Biomedical subjects
D D Bigner
Publications and source records attributed to D D Bigner.
Contained indirect viable-cell membrane immunofluorescence microassay for surface antigen analysis of cells infected with hazardous viruses.
A microtechnique for an indirect viable-cell membrane immunofluorescence titration assay was developed using Friend murine leukemia virus (F-MuLV)-producing cells and monospecific rabbit antisera to F-MuLV structural antigens. The assay was sensitive and displayed little variation within or between assays. Since moderate-risk tumor viruses, such as recently discovered primate oncornaviruses or feline leukemia virus (FeLV), may be hazardous to laboratory personnel, the assay was adapted for containment of cells infected with such viruses. Cells producing gibbon ape lymphoma virus or FeLV were grown in class III containment cabinets and transferred in sealed flasks to a class II laminar-flow cabinet, where the assay was performed. This micromethod not only conserved reagents but also minimized the numbers of moderate-risk tumor virus-infected cells handled at one time. Centrifugation was contained using custom-made devices shown to form a gas-tight seal over microtiter plates. Interspecies reactivity of monospecific rabbit antisera against F-MuLV structural antigen gp71, but not against p12, was demonstrated for surface antigens on FeLV-producing cells.
Immunobiology of primary intracranial tumors. Part 1: studies of the cellular and humoral general immune competence of brain-tumor patients.
Cellular and humoral immune parameters were evaluated in a series of brain-tumor patients, 42 with glioblastoma multiforme, 17 with other anaplastic gliomas, and 17 with meningiomas. A degree of anergy was found, which seems in the group as a whole to be proportional to the degree of anaplasia. In addition, serial bimonthyl testing in individual cases revealed further reduction in certain immune responses coincident with clinical decline.
Immunobiology of primary intracranial tumors.
The avian sarcoma virus-induced glioma model in rats was used to study the effectiveness of immunotherapy, namely, Bacillus Calmette-Guérin (BCG) with or without sarcoma cells and/or chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), upon survival times. The most consistent prolongation of survival time was produced by triple therapy: BCG + intraperitoneally administered sarcoma cells + intravenously administered BCNU.
Influence of age at inoculation on avian oncornavirus-induced brain tumor incidence, tumor morphology, and postinoculation survival in F344 rats.
Intracranial neoplasms were induced by intracerebral inoculation of a standardized, cell-free inoculum of the Bratislava-77 strain of avian sarcoma virus in F344 rats at 1, 9,97 to 99, and 528 days of age. Deaths from diseases that occur spontaneously in aged F344 rats complicated assessment of tumor incidence in rats inoculated at 528 days; 20 of 30 rats inoculated at this age developed brain tumors. All rats inoculated at age 1 day (47 rats), at age 9 days (37 rats), and at 97 to 99 days of age (41 rats) developed brain tumors. The incidence of animals developing tumors was 100% in these three groups, but the incidence of multiple tumors declined with increasing age at inoculation. The mean and variance of postinoculation survival increased from 83.8 +/- 21.5 days for rats inoculated at 1 day of age to 284.6 +/- 151.5 days for rats inoculated at 97 to 99 days of age. Poorly differentiated astrocytomas and astrocytomas of mixed morphology were common among rats inoculated as neonates. Solitary, pilocytic astrocytomas were the most common tumors among rats inoculated as adults.
Surface morphology of normal and neoplastic rat cells.
Nontumorigenic rat cells and their tumorigenic counterparts were studied with scanning electron microscopy under controlled conditions in vitro and with transmission electron microscopy after replantation in vivo to discern if external morphology reflected the cell's neoplastic state or the etiology of transformation. Interphase cells in six of seven nontumorigenic lines were flat and monolayered under confluent conditions and exhibited smooth, nonactive cell surfaces. A nontumorigenic cell line morphologically transformed with human adenovirus-2 consisted of spherical cells with blebbed surfaces. Cells from six tumorigenic lines transformed with avian sarcoma virus had highly active surfaces with many surface projections. Cells from two chemical carcinogen-transformed rat embryo lines were flat with no surface projections in subconfluent culture and rounded with only a few microvilli at high densities, but cells from a sarcoma chemically induced in an adult rat were villous. When villous cells were syngeneically replanted in vivo, they lost most microvilli. The external morphology of cells was influenced by a number of factors simultaneously, with no universal pattern associated with tumorigenic capacity or transforming agent.
Immunofluorescent analysis of expression of the RNA tumor virus major glycoprotein, gp71, on surfaces of virus-producing murine and other mammalian species cell lines.
The specificity of a single rabbit antiserum pool raised against the purified major glycoprotein, gp71, of Friend murine leukemia virus was determined for a variety of virus-producing mouse, feline, and gibbon ape cell lines by viable cell membrane immunofluorescence absorption. Among murine cells examined, Friend gp71 type specificity was shared only with Rauscher virus-producing cells, and a group specificity was present for all the murine leukemia virus-producing cells tested. Friend and Rauscher murine leukemia virus-infected cells shared interspecies cross-reactivity with feline leukemia and gibbon ape lymphoma virus-producing cells. However, Moloney, Gross, and other virus-producing murine cells shared some, but not all, of these gp71 interspecies determinants with the feline and primate cells. Immunoferritin electron microscopy localized these gp71 antigenic determinants on both virus and cell membranes.
