The strange cross-reaction of menadione (vitamin K3) and 2,4-dinitrophenyl ligands with a myeloma protein and some conventional antibodies.

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of [(3)H]-epsilon-DNP-L-lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K(3)). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 10(5) L/M (at 4 degrees C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal).

Transplantation immunity and cross-protection of two cultured murine colon lines.

Previous comparative investigations of the in vivo biology of two cultured colon tumors of BALB/c origin, C-C26 and C-C36, demonstrated different biologic activities. For elucidation of these differences, this report investigated the immunogenicity of these lines in providing protection to a subsequent challenge with different doses of tumor cells. Furthermore, the specificity of this protection was evaluated with cross-protection experiments. Our findings demonstrated these lines to be immunogenic and suggested the presence of cross-reactive tumor rejection-type antigens on these 2 cultured colon tumor lines. BALB/c mice immunized with these tumors developed resistance to challenge with the same tumor as well as to challenge with the other colon tumor lines. Further definition of these putative antigens could provide us with a better understanding for the diagnosis and treatment of colon tumors.

Characterization of tumorigenicity, mortality, metastasis, and splenomegaly of two cultured murine colon lines.

The murine colon tumors 26 and 36 have been adapted to culture in vitro from the parental, serially transplanted tumors. The biological activities of these cultured colon lines (C-C26 and C-C36) have been characterized in vivo by s.c. inoculation of serial doses of cells into syngeneic BALB/c mice. The C-C26 line is highly tumorigenic and has a low tendency (9.1%) for metastasis to the lungs. Moreover, mice inoculated with C-C26 exhibited high mortality and normal or atrophied spleens. In contrast, the C-C36 line is less tumorigenic and highly metastatic to the lungs (77.8%). Mortality was lower in animals inoculated with C-C36, and at autopsy, splenomegaly was frequently (72.2%) observed without any visible metastatic nodules in these spleens. Metastasis to the lungs was intimately associated with splenomegaly, and death followed closely. Our findings with the C-C26 and C-C36 lines agree with those reported for the parental tumors with respect to tumorigenicity, tumor growth, and mortality. However, they differ with respect to their metastatic potential, because previous reports showed this to be higher for the serially transplanted colon 26 than the colon 36 tumor cells. Furthermore, this work describes the remarkable splenomegaly observed in mice inoculated with C-C36 but not with C-C26. This finding may in turn provide us with the opportunity to compare the functional status of the spleen in these tumor-bearing animals.

Immune responses in mice infected with lactic dehydrogenase virus. IV. Functional status of the macrophage during acute LDV infection.

Macrophages from uninfected and lactic dehydrogenase virus (LDV)-infected mice were compared with respect to the affinity and number of their Fc receptors for IgG2a; no differences were found regarding these parameters. When the uptake of DNP-BGG by macrophages from uninfected and acutely LDV-infected mice was compared, again no differences were observed. However, when the per cent membrane-bound DNP-BGG was determined as a function of time after antigen uptake in these two groups, more DNP-BGG was found membrane-bound on the macrophages from the LDV-infected mice, than on uninfected macrophages. In view of the fact that humoral immunity is enhanced during acute LDV infection, these data provide a positive correlation between increased retention of membrane-bound antigen and enhanced humoral immune responses.

Immune responses in mice infected with lactic dehydrogenase virus. I. Antibody response to DNP-BGG and hyperglobulinaemia in BALB/c mice.

The humoral immune response to DNP-BGG of BALB/c mice acutely infected with lactic dehydrogenase virus (LDV) has been investigated. Virus-infected mice injected with antigen in saline exhibit a greater anti-DNP response than uninfected controls. When this antigen is presented in Freund's complete adjuvant (FCA) the anti-DNP response is greater than obtained with antigen in saline, but significant differences between infected and uninfected controls are not observed. These data are consistent with the view that acute LDV infection can have an adjuvant-like effect when this T-dependent antigen is introduced in saline. In addition, the effect of viral infection on plasma Ig class and subclass levels has been investigated. LDV infection leads to a gradual increase in plasma Ig concentration. This effect is restricted to the IgG2a subclass in most animals, but occasionally is restricted to IgG1. The mechanisms responsible for these changes have not been delineated.

Characterization of in vivo cross-reactive tumor resistance between two cultured murine colon lines.

Previous comparative investigations of the in vivo biology of two cultured colon tumors of BALB/c origin, C-C26 and C-C36, demonstrated different biologic activities. To elucidate these differences, the immunogenicity of these lines in providing protection to a subsequent challenge with different doses of tumor cells was investigated. Furthermore, the specificity of this protection was evaluated with cross-protection experiments. The findings demonstrate these lines to be immunogenic and suggest the presence of cross-reactive tumor rejection type antigens on these two cultured colon tumor lines. BALB/c mice immunized with these tumors developed resistance to challenge with the same tumor as well as to challenge with the other colon tumor line. Unexpectedly, BALB/c mice immunized with C-C26 cells and challenged with a syngeneic methylcholanthrene-induced sarcoma, Meth A, exhibited partial resistance to this tumor compared to nonimmunized controls. However, mice immunized with C-C36 cells and challenged with Meth A cells supported the same rate of growth of this tumor as unimmunized controls. These results suggest the presence of at least two different cross-reactive tumor rejection antigens. The first antigen appears to be shared between C-C26 and C-C36 cells while the second antigen may be shared between C-C26 and Meth A. Further definition of these putative antigens could provide us with a better understanding for the diagnosis and treatment of colon tumors.

Effect of splenectomy on the growth of murine colon tumors.

We have previously demonstrated a high incidence of splenomegaly in BALB/c mice bearing colon tumor C-C36, but not colon tumor C-C26. To characterize this difference, tumor development in mice splenectomized seven days before tumor inoculation was compared to that of unoperated control mice. Our findings indicate that during the early phase, tumor growth is enhanced for both C-C26 and C-C36 in the splenectomized animals. The lag period for tumor appearance was also somewhat shorter in these splenectomized animals as compared to the controls, and metastatic tendency to the lungs was unaffected by splenectomy. These findings may have useful implications for the role of the spleen in the tumor-bearing animal.