Properties of human adenoviruses 5 and 7 grown in guinea-pig cells

Summary Human adenoviruses (Ad) 5 and 7 multiplied poorly in guinea-pig cell cultures as compared to HeLa cells. Ad 7 had a modified polypeptide composition with a major 43 K component. This change persisted for up to 4 passages in HeLa cells. Ad 5 was not changed by passage in guinea-pig cells.With 2 Figures     

Human papillomaviruses and cellular oncogenes (c-myc, c-Ha-ras) in cutaneous and mucosal lesions in transplantation recipients]

Transplant recipients develop numerous benign and malignant cutaneous and mucosal lesions in which histological signs of human papillomavirus (HPV) infection are observed. To investigate the role of HPV and c-myc and c-Ha-ras cellular oncogenes' activation in transplanted patients lesions, we used in situ hybridization with biotinylated probes and Southern blot to detect HPV and oncogenes DNA. We analyzed 36 lesions from grafted patients: 11 common warts, 10 actinic keratoses, 13 squamous cell carcinomas and 2 anogenital papillomas. With in situ hybridization, HPV DNA was detected in 14/36 lesions, 10 of which contained several HPV types. Benign and potentially oncogenic HPV types were detected in warts as well as in squamous cell carcinomas. The Southern blot technique confirmed the distribution of HPV types. Group specific viral antigen was detected in 12 lesions, mainly warts. C-Ha-ras oncogene was amplified in 13 lesions and c-myc oncogene in 10 lesions, 9 of which showed both oncogene amplification. The results obtained with in situ hybridization for c-myc gene amplification were confirmed with the Southern blot technique in 11/14 cases. Ras and/or myc amplification was more frequent in squamous cell carcinomas and anogenital papillomas than in warts and actinic keratoses. The amplification was not always linked to the presence of HPV DNA; however, it was more frequent in lesions infected by potentially oncogenic HPV types than in lesions containing only benign HPV types. Myc and p21 oncoproteins were respectively localized in the nucleus and on the membrane of epithelial cells by immunofluorescence. Most lesions showed a good concordance between the detection of oncogene DNA and proteins. Our results suggest than c-Ha-ras and c-myc cellular oncogenes' activation and HPV infection could play a role in the cancerization of cutaneous lesions from transplant recipients.     

Epidermotropism of T cells correlates with intercellular adhesion molecule (ICAM1) expression in human papillomavirus (HPV)-induced lesions.

Adhesion molecules play an important role in inflammatory reactions. Among them, ICAM1, a ligand for the lymphocyte function-associated antigen (FLA1) of leucocytes, may be expressed by antigen-presenting cells and keratinocytes in various inflammatory disorders. As cell-mediated immune responses play a great role in HPV infections, we investigated the expression of ICAM1 and correlated it with the presence of LFA1-positive cells by immunohistochemistry on serial frozen sections of a series of non-regressing cutaneous and mucosal HPV-induced lesions. ICAM1 expression by keratinocytes was observed only in intensely infiltrated lesions of condylomas and laryngeal papillomas. Its induction was usually correlated with the presence of LFA1-positive cells (mainly CD8-positive cells) which were in close apposition to ICAM1-positive proliferative epithelial cells expressing also, in some cases, HLA-DR antigen. ICAM1 was not correlated with the presence of HPV DNA or viral antigen. In moderately infiltrated lesions, keratinocytes did not express ICAM1, and LFA1-positive cells were not observed in the epidermis. In all lesions, ICAM1 was more intense on endothelial cells than in normal skin; infiltrating cells (lymphocytes and dendritic cells) may also express this antigen but intraepithelial Langerhans cells were devoid of any labelling. These studies provide further evidence that T-lymphocyte mechanisms are important in the host response to HPV-induced lesions. ICAM1 expression correlates with a lesional infiltrate but not with HPV infection and probably results in a more efficient initiation of the immune reaction.     

Persistence of Adenovirus 5 in Guinea Pigs

One intracardiac inoculation of adenovirus 5 in guinea pigs leads to virus persistence in different organs, viz., 5 days in lungs and liver, 14 days in blood and lymph nodes, and 56 days or more in the spleen. After cultivation of tissue cells for 1 week, virus was recovered from blood, lymph nodes, or spleen lymphocytes, but virus could be detected directly in cells only when organs were removed within 48 h of inoculation. To determine how the virus persisted in low concentrations and as a latent infection, spleens were primarily selected for study by three techniques: homogenization of spleens, suspended Maitland fragment cultures, and in vitro cultivation of spleen cells. The last procedure showed virus in fibroblast-like cells (probably macrophages or reticuloendothelial cells) for 56 days after infection of guinea pigs. With other methods, the virus was found only within the first 2 days after inoculation.     

Malpighian epithelia and papillomavirus infections]

Human papillomaviruses (HPV) are a large group of DNA viruses, with over 60 types identified to date, which can cause the development of benign tumors in the skin and mucosal squamous epithelia. Most of these tumors regress spontaneously but some, especially in the mucosal membranes, become malignant. HPV types with a high risk for inducing malignancies (e.g. 16 and 18) are the subject of increasing interest. HPVs are both host-specific and tissue-specific: some types preferentially infect specific epithelia, giving rise to lesions with distinct topographic characteristics. HPVs are difficult to study because they do not replicate in available in vitro models. In vivo, HPVs replicate well in epithelial cells undergoing terminal differentiation, e.g. in keratinized cells. Some 40 different types have been reported in epidermal keratinocytes, the most common being types 1 and 2 which produce large amounts of viral antigens and viral particles. In contrast, HPVs replicate poorly in the weakly keratinized squamous epithelia which line the digestive, respiratory, and genital tracts. Junctional epithelia, e.g. on the uterine cervix, are especially prone to HPV infection. The most prevalent HPV types in benign genital lesions are types 6 and 11, whose characteristic features include extrachromosomal DNA and production of only small amounts of viral antigens. The profound nuclear and cytoplasmic changes induced by HPVs lead to the formation of ko?locytes which are found mainly in the granular layer of epithelia and have been especially well described in the uterine cervix and vagina. HPV epithelial tumors are squamous cell carcinomas that often harbor HPV types 16 and 18; this is especially true of cervical intraepithelial neoplasias.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early events in the interaction of adenoviruses with HeLa cells V. Polypeptides associated with the penetrating inoculum

Previous studies revealed that, during adenovirus penetration toward the nucleus, there is no obvious alteration in the appearance of inoculum particles until the uncoating phase has occurred in the vicinity of the nuclear pore complex. Early interactions of adenovirus 5 with HeLa cells were again examined on the premise that subtle, yet morphologically undetectable, changes might take place and can be associated with the removal of a specific capaid component(s). Data from autoradiography show that DNA from inoculum virions labeled with [3H]thymidine enters the nucleus rapidly and efficiently but the bulk of the [³H]leucine-labeled protein remains behind in the cytoplasm. Following isolation of ³⁵S-labeled particles from cell-virus complexes by means of sucrose gradients, analysis of virion polypeptides was conducted by polyacrylamide gel electrophoresis (PAGE). With the possible exception of minor changes in stoichiometric quantities of the fiber or penton + fiber component, the patterns of polypeptides from purified virus or from virus attached to the plasma membrane or isolated from the cytoplasm were very similar. This finding is also valid for the polypeptide spectrum obtained after analysis of intracytoplasmic virions isolated as complexes with microtubule paracrystals. These observations focus attention on the possible function of individual capaid components during internalization and vectorial transfer of the inoculum prior to uncoating.     

A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections

We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.