Dermatitis due to intravesical mitomycin C: a delayed-type hypersensitivity reaction?

Mitomycin C is an alkylating agent, used by intravesical instillation to treat carcinoma of the bladder. Repeated instillations can induce cystitis and an eczematous eruption affecting the palms, soles and face. If these effects are due to delayed hypersensitivity with sensitization to mitomycin C occurring in the bladder wall, it should be possible to demonstrate antigen-presenting cells in the bladder wall and positive patch tests to the drug. Using an immuno-alkaline phosphatase method we have identified CDI+ cells in bladder epithelium and submucosa and have demonstrated Birbeck granules in a few cells. In further support of our hypothesis it was also possible to demonstrate delayed type hypersensitivity in 13 out of 26 patients who had received mitomycin instillations by applying the allergen as a patch test. These results indicate that the eczematous eruption in this group of patients is most likely a hypersensitivity reaction and that it may be mediated transvesically.

[Effect on immunological response of host by mitomycin C adsorbed into activated carbon particles (MMC-CH) in mice].

Mitomycin C adsorbed onto activated carbon particles (MMC-CH) has been administered intraperitoneally for C57BL/6 mice. The weight of the spleen and thymus of the mice given MMC-CH was decreased lesser than those of the mice given mitomycin C aqueous solution (MMC-AQ). The number of peritoneal exudate cells (PEC) in the mice given MMC-AQ was decreased remarkably on 1st day after MMC-AQ administration and recovered within normal range on the 7th day. On the other hand, the number of PEC in the mice given MMC-CH was increased remarkably on the 1st day and then gradually decreased to normal range on the 7th day. Reactivity of spleen cells by Con A was inhibited in the spleen cells from the mice given MMC-AQ more than those from the mice given MMC-CH. Fifth percent lethal dose was 8.0mg/kg in the mice given MMC-AQ, and 18.2mg/kg in the mice given MMC-CH.

Presence of mitomycin resistant T cells in peripheral blood of normal individuals.

T cell colonies can be easily grown from peripheral blood, and are an index of cellular immunocompetence. Mitomycin-treated T cells are used as stimulator cells in mixed lymphocyte reactions and as feeder cells for growth of B cell colonies, the assumption being that mitomycin prevents proliferation of T cells. We tested this assumption by comparing the proliferation of mitomycin-treated T cells in response to stimulation with phytohemagglutinin (PHA) with that of untreated T cells in liquid cultures and in T cell colony assay. We found that incorporation of tritiated thymidine by cells from 11 healthy individuals pretreated with 25, 50 and 100 micrograms/ml mitomycin C was reduced to 13, 11 and 8%, respectively, of that of untreated cells when stimulated by an optimal concentration of PHA (10 micrograms/ml) in liquid cultures. However, parallel experiments with aliquots of the same cells showed that pretreatment with 25, 50 or 100 micrograms/ml mitomycin C merely reduced T cell colonies to 49, 45 and 45%, respectively, of untreated cells. In five additional experiments mitomycin 200 and 400 micrograms/ml reduced T cell colony numbers to 47 and 60%, respectively. Treatment of T cells with 9000 rad completely abolished T cell colony formation. Lower doses of radiation up to 6000 R did not abolish T cell colony formation, although it effectively blocked T cell proliferation to PHA in liquid cultures. 24 h preincubation of T cells with suboptimal doses of PHA and then treatment with mitomycin or radiation did not abolish T cell colony formation. T cells were recovered from the mitomycin-resistant T cell colonies and stimulated in liquid cultures with PHA, untreated or after exposure to 25 micrograms/ml mitomycin C. Incorporation of tritiated thymidine by the mitomycin-treated cells was reduced to 8% of the untreated controls. Our observations suggests: There may be inaccuracies in B cell colony assays using mitomycin-treated T cells because of significant T cell colony formation. There is a population of T cells in the peripheral blood of normal individuals which form colonies and are resistant to mitomycin.

Functional analysis of the interaction of the antigen-specific T cell receptor with its ligands.

Increasing the number of antigen-specific T cell clones in a T cell proliferation assay resulted in a shift in the antigen dose-response curves toward higher amounts of antigen (i.e., more antigen was required to achieve a given degree of stimulation). The antigen dose-response curve shifts were found to reflect the competition that occurred between the antigen-specific T cell receptors for their ligand, a combination of antigen and Ia molecule. This observation made it possible to determine whether the difference in the potency with which several synthetic cytochrome c analogs could stimulate one cytochrome c-specific T cell clone was due to a difference in the avidity of the antigen-specific receptors on the T cell clone for the different Ia molecule-antigen combinations. It was demonstrated that a single amino acid substitution at position 103 (which greatly diminished the potency of the analog) did not significantly alter the avidity of the T cell antigen-specific receptor for its ligand. In contrast, a substitution at position 99 (which resulted in a comparable decrease in potency) caused a dramatic loss of avidity. These results are consistent with the previous designation of residue 99 as one site on the antigen that contacts the T cell antigen-specific receptor, and of residue 103 as one part of the antigen that contacts the Ia molecule.

