This study shows that 10% of Panamanian women are infected with VPH. This incidence of premalign and malign infection is one of the highest in the world. It is necessary that panamanian women be educated to participate in the program of the early detection of the disease to control the incidence of cancer in the uterine cervix.

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.

A study of 759 patients with invasive cervical cancer, 1,430 controls, and 689 sexual partners of the participants who declared that they were monogamous was conducted in Colombia, Costa Rica, Mexico, and Panama from January 1986 to June 1987, to evaluate the risk factors associated with this neoplasm. The principal risk factors identified were: initiation of sexual relations by the woman at an early age, number of stable sexual partners (relationships of more than three months' duration), number of liveborn children, presence of DNA from human papilloma virus (HPV) types 16 or 18, history of venereal disease, lack of exposure to early detection programs, deprived socioeconomic conditions, and number of sexual partners of the partners of the monogamous women. Smoking increased the risk in those women who were shown to have DNA from HPV types 16 or 18. Fifty percent of the patients and 29% of the controls said they had never had a cytological examination (Papanicolaou test). No association was observed between the presence of HPV and sexual behavior. The study showed the need for further research on the possible mechanisms involved in carcinogenesis and infection. The common denominators of the risk factors mentioned are underdevelopment and poverty, which affect broad sectors of these populations. Mass detection programs targeting high-risk groups can reduce the high incidence of cervical cancer in Latin America.

A case-control study of 759 women with invasive cervical cancer and 1430 controls in four Latin American countries evaluated risk in relation to sexual behavior, histories of specific venereal diseases, and hygiene practices. Early age at first sexual intercourse and increasing number of sexual partners were associated with significantly increased risks even after adjustment for their mutual effects. Risk increased to a twofold excess among women reporting first intercourse at 14 to 15 years of age compared with 20+ years. The number of steady sexual partners was a more important predictor of risk than the number of nonsteady partners, particularly before age 30, possibly reflecting the need for prolonged or repeated exposures to a transmissible agent, or different methods of protection against sexually transmitted diseases or pregnancy. Reported frequency of intercourse was not generally associated with risk, except among women reporting increased frequencies before 20 years of age. Histories of gonorrhea or crab lice were associated with increased risk, but histories of other venereal diseases were not significant predictors. No consistently increased risks were detected for women reporting specific hygiene or douching habits, except the practice of washing the genitalia infrequently during menstruation. These results provide support for a period of increased susceptibility to carcinogens during adolescence, and suggest that this may be an important determinant of the high incidence of cervical cancer in Latin America.

A study of 759 cervical cancer patients, 1,430 controls, and 689 sex partners in four Latin American countries has made it possible to assess the influence of multiple factors upon the risk of invasive cervical cancer. The principal risk factors identified were the woman's age at first coitus, the number of her steady sex partners, her number of live births, the presence of DNA from human papillomavirus (HPV) types 16 or 18, a history of venereal disease, nonparticipation in early detection programs, and low socioeconomic status. There is good reason to believe that extensive detection programs directed mainly at high-risk groups in the Americas can reduce the high incidence of cervical cancer in this Region.Risk factors for invasive carcinoma of the cervix were analyzed in 759 cancer patients from Mexico City, Costa Rica, Panama and Bogota, Colombia, in comparison with 1430 controls and 689 male partners, by interview and analysis for human papilloma virus (HPV) types 16 and 18. Community and hospital controls were combined for the analysis, since they did not differ significantly. Average age is 1st coitus was 17.4 years for patients and 18.8 for controls. A relative risk of 1.8 resulted for women with coitus at age 14-15 compared to age 20 or more. Those with 6 or more sex partners had a risk of 1.7 compared to monogamous women, with a significant trend (p0.0001). Anal sex conferred a risk of 1.5-1.9 depending on frequency. There was a significant trend toward increasing risk for number of pregnancies up to 5.1 for 14 or more pregnancies, and especially for number of live births, with a risk of 3.7 for 12 of more. No relationship was found between risk and stillbirths, or spontaneous or induced abortions. Cesarean sections and prenatal care reduced risks. DNA from HPV types 16 or 18 was found in 62% of patients and 32% of controls. A relative risk of 1.7 was found in women who had HPV and who smoked. Risks decreased with rising educational level, and with increasing socioeconomic status. Neither oral contraception nor condoms affected risks. Factors associated with male partners related to increased risk were his number of sex partners (25 vs 5 of less) and lower education. Not having Pap smears increased risk to 2.5. Providing cervical cytology to women at risk is an obvious intervention needed to reduce the several-fold higher incidence of cervical cancer in Latin American compared to that in developed countries.

