Comparative toxicities of amphotericin B and its monomethyl ester derivative on glial cells in culture.

Amphotericin B (AmB) is a potent antifungal polyene macrolide antibiotic and is the drug of choice for the treatment of deep-seated mycotic infections. Its use is limited, owing to its nephrotoxicity, and it must be dispersed in deoxycholate for parenteral administration. In contrast, AME (the monomethyl derivative of AmB) is water dispersible, is appreciably less cytotoxic than AmB toward a variety of cell types, and is reportedly active against the acquired immunodeficiency syndrome virus (human immunodeficiency virus type 1). The latter activity has generated interest in AME as an antiviral drug. However, AME is perceived to be neurotoxic, based on the outcome of a human clinical trial of AME as an antifungal drug. AmB is not regarded as neurotoxic, presumably because any neurotoxicity in vivo is precluded by its nephrotoxicity. It was important, therefore, to determine the potential for neurotoxicity of the two agents in comparative tests, assessing the effects of their direct action against neural cells in culture. Rat cortical cells, comprising astrocytes and oligodendrocytes, were used. AME was at least 10 times less toxic than AmB and equally less toxic against several other nonneural cell types also included in these tests. Equally important, AmB disrupted the myelin sheath in these cultures, and it inhibited its generation. AME did not, even at a concentration 10 times greater than the toxic concentration of AmB. AmB is, therefore, potentially more neurotoxic than AME, contrary to current perception. AME is effective as an antifungal and antiviral drug at a concentration far below its toxic concentration for neural cells. Also, AME does not cross the blood-brain barrier appreciably, so that a therapeutic level in blood can be expected without encountering neurotoxicity.

Inhibition of HIV replication by liposomal encapsulated amphotericin B.

This report shows the potential of using a liposomal encapsulated preparation of amphotericin B (a polyene macrolide antibiotic) for the in vitro inhibition of HIV. There was no significant difference between the effective doses of the free form of drug when compared to the liposomal encapsulated preparation in inhibiting the growth of HIV. Virus expression was suppressed at a concentration of 5-10 micrograms/ml of the drugs. The liposomal preparation showed greatly reduced cytotoxicity in experiments using cultures of murine leukocytes. These results show the potential usefulness of liposomal encapsulated drugs in the treatment of patients with AIDS or AIDS related complex.

Amplification by macrophages of prostaglandin-mediated immunosuppression in mice bearing syngeneic tumors.

The role of macrophages in tumor-mediated immunosuppression was examined, using C57B1/6 strain mice bearing four different immunosuppressive transplantable syngeneic tumors (Lewis Lung Carcinoma, B16 Melanoma, and two fibrosarcomas induced by methylcholanthrene in our laboratory). When tested for immunosuppressive activity, in inhibiting the induction of antibody formation by normal spleen cells in response to SRBC in vitro, the splenic and peritoneal macrophages from tumor-bearing mice were all significantly suppressive. The degree of suppression correlated with immunosuppression in tumor-bearing mice challenged in vivo with SRBC. Direct action of tumor cells on normal splenic macrophages in vitro caused them to become suppressive, the extent of suppression dependent on the time of interaction and on the immunosuppressive activity of the tumor cells in vivo. Pretreatment of suppressive splenic macrophages with indomethacin, a potent inhibitor of the synthesis of prostaglandins (PG), reduced significantly their immunosuppressive activity. Also, peritoneal macrophages from tumor-bearing mice produced significantly more PGE in culture than control macrophages. Thus, tumor-activated macrophages, presumably those macrophages that infiltrate the tumor in a host reaction against the tumor, serve to amplify the level of immunosuppression in the host by producing relatively large amounts of PGE that is a key physiological mediator in the activation and function of suppressor T lymphocytes. The stimulation of PGE synthesis in macrophages, as a result of their interaction with syngeneic tumors, is initiated by PGE produced in relatively large amount by the tumor cells.

Factors in the production of experimental autoimmune myasthenia gravis in acetylcholine receptor immunized rabbits.

The development of experimental autoimmune myasthenia gravis (EAMG) was studied in 10 rabbits which were repeatedly injected with Torpedo acetylcholine receptor (AChR). Serum samples were obtained at various times for determination of complement fixing antibody level, serum complement level and the capacity of serum to inhibit neuromuscular transmission in amphibian muscle (passive transfer inhibiting capacity, PTIC). In seven animals the rise in level of circulating antibody occurred immediately before or in synchrony with the development of EAMG and frequently at such times serum complement rose irregularly. The PTIC was elevated during appearance of EAMG. In some animals a rise in complement fixing antibody level occurred without appearance of EAMG; in two others EAMG appeared without significant rise in antibody level. The data indicate that development of EAMG is associated with the production of antibodies which are capable of depressing neuromuscular transmission by reducing the sensitivity of the postjunctional membranes to acetylcholine. This depression can be potentiated by serum complement. Some but not all of the antibodies produced appear to fix complement when combined with Torpedo AChR. Evidence indicating possible existence of a presynaptic contribution to the development of EAMG is given.

Tumor-mediated immunosuppression: prevention by inhibitors of prostaglandin synthesis.

