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SUMMARY Activation of the cellular transcription factor nuclear factor-κB (NF-κB) by cytokines and other immunostimulants has been tightly linked with enhanced replication of human immunodeficiency virus-type 1 (HIV-1) in infected cells. Various immunomodulators are currently being examined in animal and human trials for their suitability as adjuvants in potential vaccines against acquired immunodeficiency syndrome (AIDS). It may prove to be beneficial to select adjuvants that do not induce NF-κB activation and particularly if the vaccines are to be aimed at seropositive individuals. We have examined a battery of synthetic immunostimulants of the muramyl peptide family for their ability to activate NF-κB in human and mouse cell lines. In this report, we demonstrate selective activation of NF-κB in different cell lines and by different muramyl peptides possessing immunostimulatory activities. The mechanism of such activation is apparently via production of reactive oxygen intermediates (RO1) since pretreatment of cells with antioxidants blocked subsequent activation of NF-κB. However, among all the molecules tested only one lipophilic, non-pyrogenic adjuvant active muramyl peptide showed a complete lack of NF-κB activation in all cell lines tested. This molecule could well become the adjuvant of choice in future AIDS vaccines.

Murabutide, which belongs to the immunomodulator family of muramyl peptides, was applied directly to fresh human blood to evaluate changes in leukocyte properties. After blood incubation with murabutide, lymphocytes presented a higher responsiveness to T-mitogens, and monocytes and polymorphonuclear cells exhibited an increase in their capacity to produce hydrogen peroxide. In addition, murabutide treatment enhanced phagocytic activity of neutrophils, whereas monocytes presented a decrease in this activity. Some surface markers were also investigated in the distinct leukocyte populations, After incubation with murabutide, a larger number of lymphocytes expressed Ta1 antigen (CD W26) and transferrin receptor (CD 71). In contrast, expression of interleukin-2 receptor (CD 25) was slightly decreased. Monocytes from treated blood displayed a larger number of receptors for C3bi (CD 11b), whereas the surface marker CD 14 and the class I receptor for the Fc portion of IgG were down-regulated. Activation of polymorphonuclear cells by murabutide was confirmed by the up-regulation of the C3bi receptor, Fc receptor, and CD 14 surface antigen. The effects of murabutide on leukocytes described in this paper may contribute to understanding mechanisms of the modulating activity of muramyl peptides on specific and nonspecific immunity.

We previously demonstrated that a single intradermal injection of 10 9 irradiation-killed M. vaccae , given 1 month after starting chemotherapy, caused significant changes in responses to mycobacterial antigens. Amongst 38 patients with pulmonary tuberculosis, 29% had lymphocytes responding to common mycobacterial antigens after the injection, compared with only 11 % of 49 similar patients after an injection of saline (p To increase the proportion of responders to these antigens, six modifications of the potentially immunotherapeutic injection, randomized with injections of saline, have been assessed by biochemical, clinical, haematological, immunological and radiological criteria. Subsequent lymphocyte proliferation to mycobacterial antigens enabled the modifications to be ranked in order of efficacy. Tuberculin plus murabutide plus 10 9 irradiated M. vaccae (36% of 25), an autoclaved preparation of 10 9 M. vaccae (45% of 22), and 2 x 10 9 irradiated M. vaccae (75% of 12) were the most effective. Antibody responses in several IgG subclasses to mycobacteria, but not streptococci, were also significantly increased by the most effective modifications over the 8 weeks following injection. Detailed radiological study showed that use of the autoclaved bacilli was followed by a delay in clearing of consolidation, but by better closing of cavities than was found in the control group, suggesting enhanced, or altered, immunological activity around the lesions.

Muramyl dipeptide or MDP (AcMur-L-Ala-D-iGln) is a synthetic immunoadjuvant which can also enhance non-specific resistance to bacterial infections in mice, even by the oral route. By the use of several derivatives, it has been shown that neither adjuvanticity nor pyrogenicity was a perequisite for eliciting an increased resistance, and that unwanted pharmacological effects can be eliminated by minor chemical modifications. Moreover, some lipophilic analogs or derivatives obtained by linking the glycopeptide to a carrier were found to be more active than MDP. Their effectiveness also depended on the dose and the timing of administration, and varied according to the bacterial challenge. The most appropriately timed administration of MDP and derivatives was established between one and four days before the challenge. In some cases, MDP was protective even when injected one hour after the challenge, whereas with other immunostimulants such as lipopolysaccharides or BCG, a negative phase of higher susceptibility may occur under these conditions. MDP still enhanced resistance to bacterial infections in animals with a poor immune status, like newborns or adult mice under immunosuppressive treatment. Moreover, the protective activity was not impaired after repeated injections of large doses of MDP or other adjuvant analogs, a treatment which is known to inhibit specific immune responses.

Adjuvant muramyl dipeptides enhanced the intracellular oxidative burst induced by phorbol myristate acetate in purified human polymorphonuclear (PMN) cells and in monocytes. A stronger priming effect was obtained when muramyl dipeptide was conjugated to a protein carrier. Recombinant human tumor necrosis factor (TNF) did not modify the level of intracellular NBT reduction in PMA-stimulated PMN, although it slightly increased the secretion of superoxide anion. In contrast, TNF enhanced the intracellular oxidative burst of monocytes even at the concentration of 10 pg/ml. In human PMN only, the combination of TNF and muramyl dipeptide induced a higher oxidative response than each stimulant alone.

