Enrico Mihich

Augmentation of the phagocytic activity of murine spleen cell populations induced by Adriamycin.

C57B1/6 mice were injected i.v. with Adriamycin (5 mg/kg) and various days later, spleen cells were tested for Fc dependent phagocytic activity by measuring protection of 51Cr-labelled, antibody coated SRBC from hyptonic shock. No difference in phagocytic activity was observed with spleen cells from untreated mice or mice treated with Adriamycin 1-7 days prior to killing. Increases in phagocytic activity were, however, seen with spleen cells from mice treated 9 to 13 days prior to killing. Phagocytic activity of spleen cells from mice treated 5 days prior to killing was increased after these cells were cultured for 5 days when compared to cultured cells from untreated mice. Despite depletion of mature phagocytic cells from spleen cells suspensions by silica treatment, followed 24 hours later by silica removal by density gradient centrifugation, phagocytic activity was observed with spleen cells from ADM treated mice (day-5) after 5 days in culture. No phagocytic activity was seen with spleen cells from untreated mice using the same silica treatment and culture conditions. When cultured spleen cells from untreated or ADM treated mice (day-5) were fractionated by plastic adherence, twice as many adherent cells were removed compared to controls. Their phagocytic activity (per cell) was, however, the same. Thus, ADM may increase the number of immature nonphagocytic macrophages in the spleen resulting in increased mature macrophages after culture.

Studies on the structure—activity relationship among aliphatic and aromatic bisguanylhydrazones and some related compounds

A total of 18 compounds consisting of 7 alphatic and 7 aromatic bis(guanylhydrazones), p-quinone-bis(guanylhydrazone), one monoguanylhydrazone, one diamidine and one diguanidine were studied spectrophotometrically to determine their ability to interact with native calf-thymus DNA and the possible correlation of binding with biological activity. In each case, the ability of a compound to bind to DNA correlate with its ability to inhibit the activity of DNA-dependent DNA polymerase (EC 2.7.7.7) extracted from mouse leukemia L1210 cells. For example, all the aromatic bis-guanylhydrazones and diamidine (hydroxystilbamidine), which were good inhibitors of the enzyme activity, showed a biphasic interaction with DNA. All the aliphatic compounds displayed no detectable interaction with DNA in the Tris buffer used, and were also poor inhibitors of the polymerase activity. Interaction of decamethylene diguanide (Synthalin with DNA could not be determined because the compound does not absorb light in the UV-VIS region. However, in similarity with other aliphatic compounds, this agent was a poor inhibitor of DNA polymerase reduction. The p-quinone-bis(guanyl-hydrazone) and p-phenylbenzaldehyde-monoguanylhydrazone showed only a monophasic interaction with DNA and caused an intermediate inhibition of the enzyme activity. When tested for possible anti-leukemic activity against i.p. L1210 leukemia in syngeneic DBA/2J mice, all the aromatic bis-guanylhydrazones as well as hydroxystilbamidine caused prolongation of survival of tumor-bearing mice. Among the aliphatic bisguanylhydrazones, all of which showed no binding to DNA and caused at the most only a very slight inhibition of DNA polymerase, only methylglyoxal-bis(guanylhydrazone) (CH3--G) had antileukemic activity. Synthalin also inhibited leukemia growth. Evidences presented indicate that the mechanisms of action of aliphatic and aromatic bisguanylhydrazones may be quite different. Furthermore, the ability to bind to DNA may be a useful criterion to predict the antileukemic activity of aromatic guanylhydrazones and possibly other aromatic-bis-cationic compounds, but not that of aliphatic congeners.

Effects of prostaglandins on the development of cell-mediated immunity in culture and on the cytolytic activity of in vivo-generated effector cells.

