Quantitative histology of muramyl dipeptide-potentiated induction of tumor necrosis by endotoxins

Combinations of muramyl dipeptide (MDP) and toxic or detoxified endotoxin induced necrosis and subsequent disappearance of solid Meth A tumors in syngeneic mice. Toxic endotoxin alone was far less effective. MDP and detoxified endotoxin had negligible antitumor effects of their own. These observations were confirmed by histological examination. Neither MDP nor detoxified endotoxin induced significant changes in and around the tumor by 4, 24, and 48 h after intravenous administration when compared with saline treatment. MDP amplified various effects of toxic endotoxin such as the induction of hyperemia, mitotic arrest, mast cell depletion, non-hemorrhagic necrosis and reduction in lymphocyte infiltrates, but did not affect hemorrhagic necrosis or the influx of polymorphonuclear leukocytes. The combination of MDP and detoxified endotoxin lacked the latter two effects, but the other effects were similar to, although slightly less marked than those induced by the toxic combination. Because the degree of hyperemia was proportional to the degree of subsequent non-hemorrhagic necrosis, MDP seems to potentiate necrosis by enhancing mechanisms leading to hyperemia and mast cell mediators might be involved in the latter effect. Lymphocyte influx and the therapeutic outcome are likely to be related, since exclusively therapeutic treatments reduced the influx of these cells.     

Muramyl dipeptide analogues as potentiators of the antitumor action of endotoxin

Summary The potentiation of endotoxin-induced necrosis and regression of solid syngeneic Meth A tumors in mice previously observed following administration of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was investigated further by use of various muramyl peptide analogues and two unrelated synthetic adjuvants, viz. the pluronic polyol L121 and dimethyldioctadecylammonium bromide (DDA) instead of MDP. All agents were administered in aqueous solution by the IV route. None of the muramyl peptide analogues nor L121 or DDA had any strong antitumor action of their own. Two 6-O-acylated muramyl peptides (L2-MDP and B30-MDP) and muramyl dipeptide stearoyllysine [MDP-Lys (L18)] clearly potentiated endotoxin-induced necrosis and regression. In contrast, MDP with L- instead of D-isoglutamine was completely inactive. Optimal activity of B30-MDP and MDP-Lys (L18) was only achieved by adding of suitable amounts of a nonionic surfactant. L121 and DDA could not replace muramyl peptides as potentiating agent. The combination of endotoxin, MDP, and L121 caused complete tumor regression in all mice, but was highly toxic.On the basis of the data in the literature on the biological response-modifying activities of the agents used it is concluded that the potentiating activity of muramyl peptides cannot yet be related to their immunoadjuvant action or their capacity to activate macrophages or to enhance nonspecific bacterial resistance.The work described in this paper was supported by grant UUKC 82-15 from the Koningin Wilhelmina Fonds, The Netherlands Cancer Organization     

Modification of delayed-type hypersensitivity reactions by muramyldipeptide in guinea pigs

Abstract N -Acetylmuramyl- l -alanyl- d -isoglutamine (muramyldipeptide, MDP) modulated delayed-type hypersensitivity (DTH) reactions and induced severe inflammatory lesions in guinea pigs. The animals immunized with heat-killed Mycobacterium tuberculosis were challenged with the purified protein derivative (PPD) at the flanks and the corneas to prepare DTH reactions at 2 weeks after the immunization, thereafter 24 h the animals received subcutaneous injections of MDP at the flanks of the opposite side. At the skin with the DTH reaction, increase of swelling and redness accompanied with hemorrhage and necrosis were observed. As corneal reactions in the animals that had received MDP, increase of cornea thickness, opaque and grayish-white and the projection of eyes accompanied with severe iritis were observed. Modification of the skin reaction occurred from 2 h after the MDP injection, rapidly increased to the maximum level around 10 h, maintained the level until 24 h, then slowly decreased. The polymorphonuclear leukocyte infiltration was observed from 15 min after the MDP injection, and tumor necrosis factor alpha, interleukin (IL)-1, and IL-6 levels in the serum and skin lesions increased after the MDP injection. Synthetic muramyltripeptide ( N -acetylmuramyl- l -alanyl- d -isoglutaminyl- l -lysine) also provoked definite skin reactions, while the larger peptidoglycan fragments and various inflammatory agents including cytokines so far examined were inactive in this respect. Cortisone and heparin inhibited definitely and slightly the reaction, respectively. A comparison was made with the modified DTH reaction and the necrotic reactions which we reported previously.     

