Virus-induced diabetes mellitus. XX. Polyendocrinopathy and autoimmunity.

Mice infected with reovirus type 1 developed transient diabetes and a runting syndrome. The diabetes was characterized by hyperglycemia, abnormal glucose tolerance tests, and hypoinsulinemia. Inflammatory cells and viral antigens were found in the islets of Langerhans, and virus particles were seen in alpha, beta, and delta cells. The runting syndrome consisted of retarded growth, oily hair, alopecia, and steatorrhea. Inflammatory cells and viral antigens were found in the anterior, but not posterior pituitary. Electron microscopy revealed virus particles in growth hormone (GH)-producing cells and radioimmunoassay showed that the concentration of GH in the blood was decreased. Examination of sera from infected mice revealed autoantibodies that, by immunofluorescence, reacted with cytoplasmic antigens in the islets of Langerhans, anterior pituitary, and gastric mucosa of uninfected mice. Absorption studies and enzyme-linked immunosorbent assays designed to identify the reactive antigens showed that some of the autoantibodies were directed against insulin and others against GH. Reovirus type 3, in contrast to reovirus type 1, did not induce autoantibodies to GH. By use of recombinant viruses, the segment of the reovirus genome responsible for the induction of autoantibodies to GH was identified. Virus containing the S1 gene segment from reovirus type 1, which codes for the sigma 1 polypeptide (i.e., hemagglutinin), infected cells in the anterior pituitary and induced autoantibodies to GH, whereas virus containing the S1 gene segment from reovirus type 3 failed to infect cells in the anterior pituitary and did not induce autoantibodies to GH. We conclude that reovirus type 1 infection can lead to polyendocrinopathy and autoimmunity and that the S1 gene segment is required for the induction of autoantibodies to GH.

Herpes simplex virus in mice: electron microscopy of neural spread.

Herpes simplex virus rapidly infected the trigeminal nerves of mice after intranasal inoculation. Centripetal neural spread was suggested by histologic evidence of encephalitis in the area of attachment of the trigeminal nerve. Furthermore, electron microscopy revealed virus replication primarily within Schwann cells of the trigeminal nerve, and neurons of the gasserian ganglion.

Virus-induced diabetes mellitus: reovirus infection of pancreatic beta cells in mice.

Reovirus type 3, passaged in pancreatic beta-cell cultures, produced an insulitis when inoculated into 1- to 2-week-old mice. By means of a double-label antibody technique, in which we used fluorescein-labeled antibody to reovirus and rhodamine-labeled antibody to insulin, reovirus antigens were found in beta cells. By electron microscopy, viral particles in different stages of morphogenesis were observed in insulin-containing beta cells but not glucagon-containing alpha cells. The infection resulted in destruction of beta cells, reduction in the insulin content of the pancreas, and alteration in the host's capacity to respond normally to a glucose tolerance test.

Topographical and conformational epitopes of bovine papillomavirus type 1 defined by monoclonal antibodies.

Monoclonal antibodies (MAbs) were generated against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV-1). When screened by enzyme-linked immunosorbent assay (ELISA) on intact and disrupted BPV-1, -2, and deer papillomavirus, three patterns of reactivity were defined: reactivity only with intact virus, with both intact and disrupted virus, and only with disrupted virus. On the basis of ELISA results, the topographical location and requirement for conformation for immunoreactivity of epitopes was defined as external conformational, external linear, and internal linear. Cross-reactivity of MAbs with other papillomavirus types was analyzed by immunofluorescence on warts from different species. Type-specific, BPV-1 and/or -2 cross-reactive, broadly cross-reactive, and genus-specific MAbs were identified. MAb reactivity with structural polypeptides of BPV-1 was analyzed by Western blot. MAbs reactive with epitopes defined as conformational by ELISA did not react in Western blot. All MAbs reactive in Western blot reacted with the major capsid protein (MCP) [55 kilodalton (kDa)], demonstrating that the MCP carries both type-specific and cross-reactive epitopes. Most MAbs reactive with the MCP were cross-reactive with structural polypeptides of 48 and 96 kDa, demonstrating the immunologic relatedness of these three polypeptides.