N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP), is a synthetic immunoadjuvant analogue of a bacterial peptidoglycan subunit that has a definite pyrogenic effect in the rabbit. Some adjuvant-active derivatives such as murabutide [MDP(Gln)-OnBu] or murametide [MDP(Gln)-OMe] are not pyrogenic. Murabutide did not stimulate human or rabbit cells to release endogenous pyrogen (EP), but murametide induced EP production at the same dosage levels as MDP. Moreover, plasma from rabbits treated with murametide transferred into untreated recipients elicited a febrile response typical of EP fever and comparable with that induced by plasma from MDP-treated animals. Murametide not only inhibited the central effect of EP that is generated but also the effect of an extra dose of EP administered later by the intravenous route. Moreover, pretreatment of rabbits with murametide decreased fever responses induced by certain high-molecular-weight exogenous pyrogens as mediated through the production of EP.

The effect of human recombinant tumor necrosis factor (TNF) was studied in vitro on human endothelial cells. TNF (1-1000 pg/ml) induced a dose-dependent increase in PAI level in the supernatant from 6 to 25 U/ml as estimated against urokinase. This effect was time-dependent. It was not suppressed by Polymyxin B thus excluding a possible contribution of an endotoxin contamination. Fibrinoenzymography performed after SDS-PAGE showed that this inhibitor neutralized urokinase and tissue plasminogen activator and gave rise to high molecular weight complexes. TNF (30 micrograms/kg) was also injected in rat. Blood fibrinolytic activity determined 4 hr later was decreased as estimated by the prolongation of the euglobulin clot lysis time from 37 to 188 min. Fibrinoenzymographic profile of the plasma was then characterized by a fainting of the tPA lysis band but the capacity of plasma to neutralize urokinase was not significantly modified. These results suggest that TNF could alter the fibrinolytic balance by stimulating PAI production at the endothelial level. This might be of importance in synergy with the TNF-induced procoagulant activity for promoting vascular occlusion of tumor capillaries.

Antibodies were raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of Plasmodium falciparum. The antibodies were evaluated for protective activity in a human hepatocyte culture system. They exerted their protective effect against the parasite at three points: sporozoite attachment to the hepatocyte surface, entry, and subsequent intracellular development. Inhibition of attachment and entry were found to be related to the antibody titer against the authentic circumsporozoite protein on the sporozoite surface, especially when peptides were administered with alum or complete Freund's adjuvant. Even when invasion was not totally inhibited, the presence of abnormal trophozoites and a frequent inhibition of schizont development in long-term cultures suggested continued activity of antibodies at the intracellular level after sporozoite penetration had been completed.

MDP is a synthetic copy of part of the bacterial cell wall which has been shown to be the minimal structure capable of replacing Mycobacteria in Complete Freund Adjuvant. Several hundred analogs of this molecule have been produced and have been studied in various immunopharmacological systems. Special emphasis will be given here to their pyrogenic and somnogenic effects and to certain similarities which exist between MDP and Interleukin-1.

A monoclonal anti-MDP antibody was found to bind to "Slow Wave Sleep" factor. This result confirms that this factor is a muramyl peptide and furthermore shows that it contains a structure characteristic of the synthetic adjuvant and of the bacterial cell wall, i.e. an acetylated muramic acid bound to L-alanine. This antibody was also shown to specifically inhibit a biological activity of a purified human monokine which induces fever. Because of these results and other recent observations we propose that a bacterial structure is present in certain mammalian mediators.

The visceral and lethal infection produced in BALB/c mice by Leishmania tropica (major) is accompanied by splenomegaly, anaemia and reversal of albumin-to-globulin ratio. The percentages of both B and T cells are decreased in the spleen. The spleen and lymph nodes become populated with large Ig-, Thy 1.2- 'null' cells. The similarity of some of these parameters with those produced in human kala-azar is discussed.

Muramyl dipeptide (MDP) and adjuvant-active derivatives were confirmed as unable directly to induce differentiation of mouse myeloid leukemia M1 cells. They were, however, found effective in stimulating either rabbit macrophages or human blood monocytes to produce differentiation-stimulating activity (D factor). The various conditioned media (CM) thus obtained were able to induce differentiation of the myeloblastic M1 cell line as indicated by the appearance of Fc receptors and inhibition of cell proliferation. Among the synthetic glycopeptides inducing the production of D factor, murabutide (MDP[Gln]-OnBu) was as effective as MDP, although it did not stimulate monocytes to simultaneously release endogenous pyrogen. The absence of pyrogenicity in murabutide CM was attested by IV or intracerebroventricular administration to rabbits. However, in the same CM, LAF(IL1) activity estimated by potentiation of the in vitro proliferative response to phytohemagglutinin of mouse thymocytes was usually higher than that induced by MDP.