Antibodies specific for carcinoembryonic antigen (CEA) induced polar redistribution of CEA which was expressed on the surface of human intestinal carcinoma cells grown in tissue culture. Polar redistribution occurred at 23°C and 37°C but not at 4°C and was inhibited by sodium azide and cytochalasin B. Antibodies to isoantigen A did not react with CEA at the cell surface but did react with isoantigen A which was not redistributed by the antibody. Cells which expressed both CEA and isoantigen A were used to demonstrate that polar redistribution of CEA could be induced without altering the distribution of isoantigen A. The data indicate that CEA and isoantigen A can be expressed on the same cell as separate molecules.

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.

The two types of herpes simplex virus—HSV-1 and HS V-2—are morphologically indistinguishable and consist of 150-mm particles with a DNA-containing core that is enclosed in an envelope. The two virus types can be distinguished by biologic, biochemical, and antigenic properties; however, the typical clinical lesions associated with infections by the two viruses are very similar. Generally, HSV-1 has been isolated from oral-facial lesions, as well as from cases of herpetic keratoconjunctivitis and encephalitis, while HSV-2 has been recovered from genital lesions, infants with neonatal systemic herpes, and cases of herpetic meningitis. 1 These associations of virus types with clinical diseases are not absolute. HSV-1 or HSV-2 may produce a number of clinically distinguishable illnesses. Each of these illnesses has certain epidemic-logic features that are unique. For example, Kaposi's varicelliform eruption represents a generalized cutaneous infection occurring primarily among persons suffering from atopic eczema 2–4 or Darier's disease. 5 Herpetic whitlow occurs commonly among physicians, dentists, and nurses who occupations require manual contact with individuals who may be shedding virus. 6–8 A form of cutaneous herpes has been described among wrestlers and has been called herpes gladiatorium. 9,10 Anorectal herpes has been reported with increasing frequency, and the majority of cases reported have occurred in homosexual men. 11,12 Aseptic meningitis is an occasional complication of genital herpetic infections and occurs mostly in young adults. 13–15 The vast majority of illness, however, results from infections initiated in the oral cavity or the genital tract, and these will be considered in more detail. Our knowledge of the epidemiology of HSV-1 and HSV-2 comes from studies that have varied considerably in design and in the populations examined. The epidemiology of clinical illnesses has been delineated primarily by periodically examining patients in described populations or from questionnaire surveys. Information about the occurrence of infections, many of which are asymptomatic, has come from serologic surveys or from surveys of virus shedding. Each method has limitations and the conclusions drawn from these different approaches have not always been similar. In this review, the conclusions drawn are those that are most consistent with the data available.

Sera from 231 women were used to examine their frequency of precipitation of various herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) proteins and to determine if there was a rank order of immune responsiveness of humans to these HSV antigens. Radiolabeled viral proteins were reacted with serum and immune complexes isolated with staphylococcal protein A. Individual antigens were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. As a group, these sera precipitated 31 HSV-1 and 27 HSV-2 proteins. HSV-1 polypeptides with molecular weights of 133,000, 99,000, and 82,000, as well as HSV-2 polypeptides with molecular weights of 131,000 and 101,000, were precipitated by essentially all sera that contained antibodies to HSV-1 and HSV-2. When attempts were made to order the viral proteins by constructing precipitation profiles ranking the antigens in patterns according to their frequency of precipitation, it was observed that the antigens were generally not ordered. Demographic analysis of the sera suggested that the differences in the number of proteins precipitated were associated with differences in age, education, age at first marriage, and income, which collectively may reflect the frequency of exposure to the virus.

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.