This study was undertaken in an attempt to better understand the rôle of prostaglandins in the development of the primary cell-mediated immunity (CMI) response in culture. Primary sensitization cultures with C57Bl/6 mouse spleen cells as responder cells (R) and X-irradiated P815 mastocytoma cells as allogeneic stimulator cells (S) were established in the presence and absence of test agents. The ability of effector cells to lyse P815 target cells was measured by 51Cr-release assay on day 4. 3H-Thymidine uptake into the cultured cells was also analyzed on the same day. Prostaglandins of the E series were shown to exert selective effects on the CMI response depending on dose, schedule of administration, and culture conditions. PGE1 or PGE2 enhanced the CMI response at 30 pM but inhibited it by 50% at 30 nM. At an optimal R/S ratio, 30 nM PGE1, or PGE2 always inhibited 3H-thymidine uptake more than cytotoxicity while at suboptimal ratios it inhibited both equally. PGE1 did not affect the kinetics of development of the CMI responses. PGE1 was inhibitory when added 20 h before, at the same time as or as late as 24 h after antigen. The inhibitory effect was prevented by removal of PGE1 within 24 h of addition. Pretreatment of spleen cells with PGE1 for 20 h did not affect their subsequent response to sensitization. It seems that PGE1 must be present during the early phase of lymphocyte activation and that it has a relatively long half-life in spleen cell cultures. The development of secondary CMI in culture was less sensitive to inhibition by PGE than that of the primary CMI. PGD2 and PGA2 also inhibited the induction of the primary CMI while PGF2 alpha enhanced it. On the other hand, PGI2 and TXB2 had no effect. The effect of prostaglandins on the lytic activity of the in vivo-generated effector cells was also examined. The addition of PGE2, PGI2, PGD2 or PGA2 to the 51Cr-release assay resulted in significant depression of the cell-mediated lympholysis; whereas, addition of 6-keto-PGF1 alpha, TXB2, and PGF2 alpha caused little effect. PGE2, PGA2 or PGD2 inhibited the rate of lytic activity. In order to exert this effect, these compounds must be present during the 51Cr-release assay and pretreatment of the effector cells had little effect on their cytotoxicity. These results indicate that prostaglandins have immunomodulating activity affecting the early phase of the development of the CMI response in culture; they also have the ability to inhibit the cytolytic effect of effector cells generated in vivo.

Study of the immune responses to nucleated cells. I. In vitro functional evaluation of the immune effectors responsible for complement-dependent cellular cytotoxicity.

Abstract A modification of the 51 Cr cytotoxic test has made it possible to assess under the same conditions not only cytotoxic serum antibodies, and cell-mediated immunity, but also cells releasing cytotoxic antibody. The measurement of these antibody-releasing cells was carried out with nucleated target cells, both normal and leukemic, across θ or H-2 antigenic differences. This test was found to be specific. The release of 51 Cr from the labeled target cells was proportional to the ratio of immune cells to target cells, and for a given ratio to the incubation time, 60 min usually being the optimum time at ratios of 50–100 to 1. The test was not affected by treatment of the effector cells with an anti-θ serum; however, pretreatment of these cells with an anti-IgM serum, even without complement, inhibited the test with cells taken during primary responses. Both cytotoxic IgM and IgG antibodies were detected by the assay directly without the addition of enhancing serum; discrimination between these two γ-globulins can be made by suppressing the cytotoxicity due to either Ig class consequent to the addition of the appropriate specific anti-globulin serum during the incubation.

Immunoregulation by Cancer Chemotherapeutic Agents

Considerable progress in cancer therapeutics has been achieved since the inception, in the mid 1940s, of the initial trials of antifolates, steroids, and alkylating agents, which marked the beginning of the current phase of cancer chemotherapy. Notwithstanding the demonstrated value of chemotherapy in the treatment of certain types of neoplastic disease, ways must still be identified to circumvent basic limitations before drugs can be used broadly in the effective treatment of cancer. Consequently, as discussed elsewhere (Mihich, 1978), among the main approaches in cancer chemotherapy, has been the study of the mechanisms of selective toxicity of drugs; included in these studies have been those of the interactions of drugs with host defense mechanisms. As a result of these investigations, it became apparent quite early that most anticancer drugs are potentially capable of suppressing host defense mechanisms and that in many cases this effect is a consequence of the very antiproliferative action that is the basis of the agent’s antitumor activity. In fact, despite a number of remarkably insightful experiments conducted between the mid 1950s and the mid 1960s, which indicated the unique immunomodulating potential of certain anticancer agents, the preponderance of the information obtained until the late 1970s has been restricted to their immunosuppressive properties (see Hersh, 1973; Mihich, 1975; Spreafico and Anaclerio, 1977; Bast, 1982). In light of the availability of these and other excellent reviews on the immunosuppression caused by anticancer agents (Mihich, 1979; Hersh, 1974; Bach, 1975) it is the intent of this presentation to concentrate on examples of the immunomodulating characteristics of these drugs.

