Specific immunosuppressive activity of epiglycanin, a mucin-like glycoprotein secreted by a murine mammary adenocarcinoma (TA3-HA).

Epiglycanin (Epi) is a mucin-like glycoprotein carrying multiple Thomsen Freidenreich (TF) and Tn determinants secreted by a murine mammary adenocarcinoma, TA3-Ha. As an attempt to further characterize immunoregulatory networks in the TA3-Ha animal model, we tested whether Epi causes active suppression of the cell-mediated immune response against TF determinants. In this study, we show that (a) s.c. injection of epiglycanin emulsified in either complete Freund's adjuvant or Ribi adjuvant containing trehalose dimycolate and monophosphoryl lipid A elicits a classical specific delayed-type hyperactivity response to TF haptens either naturally expressed on epiglycanin or as synthetic haptens conjugated to a protein carrier; (b) i.v. injection of as little as 500 ng of Epi induces specific immunosuppression to anti-Epi and anti-TF synthetic antigen delayed-type hyperactivity responses; (c) this immunosuppression can be abrogated by i.v. injection of cyclophosphamide prior to immunizations; (d) Epi-induced specific immunosuppression can be adoptively transferred by nylon wool-nonadherent cells 6 days following i.v. injection of Epi; (e) pretreatment of suppressor cell populations with anti-Thy-1, anti-L3T4, anti-Lyt-1, or anti-I-Jk but not anti-Lyt-2 monoclonal antibodies plus complement prior to adoptive transfer abolished immunosuppression; (f) i.v. injection of immunosuppressive amounts of Epi on Days 2 and 6 after transplantation of TA3-Ha cells increased the lethality of the tumor transplant. These results suggest that Epi-induced specific immunosuppression in the TA3-Ha animal model is mediated by Thy-1+, L3T4+, Lyt-1+2-, and I-Jk+ suppressor cells. The results are also consistent with the suggestion that this immunosuppression may enhance TA3-Ha tumor growth.

Active specific immunotherapy of a murine mammary adenocarcinoma using a synthetic tumor-associated glycoconjugate.

A synthetic tumor-associated glycoconjugate (S-TAG) "vaccine" formulation was developed for active specific immunotherapy of a murine mammary adenocarcinoma (TA3-Ha). An S-TAG composed of the Thomsen Freidenreich hapten coupled to a conventional carrier protein (keyhole limpet hemocyanin) and emulsified in Ribi adjuvant, when administered s.c. (in four doses at 3 to 6 days apart) into hosts bearing TA3-Ha tumors, provided 25% long-term survival. When administration of this synthetic glycoconjugate was preceded by treatment with cyclophosphamide (100 mg/kg i.v.), 50% long-term survival was observed for hosts in which the tumor had been established for 5 days and up to 90% long-term survival for groups of mice with tumors established for 1 to 2 days. In contrast, a significantly (P less than 0.025) lower level of survival was observed when cyclophosphamide treatment was preceded by active immunizations with the S-TAG tumor vaccine. Surviving tumor-challenged mice that had been treated with cyclophosphamide and the S-TAG vaccine had relatively good IgG antibody and delayed-type hypersensitivity responsiveness to the synthetic Thomsen Friedenreich determinants. About 30% of these animals were also able to resist and sustain long-term survival when rechallenged with a high dose (1 x 10(4] of TA3-Ha tumor cells. Lymph node cells obtained from surviving animals were highly inhibitory to tumor growth in a Winn-type assay.

In vitro interactions of immune lymphocytes and Cryptococcus neoformans.

CBA/J mice immunized subcutaneously with emulsions of heat-killed Cryptococcus neoformans in complete Freund adjuvant displayed delayed-type hypersensitivity to cryptococcal culture filtrate antigen and developed sensitized splenic lymphoid cells which inhibited the growth of C. neoformans in vitro. The in vitro assay of growth inhibition served to investigate further the kinetics of the effect of sensitized lymphoid cells on the pathogen. There was a close correlation between the delayed-type hypersensitivity response in mice and inhibition of growth of C. neoformans by lymphoid cells. Sensitized splenic lymphocytes capable of inhibiting the growth of the cryptococci were detected at day 6 after immunization and reached maximum levels by days 8 through 16. Inhibition of growth was highest with effector-to-target cell ratios of 300:1 or greater. Inhibition of growth of C. neoformans by sensitized lymphoid cells was detectable as early as 4 h after effector and target cells were mixed and increased gradually, reaching a maximum at 24 h, but dropped significantly by 48 h. By supplementing the reaction mixtures with fresh medium or additional sensitized effector cells during incubation, the inhibition of growth of C. neoformans could be maintained through 48 h. C. neoformans-sensitized effector lymphoid populations not only inhibited the growth of the pathogen in vitro but also restricted C. neoformans proliferation in various vital organs upon transfer to naive recipient animals, indicating that the in vitro growth inhibition assay may be a means of assessing the resistance of animals to C. neoformans. The effector cells from sensitized animals were nylon wool-nonadherent Thy-1+ and Ia+ lymphocytes.

Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice.

Some interrelationships among age, ambient temperature, intestinal transit, and enterotoxigenic Escherichia coli infection were studied in an infant mouse model. The transit of dye in the small intestine was accelerated during the response to heat-stable E. coli enterotoxin. Transit in the small intestine of normal mice accelerated with increased age (from less than 17 h to 8 days old) and accelerated with increased ambient temperature (from 25 to 37 degrees C). Transit was more rapid in the jejunum than in the ileum throughout the range of experimental conditions studied. E. coli strains that do not produce any of the pili known facilitate intestinal colonization were cleared from the small intestine more rapidly at 37 degrees C than at 25 degrees C. This clearance was thought to be due to accelerated transit at the higher temperature. In contrast, a strain of E. coli that produces K99 (pili previously shown to facilitate intestinal colonization in other species) was not cleared from the small intestine and colonized more intensively at 37 degrees C than at 25 degrees C. Intensified colonization by this strain was thought to be due to increased production of K99 at the higher temperature. It was suggested that sluggish intestinal transit may also be characteristic of the neonates of other species and be one of the factors predisposing them to intestinal colonization by enteropathogens. It was speculated that this predisposition may be enhanced if the neonates are chilled. However, the effect of ambient temperature on intestinal transit in homeothermic neonates such as pigs, calves, and humans may be different from that in mice because neonatal mice are poikilothermic.

Effects of age and ambient temperature on the responses of infant mice to heat-stable enterotoxin of Escherichia coli: assay modifications.

The response of infant mice to heat-stable enterotoxin from Escherichia coli was affected by the age of the mice (2, 4, 6, and 8 days) and by the ambient temperature (25, 30, and 37 degrees C) after exposure to the enterotoxin. The younger mice and/or mice held at lower temperatures tended to accumulate intestinal fluid (high gut weight/body weight ratios), but older mice and/or mice held at higher temperatures tended to respond with diarrhea and low gut weight/body weight ratios. The standard infant mouse assay forheat-stable E. coli enterotoxin can be simplified, without loss of sensitivity or reliability, by holding the mice at 37 degrees C after exposure and using diarrhea as the index of response. Diarrhea can be detected easily by incorporating dye in the inocula and (at the end of the assay) checking for dye mixed with feces on the rear quarters of the mice or on a sheet of white paper placed under them during incubation.

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene.

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP--MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS--MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-amino-acid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA-MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayed-type hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide "vaccines" could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.