Bovine papillomavirus type 1 monoclonal antibodies.

Bovine papillomavirus type 1 (BPV-1) and type 2 (BPV-2) are the etiologic agents of fibropapillomas in cattle. Polyclonal antisera produced against BPV-1 structural antigens are cross-reactive with BPV-2. In this study BPV-1 type-specific monoclonal antibodies were produced that were not reactive with BPV-2. These monoclonal antibodies could be used for identification of BPV-1 structural antigens in acetone-fixed, frozen sections by immunofluorescence and Formalin-fixed, paraffin-embedded sections by immunoperoxidase techniques. In addition, these antibodies could be used for identification and purification of BPV-1 virions by immune electron microscopy and immunoadsorption techniques, respectively.

Transmission of murine leukemia virus (Scripps) from parent to progeny mice: a comparison of assay systems.

Transmission of murine leukemia virus (MuLV) from parent to progeny C3H/St and C57BL/St mice was examined by four assay systems: 1) recovery of infectious NB-tropic MuLV from spleen cultures, 2) the radioimmunoassay for p30 antigenemia, 3) morphologic examination for lymphoma development, and 4) the indirect fluorescent antibody technique for antinuclear antibodies (ANA). Transmission of MuLV (Scripps) occurred in 90-100% of C3H/St and C57BL/St progeny nursed by mothers with p30 antigenemia. All assays except ANA were equally sensitive for the determination of MuLV transmission in C3H/St mice, but the incidence of transmission in C57BL/St mice was determined only by assays of their cultured spleens for MuLV. Incidences of ANA were increased in all generations of C57BL/St mice compared with controls; the route of transmission of MuLV (Scripps) was not a factor. Only C3H/St mice infected by virus transmitted from parent to progeny developed ANA. Infectious MuLV was invariably recovered from spleens cultured from mice with p30 antigenemia, which was present in all mice that developed lymphoma. NB-tropic MuLV was also recovered after prolonged cultivation from spleens of 75% of C57BL/St progeny mice that did not develop p30 antigenemia. These suggested that MuLV (Scripps) could exist either as a productive persistent or nonproductive latent infection in C57BL/St mice.

Immunologic relatedness of papillomaviruses from different species.

An antiserum prepared by immunization of a rabbit with sodium dodecyl sulfate-disrupted virions from a pool of plantar warts was cross-reactive with virus-positive papillomas of other animal species by both indirect immunofluorescence tests on frozen sections of wart tissues and peroxidase-antiperoxidase tests of sections of Formalin-fixed tissues. The antiserum stained plantar warts, common warts, and skin lesions of epidermodysplasia verruciformis, all from humans; bovine fibropapilloma, experimentally produced with bovine types 1 and 2; and transmissible canine oral papillomas. The staining was localized to nuclei of the upper granular layers of the peithelium and was similar in distribution to the pattern produced by antiserum specifically prepared against that papillomavirus. The antiserum did not stain virus-negative warts, or cells infected with simlan virus 40, human polyomavirus BK, and murine polyomavirus. These data suggested that papillomaviruses share a common internal antigen unrelated to a similar antigen described previously for the polyomaviruses (which include simian virus 40 and polyomavirus subgroups).

Oncogenic association of specific human papillomavirus types with cervical neoplasia.