Synergic action between tumor necrosis factor and endotoxins or poly(A·U) on cultured bovine endothelial cells

Summary In order to investigate whether direct effects on tumor vasculature may contribute to induction of necrosis of solid tumors in vivo, agents and combinations with an established different capacity to induce tumor necrosis were studied for their effects on endothelial cells in vitro. Tumor necrosis serum caused a marked inhibition of [³H]thymidine incorporation by bovine umbilical cord endothelial cells after 4h coincubation. Endotoxin was less inhibitory, whereas detoxified endotoxin and recombinant human tumor necrosis factor (rTNF) were hardly active in concentrations that can be achieved in vivo. Combinations of rTNF and (detoxified) endotoxin caused synergic inhibition. By 24h effects of the separate agents and synergic effects of the combinations were much stronger. The nontoxic dsRNA, poly(A·U), also had inhibitory activity, and acted synergistically with rTNF. Morphologically, a combination of endotoxin and rTNF but not the separate constituents induced marked cell detachment by 24 h, an indication of cell death. Whereas both endotoxin and rTNF inhibited DNA synthesis of human endothelial cells, the agents did not act synergistically on these cells. The ability of the agents and the combinations to affect endothelial cells in culture appeared to be well in line with their capacity to induce tumor necrosis. Data suggest that direct (synergic) effects on endothelium may contribute to the induction of vascular damage in tumors by (combinations of) the agents. The fact that endothelial cell death is only induced by the combinations and not by the separate agents in vivo, may be a cause of the greater therapeutic activity of the combinations in vivo. The synergy between rTNF and the other agents indicates that the agents act by different mechanisms.Supported by a grant of the Stichting Koningin Wilhelmina Fonds, Netherlands Cancer Foundation     

Synergistic action of human recombinant tumor necrosis factor with endotoxins or nontoxic poly A:U against solid Meth A tumors in mice

Summary Antitumor effects of i.v. injected human recombinant tumor necrosis factor (rTNF) against solid Meth A tumors in mice appeared to be critically dependent on the dose and were limited by its toxicity. Extensive necrosis and complete cures were only induced by doses having untoward effects, such as diarrhea, hypothermia, ruffled fur, and lethargy. Murine tumor necrosis serum (TNS, 0.5 ml) had about the same antitumor potential and induced all side effects except diarrhea. More extensive necrosis and approximate doubling of the incidence of complete regression in the absence of gross side effects were observed upon administration of a low dose of rTNF combined with detoxified endotoxin, nontoxic poly A:U, or submicrogram doses of toxic endotoxin. The separate constituents had little antitumor effects, if any at all. Increasing the dose of toxic endotoxin resulted in a further potentiation of necrosis, overt toxicity, but no cures. Muramyl dipeptide and interferon / did not potentiate effects of rTNF. In vitro growth of Meth A cells was not inhibited by toxic endotoxin, rTNF or the combination, although TNS was highly inhibitory. Data show that therapeutic effects of rTNF and its synergy with endotoxin are not due to direct effects on the tumor cells and that the extent of prompt in vivo tumor necrosis does not predict the course of tumor growth. Therapeutic effects of both TNS and toxic endotoxin probably involve a synergy between low levels of TNF and other factors/effects induced by endotoxin. Detoxified endotoxin and poly A:U probably induce the latter effects and little or no TNF, so explaining the absence of side effects, their weak antitumor potential, and their powerful synergistic action with rTNF. A role for interferon / as an induced synergistic factor is not likely. Muramyl dipeptide and TNF might share properties needed for synergy with endotoxins. Present address: Department of Immunotoxicology, State University of Utrecht, Biltstraat 172, 3572 BP Utrecht, The Netherlands     

Antitumour activity of endotoxin, concanavalin A and poly I: C and their ability to elicit tumour necrosis factor, cytostatic factors, and interferon in vivo

Summary Concanavalin A, endotoxin, poly I : C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A : U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I : C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I : C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I : C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator.From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.     

