The combined treatment of human peripheral blood mononuclear cells with thymolymphotropin and interleukin 2 increases PPD-driven T-cell proliferation and IL-2 induced cellular cytotoxicity against HIV-infected cells.

Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).

Activation of macrophage cytostatic and cytotoxic activity in vitro by liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL).

The ability of liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL), to induce peritoneal macrophage cytostatic activity and alveolar macrophage cytotoxic activity toward tumor cell targets in vitro was determined. MTP-CHOL was shown to be efficiently incorporated and subsequently retained in distearoylphosphatidylcholine/phosphatidylserine liposomes (DSPC/PS; 7:3 molar ratio), whereas hydrosoluble muramyl dipeptide (MDP) was rapidly lost due to leakage. Liposomes containing MTP-CHOL were able to stimulate mouse peritoneal macrophage cytostatic activity under conditions where free MDP was without effect. MTP-CHOL incorporated into liposomes was approximately eightfold more effective than liposomes containing entrapped MDP and 7,400-fold more effective than free MDP in inducing rat alveolar macrophage cytotoxic activity. These results provide evidence that the coupling of MDP to a lipophilic molecule, cholesterol, results in the formation of a viable liposome formulation that is a potent inducer of macrophage-mediated antitumor activity.

In vitro inhibition of murine B-cell tumor growth by MDP, MDP(D-D) and Vaccin is mediated by macrophages.

The effect of two muramyl dipeptides, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and N-acetylmuramyl-D-alanyl-D-isoglutamine (MDP(D-D)), on the in vitro growth of two murine ascitic plasmacytomas, MOPC 173 and TEPC 15, and on an ascitic lymphoma cell line, ABPL2, was studied. The ability of the muramyl dipeptides to inhibit tumor cell growth was compared with a sonicated antigenic preparation of Mycobacterium vaccae (Vaccin), known to have macrophage stimulation activity. The growth of all three ascitic cell lines was inhibited by both muramyl dipeptides and Vaccin. Macrophage-depletion of the ascitic cell populations led to an increase in cell growth of TEPC 15 and MOPC 173, and a decrease in ABPL2. A reduction or loss of the inhibitory activity of the muramyl dipeptides or Vaccin was also observed, and no inhibitory activity was found when the tumor cell lines were cultured in vitro to render them macrophage-free. The inhibitory activity of MDP or MDP(D-D) was restored when purified ascitic macrophages were added to the in vitro cultured cell lines. It was demonstrated that a minimum number of macrophages were necessary for the expression of inhibitory activity. Indomethacin, a PG-synthetase inhibitor, was found to act in a synergistic manner with MDP and MDP(D-D) in inhibiting TEPC 15, but antagonized the effect of these two agents on ABPL2. The lymphoma cell line ABPL2 appeared to be the most sensitive to inhibition by MDP(D-D), a nonpyrogenic, adjuvant-inactive stereoisomer of MDP.

Activation of alveolar macrophage tumoricidal activity and eradication of experimental metastases by freeze-dried liposomes containing a new lipophilic muramyl dipeptide derivative.

The ability of a member of a new class of lipophilic muramyl dipeptide (MDP) derivative, muramyl dipeptide-glyceryldipalmitate (MDP-GDP), to induce alveolar macrophage cytotoxic activity in vitro towards B16 melanoma cells when incorporated into two types of liposome was studied. MDP-GDP incorporated into conventionally prepared liposomes formulated from distearoylphosphatidylcholine and phosphatidylserine (7:3 molar ratio) was 10-fold more effective than liposomes containing MDP, and 7000-fold more effective than free MDP in inducing macrophage cytotoxic activity. MDP-GDP incorporated into freeze-dried liposomes was 50,000- to 100,000-fold more effective than free MDP in inducing such activity. Freeze-dried liposomes containing MDP-GDP were efficiently localized in the lungs of normal mice, and induced cytotoxic activity in the alveolar macrophages. Such liposomes were able to significantly reduce the pulmonary metastatic burden of mice carrying the B16 melanoma. These data provide evidence that this class of lipophilic MDP derivative, when incorporated into freeze-dried liposomes, is a potent inducer of macrophage cytotoxic activity in vitro and in situ, and has antitumor activity in vivo. In addition, the use of a freeze-drying procedure allows the preparation and long-term storage of reproducible liposome formulations.

The immunomodulatory activity of the plant proteins Momordica charantia inhibitor and pokeweed antiviral protein.

The immunological activity of Momordica Charantia inhibitor (MCI) and of Pokeweed antiviral protein (PAP-S), 30,000 daltons plant proteins possessing close similarity to Ricin A chain as inhibitor of protein synthesis, was investigated in mice. In vivo, single nontoxic injections of microgram amount of these substances delayed H2-incompatible skin allograft rejection, splenocyte responsiveness to ConA and PHA, but not to LPS, and abrogated the PFC response to a T-dependent (SRBC) antigen while totally sparing that to a T-independent (S III) stimulus. Injection of these substances could also reduce NK cell activity while increasing macrophage-mediated spontaneous cytotoxicity. In vitro, MCI and PAP-S at non-cytotoxic concentrations inhibited lymphoid cell responsiveness to PHA and ConA, but not to LPS, and markedly enhanced macrophage-dependent cytotoxicity.

In vitro generation of nonspecific suppressor cells and their characterization.

In vitro preculture of C3H/He splenocytes for 48 to 96 hr induces aspecific suppressor cells evaluated by their ability to reduce 3H-TdR uptake by fresh syngeneic splenocytes stimulated by different amounts of Concanavalin A (Con A) in vitro. This suppressive activity is obtained in medium containing 10% heat-inactivated fetal calf serum (FCS) or similar amounts of heat-inactivated bovine serum, syngeneic, or allogeneic murine sera but not by unheated FCS. Suppressive activity is resistant to mitomycin C and radiation up to 5,000 R. Exposure of precultured cells to carbonyl iron or plastic adherence, but not to anti-Thy 1.2 serum plus complement, results in removal of the suppressive activity.

Suppression of mitogen responses and graft-versus-host reaction by splenocytes from mice bearing Lewis lung carcinoma.

The presence of suppressor cells in the spleens of C57BL/6 mice bearing Lewis lung carcinoma was investigated with the use of the in vitro lymphoproliferative response to mitogens and the graft-versus-host reaction (GVHR) as test systems. Splenocytes from tumor-bearing mice showed a lower response to mitogens when obtained 15-27 days after tumor transplant. In parallel, these cells were capable of suppressing the response of normal spleen cells to mitogens and their capacity to mount a GVHR in allogeneic hosts. Treatments with procedures known to remove adherent phagocytes, but not treatments with anti-Thy 1.2 serum plus complement, removed the suppressive activity observed.