Molecular hybridization analysis of human papillomavirus (HPV) DNA from 190 cervical biopsy specimens from women in the United States, Brazil, and Peru revealed viral sequences in 2 (9%) of 23 biopsy specimens of normal mature squamous epithelium, 7 (44%) of 16 biopsy specimens of metaplastic squamous epithelia, 60 (77%) of 78 cervical intraepithelial neoplasia (CIN), 57 (89%) of 64 invasive squamous carcinomas, and 8 (89%) of 9 endocervical adenocarcinomas. HPV typing by DNA hybridization revealed HPV 6 and HPV 11 sequences in metaplastic squamous epithelia, CIN I, and CIN II, but not in CIN III lesions or invasive carcinomas. HPV 16 was detected in metaplastic epithelium and in nearly half of the invasive squamous carcinomas and adenocarcinomas. It was present in 31% of CIN lesions, increasing in frequency with the severity of CIN from 20% of CIN I to 50% of CIN III. HPV 16 showed a striking difference in geographic distribution, being detected in 36% of the carcinomas from the United States compared to 64% of the carcinomas from Brazil and Peru. HPV 18 was found in metaplastic epithelia and in 17% of carcinomas but in only 1% of CIN lesions. HPV 31 was not found in metaplastic epithelium but was present in 6% of carcinomas and in 18% of CIN lesions. In addition, a group of uncharacterized HPVs, not corresponding to any of the probes used, was found in 5% of normal and metaplastic epithelia and in 18% of CIN and 19% of invasive cancers. These results suggest that individual HPV types that infect the cervix have varying degrees of oncogenic association. HPV 6 and HPV 11 appear to have very little oncogenic association, HPV 31 has low oncogenic association, and HPV 16 and HPV 18 have high oncogenic association.

Transmission of murine leukemia virus (Scripps) from parent to progeny mice as determined by p30 antigenemia.

All mice of C57BL/St, C3H/St, BALB/cSt, NZB/Scr, and NZW/Lac strains developed high levels of p30 antigenemia after inoculation at birth with murine leukemia virus (Scripps). Transmission of virus from neonatally infected parents to their progeny for three successive generations, as evidenced by development of p30 antigenemia, varied among the five strains. Through the three generations, 100% transmission occurred in C3H/St and BALB/cSt mice, 50 to 61% transmission occurred in C57BL/St and NZW/Lac mice, and 11% transmission to the first generation, with no subsequent transmission, occurred in the NZB/Scr mice. Transmission appeared to occur readily via the milk in all strains. Intrauterine events also played a role with evidence of some viral transfer prior to birth in the C3H/St strain or, conversely, the development of resistance to infection prior to birth in C57BL/St mice. The occurrence of litters from infected parents containing both normal offspring and offspring with elevated p30 appeared to be the result of variable resistance in the intact offspring, perhaps as a result of intrauterine events, and not related to cellular resistance observable in tissue culture or to dominant genetic factors.

Virus-induced diabetes mellitus. XI. Replication of coxsackie B3 virus in human pancreatic beta cell cultures.

The capacity of Coxsackie B3 virus to infect insulin-containing beta cells was studied in human pancreatic cell cultures. Antibody to Coxsackie B3 virus was labeled with fluorescein isothiocyanate, and antibody to insulin was labeled with rhodamine. By use of a double-label antibody technique, three populations of cells were identified: uninfected insulin-containing beta cells, which stained only with rhodamine-labeled anti-insulin antibody; Coxsackie-infected (noninsulin-containing) cells, which stained only with fluorescein-labeled anti-Coxsackie antibody; and Coxsackie-infected insulin-containing beta cells, which stained with both antibodies. Radioimmunoassay showed that intracellular immunoreactive insulin decreased rapidly beginning at 24 hours after infection, and the decrease in insulin roughly paralleled the increase in viral titer. It is concluded that, under in vitro conditions, human beta cells are susceptible to Coxsackie B3 virus.

Prevalence of antibodies against virus-like particles of Epidermodysplasia verruciformis-associated HPV8 in patients at risk of skin cancer.

There is increasing evidence for widespread occurrences of infection with Epidermodysplasia verruciformis-related human papillomaviruses, both in the general population and in immunosuppressed patients. In order to test for the prevalence of antibodies directed against the native L1 epitopes exposed on the surface of the virions, we have established an IgG-specific enzyme-linked immunosorbent assay with L1 virus-like particles of the Epidermodysplasia verruciformis-specific human papillomavirus 8 as antigen to screen 567 representative serum samples from the general population and immunosuppressed/dermatologic patients. Among healthy European donors (n = 210), 7.6% were found to be seropositive. In a group of renal transplant recipients (n = 185) the antibody prevalence was elevated to 21.1%, irrespective of the presence or absence of skin cancer. High positivity rates could be detected among (i) immunocompetent patients with nonmelanoma skin tumors (45.6%, n = 79) and (ii) Psoralene/UVA treated psoriasis patients (42.9%, n = 42). In contrast, anti-human papillomavirus 8-virus-like particle antibodies were found in only 6.8% of Hodgkin lymphoma patients (n = 44).