In vivo effects of toxic and detoxified endotoxin alone or in combination with muramyl dipeptide on lymphoid and non-lymphoid cells in the spleen of Meth A sarcoma-bearing mice

In this study we confirmed that combinations of toxic or detoxified endotoxin with muramyl dipeptide (MDP) induced much more necrosis of transplanted Meth A sarcoma in mice than toxic endotoxin alone. Detoxified endotoxin and MDP alone had little antitumor effects. We investigated whether these divergent antitumor effects could be related to histopathological changes in the white pulp of the spleen of Meth A sarcoma-bearing mice. Toxic endotoxin reduced the T:B cell compartment ratio in the splenic white pulp by increasing the size of the B-cell compartment while leaving the size of the T-cell dependent inner PALS unaffected. The number of the T-lymphocytes in this area, however, was reduced. The border of B-lymphocytes in the marginal zone was markedly narrowed and the number of marginal metallophils along the inner border of the marginal sinus was decreased. None of these changes were observed after treatment with detoxified endotoxin or MDP. Addition of MDP to either endotoxin did not change their effects. The histopathological changes in the lymphoid and non-lymphoid compartments of the splenic white pulp are apparently exclusively induced by toxic endotoxin. As the antitumor activity of both toxic and detoxified endotoxin combined with MDP are about equal and more powerful than the activity of toxic endotoxin alone, it is concluded that these antitumor effects cannot be related to changes in the white pulp of the spleen.     

Antitumor effects of endotoxin against solid murine Meth A tumors of different ages

Mice with 3-, 6-, 9- and 15-day Meth A tumors in the skin were injected intravenously with endotoxin to study age-dependent induction of tumor necrosis and reactive changes in draining lymph nodes. By 24 h after treatment with endotoxin, macroscopic necrosis was seen in 9-day, and to an even greater extent in 15-day tumors; microscopy showed extensive necrosis in 9-day tumors and some necrosis in 6-day tumors. The necrosis was predominantly coagulative, but the 9- and 15-day tumors showed a rim of hemorrhagic necrosis near the skin with surviving tumor tissue located at the lateral and basal margins. All the tumors showed about equal hyperemia by 4 h and mitotic arrest by 4 and 24 h. Depletion of mast cells, which were most numerous in the 9-day tumors, was seen on the dermal aspects by 24 h. Endotoxin did not change the morphology of the lymph nodes and disseminated tumor cells within 24 h. Endotoxin is known to cure 9-day Meth A tumors, and the extent of the rapid necrosis induced is clearly the crucial factor. Necrosis, however, is not a direct consequence of early hyperemia or mitotic arrest and other factors related to the age and site of the tumor, apparently affect whether or not necrosis ensues.     

Role of the hepatic function in the development of the pyrogenic tolerance to muramyl dipeptide

We have demonstrated that the hepatic function may have an important role in the development of tolerance to the pyrogenic effect induced by endotoxin. To further investigate if the role of the hepatic function in the development of tolerance also extends to that induced by other pyrogenic stimuli, we investigated the effect of galactosamine, a specific inhibitor of the hepatic protein synthesis, on the development of tolerance to the pyrogenic effect induced by muramyl dipeptide (MDP) in rats. Pyrogenic tolerance was observed after the second intravenous or intraperitoneal injection of MDP (500 microgram/kg), 24 h after the first injection, similar to what was observed with endotoxin. Pyrogenic tolerance was abolished when galactosamine (300 mg/kg ip) was injected simultaneously with MDP (500 microgram/kg iv) on the first day. When uridine (600 mg/kg ip) was administered simultaneously with galactosamine (300 mg/kg ip) and the first injection of MDP (500 microgram/kg ip), pyrogenic tolerance was again observed after the second injection of the peptidoglycan. In conclusion, the hepatic function may not be important only for the development of tolerance to endotoxin, but also to a totally different pyrogenic stimulus such as MDP.     

