Sleep-promoting effects of intraperitoneally administered uridine in unrestrained rats

An intraperitoneal injection of 0.1 nmol uridine in rats resulted in a transient excess slow-wave sleep if administered shortly before onset of the dark period. The sleep latency was remarkably shortened. A small dose (0.01 nmol) and larger doses (1, 10, 100 nmol) caused no effect. Uridine at a dose of 0.1 nmol was entirely ineffective if injected shortly before onset of the light period, while it resulted in transient excess paradoxical sleep if injected at an early phase of the light period. It is concluded that uridine, if timely administered through a systemic route, may pass the blood-brain barrier to modulate sleep in rats.     

Pharmacokinetics and feeding responses to muramyl dipeptide in rats

N-acetyl-muramyl-L-alanine-D-isoglutamine or muramyl dipeptide (MDP) is the minimally active subunit of bacterial peptidoglycan. During a systemic infection, the involvement of MDP has been demonstrated in food intake depression by the macrophage hydrolysis of Gram-positive bacteria. Under normal conditions, mammals are constantly exposed to the release of endogenous MDP from degraded gut flora and that of exogenous MDP from the diet. However, MDP digestion and absorption in the gastrointestinal tract are not fully understood, and their physiological significance needs to be clarified. After gavage (1.5 mg/kg), very low levels of MDP were found in the systemic circulation of rats and feeding patterns were not altered. In contrast, after the intraperitoneal injection of a similar dose, a depression in food intake was observed. The rats reduced their meal frequency and constant feeding rate, showing signs of satiety. The behavioral satiety sequence (BSS) was modified by behavioral changes, similar to those which appear during sickness, such as an increase in resting and a reduction in grooming. Our data suggest that the hypophagic effect of MDP may result from satiety and sickness behavior.     

Effects of delta-sleep-inducing peptide in cerebral ischemia in rats

Experimental studies were carried out to investigate the neuroprotective effects of delta-sleep-inducing peptide in animals with cerebral ischemia induced by bilateral compression of both carotid arteries, and to compare the efficacy of this peptide with that of MK-801. These studies led to the conclusion that the peptide had pronounced anti-ischemic effects, which were evident within 24 h and consisted of reductions in the severity of postural abnormalities in rats with bilateral cerebral ischemia, along with a reduction in lethality. Comparison of the efficacies of peptide and MK-801 showed the peptide to have the greater neuroprotective effect. These results are regarded as providing an experimental basis for using the peptide as a therapeutic agent in patients with stroke. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 3, pp. 95–99, March, 1997.     

Effects of delta-sleep-inducing peptide on NMDA-induced convulsive activity in rats

Acute experiments on rats showed that the ED₁₀₀ of NMDA for induction of clonic convulsions was 0.53 μg, while the ED₁₀₀ of NMDA for inducing tonic extension of the forelimbs was 5.02 μg/animal. Determination of these parameters after administration of delta-sleep-inducing peptide (100 μg/kg, i.p.) revealed 2.3- and 4.46-fold increases. These results provide evidence for a neuroprotective role of delta-sleep-inducing peptide in relation to excitatory amino acid receptor agonists. Department of Normal Physiology, State Medical University, 2 Valikhovskii Lane, 270100 Odessa, Ukraine. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 9, pp. 32–36, September, 1997.     

Biologically active antibodies elicited by a synthetic circumsporozoite peptide of Plasmodium knowlesi administered in saline with a muramyl dipeptide derivative.

A synthetic peptide whose sequence was derived from the circumsporozoite protein of Plasmodium knowlesi coupled to bovine gamma globulin has been shown to be immunogenic when administered with Freund complete adjuvant. The present experiments were designed to test the immunogenicity of the peptide when attached to a tetanus toxoid carrier and administered with alum or murabutide, both acceptable clinical adjuvants. In both cases, the use of an adjuvant increased the levels of circulating anti-peptide antibodies over those observed when no adjuvant was used. However, when the antisera were tested for reactivity with the native protein, animals of the group receiving the conjugate associated with murabutide always had titers greatly exceeding those observed in animals that received the conjugate with alum. Moreover, the sera of the murabutide-treated group were shown to be more active in eliciting shedding of the circumsporozoite protein than were sera of animals of the Freund complete adjuvant-treated group. The use of tetanus toxoid in secondary immunizations could be eliminated when the mice primed with peptide-tetanus toxoid and murabutide were boosted with a polymer of the peptide. The results indicate that the synthetic malarial peptide-tetanus toxoid conjugate is capable of stimulating high levels of biologically active antibodies only when administered with murabutide.     

Anti-inflammatory effects of muramyl dipeptide in experimental models of acute inflammation

Synthetic muramyl dipeptide,N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), has been tested for activity against acute inflammation. In all models employed (Bayol + Arlacel paw oedema in rats, carrageenan pleurisy in rats, and carrageenan of dextran paw oedema in mice), MDP given in admixture with the phlogistic agents significantly lowered the resulting inflammatory reaction by about 30–40%. 0.1–0.2 mg of MDP per animal was applied. The mechanism of anti-inflammatory activity of this substance remains unknown.     

