Immunostimulating lipopeptide,LtriP (RP 56142): Comparison of the effect on hepatic cytochrome P 450 modulation and radioprotection in male and female of three mouse strains

The sex-dependent effect of lauroyl-L-Ala-D--Glu-L,L-A₂pmNH₂ (LtriP, RP 56142) on hepatic microsomal cytochromes P 450 (cyt P 450) was studied in three mouse strains NMRI, C3H/OuJ and C3H/HeJ. In NMRI and C3H/OuJ, strains which are responsive to bacterial lipopolysaccharides (LPS-responsive), regardless of the sex of the mouse, significant decrease in the amount of cyt P 450 was observed after LtriP treatment, with a concomitant reduction in ethoxyresorufin-O-deethylase (cyt P 450 1A-dependent) and 7-ethoxycoumarin-O-deethylase activities. This was not seen in C3H/HeJ (LPS-hyporesponsive) mice. These effects may be related to LtriP-dependent cytokine induction, since neither LtriP nor LPS stimulated interleukin-1 (IL-1) secretion by C3H/HeJ macrophages. 11- and 12-hydroxylations (11- and 12-OH) of lauric acid were compared in C3H/OuJ and C3H/HeJ mice. LtriP depressed the total enzymatic conversion of lauric acid in the two strains without modification of the 11/12-OH ratio for C3H/OuJ or male C3H/HeJ mice. However, in females C3H/HeJ mice this decrease was particularly significant and concerned especially the 12-OH activity (a marker of cyt P450 4A family). Although males of the three strains were more sensitive to irradiation than females, LtriP exerted a sex-independent radioprotection on NMRI and C3H/OuJ mice. Its radioprotective effect was illustrated by the preservation of all the enzymatic activities studied in treated NMRI mice, contrary to irradiated control animals. In contrast, for the C3H/HeJ strain, males were not protected by LtriP treatment and, furthermore, females showed a marked sensitization to irradiation.The effects in CH3/HeJ strain implicate LtriP in the control of cyt P 450 induction and of sensitivity to irradiation independently of IL-1 induction.     

2-Carboxyethylgermanium sesquioxide,a synthetic organogermanium compound,as an inducer of contrasuppressor T cells

2-Carboxyethylgermanium sesquioxide (Ge-132), a synthesized organogermanium compound with immunomodulaing activities, was shown to be an inducer of anti-suppressor T cells in normal mice. The suppressor cell activity of T6S cells, a clone of burn-induced CD8⁺ IL-4-producing suppressor T cells, was clearly inhibited when a mixed lymphocyte-tumor cell reaction of the clone was conducted with splenic mononuclear cells from mice treated orally with a 100 mg/kg dose of Ge-132. The activity of anti-suppressor cells was demonstrated in spleens of mice 2 days after treatment with Ge-132 and reached its peak on day 3. The anti-suppressor cells induced by the compound were of a contrasuppressor T cell-linage, because they were characterized as CD4⁺ CD28⁺ TCR/⁺ Vicia villosa lectin-adherent T cells. These cells produced IFN- but did not produce IL-2, IL-4, IL-6 or IL-10 in their culture fluids. CD4⁺ anti-suppressor T cells induced by Ge-132 may be different from other subsets of CD4⁺ T cells because Th1 and Th2 cells generated in our laboratory did not adhere toVicia villosa lectin-coated petri dishes, and each produced specific cytokines. Th1 cells produced IFN- and IL-2 while Th2 cells produce IL-4 and IL-10 in vitro. These results suggest that Ge-132 may be useful as an inducer of contrasuppressor T cells in immunocompromised individuals bearing suppressor T cells. To eliminate suppressor T cells from immunocompromised hosts may result in improved resistance from various opportunistic infections.     

Mitogenicity of autolysates ofTrypanosoma congolense

Summary Autolysates ofTrypanosoma congolense, in subcytotoxic amounts, were found to be highly mitogenic in vitro for the spleen cells of normal mice. Significant amounts of [³H]-thymidine were also incorporated by the responding spleen cells of nu/nu (athymic) mice. In contrast, the spleen cells of cyclophosphamide-treated mice were unresponsive. The findings suggest that a potent B-cell-mitogen is generated by the autolysingT. congolense organism.Acknowledgments. This investigation was supported by the International Development Research Centre, Canada.     

Effect of lymphokines produced in a grafted system on production of prostaglandins by macrophages]

Mice peritoneal macrophages cultured in vitro release a prostaglandin-like activity as evaluated by a bio-assay using Rat stomach fundus. The prostaglandin release is greatly increased when macrophages are incubated with the supernatant of mixed lymphocyte cultures between recipient and donor of a skin allograft. This phenomenon was found in all strains of Mice tested except C3H/HeJ Mice, a strain already known for its defective responsiveness to bacterial lipopolysaccharide (LPS).     