Pichinde virus causes a lethal disease after i.p. inoculation of adult MHA hamsters; other strains of Syrian hamsters are resistant to this lethal infection. During studies of cell-mediated immune responses to Pichinde virus, it was noted that MHA hamsters survived infection when the virus was given in the footpad. However, unlike the resistant LSH and LVG strains of hamsters, the MHA hamsters did not manifest a footpad swelling response. Failure of the MHA hamster to respond to a footpad inoculation of Pichinde virus was shown to be virus-specific and appeared to be mediated by a cell-associated suppressor mechanism.

Mutations in the DNA polymerase locus of phage, bacteria, and eukaryotic cells may change the mutation rates at other loci of the genome. We used resistance to phosphonoacetate to select mutants of herpes simplex virus with mutated DNA polymerase and then determined the reversion frequency of viral thymidine kinase mutation in mutants and recombinants. The results obtained indicate that mutations causing resistance to phosphonoacetate do not affect the mutation rate of the viral genes. This finding is consistent with the existence of two functional regions in the DNA polymerase molecule, one involving the pyrophosphate acceptor site and responsible for resistance to phosphonoacetate and another involved in the editing ability and recognition specificity of the enzyme.

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibility to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.

A double-antibody enzyme immunoassay was developed for the identification and typing of herpes simplex virus (HSV) by employing a polyclonal rabbit capture antiserum together with type-common and type 2-specific monoclonal antibodies as detectors. The test successfully identified 45 type I isolates and 30 type 2 isolates as HSVs. Compared with immunofluorescent staining and restriction endonuclease analysis, enzyme immunoassay correctly typed 45 type 1 and 30 type 2 HSV isolates. Enzyme immunoassay was 100% sensitive for identification of HSV as compared with cell culture and 100% specific for typing as compared with immunofluorescence and restriction endonuclease analysis. Electron microscopy analysis suggested that approximately 10(6) virus particles were required for the identification and typing of HSV by enzyme immunoassay.

Two inbred strains of Syrian hamster have been shown to display differences in splenic NK cell activity both endogenously and after infection with Pichinde virus. On further investigation of these differences, it was found that the spleens and thymuses of the high NK strain, MHA, displayed greater cellularity than those of the low NK strain, LSH. Additionally, thymocytes from MHA hamsters were found to proliferate to a greater extent than those of LSH hamsters in response to Con A-induced conditioned medium plus lectin. Splenocytes from MHA hamsters showed high levels of lymphokine-activated killer cell (LAK) activity after culture in conditioned medium, in contrast to LSH splenocytes cultured under the same conditions. By limiting dilution analysis, spleens from MHA hamsters were found to contain a greater frequency of LAK precursors than those from LSH hamsters. The data suggest that the relative concentrations of lymphopoietic cells is genetically determined.

SummaryThe PHA-stimulated leukocyte cultures from healthy donors were inoculated with representatives from picornavirus, picodnavirus, arbovirus, myxovirus, paramyxovirus, rhabdovirus, reovirus, adenovirus and papovavirus groups. Cultures were harvested after 6 days of incubation and assayed for virus multiplication and for DNA synthesis of the leukocytes by uptake of thymidine-3H. Virus replication and inhibition of the PHA response of the lymphocytes by these viruses were limited primarily to viruses which replicated and assembled in the cytoplasm of the cell. Leukocytes from 15 patients with viral hepatitis were studied, and in 8 of them the cells were hyporesponsive to PHA stimulation after 3 days of incubation. This pattern was observed in all samples taken within the first week after onset of jaundice. Sera from patients with viral hepatitis when added to normal leukocyte cultures showed some inhibition of the PHA response, but the effect could not be serially passed to fresh cultures. The same patt...

The congenital rubella syndrome is now recognized as a generalized systemic disease. This report reviews the pathological changes in 18 fatal cases and compares the results with previous reports. Chronic inflammation was prominent in the leptomeninges, lung, and uveal tract of the eye in most of the cases. In fewer cases, the kidney, liver, peritoneum, and testes were chronically inflamed. Noninflammatory lesions included cardiovascular anomalies, especially patent ductus arteriosus and nodular sclerosis of the cardiac valves, growth aberrations in the long bones and ribs, precocious development of lymphoid germinal centers, and adrenal cortical cytomegaly. Infection of clones of cells in the embryo causes faulty cell multiplication, and this results in the lesions and in generalized retardation of growth.