Differing contribution of various effector cells in the elimination of syngeneic or allogeneic cells.

The growth of Meth A (MA) tumors was suppressed in tumor-antigen-specific manner in BALB/c mice immunized with mitomycin C-treated MA (MMC-MA) cells in saline or in Freund's complete adjuvant (FCA). The antitumor activity of their peritoneal exudate cells (PEC) was detected by in vivo neutralization test and in vitro cytostasis assay but not by in vitro cytolysis assay. Positive delayed footpad reaction was elicited by footpad injection of MMC-MA cells in such immunized mice, before or after the inoculation of viable MA cells. Expression of cytostatic activity in PEC required the interaction of non-adherent and adherent cells. Normal PEC could exert the cytostatic activity in the presence of a nonadherent population of immune PEC. These findings suggest that cytostatic macrophages are activated after the interaction between sensitized lymphocytes and tumor-specific antigens and that they play an important role in the principal effector mechanism in this syngeneic system. On the other hand, immunization with MMC-MA in FCA or viable MA cells also induced PEC the antitumor activity detected by in vivo neutralization test in allogeneic C57BL/6 hosts. Immunization with viable MA cells induced not only cytolytic but also cytostatic activity, whereas, immunization with MMC-MA in FCA induced cytostatic activity but not cytolytic activity. In contrast to a syngeneic system, cytolytic activity was effectively induced against allogeneic MA cells together with cytostatic activity. We conclude that there are various effector cells contributing to the elimination of syngeneic or allogeneic murine tumor cells.

Mode of action of polyethylene glycol 6000 in potentiating the in vitro generation of antitumor cytotoxicity by MOPC-315 tumor bearer spleen cells.

Some of the possible mechanisms by which polyethylene glycol (PEG) augments the ability of MOPC-315 tumor bearer spleen cells to mediate in vitro antitumor cytotoxicity were evaluated. The level of antitumor cytotoxicity obtained in 5-day cultures of tumor bearer spleen cell suspensions correlated inversely with the percentage of Trinitrophenol (TNP)-rosettable cells (presumably metastatic tumor cells) present in the spleen. The kinetics of decrease in the percentage of TNP-rosettable cells coincided with the appearance of antitumor cytotoxicity. In addition, PEG was shown to interfere with the ability of viable tumor cells to suppress the in vitro generation of antitumor cytotoxicity in normal spleen cells cultured with mitomycin C-treated tumor cells. However, the decrease in the content of TNP-rosettable cells and the concurrent increase in the level of antitumor cytotoxicity were not due to direct cytotoxic and/or cytostatic effects of PEG on tumor cells. Spleen cells cultured in the presence of PEG had an increased rate of [3H]thymidine incorporation and proliferation compared to spleen cells cultured in the absence of PEG. However, the PEG-induced decrease in the percentage of TNP-rosettable cells either preceded or occurred at the same time that the PEG-induced increase in spleen cell number was observed. Therefore, spleen cell proliferation can at best explain only partially the PEG-induced decrease in the content of TNP-rosettable cells, and other mechanisms for the decrease must be considered.

Immune responses to naturally occurring rat sarcomas.

Attempts were made to induce immunity to 5 spontaneous rat sarcomas transplanted into syngeneic recipients. Rats were immunized by surgical removal of growing tumour transplants or by treatment with attenuated tumour, followed by challenge with tumour cells in suspension. Two tumours wee apparently not immunogenic, but a low level of immunity was induced against 2, and weak evidence of immunity was observed with another. Induced immunity was individually specific rather than cross-reactive. It is concluded that, contrary to some reports, some spontaneous animal tumours are immunogenic in the strain of origin.

Effect of OK-432 on immunization with mitomycin-C-treated L1210 cells.

The pretreatment of B6D2F1 mice with OK-432 alone was not so effective to retard the growth of L1210 leukemia, and the leukemic cell was poorly immunogenic in the mice. However, when OK-432 was given by ip injection in combination with mitomycin-C-treated L1210 cells, the growth of L1210 leukemia was significantly retarded and some mice did not "take" the leukemia. Meanwhile, BCG was also effective in this respect. Differences in properties as an immunopotentiator between OK-432 and BCG were suggested by the experiments of in vitro cytotoxicity test of spleen cells.