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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.

Azidothymidine (AZT) is the only drug currently approved for treating HIV infections. AZT treatment results in decreased mortality and frequency of opportunistic infections but is associated with bone marrow depression or failure (Pizzo 1988). The termination of AZT treatment results in increased levels of viral core protein p24 and decreased numbers of CD4+ cells (Jackson 1988).

It has repeatedly been observed that various bacterial preparations could increase the host's resistance to tumors. It has also been shown that after nonspecific activation by BCG (bacillus Calmette-Guérin), peritoneal macrophages could inhibit in vitro the growth of neoplastic target cells. In the present study a fraction extracted from Myobacterium smegmatis and referred to as interphase material was tested in view of measuring its ability to activate macrophages in vitro and in vivo. This preparation was previously shown to protect mice against a syngeneic leukemia and to increase the immune response of the guinea pig. Other water-soluble adjuvants devoid of demonstrable antitumor activity in vivo were also assayed. The results argue in favor of a correlation between adjuvant activity and the capacity of activating macrophages. Moreover, interphase material administered in vivo consistently induced stronger and more persistent stimulations of macrophages than the other preparations assayed.

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.

Injection of MDP into mice induces a rapid elevation of monocyte-macrophage CSA in the serum. This effect can also be observed in LPS-hyporesponsive C3H/HeJ mice. MDP and LPS induce CSA synergistically in normal mice. In contrast to the tolerance that is rapidly observed after repeated administration of LPS, MDP does not lose its capacity of inducing serum CSA after repeated injections. Repeated daily injections of MDP also fail to induce tolerance to the LPS-CSA inducing effect. Furthermore, whereas mice rendered tolerant to LPS become hyporesponsive to many other bacteria or bacterial products, they remain responsive to MDP. These data showing that MDP can act synergistically with another CSA inducer, can be injected repeatedly, and can stimulate mice unresponsive to LPS suggest potentially important in vivo applications.

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.

Leishmania tropica causes a lethal visceral disease in highly susceptible BALB/c mice, with many immunopathologic features resembling those in human kala-azar. The responses to thymus-independent antigens of Type 1 and 2 (TI-1, TI-2) were compared in infected mice of susceptible BALB/c and resistant C57BL/6 strains at various times after infection. The infected BALB/c mice had an augmented response to both types of antigens at 45 days after infection. Later (day 76), the response to trinitrophenylated lipopolysaccharide (TNP-LPS, a TI-1 antigen) was diminished but that to dinitrophenylated Ficoll (DNP-Ficoll, a TI-2 antigen) remained statistically above the response of uninfected mice. The response of the resistant strain to either antigen was not modified as a result of the infection. Both strains showed significant polyclonal activation, which was considerably greater in the BALB/c than in the C57BL/6 mice. The observations presented here are in contrast to the widely held belief that a generalized nonspecific immunosuppression occurs in L. tropica infected BALB/c mice.

Two purified peptidoglycans extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

A wide range of adjuvants including alhydrogel, saponin, Corynebacterium parvum, DDAB, Pfizer CP-20,961, oil adjuvants and several MDP analogues have been compared for their adjuvant activity in protecting mice against lethal Trypanosoma cruzi infection following immunization with a T. cruzi 90K cell surface glycoprotein. Only saponin was found to be effective. Promotion did not correlate with the ability to promote a particular Ig isotype; however, saponin was unique in its ability to promote cell-mediated immunity against the 90K glycoprotein.

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.

