Effect of a fungal Cu/Zn superoxide dismutase on the cell-mediated immune response in Graffi tumor bearing hamsters

The antibody-dependent cell cytotoxicity (ADCC) of spleen lymphocytes, isolated from hamsters with progressing myeloid Graffi tumor, was studied. The effect of the application of Cu/Zn superoxide dismutase, isolated from the fungal strain Humicola lutea (HL SOD), before and during tumor transplantation on the lymphocyte ADCC was examined. Myeloid Graffi tumor cells as target cells were used. Antibodies from a rabbit hyper-immune anti-tumor Graffi cells serum, or from tumor-bearing hamsters serum were used in the test. The leukocyte adherence inhibition (LAI) in the presence of tumor antigen was examined also during tumor progression. ADCC of the spleen lymphocytes, determined by both, rabbit and hamster anti-tumor antibodies, decreased during tumor progression. The optimum treatment of the animals by HL SOD induced a 20-30% increase of lymphocyte cytotoxicity against myeloid Graffi tumor cells. Cytotoxicity in presence of tumor bearing hamsters serum was twofold lower as compared to that one determined in the presence of rabbit hyper-immune anti-myeloid Graffi tumor cells serum. Leukocyte adherence inhibition (LAI) index in the presence of tumor antigen increased during tumor development in the groups of treated and untreated animals. The LAI indices of HL SOD-treated tumor-bearing hamsters were lower than that of untreated animals with tumors, what can be explained by a higher adherence ability of leukocytes induced by HL SOD treatment (in formula for calculation of LAI index the adherence value is in the denominator). The results show the beneficial effect of HL SOD on the cell-mediated immune response of myeloid Graffi tumor bearing hamsters, what is probably due to the participation of the enzyme in the host's oxidant-antioxidant balance.     

BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes

Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host.     

Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells

Feldman, Lawrence A. (Baylor University College of Medicine, Houston, Tex.), Janet S. Butel, and Fred Rapp. Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells. J. Bacteriol. 91:813-818. 1966.-Adenovirus types 2, 7, and 12 undergo an abortive growth cycle in green monkey kidney cells; they induce the formation of adenovirus tumor antigen, but synthesis of adeno capsid antigen and infectious adenovirus was observed only when cultures were concomitantly infected with a simian papovavirus (SV40). Several other viruses, including herpes simplex and measles which replicate in monkey cells, and rabbit papilloma and human wart papovaviruses which do not, failed to stimulate adenovirus replication in the monkey cells. Adenovirus tumor antigen was detected 8 to 10 hr postinfection by immunofluorescent techniques. The antigen induced by adenovirus types 2 and 7 appeared as intranuclear masses; adenovirus type 12 tumor antigen also appeared as cytoplasmic and nuclear flecks. Sera from hamsters bearing tumors induced by adenovirus type 12 cross-reacted with tumor antigens induced by types 2 and 7 but not with antigens induced by SV40.     

Forssman antigen and phase specific fetal antigens: an evaluation of their role in SV40 tumor immunity.

Forssman heterophile antigen was detected on hamster fetal cells which had been previously shown to be capable of eliciting transplantation resistance to syngeneic hamster SV40 tumors. The expression of Forssman antigen continued throughout fetal development and could be detected postpartum in the tissues of neonate hamsters. In contrast, fetal antigen(s) evoking immunity to SV40 tumors was also present on early fetal cells but, unlike Forssman antigen, was not expressed after the tenth day of gestation. Immunization of hamsters with guinea pig kidney cells or sheep erythrocytes which carry Forssman antigen failed to demonstrate significant protection against SV40 tumor development. Again by contrast, immunization with fetal cells was effective in evoking tumor immunity. Evaluation of serological responses to the FORSSMAN A ANTIGEN IN HAMSTERS INDICATED THAT THE HEMOLYTIC REACTIVITY PRODUCED BY IMMUNIZATION WITH GUINEA PIG KIDNEY CELLS OR SHEEP ERYTHROCYTES WAS ELICITED AGAINST ISOANTIGENS AND NOT THE Forssman antigen. A response to the Forssman determinant could only be detected in the serum from animals receiving exhaustive hyperimmunization with fetal cells or SV40 tumor cells. These data would eliminate a possible role of the Forssman heterophile antigen in the tumor protection evoked by immunization with fetal cells bearing embryonic antigens.     

Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 x 10(4) to 4 x 10(4), which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Possibility of detecting human embryonal leukemia antigen]

A possibility of detecting embryonic leukemic antigen on human leukemic blast cells in an acute human leukemia cytotoxicity test with the sera and 7S and 19S serum immunoglobulins of the placental blood was studied. The presence on the blast cells of patients suffering from acute leukemia of an antigen detectable by antibodies of placental blood (of parturients) was demonstrated; this antigen was absent on the leukocytes of healthy donors.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

The role of surface structures of Streptococcus agalactiae in adhesion to epithelial cells

Induced mutants of S. agalactiae which differed in surface structures were used for the study. The aim of using them was to try to correlate the presence of defined structures or surface properties with the ability of group B streptococci to attach to epithelial cells. The presence of protein antigen R conditioned strong binding of S. agalactiae cells to hydrophobic gel. Strains bearing clumping factor (CF) showed high surface hydrophobicity and presented compact growth in serum soft agar. However, there was no correlation between high surface hydrophobicity and the ability to adhere. Fibrinogen binding decreased the attachment to epithelial cells of CF-positive strains. Preincubation of bacterial cells with lectin (ConA) did not influence the attachment of S. agalactiae strains with protein surface antigen but increased the adhesion of the strains with polysaccharide antigen or untypable.     

Alternate Changes of Surface Antigen(s) in Adenovirus Type 12-Transformed and Tumor Cells1

Alternate changes of specific surface antigen(s) (S antigen) were examined in transformed and tumor cells induced by human adenovirus type 12. All of the hamster and mouse cells transformed in vitro showed ring-form membrane fluorescence staining by anti-S antigen rabbit sera, whereas tumor cells, either induced by the virus in vivo or produced by inoculation with the S(+)-transformed cells, did not show any fluorescence. When the S(–) tumor cells were serially subcultured in vitro, all of them converted to S(+) cells, although more than ten subcultures were necessary. For the S(+) cells to form tumors in hamsters about ten times as many cells were necessary as the S(–) cells. This difference became greater when tumor formation was tested in preimmunized hamsters, while little, if any, when tested in X-irradiated hamsters. In addition, immunogenicity of the S(+) cells was suggested to be higher than that of the S(–) cells. These findings indicate that the S(+) cells are more immunosensitive and immunogenic than the S(–) cells, and that in vivo conversion from S(+) to S(–) may be due to selection of S(–)-mutant cells. In vitro conversion from S(–) to S(+) was also suggested to be due to the appearance of S(+)-mutant cells.     

Protein kinase activity immunoprecipitated from adenovirus infected cells by sera from tumor-bearing hamsters.

Earlier, we reported (N. J. Lassam, S. T. Bayley, F. L. Graham, and P. E. Branton, Nature (London) 277:241-243, 1979) detecting protein kinase activity when cytoplasmic extracts of human adenovirus type 5 (Ad5)-infected KB cells immunoprecipitated with 14b antitumor serum directed against the transforming proteins of Ad5, were incubated with [gamma-32P]ATP. Here we show that in the in vitro assay this kinase phosphorylated both the heavy chain of immunoglobulin G and polypeptide than comigrated on sodium dodecyl sulfate gels with the 58,000-dalton Ad5 antigen. It also phosphorylated added histone H3. Evidence is presented that the protein kinase activity found with extracts from Ad5-infected cells is not due to nonspecific trapping of cellular enzymes in immune complexes, but to an enzyme which is distinct from kinases detected at background levels in controls. Serine and threonine were the major phosphorylated amino acids, and essentially no phosphotyrosine was detected. Protein kinase activity detected in Ad12-infected cells immunoprecipitated by an antiserum derived from hamsters bearing Ad12-induced tumors appeared to be immunologically distinct from that immunoprecipitated from Ad5-infected cells by 14b serum.     

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.     

The immunoglobulins of carcharhine sharks: a comparison of serological and biochemical properties

The immunoglobulins of three carcharhine sharks were isolated from serum by means of salt precipitation and gel chromatography. The Galapagos shark (Carcharhinus galapagensis), the sandbar shark (Carcharhinus plumbeus) and the tiger shark (Galeocerdo cuvieri) each contained high molecular weight (18S) and low molecular weight (7S) IgM-like molecules as the major serum immunoglobulins. Both within and between species 18S and 7S immunoglobulins closely resemble each other in antigenic character, polypeptide chain composition, chain mass, amino acid composition, carbohydrate content and amino-terminal sequence. These results suggest that the immunoglobulins of carcharhine sharks have undergone little structural divergence during their evolution.     