Modification by biological products of the generation of suppressor cells in culture

This report describes the effects of prostaglandin E2 (PGE2), indomethacin, and human prealbumin on the generation of culture-induced and allo-antigen-induced suppressor cells. The ability of the suppressor cells to affect cell-mediated immunity (CMI) generation cultures was assessed by 3H-thymidine uptake and cell-mediated lympholysis (CML). The generation of culture-induced suppressor cells is dependent on the fetal calf-serum (FCS) used in the medium and at least 4 days are necessary for their generation. Suppression is totally abolished by 2,000r X-irradiation of suppressor cells prior to their testing in CMI generation cultures. Spleen cells cultured in the presence of 0.03 to 3 microM PGE2 are not suppressive, while 3 nM PGE2 only partially abolishes their suppressive activity. Indomethacin has little effect on the development of this suppressor cell activity. Spleen cells cultured in the presence of human prealbumin have augmented cellular proliferation but do not develop suppressor cell activity. Alloantigen-activated cells added to CMI generation cultures suppress cellular proliferation (3H-thymidine uptake), but suppress CML development only after X-irradiation. PGE2 inhibits the proliferation of alloantigen-activated cells in a dose dependent manner. The ability of PGE2 to abolish their suppressive activity (after X-irradiation) in CMI generation cultures is directly proportional to its effects on cell proliferation. Indomethacin augments the proliferation of alloantigen-activated cells but does not further augment suppression. Human prealbumin augments the cellular proliferation of alloantigen-activated suppressor cell culture systems, but does not affect the generation of alloantigen-activated suppressor activity.

The effects of dichloro-trans-dihydroxy-bis-isopropyl-amine-platinum IV on the primary cell-mediated cytotoxic response.

The effects of diamino-dichloro-cis-platinum II (DDP) and dichloro-trans-bis-isopropyl-amine-platinum IV (DDIP or CHIP) on the primary cell-mediated cytotoxic (CMC) response developed in a mixed lymphocyte tumor cell culture (MLTC) were investigated. DDP as well as CHIP added on day 0 augmented the CMC response when spleen cells from C57Bl/6 mice were cultured for 4 days with supraoptimal numbers of irradiated (4000 rads) allogeneic P815 mastocytoma cells. A similar effect was observed when mice were treated with either of the two platinum compounds 1 or 5 days prior to spleen removal and sensitization of the spleen cells in a MLTC. Augmentation was not observed at optimal or suboptimal R:S. When spleen cells were preincubated with CHIP, only inhibitory effects on the CMC response could be demonstrated. Decreasing Cl- -ion concentrations in the preincubation medium increased the inhibitory effect of CHIP. After CHIP administration to spleen donor mice, the development of antigen specific suppressor cells was inhibited only at doses higher than those required to inhibit the development of nonspecific suppressor cells. Spleen responder cells from CHIP-treated donors exhibited a lower sensitivity to both antigen-specific and nonspecific suppressor cells than spleen responder cells from untreated control donors.