Non-specific immunostimulants were used in an attempt to protect baboons from infection by schistosomes. Subcutaneous vaccination with cord factor (4·50 mg) and muramyl dipeptide (4·56 mg) 6 days before percutaneous exposure to 3000Schistosoma haematobiumcercariae/baboon (c.p.b.) failed to protect naive baboons: baboons with a 7-month-old, 5000 c.p.b.S. haenzatobiumprimary infection had developed too strong a natural immunity to detect any protection attributable to vaccination. Subcutaneous vaccination with 0·4 ml of Bacillus Calmette-Guerin (BOG, 1–8 x 108colony forming units7sol;ml) 4 days before exposure to 1000 c.p.b.S. mansonigave a significant (38%) reduction in worm load compared with controls. However, vaccination with 0·8 (intramuscular) and 0·2 (intradermal) ml of BOG 11 days before exposure toS. mansoni800 c.p.b. did not protect naive baboons, nor did it significantly reduce challenge worm recovery from baboons with a 13-week-old, 500 c.p.b.S. mansoniprimary infection. Obvious pathology was seen at the site of vaccination in the first but not the second BCG experiment. These results partly support the findings in mice that non-specific macrophage and monocyte activators give partial protection against schistosome infections but they also illustrate that rodents and primates do not necessarily react identically. Hence, findings from rodent models should be extrapolated to man with some caution.

Synthetic antigens have been shown, in experimental models, to induce protective immunity against a variety of pathogens. These studies have demonstrated that, due to their low immunogenicity, these synthetic antigens required conjugation to carrier molecules. Therefore, the choice of appropriate carriers for human immunization by future synthetic vaccines is a major issue. Tetanus toxoid is generally considered to be an effective potential carrier devoid of side-effects. However, the present study performed in mice with two synthetic vaccine models demonstrates that the immune response against the synthetic epitopes conjugated to tetanus toxoid can be suppressed by pre-existing immunity against this same carrier. Because most humans have been exposed to this antigen, this effect may have important implications for the development of synthetic vaccines.

A water-soluble extract of Nocardia opaca (NWSE) induced a very strong stimulation of rabbit and mouse spleen lymphocytes and a weak, but significant, increase of thymidine incorporation by human peripheral lymphocytes and monkey blood, or spleen cells. As was demonstrated by the migration inhibition factor assay, the strong mitogenic activity observed with mice spleen cells was not related to a state of natural impregnation. In the same experiments, the various lymphocytes were also incubated with M. smegmatis water-soluble adjuvant (WSA) and S. enteritidis lipopolysaccharide (LPS). No mitogenic activity was shown with WSA even when this extract was incubated with the lymphocytes of human tuberculin responders, showing that this preparation is free of tuberculoprotein contaminants. Finally, it must be noted that, contrary to what is observed in mice, LPS did not induce thymidine incorporation by monkey or rabbit spleen cells.

Two muramyl dipeptides, N-acetylmuramyl-L-alanyl-D-isoglutamine and its adjuvant-inactive isomer N-acetylmuramyl-D-alanyl-D-isoglutamine, were examined for their ability to protect mice carrying the CBA/N immune deficiency gene (xid) against lethal bacterial challenge. Prophylactic treatment with N-acetylmuramyl-L-alanyl-d-isoglutamine gave significant protection against Streptococcus pneumoniae, Salmonella typhimurium, and Salmonella enteritidis infection. N-Acetylmuramyl-D-alanyl-D-isoglutamine was unable to confer protection. Incorporation of the lipophilic glycerol dipalmitate derivatives of the two muramyl dipeptides within liposomal carriers resulted in a significant enhancement of anti-infectious activity, both with respect to number of survivors and length of survival. Liposomal muramyl dipeptides were 10- to 15-fold more potent than free muramyl dipeptide; enhanced potency was most evident with N-acetylmuramyl-D-alanyl-D-isoglutamine. Prophylactic treatment with liposomes containing the lipophilic muramyl dipeptides resulted in enhanced clearance of bacteria from the blood (greater than 3-fold increase in rate) when compared with that of hydrosoluble N-acetylmuramyl-L-alanyl-D-isoglutamine, indicating a correlation between reticuloendothelial stimulation and anti-infectious activity.