Changes in the cell membranes after infection or transformation of cells by oncogenic viruses have been demonstrated using a plant agglutinin, Concanavalin A (Con. A) isolated from Jack beans. Upon infection or transformation of cells by oncogenic viruses, the cells agglutinate in the presence of Con. A (Inbar & Sachs, 1969; Ben-Bassat, Inbar & Sachs, 1970; Benjamin & Burger, 1970). Moore & Temin (1971) reported a lack of correlation between transformability by RNA tumor viruses and agglutinability of cells by Con. A. Recent evidence (Cline & Livingston, 1971; Ozanne & Sambrook, 1971; Arndt-Jovin & Berg, 1971; Inbar, Ben-Bassat & Sachs, 1971) has suggested that both normal and transformed cells have the same number of Con. A binding sites but agglutinate differentially to a given concentration of Con. A. Nicolson (1971) showed that single Con. A binding sites at the surface of normal cells are randomly distributed whereas in transformed cells the Con. A sites are present in clusters.

Rubella virus was centrifuged in sucrose density gradients. One of two densities could be ascribed to the virus, depending upon the suspending medium used. The virus was found at a density of 1.16 g/cm 3 after centrifugation for 18 hr in sucrose gradients prepared in distilled water. By contrast, when the sucrose gradients were prepared in tris(hydroxymethyl)aminomethane (Tris)buffer containing ethylenediaminetetraacetic acid (EDTA), the virus was found at a density of 1.18 g/cm 3 after 18 hr of centrifugation. The virus banded at this higher density after only 2 hr of centrifugation when pretreated by overnight incubation in the Tris-EDTA buffer. A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1.16 g/cm 3 after 2 hr of centrifugation to the higher 1.18 g/cm 3 density after 18 hr. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.16 g/cm 3 density. The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. However, it is possible that the two densities observed were a reflection of the existence of rubella virus in different hydration states in the presence and absence of Tris buffer containing EDTA.

SummaryThe hemadsorption-negative plaque assay technique has provided a means of quantitatively studying rubella virus neutralization. A 3-day assay for rubella antibodies has been developed which is sensitive and reproducible. The addition of fresh guinea pig serum enhanced rubella antibody titers up to 16 times those obtained in heated sera. The factor or factors responsible for the enhancement of antibody titers have the properties of complement.The authors gratefully acknowledge the able assistance provided by Miss Marian Moore and Mrs. Marjorie Burkhardt. The helpful advice and consultation of Dr. Gordon R. Dreesman is deeply appreciated.

Summary Sucrose density-gradient centrifugation was used for the study of serum IgM and IgG antibodies in rubella infection in early pregnancy. Seventeen women with proven rubella in the first trimester of pregnancy were tested and all were found to have an IgM antibody response to the virus. IgM antibody was present for 1 mth following the onset of illness but not later, and it was absent in women who did not have acute rubella. With one exception, IgM antibodies were not found in women who had recently given birth to an infant with congenital rubella. The technique involved the collection of six fractions from the density gradient. The second fraction, which contained IgM but was free from IgG, was tested for rubella antibody by means of an HI test. Examination of this one fraction from a single serum collected within 1 mth of the onset of illness was sufficient to confirm or exclude a diagnosis of rubella.

The failure of type 1 herpesvirus to form plaques in chick embryo cells distinguishes the type 1 virus from the type 2 virus. Herpesvirus type 2 forms plaques in these cells at titres equal to those obtained in rabbit kidney cells, while most type 1 strains fail to form plaques in chick embryo cells (Figueroa & Rawls, 1969). However, most type 1 virus isolates contain a small proportion of virus which does form plaques in chick embryo cells, and this proportion increases with passage of the isolates in tissue culture. The type 1 variants capable of forming plaques in chick embryo cells retain the biological and antigenic characteristics of the parental type 1 virus, but acquire a relative resistance to the DNA inhibitor ara-A (Lowry, Melnick & Rawls, 1970). The present study was undertaken to characterize the type 1 abortive infection in chick embryo cells.