Selective effect of burn injury on splenic CD11c+ dendritic cells and CD8α+CD4−CD11c+ dendritic cell subsets

Burn injury causes an immunosuppression associated with suppressed adaptive immune function. Dendritic cells (DCs) are APCs for which signaling via their Toll-like receptors (TLRs) induces their maturation and activation, which is essential for the adaptive immune response. In this study, we examined if burn injury alters the TLR activity of splenic DCs. After injury, we noticed that DC functions were impaired, characterized by a suppressed capacity to prime naive T cells when triggering the TLR4 signaling cascade using specific ligands (LPS or rHSP60). The observed perturbations on LPS-primed DCs isolated from burned mice exhibited significantly diminished IL-12p40 production and enhanced IL-10 secretion-associated impairment in mitogen-activated protein kinase activation. Interestingly, we observed a decrease of TLR4/MD-2 expression on the CD8α⁺ DC subset that persisted following LPS stimulation. The altered TLR4 expression on LPS-stimulated CD8α⁺ DCs was associated with reduced capacity to produce IL-12 after stimulation. Our results suggested that TLR4 reactivity on DCs, especially CD8α⁺ DCs, is disturbed after burn injury.     

Efficacy of tumor cell vaccine after incorporating monophosphoryl lipid A (MPL) in tumor cell membranes containing tumor-associated ganglioside

Murine B16 melanoma expresses the ganglioside. GM₃. GM₃ shed from tumor cells is immunosuppressive and promotes tumor growth¹. Reduction or elimination of the shed GM₃ could be therapeutic, and the anti-GM₃ antibodies may reduce and clear the shed ganglioside. To test this hypothesis, mice were challenged with tumor cells, with or without inducing anti-GM₃ antibody response. Since gangliosides are poor immunogens and T-cell independent antigens, an adjuvant (monophosphoryl lipid A (MPL), a non-toxic lipid A ofSalmonella), directed against B-cells, was employed. MPL was incorporated onto liposomes and into the surface membrane of B16 mouse melanoma cells; both are rich in GM₃. C57BL/6J mice immunized with MPL-liposomes or MPL-B16 cells responded with elevated levels of anti-GM₃ IgM. Non-immunized mice or mice immunized with B16 cells alone or ganglioside GM₃ alone (without MPL) elicited poor anti-GM₃ IgM response, confirming the GM₃'s immunologic crypticity and MPL's immunopotentiating effect. MPL's immunopotentiating effect was improved by coupling it to melanoma cell membranes C57BL/6J mice were immunized with irradiated B16 alone or MPL alone or MPL-conjugated irradiated B16. After three weekly immunizations, each mouse received a challenge dose of viable syngeneic B16. Neither MPL alone nor B16 alone had a significant effect on tumor growth or host survival; however, administration of MPL-conjugated B16 cells significantly prevented tumor growth and prolonged survival. Our results indicate that MPL-incorporated B16 cells augment the anti-GM₃ IgM response, which may reverse GM₃-induced immunosuppression by eliminating tumor-derived GM₃, and restore immunocompetence.     

Identification and characterization of a highly thermostable bacteriophage lysozyme

Pseudomonas aeruginosa bacteriophage KMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the KMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P. aeruginosa PAO1 and Escherichia coli WK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100°C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology.Received 13 July 2004; received after revision 31 August 2004; accepted 6 September 2004     

A rapid method of following the spontaneous regression of experimental leukemias

Summary Transplantation of virus and chemically induced leukemias from C3H/He-mgxAKR/F₁ hybrid mice into C3H/He-mg males induced leukemias in the latter, which was followed by a spontaneous regression of the disease within a few days. The regression of leukemia could easily be followed by measuring the changes in the pyruvate kinase activity of para-aortic lymph node cells.     

Periplasmic lysozyme inhibitor contributes to lysozyme resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The product of the Escherichia coli ORFan gene ykfE was recently shown to be a strong inhibitor of C-type lysozyme in vitro. The gene was correspondingly renamed ivy (inhibitor of vertebrate lysozyme), but its biological function in E. coli remains unknown. In this work, we investigated the role of Ivy in the resistance of E. coli to the bactericidal effect of lysozyme in the presence of outer-membrane-permeabilizing treatments. Both in the presence of lactoferrin (3.0 mg/ml) and under high hydrostatic pressure (250 MPa), the lysozyme resistance of E. coli MG1655 was decreased by knock-out of Ivy, and increased by overexpression of Ivy. However, knock-out of Ivy did not increase the lysozyme sensitivity of an E. coli MG1655 mutant previously described to be resistant to lysozyme under high pressure. These results indicate that Ivy is one of several factors that affect lysozyme resistance in E. coli, and suggest a possible function for Ivy as a host interaction factor in commensal and pathogenic E. coli.Received 12 February 2004; received after revision 11 March 2004; accepted 16 March 2004     