The binding of the synthetic immunoadjuvant N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP, for muramyl dipeptide) to human and rabbit sera containing peptidoglycan (PG) antibodies was investigated. Studies were performed by employing the corresponding 14C or 125I-labeled compounds in Farr-type binding and inhibition assays. Whereas MDP did not react with naturally occurring or experimentally induced PG-antibodies, the analog of MDP MDP-L-Lys-D-Ala did bind to hyperimmune rabbit anti-PG sera, but not to the human sera tested. These studies indicate that the radioimmunoassays employed basically are applicable for the selection of nonimmumogenic MDP analogs possessing immunoadjuvant activity.

Lipopolysaccharide (LPS) is a well known interleukin-1 inducer. Polymyxin B sulphate (PMB) is a cyclic antibiotic which neutralizes different biological properties of LPS. Under the conditions used in our laboratory, we were able to show that PMB alone could stimulate human monocytes to produce and release interleukin-1 activity, and that PMB was unable to inhibit the production of interleukin-1 by human monocytes stimulated with LPS. On the contrary, a synergistic effect was obtained which was not observed with another stimulant such as muramyl dipeptide.

Adjuvants are largely used in human and veterinary medicine with conventional vaccines. Vaccines of the future could be synthetic immunogens which would require the addition of more immunomodulators. Muramyl peptides (MDP) are synthetic glycopeptides which can substitute mycobacteria in FCA and are also adjuvant-active in saline. A derivative, Murabutide, is currently undergoing clinical trials and has been found efficient in several experimental models of conventional and synthetic vaccines. For example, when a Plasmodium knowlesi sporozoite peptide conjugated to tetanus toxoid was administered in saline with Murabutide higher titers of biologically active antibodies were obtained than when the same conjugate was given in FCA. Coupling of MDP or derivatives to the immunogens enhanced their activity and allowed the use of carriers to be diminished or even avoided. In the same malarial model the linkage of a Murabutide derivative to the conjugate stimulated the response to the peptide and decreased the response to the carrier. Promising results were obtained when several peptides copying structures of different pathogens were associated in the same molecule by covalent linkage and administered with Murabutide in saline: biologically active antibodies were demonstrated to three of the corresponding pathogens.

Muramyl peptides are endowed with numerous modulatory effects on the immune and nervous systems. Studies with synthetic muramyl dipeptide (MDP), the smallest unit of bacterial cell walls that can replace Mycobacteria in Freund's complete adjuvant, revealed that this glycopeptide can regulate several functions of cells involved in the immune response. The adjuvanticity of MDP and the MDP-induced activation of macrophages against tumors were found to be potentiated in vitro and in vivo with monoclonal anti-MDP antibodies. When used on immunoadsorbent columns, the anti-MDP antibodies removed the somnogenic and pyrogenic activities contained in supernatants of stimulated rabbit peritoneal macrophages. Based on these data a hypothesis is put forward to explain the immuno- and neuro-modulatory effects of muramyl peptides.

In the present study, well-defined immunomodulatory synthetic glycopeptides were used to investigate putative regulatory mechanisms of in vitro IL 2 production by normal human peripheral blood mononuclear cells. MDP (muramyl dipeptide) and two of its structural analogs, murabutide and MDP-DD, were shown to inhibit the in vitro PHA-induced IL 2 production in a majority of normal individuals tested. Involvement of prostaglandins in such an inhibitory effect was suggested by the fact that indomethacin completely abrogated the MDP-induced suppression. There was, however, some evidence indicating that the inhibition induced by the synthetic glycopeptides and that induced by PGE2 were somewhat different. Indeed, although the PGE2-induced suppression of IL 2 production was completely reversed by preirradiation of PBMNC, this was not observed for the MDP-dependent inhibition. In addition, PMA was able to abrogate the suppression induced by MDP, whereas it increased that of PGE2. From these data we propose that at least two independent pathways in the regulation of human IL 2 production exist: a one-signal pathway already described in which PGE2 directly triggers a radiosensitive suppressor T cell subset; and a second pathway with two signals, one given by PGE2 and a second one given by agents such as muramyl peptides. These two signals are required to activate a radioresistant suppressor cell subset.

Muramyl dipeptide (MDP) is a low molecular (less than 500 daltons) synthetic analogue of a streptococcal peptidoglycan subunit. It was demonstrated to be the minimal structure essential for eliciting the adjuvant effect ascribed to Freund's complete adjuvant (FCA). MDP was likewise shown to represent the minimal requirement for peptidoglycan-induced changes in temperature and it was at first concluded that there was an almost perfect correlation between pyrogenicity and adjuvant activity. However, other experiments showed that certain MDP derivatives were as adjuvant active without eliciting a febrile response in the rabbit. Moreover an anti-inflammatory substance such as indomethacin completely inhibits the pyrogenic effect of MDP but does not affect or even enhance various of its immunostimulant properties. Inhibition of MDP-induced fever is not associated with modification of leucopenia or production of circulating endogenous pyrogens observed after MDP injection. Indomethacin incubated with peritoneal macrophage cultures at a dose which blocks prostaglandin synthesis, does not decrease the production by MDP of endogenous pyrogens.