Role of the functional state of the liver RES in the pathogenesis of toxic liver injury

In rats to which E. coli endotoxin (250 micrograms/kg i.p.) was administered 24 h before they were given tetrachlormethane (CCl4) (1.5 ml/kg intragastrically), stimulation of liver DNA synthesis was observed during the first 48 h after administration of the hepatatoxin. In experimental rats to which prodigiosan (a Serratia marcescens polysaccharide, 250 micrograms/kg i.p.) was administered 24 h before CCl4 (1.5 ml/kg i.p.), liver damage 24 h after CCl4 poisoning was expressed less--judging from the size of liver necrosis and the size of glycogen-free zones in the liver lobules than in the controls. To elucidate the role of activated macrophages in the induction of liver resistance to CCl4, liver injury caused by this hepatotoxin was compared after the pre-administration of protein extract from the Kupffer cells or hepatocytes of prodigiosan-stimulated rats. In rats given the larger dose of Kupffer cell extract (6 mg/ml i.p.), the necrotic foci formed after the administration of CCl4 were significantly smaller. The results confirm the conception that liver macrophages participate in the development of resistance to CCl4.     

Hormone-dependent changes of blood vessels in DMBA-induced rat mammary carcinoma and its regression studied by 3H-thymidine autoradiography

Using DMBA-induced rat breast cancer, the changes in the histology and proliferative activity underlying the phenomenon of tumor regression by hormone therapy were studied by 3H-thymidine autoradiography. The control tumor was found to essentially consist of two histologically different areas, medullary (A area) and tubular or cystic (B area). The cancer cells in the A area were homogeneously proliferating with a cell cycle time of 51h, and among those in the B area, 65% were proliferating with a cell cycle time of 81h while 35% were non-proliferating. Among the various-kinds of hormone therapies, ovariectomy plus male sex hormone administration was most effective in inducing tumor regression. In the regressed tumor, the A area was greatly diminished due to central necrosis and replaced by cystic B area. In the remaining A area, the cell cycle time was lengthened to 97h, and that for the proliferating cells in the B area was as long as 118h. The most striking histological change after ovariectomy plus male sex hormone administration was the diffuse necrosis of the capillary endothelial cell within 24h, followed by hemorrhage, central necrosis in the A area (1W), and final stage of fibrosis (2W). The tumor administered with female sex hormone after ovariectomy showed a rebound growth from the regression, due to the initial reactivation of the endothelial cell proliferation and following stimulation of cancer cell mitotic activity. From these observations, it is concluded that the capillary endothelial cells in DMBA-induced rat breast cancer are estrogen dependent, and that the tumor regression induced by decreased estrogen-level is attributable to the massive necrosis from capillary insufficiency and anoxia.     

Effect of verapamil, cromoglycate and dimedrol on the survivability of mice in endotoxic shock

The trial has been performed on blocking the potentiation of the lethal action of Sh. Sonnei endotoxin isolated using the agents possessing antiaggregation and antimediator properties--verapamil, cromoglycate and diphenhydramine. It was shown that LD50 of endotoxin increased 2-2.5-fold, provided there were 3 drug injection 1, 4 and 9 hours after endotoxin administration. The survival of mice at 100% level of mortality was also increased 2-2.5-fold. With endotoxin administered together with the first drug injection, the drugs were ineffective. In case endotoxin was administered 3 times at a 4-hour interval prophylactic and therapeutic effects of the drugs and their combinations have been observed.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.     

Reduced lipopolysaccharide (LPS)-induced nitric oxide production in peritoneal macrophages and inhibited LPS-induced lethal shock in mice by a sugar cane (Saccharum officinarum L.) extract

A sugar cane extract (SCE) has been found to have an immunostimulating effect in several animals. Lipopolysaccharide (LPS) is known to induce endotoxin shock via the production of inflammatory modulators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO). We examined in the present study the effects of SCE on the TNF-alpha and NO production in LPS-stimulated mice peritoneal cells and the endotoxin shock in mice. The supplementation of SCE to peritoneal macrophages cultured with LPS resulted in a significant decrease in NO production. All the mice injected intraperitoneally with LPS and D-galactosamine (LPS+GalN) died within 24 h. However, a peritoneal injection, but no intravenous or oral administration, of SCE (500-1,000 mg/kg) at 3 to 48 h before the LPS+GalN-challenge resulted in a significantly improved survival rate. These results suggest that SCE had a protective effect on LPS-induced endotoxin shock via one of possible mechanisms involving the suppression of NO production in the mouse peritoneal cavity.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Effects of ibuprofen and pentoxifylline on the cardiovascular response of normal humans to endotoxin.