Multiplicity of uses of monoclonal antibodies that define papillomavirus linear immunodominant epitopes.

During the last 10 yr, we have derived monoclonal antibodies from animals immunized with denatured bovine papillomaviruses type 1 major capsid (L1) protein, mapped their corresponding immunodominant epitopes to within a single amino acid (aa), and compared the reactivity of authentic L1 proteins to the predicted response by collinear analysis of the aa sequences of the same and other papillomaviruses (PVs). The data obtained from this approach has provided us with new insights into the sensitivity and specificity of the antibody response to viral proteins. We have included here some observations and conclusions that appear to be generic for the immune response, some of which might have applications for working with linear epitopes in other experimental systems.

Papillomavirus infection of the cervix. II. Relationship to intraepithelial neoplasia based on the presence of specific viral structural proteins.

Three hundred twenty-two cases of cervical dysplasia (mild, moderate, and severe) and carcinoma in situ (CIS) were examined for the presence of papillomavirus structural antigens with a peroxidase-antiperoxidase method on formalin-fixed, paraffin-embedded tissue. The primary antiserum, prepared from purified, detergent-disrupted bovine papillomavirus type 1 virions, is broadly reactive against the genus-specific (common) antigen(s) of the papillomaviruses. Using the peroxidase-antiperoxidase technique on cervical tissue obtained from biopsy, conization and hysterectomy specimens, papillomavirus structural proteins were identified in association with mild dysplasia in 65 of 152 (43%) cases, with moderate dysplasia in 12 of 82 (15%) cases, with severe dysplasia in eight of 47 (17%) cases, and with CIS in four of 41 (10%) cases. Papillomavirus antigens were found directly within the lesion in all the cases of mild and moderate dysplasia but in only two instances of severe dysplasia and in none of the examples of CIS. In the remaining 10 cases of severe dysplasia and CIS associated with the presence of papillomavirus antigens, cells containing papillomavirus structural proteins were present in areas of moderate dysplasia immediately adjacent to the high-grade lesions in seven instances and in areas of mild or moderate dysplasia not directly in contact with the high-grade lesions in three. Among the 12 high-grade lesions associated with the presence of papillomavirus antigens, a morphologic transition from areas of moderate dysplasia containing papillomavirus antigens to the areas of severe dysplasia and CIS was present in five instances. The results of this study, therefore, provide direct evidence demonstrating the relationship of papillomavirus to intraepithelial cervical neoplasia ranging from mild dysplasia to severe dysplasia and CIS.

The role of human papillomaviruses in the pathogenesis and histologic classification of precancerous lesions of the cervix.

It is clear that the relation between HPV infection and cervical neoplasia is more complex than initially realized. Preliminary molecular virologic data suggest preferential distributions of low- and high-risk HPV types in CIN that tend to correlate with the morphologic appearance. Thus, mild and moderate dysplasias (CIN I and II) contain a diverse distribution of HPV types, including a minority that have a high risk of malignant potential. HPV, therefore, appears to play a major role as a promoter. Neoplastic transformation is probably determined by specific HPV types but, in addition, requires initiation by some other carcinogenic stimulus, e.g., HSV II, cigarette smoking. Despite numerous studies, performed during the past 30 years, the long-term behavior of dysplasia remains uncertain. The natural history of HPV-associated lesions is unknown. Until this information is available, it is recommended that the conventional dysplasia--CIS or CIN nomenclature be used. The presence of associated viral changes can be considered and added to the diagnosis, e.g., "moderate dysplasia (CIN II) with evidence of papillomavirus infection." Treatment should be the same for all intraepithelial lesions, regardless of the presence of morphologic evidence of HPV. In the future, it may be necessary to modify the classification of precancerous lesions of the cervix if it is shown that a specific HPV type induces a characteristic morphologic alteration or that the HPV type, in and of itself, has greater prognostic significance. Until then, confusion will be minimized and management optimized if the conventional dysplasia--CIS or CIN nomenclature is employed.

Major histocompatibility complex and human papillomavirus type 16 E7 expression in high-grade vulvar lesions.