A study of 13 patients with laryngeal papillomas is presented, with an analysis of epidemiologic factors, electron microscopic study of the lesion, and therapeutic trial of frequent microlaryngoscopic surgery and application of idoxuridine. Five of the nine children in this group were delivered by mothers who had condyloma accuminatum at time of delivery. These five children developed symptoms and required surgery much earlier than the other four. This correlation is shown to be statistically significant. The electron microscopic study of tissue specimens from all our patients failed to find any actual virus particles. The comparison study with skin and genital warts, however, showed ultrastructural details linking the three lesions. Results of the therapeutic program showed an overall improvement in frequency of recurrence, but no complete cures. This improvement is felt to be related to frequent complete surgical excision, and not to application of idoxuridine.

A modification of the macrophage migration inhibition test using infected monolayers as a source of antigen was applied to the in vitro study of cellular immunity to fibroma virus infection. The capacity of infected monolayers or infected cell homogenates to inhibit migration of peritoneal exudate cells from fibroma virus-infected rabbits paralleled the onset of a cutaneous delayed hypersensitivity response and resistance to re-infection in the intact animal. Studies using antigens prepared at different intervals after infection of cells with the virus suggest that the test is measuring specific antigen(s) associated with the cell surface and probably not a component of the intact infectious virion. Immune serum did not prevent the antigen from inhibiting migration of sensitized cells, but instead enhanced the inhibition. The data obtained demonstrate that the macrophage migration test has practical application in the study of cell-mediated immunity to animal virus infections.

A cell wall deficient form of an alpha-haemolytic streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple's disease. Serological cross reactivity was shown between the organism and the material within Whipple's disease macrophages positive for diastase-resistant periodic acid-Schiff (D./P.A.S.). In vitro studies characterized the organism as a facultative intracellular parasite which caused the accumulation within cells of D./P.A.S.-positive material. These results suggest that a pathogenic bacterium is the essential aetiological agent and that the culture of Whipple's disease tissues in hypertonic media may have practical value.

The pathogenesis of persistent virus excretion in infants with the congenital rubella syndrome remains an immunological paradox. The presence of circulating 19S rubella-virus-neutralising antibody indicates that an immune response has been provoked by the virus and that tolerance in the usual sense of the word is not present. From the existing knowledge of those host factors involved in resistance to virus infection, a defect in cellular immune functions is postulated as underlying the failure of the tissues of some infants with the congenital rubella syndrome to terminate virus excretion. Attempts to demonstrate specific cellular immunity to rubella virus in vitro were unsuccessful, probably for the same reasons that cellular immunity may be deficient in vivo. Rubella-virus infection of human lymphocytes results in inability of these cells to undergo the metabolic changes requisite to their recruitment for immunological functions.

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.

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.

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.

The incidence of mumps in vaccinated and nonvaccinated schoolchildren was studied after a recent epidemic. Information was collected by telephone interviews with the parents and a review of the physicians' records. The vaccine appeared to be effective, for the incidence of mumps in the 145 vaccinated children--5.5%, or 8 cases--was significantly less (P less than 0.001) than the incidence in the 350 children considered susceptible to infection--21.7%, or 76 cases. The percentage of children who had been immunized decreased with increasing age, and acquisition of immunity through natural infection had the reverse trend; thus, the proportions of children susceptible to infection in each age group were about the same, and the age-specific attack rates were similar. Although the mothers were accurate in indicating absence of vaccination, they incorrectly indicated vaccination of their children 43.0% of the time; this error in reporting could influence vaccine administration in older children. Our findings suggest that mumps vaccination may substitute for natural illness in immunizing populations, and that more extensive use of the vaccine over a broader age range is required to prevent similar epidemics in the future.

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.

The Pichinde virus RNA did not possess the following characteristics of eucaryotic mRNA: polyadenylic acid sequence, capped methylated structure, and ability to direct protein synthesis in vitro. Polysomal RNA extracted from cells infected with Pichinde virus reannealed with 32P-labeled virus RNA, protecting about 60% of the latter against RNase degestion. The polyadenylic acid-containing polysomal RNA also reannealed to the 32P-labeled virus RNA to approximately the same extent. These indicate that the major part of the genomic RNA of Pichinde virus is negative stranded.

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.