Epithelioid granuloma induced by muramyl dipeptide in immunologically deficient rats.

When WKA rats were either neonatally thymectomized or injected with anti-rat thymocyte sera, their T-cell functions were effectively suppressed. When neonatally thymectomized plus anti-rat thymocyte serum-treated rats were injected with non-immunogenic muramyl dipeptide in water-in-oil emulsion, they produced massive epithelioid granulomas. Essentially, no morphological difference was noticed between granulomas induced in untreated rats and in thymectomized plus anti-rat thymocyte serum-treated rats. These findings strongly suggest that muramyl dipeptide-induced epithelioid granulomas required no T cells for their formation. In contrast, the induction of adjuvant arthritis appeared to depend on the presence of T cells.     

Opposite effects of the synthetic immunomodulator, muramyl dipeptide, on rejection of mouse skin allografts

The influence of muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine) on the rejection of mouse skin allografts was investigated. While the 0.1-mg dose administered on days 7, 6, 5 prior to transplantation caused significant prolongation of the graft survival, the 0.5-mg dose administered on days 3, 2, 1 prior to transplantation resulted in remarkable augmentation of the graft rejection. The present results support the view that muramyl dipeptide can induce both stimulatory and suppressive immune mechanisms, depending on the treatment regimen.     

Lipophilic derivative of muramyl dipeptide is more active than muramyl dipeptide in priming macrophages to release superoxide anion.

Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement.     

Comparison of the effects of bacterial lipopolysaccharide and muramyl dipeptide on food intake

For further characterization of the mechanism involved in the anorexia during bacterial infection, we investigated whether muramyl dipeptide (MDP), the minimal immunologically active structure of gram-positive bacterial cell walls, affects rats' food intake in the same way as lipopolysaccharide (LPS) from E. coli. MDP (1.6 mg/kg body weight = b.wt.) injected intraperitoneally (IP) reduced food intake by decreasing meal frequency without affecting meal size. Indomethacin (2.5 mg/kg b.wt., IP) but not verapamil (5 mg/kg b.wt., IP) attenuated the hypophagic effect of MDP. In further experiments, MDP and LPS (100 micrograms/kg b.wt., IP) both inhibited gastric emptying and indomethacin failed to block this effect of LPS. Hepatic vagotomy did not attenuate the hypophagic effects of MDP or LPS. LPS reduced water intake only when food was available, but reduced food intake also during water deprivation. MDP did not affect water intake. MDP and LPS both had an aversive effect, but LiCl, which was also aversive, failed to reduce feeding under the conditions tested. This questions the role of a conditioned taste aversion in the hypophagia induced by MDP or LPS. The results suggest that a stimulation of eicosanoid synthesis contributes to MDP-induced hypophagia and may therefore also contribute to the anorexia during infection. In contrast, an inhibition of gastric emptying, an activation of hepatic satiety signals or a reduction of water intake, does not seem to be crucial for the hypophagic effects of MDP or LPS.     

Arthritogenic activity of a synthetic immunoadjuvant, muramyl dipeptide.

A synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, dissolved in saline only and applied subcutaneously to rats of the Lewis inbred strain, produced arthritis, clinically manifest by hind feet paresis but without apparent paw swelling in most cases. Histologically, the disease was characterized by edema and hyperemia of connective tissues, joint synovias, and tendon sheaths, with massive accumulation of inflammatory cell infiltrates composed mainly of lymphoplasmocytes and partly of neutrophil leukocytes. Fibrin exudation and fibrinoid necrosis in connective tissues were observed in the most severe cases. Synovial layers of the talocrural joint, especially on their villi, exhibited marked swelling or cell desquamation of the inner zone. Clinical symptoms of the disease disappeared spontaneously within 5 days after cessation of the treatment; also, histological examinations showed that the effects were reversible. Our results prove that (i) muramyl dipeptide is the principal substance involved in the production of arthritis, (ii) there is no necessity for the presence of additional mycobacterial cell wall components, and (iii) the involvement of the oil moiety is not requisite for the production of arthritis.     

Biphasic effects of muramyl dipeptide or lipopolysaccharide on superoxide anion-generating activities of macrophages

The superoxide anion (O2-)-generating activity of guinea pig macrophages stimulated by wheat germ agglutinin (WGA), immune complexes, or phorbol myristate acetate (PMA) was studied after short- and long-term exposures of the cells to muramyl dipeptide (MDP) or lipopolysaccharide (LPS). Neither MDP nor LPS alone induced O2- release in macrophages. Short-term (30 min) exposure to these agents caused the enhanced release of O2- in response to WGA or immune complexes, though the PMA-induced O2- generation was not affected. On the other hand, long-term exposure (more than 24 h) to MDP or LPS progressively enhanced O2- generation of the cells induced by WGA, immune complexes, or even PMA. These results suggest that the mechanism for O2- generation of macrophages stimulated by WGA or immune complexes differs from that stimulated by PMA and that the differences also exist between short- and long-term exposure to MDP or LPS.     

