Louis Chedid

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-l-alanyl-d-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

Abstract N -Acetylmuramyl- l -alanyl- d -isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F 1 hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.

Nonspecific activation of murine spleen cells in vitro by a synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine).

Abstract We reported previously that synthetic N -acetyl-muramyl- l -alanyl- d -isoglutamine (MDP) displayed marked adjuvant activity but was devoid of mitogenicity in vitro . The data reported here establish that, under different cultural conditions, thymidine uptake and blast cells can be increased by MDP in spleen cells of DBA/2 and Balb/c mouse strains. Optimal responses were obtained on culture in a serum-free medium supplemented with 2-mercaptoethanol for 4 or 5 days. This effect was also obtained with spleen cells of Balb/c nude mice. When the synthetic MDP was compared to a natural water-soluble adjuvant (neo-WSA), extracted from Mycobacterium smegmatis cells, both were found to stimulate [ 3 H] thymidine incorporation by mouse spleen cells. However, with the neo-WSA, the effect peaked on Day 2 and was weak or absent on Days 4 and 5. When the cells were cultured in a medium containing fetal calf serum, neo-WSA activation was completely abolished, while MDP-mediated stimulation was decreased.

Stimulation of non-specific resistance to infections by synthetic immunoregulatory agents@@@Stimulation der unspezifischen Infektionsabwehr durch synthetische immunregulierende Substanzen

SummaryMuramyl 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.ZusammenfassungMuramyl-Dipeptid oder MDP (AcMur-L-Ala-D-iGln) ist ein synthetisches Immunadjuvans, das sogar bei oraler Verabreichung die unspezifische Abwehr gegen bakterielle Infektionen bei Mäusen verstärken kann. Durch Anwendung verschiedener Derivate wurde belegt, daß weder Adjuvans-Aktivität noch Pyrogenität die Voraussetzung für eine Steigerung der Abwehr sind und daß unerwünschte pharmakologische Wirkungen durch geringfügige chemische Modifikationen ausgeschaltet werden können. Es fand sich außerdem, daß einige lipophile Analoga oder Derivate, die durch Bindung des Glykolipids an einen Carrier gewonnen wurden, aktiver sind als MDP. Ihre Wirksamkeit war ebenfalls dosisabhängig und wurde vom Zeitpunkt der Applikation beeinflußt; sie variierte außerdem entsprechend der Größe der bakteriellen Belastung. Als günstigster Applikationszeitraum für MDP und seine Derivate hatte sich Tag eins bis vier vor dem Setzen der Infektion erwiesen. In manchen Fällen wirkte MDP sogar protektiv, wenn es eine Stunde nach Infektion injiziert wurde, während unter dieser Voraussetzung mit anderen Immunstimulantien wie LPS oder BCG eine negative Phase mit erhöhter Anfälligkeit auftreten kann. Bei Tieren mit schwacher Abwehrlage wie neugeborenen Mäusen oder erwachsenen Tieren nach immunsuppressiver Behandlung wurde die Resistenz gegen bakterielle Infektionen durch MDP noch erhöht. Nach wiederholter Injektion von MDP oder Analoga mit Adjuvans-Aktivität in hohen Dosen (die bekanntlich spezifische Immunreaktionen hemmen) wurde außerdem die protektive Wirkung nicht beeinträchtigt.

Changes in Rabbit Febrile Responses to Muramyl Dipeptide (MDP) After Coupling to a Synthetic Carrier

Muramyl dipeptide (MDP) is a small molecular weight synthetic glycopeptide (< 500), which has been shown to be an immunoadjuvant, and to induce a biphasic febrile response in the rabbit—probably via the release of endogenous pyrogen—accompanied by a marked leukopenia. Macromolecularization by coupling to a synthetic carrier (MW = 60,000) potentiates the immunostim‐ulant properties of MDP but also its pyrogenicity. The present study demonstrates that such a conjugate induced the release of endogenous pyrogen in vivo and in vitro at lower dosage levels than free MDP. Further experiments showed that there existed several differences between free and conjugated MDP. Thus, after intravenous administration of the conjugate, the fever pattern was monophasic with a prompt defervescence and not accompanied by leukopenia at dosage levels inducing similar increase in body temperature. In addition, when fever was recorded after intracerebroventricular administration, the increase in sensitivity was much greater in the case of free MDP than of MDP‐A‐L.

On the Nature of Some Nonspecific Host Responses in Endotoxin-Induced Resistance to Infection

“Tolerance” to endotoxins can be established by a previous injection of an unrelated O antigen which increases the host’s resistance and his capacity for clearing the second dose of endotoxin. It has been shown that these responses are nonspecific, i.e., not mediated by antibodies, but are produced by degradation and detoxification of endotoxic lipopolysaccharides [1, 2, 3].

Production of lymphocyte activating factor (LAF) in the absence of endogenous pyrogens (EP) by rabbit or human leukocytes stimulated by a muramyl dipeptide derivative

N-acetylmuramyl-L-alanyl-D-isoglutamine or MDP is a synthetic immunoadjuvant capable of inducing a febrile response in the rabbit and of eliciting production of endogenous pyrogen (EP) in vivo and in vitro. It was also shown to stimulate in vitro lymphocyte activating factor (LAF) release. After conjugation to a synthetic carrier, the pyrogenic effect of MDP is strongly enhanced. Recently, adjuvant active derivatives of MDP devoid of pyrogenicity such as MDP(Gln)-OnBu (AcMur-L-Ala-D-Gln-alpha-nbutyl ester) have become available. In the present study, their stimulating properties on rabbit peritoneal exudate cells or human peripheral blood mononuclear cells were simultaneously evaluated by testing lymphocyte activating property and pyrogenic effect of the culture supernatant. In both types of cell cultures, whereas MDP elicited dual effects, the nonpyrogenic analog MDP(Gln)-OnBu produced LAF without detectable pyrogenicity even when a very sensitive assay (intracerebroventricular administration) was used. In contrast, conjugated MDP was capable of inducing the production of endogenous pyrogen in rabbit cell cultures with no or little LAF release. These results argue against the possibility that a single factor is responsible for both LAF and EP activities.