Endotoxin is a major mediator of the life-threatening cardiovascular dysfunction that characterizes Gram-negative sepsis. In animal models of endotoxemia, pretreatment with ibuprofen or pentoxifylline attenuates some of these cardiovascular changes. To evaluate the effects of these agents on the human cardiovascular response to endotoxemia, hemodynamic variables were measured serially in 24 normal subjects who were given intravenous endotoxin. The subjects were randomized to receive oral ibuprofen (n = 9), pentoxifylline (n = 10), or no medication before endotoxin administration (n = 5). The subjects were volume loaded 3-5 h after endotoxin administration, and hemodynamic measurements were reassessed. Core temperature after endotoxin alone or endotoxin-pentoxifylline approached a maximum at 3 h (greater than or equal to 38.6 degrees C), while the endotoxin-ibuprofen group remained afebrile. At 3 and 5 h, all three groups had significant increases in heart rate, cardiac index, oxygen delivery, and oxygen consumption, while systemic vascular resistance index decreased significantly from baseline. The oxygen extraction ratio remained unchanged. After volume loading, the left ventricular ejection fraction and left ventricular end-diastolic and end-systolic volume indexes did not differ among the groups. The hyperdynamic cardiovascular response to endotoxin in humans occurs in the absence of fever and is not significantly ameliorated by oral cyclooxygenase or phosphodiesterase inhibition.     

Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress

Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS.     

Circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral E. coli challenge.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10(7), 2.5 x 10(8), 2.5 x 10(9) colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain IL-1beta levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.     

Stimulation of Nonspecific Host Resistance to Infection Induced by Muramyldipeptides

The effect of muramyldipeptide (MDP), N-acetylmuramyl-l-alanyl-d-isoglutamine [MDP(Ala)], and its analogs on bacterial infection was studied using the experimental model of sepsis infection in mice. Injection of MDP(Ala) gave mice definitive protection against E. coli infection, but only partial protection against P. aeruginosa or K. pneumoniae infection. Several factors influencing the protective activity of MDP (Ala) on E. coli infection were studied, and it was demonstrated that the activity was induced by various routes of administration of MDP(Ala), including the oral route, and was markedly influenced by the bacterial inoculum size. It was also shown that the effective dose of MDP(Ala) was 100 μg per mouse for intraperitoneal, intravenous or subcutaneous injections and 1,000 μg per mouse when administered orally. Furthermore, the optimal interval between MDP-treatment and infection was 24 hr when the treatment was carried out before infection. Clearance of bacterial cells in blood was observed after E. coli infection in mice treated with MDP(Ala). The efficacy of MDP(Ala) and two analogs, N-acetylmuramyl-l-valyl-d-isoglutamine [MDP(Val)] and N-acetylmuramyl-l-seryl-d-isoglutamine [MDP (Ser)], was evaluated for the E. coli infection; MDP(Val) was proven to be slightly less active than MDP(Ala), and MDP(Ser) to be the least effective, although MDP(Val) or MDP(Ser) was reported to have higher adjuvanticity than MDP (Ala) for the development of delayed-type hypersensitivity.     

Recombinant tumor necrosis factor depresses cytochrome P450-dependent microsomal drug metabolism in mice

The effect of recombinant tumor necrosis factor on liver cytochrome P450 and related drug metabolism enzymes was investigated. Treatment of mice with tumor necrosis factor caused a marked depression of cytochrome P450 and some drug metabolizing enzymes (ethoxycoumarin deethylase and arylhydrocarbon hydroxylase) in the liver and many other organs. This effect was maximal 24-48 h after treatment and was dependent on the dose of tumor necrosis factor administered. Depression of liver drug metabolizing enzymes was also observed in the endotoxin-resistant C3H/HeJ strain of mice, thus ruling out that this effect may be due to minor endotoxin contamination of recombinant tumor necrosis factor. These data indicate that depression of liver drug metabolism might be an important side effect of tumor necrosis factor, and suggest a role for this macrophage product as an endogenous regulator of liver metabolism.