Immunofluorescent analysis of expression of the RNA tumor virus major glycoprotein, gp71, on the surfaces of normal murine cells.
The expression of the major glycoprotein, gp71, of murine leukemia virus was studied on the surfaces of a variety of normal murine cell lines with a monospecific rabbit antiserum raised against purified Friend murine leukemia virus gp71. Using viable cell membrane immunofluorescence, most established and early passage normal murine cell lines were significantly reactive with the antiserum, irrespective of neoplastic transformation, strain genotype, or whether they were of embryonic or adult tissue origin. The only murine cells tested not detectably expressing gp71 determinants were BALB/3T3 lines. Although some Friend gp71 interspecies reactivity was discernible on normal murine cells, the principal reactivity was shown to be group specific. Fresh thymocytes from BALB/cJ (GIX-), C57BL/6J (GIX-), and 129/J (GIX+) mouse strains were also reactive with Friend gp71 antiserum, and this activity, as well as that of an antiserum prepared against purified AKR gp71, were also group specific. An activity discriminating GIX+ from GIX- thymocytes was not observed with either Friend or AKR gp71 antisera.
Antigens shared by rat schwannomas, neuroblastomas, and a testicular interstitial cell carcinoma.
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Surface antigenic characteristics of human glial brain tumor cells.
The surface antigenic characteristics of human glial brain tumor (HGBT) cells were studied by complement-dependent cytotoxic antibody assays and indirect membrane immunofluorescence. Eight permanent, well-characterized cell lines derived from human gliomas were used for analysis with antisera raised by hyperimmunization of nonhuman primates (Macaca fascicularis) with glioblastoma multiforme tissue or established HGBT cells lines. Exhaustive absorption of these antisera to remove predominantly antispecies activity rendered HLA nonreactive "preabsorbed" antisera, which reacted with a large panel of gliomatous and nongliomatous human tumor cells; 1 carcinoma, 2 sarcomas, 2 melanomas, 1 neuroblastoma, and 8 HGBT cell lines. Four lymphoblastoid lines and 2 carcinomas were unreactive. After further absorption with a human osteogenic sarcoma cell line, the antisera demonstrated significant levels of reactivity for 8 tested HGBT cell lines and no longer reacted with the nongliomatous cultured tumor cells lines. Therefore, extensive absorption of nonhuman primate anti-human glioma sera removed all activity for the nongliomatous cell lines tested, but it left significant reactivity against a glial tumor cell line-associated antigen(s) present on all 8 human glioma cell lines tested.
Cellular immunity in rats with primary brain tumors: inhibition of macrophage migration by soluble extracts of avian sarcoma virus-induced tumors.
Rats bearing primary tumors of the brain induced by avian sarcoma virus (ASV) were studied with the migration-inhibition factor (MIF) assay for the presence of cell-mediated immunity to tumor-associated antigens. Astrocytomas and sarcomas of the brain were induced in 34 neonatal F344 rats by the intracerebral inoculation of Bratislava-77 ASV. At weekly intervals from 4 to 9 weeks after the inoculation with virus, peritoneal exudate cells (PEC) from rats bearing brain tumors were tested an an MIF assay against soluble and KCl-treated extracts of a syngeneic, ASV-induced sarcoma. Incubation of the PEC with a soluble extract of syngeneic liver or with a KCl extract of a syngeneic, chemically induced tumor served as controls. Of 14 rats tested against the soluble tumor extract, 6 (43%) had statistically significant inhibition of migration (P less than or equal to 0.05). Of 23 animals tested against the KCl extract, 16 (70%) had significant inhibition. Immunity to the KCl extract was significant in most rats at each period. Ten rats were tested against a KCl extract of a hamster ASV-induced tumor; 7 gad significant inhibition of migration. None of 3 tested against a soluble extract of a syngeneic, chemically induced tumor had significant inhibition. Rats bearing ASV-induced brain tumors displayed cell-mediated immunity to tumor-associated antigen or antigens of ASV-induced tumors, which could be solubilized.
Sequential adsorption analysis of antibodies to neuroectodermal cells by an indirect quantitative method.
An indirect radioimmunoassay was developed for the sequential adsorption analysis of rabbit antibodies raised against rat neuroectodermal cells. The quantitativee relationship between primary adsorbed rabbit antitumor antibodies and secondary adsorbed goat antibodies to rabbit IgG was explored by paired radioiodine analysis. In the final indirect method, the amount of unlabeled rabbit antibody removed by cultured monolayers of cells at each step in a sequence of cells could be determined from an equation relating the unlabeled amount to values of bound 125I-labeled goat antirabbit IgG. To obtain the total quantity of rabbit antibody in a particular antiserum reagent, a sequential adsorption analysis was done with successive steps of homologous cells. To obtain the amount of antibody that was cross-reactive for other cell lines, we included those lines in the first several steps of the sequence. The sequential adsorption profile was considered as a more important indicator of the quality of a particular antiserum reagent than was the total amount of antibody contained in it. Neuroectodermal cell lines used as illustrative examples included subclones of the C6 astrocytoma and of the RN-2 schwannoma.