The addition of MC16 tumor cells (a prostaglandin E2-producing cell line induced in C57BL/6J mice by methylcholanthrene) to cultures of cells to sheep red blood cells. This inhibition can be blocked by adding to the cultures prostaglandin synthetase inhibitors, such as indomethacin, flufenamic acid and aspirin. These MC16 tumor cells are also immunosuppressive in vivo. Mice bearing the syngeneic MC16 tumor become unresponsive to sheep red blood cells as the tumor grows. As in the in vitro test system, inhibitors of prostaglandin synthetases seem to block the immunosuppressive activity of MC16 cells in vivo since tumor-bearing mice, treated therapeutically with indomethacin, responded normally in their production of antibody to sheep red blood cells.

[Changes in the membrane receptors of lymphocyte populations during immunization]. / Modificazioni der recettori di membrana di popolazioni dinfocitarie in corso di immunizzazione.

A population of splenic lymphocytes has been studied as well as their surface antibodies and their affinity for antigen in different days and periods of immunization. Fractionation of a specific population of lymphocytes having an affinity for antigens coated on glass beads were accomplished. The other aspecific population did not bind ot the glass beads and was not retained on the column. During the primary immuno-response the number of lymphocytic cells with surface antibodies specific for antigen was higher than in the booster response: in secondary response only the total number of antibody forming-cells increases while the number of lymphocytes with surface antibodies is constant. The authors suppose that the surface of spleen lymphocytes is modificated in different way at different time, by immunizing antigen.

Subversive activity of syngeneic tumor cells as an escape mechanism from immune surveillance and the role of prostaglandins.

Mice bearing a syngeneic tumor become increasingly immunodepressed during growth of the tumor, being unable to develop both cellular and humoral immunity to a histoincompatible tumor allograft and to reject the allograft. This failure to reject a strongly antigenic tumor allograft suggests that immunodepression associated with growth of a weakly antigenic syngeneic tumor provides the syngeneic tumor with an escape mechanism. This immunodepression is also manifest by the suppression of the response of spleen cells to mitogen stimulation by syngeneic tumor cells, both in vivo and in vitro. T cells that are stimulated by PHA, a T-cell mitogen, are the primary targets, and their suppression is the result of the direct subversive activity of the tumor cells. Subversion of T cells by tumor cells seems to be mediated through the prostaglandin pathway, because the prostaglandin PGE2 is itself suppressive, and an antagonist of PGE2 and an inhibitor of prostaglandin synthetases both inhibit the subversive activity of tumor cells. Several tumor cell lines tested, of different etiology and histologic type, all were subversive. This suggests that this subversive activity may be a general property of tumor cells and may be a key element in their ability to thwart the immunological system of the host. For this reason, any therapeutic regimen of cancer, based on immunostimulating drugs, should include drugs that can inhibit active subversion of the immune system by the tumor itself. Antagonists of prostaglandins and inhibitors of prostaglandin synthetases show promise in this regard.

Subversion of immune system by tumor cells and role of prostaglandins.

Mice bearing syngeneic tumors, chemical and virus-induced, became immunologically unresponsive to sheep erythrocytes. The increase in the degree of unresponsiveness with tumor growth suggested a causal relationship. Immunosuppression was in fact caused by the tumor cells because the addition of tumor cells to in vitro cultures of spleen cells and sheep erythrocytes resulted in suppression of antibody response. Suppression was dose dependent with a ratio of 1 to 1000 of tumor cells to spleen cells sufficient to produce significant suppression. Prostaglandins were found to have a role in immunosuppression by tumor cells in that PGE2 was itself immunosuppressive and in that indomethacin and aspirin, inhibitors of prostaglandin synthetases, blocked immunosuppression in vitro and retarded tumor growth in vivo. These findings suggest that tumors, although antigenic, may be able to escape immuno-sureillance by their host by means of subverting the immune system. Thus, success of immunotherapy may well depend on our ability to prevent or block the immunosuppressive activity of tumors.

Cyclic AMP and immune responses: changes in the splenic level of cyclic AMP during the response of mice to antigen.

Intravenous injection of sheep erythrocytes into normal immunologically competent C57BL/6J mice results in significant and characteristic changes in the splenic level of 3':5'-cAMP with initiation of the immune response and proliferation of antibody-forming cells. The level increases 2- to 3-fold initially, peaks at 2 min, and returns to base level in an hour. Between 2 and 5 days there is a decrease, followed by a peak when the rate of proliferation of antibody-forming cells is maximal. Changes in splenic level of cAMP are thus transitory and biphasic, and they occur only in response to foreign substances that are immunogenic, such as heterologous erythrocytes, and not to antigenically inert carbon particles. They are also dependent upon the dose of immunogen. Moreover, the double-stranded hybrid of polyadenylate and polyuridylate, which acts synergistically with antigen in stimulating endogenous cAMP, is immuno-enhancing if given with sheep erythrocytes when the cAMP level is increasing, and immunosuppresive if given when cAMP is decreasing. These data provide direct evidence for a role of cAMP as a mediator in the activation and proliferation of immunocytes stimulated by antigen. With knowledge of the transitory and biphasic nature of the cAMP response induced by antigen, one can avoid indiscriminate use of drugs that modify the level of endogenous cAMP and instead employ them rationally in controlling the immune response, enhancing or suppressing it as desired.