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.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti-RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

Resistance to schistosomiasis in mice can be acquired either specifically, by primary infection with Schistosoma mansoni, or nonspecifically, by treatment with a variety of unrelated agents such as bacille Calmette-Guérin. Several immunoadjuvants related to mycobacteria were examined for their ability to induce resistance to schistosomiasis. Natural cord factor (6,6'-trehalose dimycolate), a 100-carbon synthetic cord factor analogue, and dipalmitate trehalose induced significant protection. Trehalose dibehenate and muramyl dipeptide did not induce consistent protection. Since protection acquired by primary schistosomal infection or by any of these potentiating agents is partial, their possible additive effect was evaluated. The resistance of mice with schistosomiasis that were injected with trehalose dipalmitate and challenged with schistosomal cercariae was increased, as assayed by recovery of schistosomula from the lungs and of adult worms from the portal system. Thus, these synthetic adjuvants not only induce partial protection against schistosomiasis, but also significantly enhance acquired immunity in mice with primary infections.

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.

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 nonimmunogenic MDP analogs possessing immunoadjuvant activity.

In a previous study we demonstrated that lipopolysaccharide failed to elicit nonspecific resistance in C3H/He lipopolysaccharide low-responder mice against Klebsiella infection in contrast to its activity in a closely related histocompatible high-responder subline, C3HeB/Fe. Complete restoration of lipopolysaccharide-induced protection against 10(5) Klebsiella was obtained in the present study by transferring bone marrow from high-responder mice to the highly deficient C3H/He mice. The ability of C3H/He mice to clear and destroy bacteria in 5 h was also transferred by C3HeB/Fe marrow cells. In contrast, when high-responder C3HeB/Fe mice were reconstituted with low-responder bone marrow, the clearance and destruction of K. pneumoniae were similar to what is observed in the high-responder strain, but survival was only temporary. Collectively, our data show that the failure of C3H/He mice to respond to lipopolysaccharide with nonspecific immunity is due to a defect in two types of bone-marrow-derived cells--radioresistant and radiosensitive.

A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), was previously shown to enhance polyclonal antibody response in murine spleen cell cultures. When MDP was added to the culture together with a potent murine B cell mitogen (such as bacterial lipopolysaccharide (LPS)), it inhibited completely the LPS-induced polyclonal activation without affecting either the 3H-thymidine incorporation or the number of blast cells in cultures. Strong suppression of mitogen-induced polyclonal activation by MDP was obtained by using a large range of cell concentrations in cultures and over various dosage levels of the stimulating mitogens (LPS and NWSM). An inhibition could be obtained even when MDP was added 24 hr after the addition of the mitogen, and highly significant suppression was observed in the absence of cell division in the cultures.

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.

Digestion by lysozyme of delipidated cells of Mycobacterium smegmatis liberates a water-soluble immunoadjuvant fraction which is chemically very similar to the water-soluble adjuvant (WSA) obtained previously from purified cell walls, but which contains somewhat more non-peptidoglycan amino acids. The yield of peptidoglycan-arabinogalactan complex is about 10 times greater starting from whole cells than from cell walls. The main biological properties of this “neo-WSA” are described: it increases circulating antibodies to ovalbumin in guinea pigs, it does not produce polyarthritis in rats or induce hypersensitivity to tuberculin, it does not increase susceptibility to histamine or hyperreactivity to endotoxin, and does not produce spleen and liver hypertrophy. Analogous immunostimulant fractions have also been obtained from delipidated cells of Nocardia opaca by lysozyme treatment.

Summary The bactericidal effect of normal mouse serum, generally ascribed to natural “O” antibodies, can be removed by absorption with rough microorganisms. Passive hemagglutination tests disclose the presence of thermolabile, non-agglutinating “R” antibodies in normal mouse serum; titers in germ-free animals are much lower. Horse hyperimmune serum against rough S. typhimurium protects mice against infection with unrelated virulent smooth K. pneumoniae. Provided the Klebsiellae are preincubated in normal mouse serum bactericidal activity can also be demonstrated in vitro. This activity does not require the presence of complement and can be removed either by the same Klebsiella strain or by rough strains of Salmonellae but not by the smooth strain of Salmonellae from which these mutants originate. Although these effects would most easily be accounted for by specific antibodies it is also possible that they are instead attributable to a serum protein capable of charge interaction with rough antigens. In any event these findings, together with previous observations concerning degradation of somatic antigen and phenotypic modification of Klebsiellae recovered from endotoxin-treated mice, have led to the development of a new concept of natural immunity to Enterobacteriaceae. In this hypothesis, enzymatic factors capable of attacking cell wall components unmask R antigenic sites common to many bacterial strains and species. Thereafter the presence of a few types of “R” antibodies or of serum factors reacting with rough antigens have the capability of coping, like masterkeys, with a very wide range of infections due to serologically unrelated organisms.