Pichinde virus causes a lethal disease after i.p. inoculation of adult MHA hamsters; other strains of Syrian hamsters are resistant to this lethal infection. During studies of cell-mediated immune responses to Pichinde virus, it was noted that MHA hamsters survived infection when the virus was given in the footpad. However, unlike the resistant LSH and LVG strains of hamsters, the MHA hamsters did not manifest a footpad swelling response. Failure of the MHA hamster to respond to a footpad inoculation of Pichinde virus was shown to be virus-specific and appeared to be mediated by a cell-associated suppressor mechanism.

A prospective study of premature infants admitted to an intensive care neonatal unit was undertaken to evaluate the relative role of blood transfusions and maternal exposure in infections caused by cytomegalovirus (CMV), varicella zoster virus (VZV) and herpes simplex virus (HSV). During the first year of life, 13.7%, 4.8% and 4% of the infants became infected with CMV, VZV and HSV, respectively, as determined by virus isolation or seroconversion. Transfusion in the newborn period did not influence the rates of VZV or HSV infections. A higher rate of CMV infections occurred in transfused (18.8%) than in non-transfused (11%) infants but the difference was not statistically significant. A significant correlation was found between maternal antibody titers and the rate of CMV infection in infants whether or not transfusion occurred. An even stronger association was found between breast feeding and CMV infections. The risk of alloimmunization to erythrocyte and leucocyte antigens was also assessed; no evidence of increased alloimmunization to these antigens was associated with transfusion.

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.

Three RNA polymerase activities were found and associated with purified Pichinde virus, a member of the Arenaviridae. A heat-labile polymerase activity which required all four ribonucleoside triphosphates for optimal activity co-sedimented on sucrose gradient centrifugation with the viral ribonucleoprotein complex from detergent-disrupted virus preparations. This enzyme synthesized heteropolymers which represented about 23% of the genome RNA as determined by nucleic acid hybridization. Two relatively heat-stable polymerase activities which differed in their cation requirement and substrate specificity were recovered with the virus-associated ribosomes. These polymerase activities synthesized homopolymers of limited chain length: in the presence of 10 mM Mg2%, polyuridylic acid was made, whereas in the presence of 1 mM Mn2%, polyadenylic acid was made. The addition of complementary RNA synthesized with the viral transcriptase in vitro to the reaction mixture containing the polyadenylic acid polymerase activity resulted in the terminal addition of polyadenylic acid to the complementary RNA. The possible function of the ribosome-associated polymerase activities in the replication of the virus is discussed.

Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited number of ulcerative lesions revealed that indirect immunofluorescence and virus isolation were comparable in detecting herpes simplex virus. The results indicate that monoclonal antibodies can be used to accurately identify and type isolates of herpes simplex virus.

Cells from rabbit spleens, bronchial washings (BW) and bronchus-associated lymphoid tissues (BALT) were examined for their ability to lyse cells infected with herpes simplex virus (HSV). Specific lysis of HSV-infected cells was mediated by BW cells as early as 4 days after intratracheal infection of the rabbits with the virus whereas lysis by spleen cells and BALT cells was not detected until 7 or more days after infection. Lysis by spleen cells was initially detected 7 days after intraperitoneal injection of the virus but lysis by BW and BALT cells was not observed until 14 days after infection. Although spleen, BW, and BALT cells could lyse antibody-coated target cells, antibodies detectable by antibody-dependent cellular cytotoxicity could not be detected in bronchial washings until 7 or more days after infection. The data suggest that cells capable of direct cytotoxicity of virus-infected cells appear within the bronchus after local infection by the virus.

Antibodies to herpes simplex virus type 1 and type 2 were detected in the sera of rabbits by release of histamine from basophils sensitized in vitro with the sera. The time course of the appearance of the antibodies, the dose-response curve of the release of histamine in relation to antigen concentration, the sedimentation characteristics of the antibodies in sucrose gradients, and the ability to destroy the sensitizing capacity of the sera with heat suggest that the antibodies being assessed were of the IgE class. These antibodies were induced in animals injected intradermally, intramuscularly, and i.p. with live virus. The antibodies were detected 1 week after primary injection and a similar time course of antibody appearance was observed after a second or third injection. The same cross-reactivity between type 1 and type 2 virus observed with IgG antibody was also observed with IgE antibody.