Stimulatory effects of muramyl dipeptide upon neutrophils isolated from a local bacterial infection

This study examined the effects of muramyl dipeptide (MDP) in vivo upon the local inflammatory response to a bacterial challenge. In addition to quantitative bacteriology of the tissues surrounding an infected suture, polymorphonuclear leucocytes (PMN) involved in the local inflammatory response were extracted and estimations made of their number, viability and phagocytic activity. Fewer bacteria were recovered from the muscle around the suture in MDP-treated animals compared to placebo-treated controls (P less than 0.02), although there was no difference in the number of bacteria on the suture itself. Polymorphonuclear leucocytes were present in greater numbers (P less than 0.01), more PMNs were viable (P less than 0.01) and more PMNs had visibly phagocytosed bacteria (P less than 0.01) in the MDP group compared to the placebo group. These data indicate that MDP enhances the local inflammatory response to infection with increased influx, viability and phagocytic activity of PMNs, resulting in improved local control of a test bacterial challenge.     

The effects of immunomodulating peptidoglycan monomer and muramyl dipeptide on hepatic microsomal UDP-glucuronyltransferase and beta-glucuronidase

The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT1) and testosterone for the second functional form (GT2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal beta-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of beta-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.     

Pharmacokinetics and metabolism of muramyl dipeptide and nor-muramyl dipeptide [3H-labelled] in the mouse

The fates of ³H-muramyl dipeptide (MDP) and ³H-nor-MDP have been investigated after intravenous (i.v.), intraperitoneal, and subcutaneous injection of a range of doses in the mouse. After i.v. injection both compounds were cleared rapidly from the circulation, distributed initially to the tissues, and finally excreted largely intact in the urine. Most of the tissues contained intact material at 2 min after injection, but the much lower levels of radioactivity persisting at 1 h had undergone considerable metabolism (except in intestine, where some intact material persisted for as long as 24 h). Some accumulation of radioactivity was observed in liver and kidney and there were quantitative and qualitative differences between the two compounds. Characterisation of some of the metabolites in these tissues was undertaken, and the deamidated muramyl dipeptide was tentatively identified which is known to have some biological activity. The mechanism of the biological effects, which may be expressed over a relatively long time period, remains to be explained in view of the rapid excretion of most of the dose.     

T-cell-dependent immunobiological activity of a desmuramyl analog of muramyl dipeptide,adamantylamide dipeptide (AdDP), and D-isoglutamine

The present experiments demonstrate that, similar to immunomodulatory muramyl dipeptide, its desmuramyl analog adamantylamide dipeptide is able to induce mild and fully reversible paw edema in mice. This effect is an immune-related phenomenon depending on the activation of T-cell/macrophage interactions and on production of prostaglandins. Possible involvement of certain immunoregulatory/inflammatory cytokines (e.g. IL-1, IL-2) has been suggested. The most probable intrinsic moiety of the adamantylamide dipeptide molecule responsible for triggering the edema formation is obviously D-isoglutamine.     

Some characteristics of inflammation induced by muramyl dipeptide,endotoxin, and concanavalin A

Inflammatory reactions induced by muramyl dipeptide (MDP), endotoxin, and concanavalin A (Con A) were examined in the skin of rabbits. The neutrophil influx induced by MDP peaked at 2 h and declined to a low level by 4 h, thus resembling the response induced by endotoxin (2). MDP and endotoxin exhibited homologous desensitization when extant lesions were restimulated at 6 h. These agents did not, however, induce heterologous desensitization. Con A induced a biphasic influx of neutrophils into inflammatory lesions with peaks at 2 h and 12 h. Neither the first nor second peak exhibited desensitization to homologous restimulation; however, the cell influx in restimulated lesions assumed a monophasic character peaking at 3 h. Con A lesions were not desensitized to restimulation with MDP or endotoxin. The results suggest that these chemotaxinogens rely on different endogenous mediators to induce an inflammatory response. The protracted second period of neutrophil infiltration of Con A-induced lesions and the failure of desensitization of this response to develop suggest that the mediator of Con A-induced inflammation may play an important role in the sustained recruitment of neutrophils in some inflammatory diseases.     

Stimulatory effects of muramyl dipeptide upon neutrophils isolated from a local bacterial infection.

This study examined the effects of muramyl dipeptide (MDP) in vivo upon the local inflammatory response to a bacterial challenge. In addition to quantitative bacteriology of the tissues surrounding an infected suture, polymorphonuclear leucocytes (PMN) involved in the local inflammatory response were extracted and estimations made of their number, viability and phagocytic activity. Fewer bacteria were recovered from the muscle around the suture in MDP-treated animals compared to placebo-treated controls (P less than 0.02), although there was no difference in the number of bacteria on the suture itself. Polymorphonuclear leucocytes were present in greater numbers (P less than 0.01), more PMNs were viable (P less than 0.01) and more PMNs had visibly phagocytosed bacteria (P less than 0.01) in the MDP group compared to the placebo group. These data indicate that MDP enhances the local inflammatory response to infection with increased influx, viability and phagocytic activity of PMNs, resulting in improved local control of a test bacterial challenge.