Cellular immunity for cancer chemoimmunotherapy—an overview

In recent years, more knowledge has been acquired on the molecular and cellular mechanisms of immune responses to tumors, of tumor-induced suppression of immunity which conditions at least in part tumor escape from innate and/or induced immunity, and of immunologic tolerance to tumors. Based on this increased knowledge and on the information being acquired on the effects of vaccines, passive transfer of cells, antibodies, and the appropriate use of immunomodulating drugs, it seems reasonable to postulate that in the foreseeable future immunomodulation-dependent treatments may be developed which may offer additional opportunities for therapy in the clinics. This Symposium in Writing is focused on the identification of possible new sites of intervention toward achieving a modulation of antitumor immunity which may provide therapeutic advantages; particular emphasis is given to the exploitation of cellular immunity. Current relevant information on T-cell activation mechanism, lymphocyte trafficking, and interactions of anticancer drugs with vaccine treatment is reviewed. The activation of T cells requires costimulation by at least two signals which are also needed to sustain and augment T-cell responses. The mechanisms of stimulation of these responses are discussed herein by Vesosky and Hurwitz, who considered the large number of costimulatory molecules relevant to the activation of T cells, as well as that of B cells, macrophages, and dendritic cells. Indeed a knowledge of the mechanisms of antigen presentation and of T-cell activation is critical for any attempt to explore new approaches directed toward an augmentation of antitumor immune responses. Recently, clinical trials based on the experimental work of Allison’s group [1, 2] have been carried out. The CTLA-4–mediated inhibition of T-cell activation is reduced with anti–CTLA-4 antibody, and vaccination is applied using suitably modified tumor cells. Indeed Hurwitz’s contribution reviews the different costimulatory receptors that may provide sites of intervention toward increasing antitumor immune responses; multiple signals stemming from these molecules, some of which are not redundant, also provide opportunities for their exploitation through combination regimens. Activated T cells must be able to reach target tumor cells in order to exert their action. As discussed by Chen, Wang, and Evans, in some cases the tumor microvasculature represents a barrier impeding the access of activated T cells to tumor cell populations. The molecular and cellular mechanisms of leukocyte migration into tumor are implemented through a network of cytokines and chemokines, also involving related receptors and signaling pathways, which condition various phases of the migration process. A cascade of wellregulated phenomena can be altered in tumors and offer therapeutically oriented possibilities of intervention. As discussed by Evans, those include the appropriate exploitation of proinflammatory cytokines and/or biophysical stress responses. Studies using the pancreatic model developed by Hanahan [3] have shown that the impediments to T cells’ access to tumor cells can be reduced by X-irradiation [4, 5]; in other models feverrange thermal therapy was found to facilitate leukocytes’ migration into tumor [6]. During the past several years increasing evidence has been accumulated indicating that certain anticancer drugs, under appropriate conditions, may have immunomodulating effects which can be exploited therapeutically [7, 8]. An example is provided by studies carried out in this laboratory with doxorubicin (DOX). This antibiotic was found to induce activation/differentiation of cells of the monocytic-macrophage type and stimulation of T-cell responses [8]: increased production of interleukin 1, interleukin 2, and TNF-a, and modulation of natural killer (NK) cell function [8]. The mRNA of Cancer Immunol Immunother (2003) 52: 661–662 DOI 10.1007/s00262-003-0423-6

Immunosuppressive effects of methylglyoxal-bis(guanylhydrazone) on mouse bone marrow and spleen cells and their antagonism by spermidine.

Abstract Changes in immunological function and in the activity of S-adenosylmethionine decarboxylase were studied in bone marrow and spleen cells from mice immunized with sheep red blood cells and given multiple doses of methylglyoxal-bis(guanylhydrazone) (CH3-G) or CH3-G in combination with spermidine. Inhibition by CH3-G was found in the complement-dependent and complement-independent immune responses of cells from both tissue sources; however, this inhibition was greater and more persistent in bone marrow than in spleen cells. Co-administration of spermidine with CH3-G completely prevented this inhibition in the spleen at all the time points studied, whereas in the bone marrow prevention appeared later and was lower in magnitude. The increase in the activity of S-adenosylmethione decarboxylase, a key enzyme in spermidine biosynthesis, was greater in bone marrow than in spleen after the administration of CH3-G. Co-administration of spermidine with CH3-G consistently reduced this increase in enzyme activity in spleen but not in bone marrow. In addition to prevention, appropriate scheduling of spermidine subsequent to CH3-G resulted in reversal of both the immunosuppression and the increase in S-adenosylmethionine decarbooxylase activity produced by CH3-G in spleen cells.

Time limitations in the reversal by citrovorum factor of methotrexate-induced immunosuppression in mice☆

Abstract Methotrexate was found to inhibit both the complement-dependent and the complement-independent cellular cytotoxicity of spleen cells from mice immunized with sheep erythrocytes. The inhibition was essentially complete when a single dose of 100 mg/kg (about one-half the ld 50 ) was given 2 days after antigen administration, and the response was measured on day 4. Recovery from this inhibition was incomplete on days 5–7 and complete on day 14. The inhibition measured on day 4 could be partially prevented by citrovorum factor (200 mg/kg), but only if this compound was administered 1–5 hr before methotrexate. In contrast, the residual inhibition measured during recovery, on day 7, was completely reversed if citrovorum factor was injected even as late as 6 hr after methotrexate on day 2. The data suggest that protection of the majority of the rapidly proliferating, antigen-stimulated, spleen lymphoid cells from the effects of methotrexate can be provided only by preloading these cells with citrovorum factor.