The fine structure of intracranial neoplasms induced by the inoculation of avian sarcoma virus in neonatal and adult rats.
Groups of F-344 rats were inoculated with the Bratislava-77 strain of avian sarcoma virus (B-77 ASV) within 24 hours of birth, at 9 days of age, or between 97 and 119 days of age. Intracranial tumors developed in each age group. Multiple tumors with mixed histologic patterns developed in rats inoculated at 1 or 9 days of age. Solitary tumors with a uniform histologic pattern developed in rats inoculated as adults. On the basis of light and electron microscopic study, the majority of tumors in each age group were classified as astrocytomas and divided into either poorly differentiated, gemistocytic, pilocytic, or polymorphic varieties. The polymorphic astrocytomas were most common among neonatally inoculated rats, while the pilocytic astrocytomas were most common among rats inoculated as adults. Ultrastructural characteristics of astrocytes, including gap junctions and 7- to 9-nm filaments, were present in the majority of tumors in each age groups. Astrocytomas induced in adult rats were remarkable for the presence of extensive basement membrane alone the astrocytic cell surfaces. Intracytoplasmic virus-like particles (R particles) were common in the tumor cells. These virus-like particles are morphologically distinct from C-type B-77 ASV, and no morphologic evidence of C-type virus replication was observed in any of the tumors.
Virus-induced experimental brain tumors and putative associations of viruses with human brain tumors: a review.
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R-type virus-like particles in avian sarcoma virus-induced rat central nervous system tumors.
Morphologically distinct virus-like particles (VLP), similar to R-type VLP, were observed by electron microscopy in experimental rat central nervous system tumors induced with the B-77-C strain of avian sarcoma virus (ASV). R-type VLP have a characteristic internal radial structure and were observed previously only in hamster cells and in an established bovine cell line. They were not observed in the B-77 ASV inoculum used to induce the rat tumors or in the B-77 induced hamster glioma cells from which the B-77 was rescued. Nevertheless, the genome of an endogenous hamster R-type particle also might have been rescued and carried in the B-77 inoculum. Alternatively, R-type VLP may exist in a number of animal species, including the rat, and may be expressed in certain conditions such as neoplastic transformation.
The induction of intractranial neoplasms by the inoculation of avian sarcoma virus in perinatal and adult rats.
The induction of intracranial neoplasms following the intracerebral inoculation of avian sarcoma virus (ASV) in neonatal mammals is well established. The present study demonstrates the susceptibility of adult rats and compares the incidence and morphology of tumors induced by a uniform inoculum of the Bratislava-77 strain of ASV in adult, neonatal, and fetal Fischer 344 rats. Post-inoculation mortality varied significantly between groups inoculated at 1, 10 and 100 days and was most precipitous in perinatally inoculated rats. Percentage of tumor induction declined from 100% among rats inoculated at 1 day of age to 50% among rats inoculated at 100 days of age. The mean number of tumors/animal was inversely proportional to the logarithm of the age at inoculation. A large majority of tumors in each group were glial; the remainder were mesenchymal and mixed glial and mesenchymal. Neuroglial tumors included: mixed gliomas with oligodendroglial and astrocytic elements; and gemistocytic, pilocytic, fibrillary, anaplastic and protoplasmic astrocytomas. Tumors induced in perinates were more heterogeneous in histological pattern while tumors induced in perinates were more heterogeneous in histological pattern while tumors induced in older animals tended to be purely astrocytic and of uniform cell type. Mesenchymal tumors occurred primarily in the meninges and were common among animals inoculated perinatally but were rare among animals inoculated as adults. No neuronal tumors were encountered even among rats inoculated as early as 16 days of gestation.
Expression of murine leukemia virus structural antigens on the surface of chemically induced murine sarcomas.
Cultured cells of different chemically-induced C57BL/6N murine sarcomas produced variable amounts of infectious murine leukemia virus (MuLV) and contained proportional amounts of MuLV structural components as determined by radioimmunoassay. Monospecific antisera directed against the major MuLV glycoprotein (gp71), the major internal antigen (p30), and the ribonucleoprotein (p10) were capable of mediating tumor cell lysis in the presence of complement, suggesting that these viral structural components were localized at least in part to the cell surface. Membrane immunofluorescence studies with MuLV p30 antiserum confirmed surface localization. Addition of MuLV p30 polypeptide to normal cells and tumor cells enhanced the cytotoxicity of MuLV p30 antiserum. Studies are presented which suggest that the presence of MuLV structural components on cell surfaces can be independent of virus production and cellular transformation.
Improved techniques for staining and enumerating focus formation in viral infectivity assays.
Improved techniques consisting of glutaraldehyde fixation, Giemsa staining, and colony counter enumeration of foci in viral infectivity assays were compared to microscope scanning and lantern slide projection methods. These techniques broadened the scope of application to potentially hazardous viruses and increased the efficiency of assaying.