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-l-alanyl-d-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH ± MDP-primed mice and a fixed number of haptenprimed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.

Modulation of myelopoiesis by three synthetic muramyl peptides was investigated in vivo. Two adjuvant-active compounds (N-acetylmuramyl dipeptide [MDP] and MDP-butyl-ester) elicited significant responses in DBA/2 mice characterized by a rise in the level of monocyte-macrophage colony-stimulating activity in serum, a proliferation of multipotential stem cells in the bone marrow, and an expansion of granulocyte-macrophage progenitors in the spleen. In contrast, the adjuvant-inactive stereoisomer MDP(D-D) induced only low levels of circulating colony-stimulating activity. Thus, MDP or MDP-butyl-ester injection could induce a greater number of macrophages and therefore enhance both specific and nonspecific immunity.

N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), an apparently nonimmunogenic bacterial peptidoglycan-derived small peptide, was found to induce a polyarthritis the rat similar to that induced by Freund's complete adjuvant when injected in the form of an oil emulsion. An oil emulsion of its isomer, N-acetylmuramyl-L-alanyl-L-isoglutamine, which unlike MDP has no immunostimulatory activity, failed to induce the disease.

The pyrogenicity in the rabbit and the ability to stimulate nonspecific resistance to bacterial infection in the mouse of muramyl dipeptide (MDP), a synthetic immunoadjuvant, are known to be enhanced when the glycopeptide has been conjugated to a carrier. The effects of indomethacin on fever induced by MDP or its conjugated derivative were studied. Indomethacin reduced febrile responses to MDP or its derivative, although it did not decrease production of endogenous pyrogen in vivo or in vitro. When incubated with rabbit peritoneal cells, indomethacin suppressed the elevation in prostaglandin levels usually induced by MDP. When administered to mice, indomethacin alone stimulated resistance to bacterial infection and, under appropriate conditions, had a strong synergistic effect when combined with free or conjugated MDP. The results demonstrate that neither pyrogenicity nor increased prostaglandin levels are prerequisites for immunopotentiation by synthetic glycopeptides.

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.

The use of mycobacteria in cancer therapy, either as systemic or as local immunoadjuvants, has been proposed [9, 11, 16]. These microorganisms can enhance the resistance of the host to a variety of attacks (bacterial, protozoal, viral or tumoral) by nonspecific mechanisms. Because they are powerful immunoadjuvants, they can also potentiate specifically the immune response to a given antigen. This capacity may be particularly valuable in the case of neoantigens that are weak immunogens. In recent years, both approaches have been used clinically and experimentally [11, 13, 20]. BCG (Mycobacterium tuberculosis, bacillus Calmette-Guerin), Hemophilus pertussis and Corynebacterium parvum are amongst the most popular candidates for clinical and animal experimentation in this field. H. pertussis and more recently C. parvum have been shown to increase the susceptibility of mice to histamine [3, 14] and may therefore expose the host to a wide variety of allergic and physiological accidents. Although BCG lacks this histamine-sensitizing activity, it is still capable of inducing many of the noxious effects observed after the injection of H. pertussis or C. parvum. Thus, killed BCG administered in saline sensitizes to tuberculin, produces lymphoid hyperplasia and increases the reactivity of mice to endotoxins [8, 18]. The decreased resistance towards lipopolysaccharides may be the cause of a great number of pathophysiological disturbances. Mycobacteria administered as a water-in-oil emulsion also produce allergic polyarthritis in the rat, a response which has been considered to indicate autoimmune disease [17]. Several investigators have suggested that insoluble Wax D extracted from virulent strains was the immunoadjuvant component of mycobacterial cells and that this substance is also responsible for allergic polyarthritis [17, 19].