To determine whether expression of the human papillomavirus (HPV) type 16 E7 open reading frame influences expression of major histocompatibility complex (MHC) antigens on the surface of squamous epithelial cells, serial frozen sections from seven HPV type 16-positive, high-grade vulvar intraepithelial neoplasia (VIN 2-3) lesions were tested for viral transcription by RNA-RNA in situ hybridization, for MHC expression by immunohistochemical staining with antibodies to MHC class I and II molecules, and for keratinocyte differentiation by immunohistochemical staining with anti-filaggrin and cytokeratin 10 antibodies. Despite the histologic appearance of high-grade VIN lesions, expression patterns of cytokeratin 10 and filaggrin suggested a certain degree of keratinocyte differentiation in all specimens. These differentiation markers were especially prominent in parakeratotic and hyperkeratotic superficial areas, which did not express MHC antigens or contain E7 mRNA. Expression of MHC class I molecules within dysplastic tissues was greater than within HPV type 16-negative, normal vulvar epithelium from the same patients. In five of the VIN 2-3 specimens anti-MHC class I antibodies reacted more strongly with cells of the basal and suprabasal layers than with cells of the epithelial surface. In one lesion basal cells stained less intensively than surface cells, whereas in another specimen all epithelial layers were equally MHC class I positive. Staining with anti-MHC class II antibodies was generally restricted to isolated foci, representing invading lymphocytes, tissue macrophages, and Langerhans cells. In two lesions, however, there was heterogeneous keratinocyte expression of MHC class II proteins, perhaps due to inflammation. Major histocompatibility complex antigen detection was independent of the presence or distribution pattern of E7-specific transcripts. Hence, a correlation between MHC and E7 expression appears unlikely in warty VIN lesions.

Prospects for a vaccine against human papillomavirus.

OBJECTIVE: To summarize existing data regarding the feasibility of developing strategies for prophylactic and therapeutic vaccination against human papillomavirus (HPV) infection. DATA SOURCES: We used the Medline data base and reference lists of articles to identify English-language papers that evaluate strategies for prophylactic and therapeutic vaccination against HPV infection. METHODS OF STUDY SELECTION: Our search uncovered several reports of systems that produce recombinant HPV major capsid proteins as antigens for biochemical, molecular, and immunologic studies and investigations that evaluate cell-mediated immune responses to HPV-induced, tumor-associated peptides. DATA EXTRACTION AND SYNTHESIS: Recombinant HPV major capsid proteins, which self-assemble into virus-like particles, are produced in quantity, mimic the conformation of native virions, react with neutralizing antibodies, and are type-specific. Human papillomavirus early viral peptides induce cytotoxic T lymphocyte responses that retard tumor progression and protect against tumor development after challenge in animal models. CONCLUSIONS: Recombinant papillomavirus virus-like particles are highly antigenic, protective in animal models, lack potentially carcinogenic viral DNA, and are, therefore, ideal candidates for a prophylactic vaccine against HPV infection. Immunization with HPV tumor peptides may be beneficial in tumor prevention, regression, and rejection. Vaccines against HPV infection can be important in reducing the incidence of cervical dysplasia and carcinoma worldwide, particularly in developing countries.

Human papillomavirus infection of the cervix: relative risk associations of 15 common anogenital types.