Detection and Assay of Avian Tumor Virus Group-Specific Antigen and Antibody by the Paired Radioiodine-Labeled Antibody Technique

The paired radioiodine-labeled antibody technique (PRILAT) was applied to the detection and quantitation of avian tumor virus group-specific (gs) antigens and antibody. The technique proved to be specific, repeatable, and appreciably more sensitive than the microcomplement-fixation test for avian leukosis (COFAL). The PRILAT facilitated direct measurement of comparative antigen content of several types of transformed, neoplastic, or virus-infected cells and the magnitude of nonspecific antibody binding by appropriate control cells. The versatility of the technique was illustrated by application to the detection and quantitation of gs antibody content of chicken, turkey, pigeon, and hamster sera. Antibodies were detected in COFAL-negative sera from hamsters bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Sera from chickens bearing similar tumors were not positive for gs antibodies, although sera from turkeys and chickens immunized with avian leukosis virus did contain gs antibodies.     

Combined S99/RB51 antigen for complement fixation test for serological diagnosis of brucellosis in cattle and sheep

AIMS: To assess the efficiency of a single antigen for the complement fixation (CF) test, prepared by combining Brucella abortus smooth strain 99 (S99) with Brucella abortus rough strain RB51(RB51), in detecting cattle and sheep infected or vaccinated with Brucella spp. METHODS AND RESULTS: Serum samples from B. abortus-infected and RB51-vaccinated cattle were tested by the CF test using S99, RB51 and the combined S99/RB51 as antigens. Likewise, serum samples from Brucella melitensis-infected, RB51-vaccinated and Brucella ovis-infected sheep were tested by the CF test using S99, RB51, hot saline (HS) and combined S99/RB51 as antigens. Comparative analysis of the CF results showed that no reduction of sensitivity or specificity occurs when S99/RB51 antigen is used instead of specific antigens used separately. CONCLUSIONS: The results of this study indicated that combined S99/RB51 antigen used in the CF test, because of its specificity and sensitivity, could be used in animal brucellosis surveillance systems to improve the efficiency of the preliminary screening of herds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes an improved antigen for the CF test for the epidemiological survey of animal brucellosis. It could represent advantages over standard protocols because of its ability to detect antibody responses following infection or vaccination withBrucella strains of rough and smooth phenotype.     

Studies on Nondefective Adenovirus-Simian Virus 40 Hybrid Viruses I. A Newly Characterized Simian Virus 40 Antigen Induced by the Ad2+ND1 Virus

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen-the SV40 "U" antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T(+)U(+) sera) and those that react with SV40 T antigen only (T(+)U(-) sera). SV40 U-specific sera from monkeys immunized with Ad2(+)ND(1)-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T(+)U(+) hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2(+)ND(1) virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Golden hamsters were used as hosts in this work, and mice of various strains as donors of antigens. 1) There were no strain differences in immunogenicity of erythrocytes from C57BL/6, AKR, SL and CF1 mice. 2) Primary intravenous immunization with mouse erythrocytes (MRBC) induced the production of hemolysin plaque-forming cells (PFC) in a large number, but elicited only in a negligible titer production of 2-mercaptoethanol-resistant antibody. 3) 2-Mercaptoethanol-resistant antibody was produced more efficiently in hamsters pre-sensitized with mouse lymph node (MLN) cells rather than those pre-immunized with MRBC after a booster with MRBC. 4) Numbers of PFC in pre-sensitized hamsters were three-times that of the non-sensitized hamsters after a booster with MRBC, when pre-sensitization was performed intradermally with a small number of MLN cells. 5) Average diameter of the hemolysin plaques in pre-sensitized hamsters was one and a half times larger than that in non-sensitized hamsters. Conclusions agree well with the results in our previous papers that the reversed combination of hosts and antigen donors employed support the concept that certain processes required for delayed hypersensitivity contributed to antibody production under a condition suitable for antibody response.     

Studies on Complement-Fixation Reaction in Equine Infectious Anemia

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

Lymphotropic papovavirus transformation of hamster embryo cells

Hamster embryo cells were transformed by African green monkey lymphotropic papovavirus (LPV). The transformed cells contained intranuclear T-antigens demonstrable by fluorescent antibody staining with hamster anti-LPV serum. Analysis of uncloned and cloned lines of transformed cells for LPV sequences revealed that the viral DNA was present as free nonintegrated and integrated genomes; there were approximately 10 copies of free DNA and about one to two copies of integrated genomes per cell. The cells were highly tumorigenic when inoculated into hamsters and produced progressively growing tumors in 100% of newborn or 10-day-old hamsters that were inoculated with LPV-transformed cells. The serum from tumor-bearing hamsters reacted with LPV-transformed cells and also showed a weak reaction with simian virus 40-, BK virus-, and JC virus-transformed cells, thereby showing an antigenic relationship with the T-antigens of other primate polyomaviruses. The large T-antigen of LPV was found to be an 84,000-molecular-weight protein which was immunoprecipitated by hamster anti-LPV (antiviral) as well as by tumor serum.     

Studies on complement-fixation reaction in equine infectious anemia. II. Identification of complement-fixing inhibitors.

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.