During the years 1982-1989, 2627 women were recruited into eight studies analyzing the relationship between human papillomavirus (HPV) infection and cervical neoplasia. Subsequently, each individual was assigned as either a case or control, and each cervical sample was rescreened for HPV DNA by low-stringency Southern blot hybridization. Positive samples were retested at high stringency with specific probes for HPVs 6/11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 51, 52, 56, and (in most instances) 58. Most cases (153 cancers, 261 high-grade and 377 low-grade squamous intraepithelial lesions) had target or cone biopsies; all 270 borderline atypia subjects and more than 85% of the 1566 normal controls had cytology plus colposcopy/cytology. Scientists performing HPV testing were masked to the clinical diagnoses. Human papillomavirus DNA was detected in 79.3% of specimens from women with definite cervical disease (627 of 791), in 23.7% of borderline atypia subjects (64 of 270), and in 6.4% of normal subjects (101 of 1566). Graphic analysis of odds ratios at each point in the diagnostic spectrum defined four categories: 1) "low risk" (HPVs 6/11, 42, 43, and 44), present in 20.2% (76 of 377) of low-grade lesions but absent in all 153 cancers; 2) "intermediate risk" (HPVs 31, 33, 35, 51, 52, and 58), detected in 23.8% (62 of 261) of high-grade squamous intraepithelial lesions but only 10.5% (16 of 153) of cancers; 3) "high risk/HPV 16," associated with 47.1% of both high-grade intraepithelial lesions (123 of 261) and cancers (72 of 153); and 4) "high risk/HPV 18" (HPVs 18, 45, and 56), found in 26.8% (41 of 153) of invasive carcinomas but only 6.5% (17 of 261) of high-grade intraepithelial lesions. The presence of an oncogenic HPV type conferred relative risks ranging at 65.1-235.7 for the occurrence of a high-grade lesion and 31.1-296.1 for an invasive cancer.

Verrucous carcinoma of the vulva associated with an unusual type 6 human papillomavirus.

An unusually rapid growing vulvar verrucous carcinoma found associated with human papillomavirus type 6 is described. Both human papillomavirus structural antigens and deoxyribonucleic acid (DNA) sequences were detected in the tumor biopsy. The human papillomavirus genome was isolated and compared with the genome of human papillomavirus type 6b, which was cloned from an anogenital wart. The two genomes were extremely homologous in genomic organization and DNA sequence, except for a region on the viral genome containing the virus control elements. A discussion of this observation and how it could relate to the importance of viral subtypes follows.

Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix.

Restriction enzyme digestion and Southern blot hybridization were used to analyze deoxyribonucleic acid (DNA) extracted from exfoliated cervical cells for the presence of human papillomavirus sequences and these results were correlated with cytologic findings on Papanicolaou smears. Specimens (N = 204) were obtained from a nonselected population of women undergoing routine cytologic screening and human papillomavirus DNA sequences were detected in 33 (16%) women. Thirteen smears contained atypical squamous cells, ranging from very mild dysplasia to moderate dysplasia; all showed associated morphologic evidence of human papillomavirus infection characterized by koilocytosis, nuclear enlargement, wrinkling, and hyperchromasia, and human papillomavirus DNA was demonstrable in 12 (92%) smears. Of the remaining 191 samples with normal cytology, 21 (11%) also contained human papillomavirus DNA sequences. Reevaluation of the smears from these women resulted in a revision of the cytologic diagnosis to very mild dysplasia in four cases. These data suggest that human papillomavirus infection occurs more frequently than predicted by cytologic screening.

Pathogenesis of Pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study.

Pichinde virus has been adapted to produce lethal infection of Strain 13 guinea pigs. Viral replication and presence of viral antigen in frozen tissues stained by immunofluorescence has been previously described. Further investigation into the pathogenesis of this disease has been hampered by the lack of a light microscopic method for correlating histologic lesions and the presence of Pichinde viral antigens. For this purpose, we developed a sensitive immunocytochemical technique for staining Pichinde viral antigens in formalin-fixed, paraffin-embedded tissue. Enhancement of the immunocytochemical staining with nickel chloride markedly improved detection of viral antigens. We examined frozen and formalin-fixed tissues from Strain 13 guinea pigs for viral antigens by light microscopy and immunocytochemistry at various intervals after infection with Pichinde virus. Progressive involvement of different tissues correlated with organ injury measured by serum biochemical abnormalities. Pichinde viral antigen was first detected in splenic macrophages five days after infection and their subsequent destruction facilitated persistent viremia. The inability to clear virus led to multiple organ infection and vascular involvement. Ensuing infections involved particularly the liver, spleen, adrenal glands, lungs, and intestines. Gastroenteritis developed, with extensive involvement of the muscularis mucosa throughout the gastrointestinal tract. Water and food intake decreased rapidly after day 8, leading to marked weight loss. Fatty changes of the liver suggested metabolic derangement that was further exacerbated terminally by adrenal infection and pulmonary impairment.