Transgenic and mutant animal models to study mechanism of protection of red cell genetic defects against malaria

Malaria, caused by members of the genusPlasmodia, is still the most prevalent parasitic disease in the world. In an attempt to understand genetic factors conferring resistance to malaria, mouse models of thalassemia, sickle trait, and ankyrin and spectrin deficiency were studied during infection with species of malaria infectious to rodents. Although growth ofP. falciparum is not inhibited in thalassemic erythrocytes in culture, mice carrying a -thalassemia mutation were protected fromPlasmodium chabaudi adami, supporting epidemiologic findings. Transgenic mice expressing ^s hemoglobin were also significantly protected from two species of rodent malaria. Importantly, a significant role for the spleen in protection in the ^s transgenic mice was found. Finally, mice deficient in spectrin and ankyrin were studied with respect to their ability to support the growth of malaria. It was found that spectrin deficient mice were almost completely refractory toP. chabaudi adami andP. berghei. These models will allow further study of host factors in resistance to malaria.     

Evidence for a role of IFN γ in control ofListeria monocytogenes in T cell deficient mice

Summary The role of interferon (IFN) in controlling chronic infections ofListeria monocytogenes (Listeria) was studied in athymic C57BL/6nu/nu mice, and by treating thymectomized C57BL/6 +/+ mice with monoclonal rat CD4 and CD8-specific monoclonal antibodies (Mab). Mice treated with a combination of the two T cell subset antibodies were similar to athymic, nude mice in being able to contol Listeria infection, keeping the titers below 3–5 log₁₀ bacteria per organ, but they could not eliminate them completely. Treatment with antibodies to IFN of nude or CD4⁺ + CD8⁺-T cell-depleted mice suffering from chronic Listeria infection caused a marked increase of Listeria titers, in liver and spleen. This result implies a role of IFN in maintaining anti-Listeria resistance in mice lacking mature T cells.     

Bacterial lipopolysaccharide (LPS) enhances concanavalin A reactivity of thymocytes from the low-LPS-responder mouse strain C3H/Hej.

The capacity of LPS to enhance Con A reactivity of thymocytes was studied comparatively in the low-LPS-responder C3H/Hej mice and the high-LPS-responder CBA mice. The extent of synergism LPS + Con A was found similar in both strains.     

Alternariol,a dibenzopyrone mycotoxin ofAlternaria spp., is a new photosensitizing and DNA cross-linking agent

Summary The mycotoxin alternariol (3,4,5-trihydroxy-6-methyldibenzo [a] pyrone) but not alternariol monomethyl ether (3,4-dihydroxy-5-methoxy-6-methyldibenzo [a] pyrone) is phototoxic toEscherichia coli in the presence of near UV light (320–400 nm). The phototoxicity bioassays with a DNA repair-deficient mutant ofE. coli suggested that DNA may be the molecular target for photo-induced toxicity of alternariol. Interactions between alternariol and double-stranded, supercoiled DNA suggest that alternariol interacts with DNA by intercalation. No DNA breakage was detected in this system; however, alternariol forms a complex and cross-links double-stranded DNA in near UV light. These results suggest that alternariol is a new phototoxic, DNA-intercalating agent and is a DNA cross-linking mycotoxin in near UV light.Acknowledgment. Dr Albert Stoessl (Agriculture Canada, London, Ontario, Canada) generously provided a mixture of alternariol and alternariol monomethyl ether, and made many helpful suggestions. Dr Ashwood-Smith (University of Victoria, Victoria, British Columbia, Canada) kindly supplied the microorganisms through Dr G.H.N. Towers (University of British Columbia, Vancouver). We gratefully acknowledge the gifts. It is a pleasure to acknowledge the able assistance of Mr S. Tallevi.     

Effect of actinomycin D onTrypanosoma cruzi

Summary Viable metacyclic forms ofT. cruzi, Y strain, treated with an adequate dose of actinomycin D (50 g Act-D/ml/10⁷ parasites/ml for 72 h at 28° C) showed the following properties: 1) they lost their ability to replicate in culture medium, in blood and in tissues of normal mice and were no longer able to incorporate tritiated thymidine; 2) they could no penetrate into Vero cells and could not replicate inside normal macrophages; 3) they retained their immunogenicity and the ability to protect mice against a virulent infection; 4) they did not induce histological lesions as described in chronic experimental Chagas' disease.     

Modulation of antibody synthesis by an anti-tumour alga

Summary An unicellular alga,Chlorella pyrenoidosa, which had been reported to protect C₃H mice against sarcoma BP8, is shown, when injected in Freund's incomplete adjuvant, to modulate the antibody synthesis induced by immunization with a hapten-carrier complex.C. pyrenoidosa appeared to be able to initiate an antigenic competition between hapten and carrier determinants of the antigen molecule during antibody synthesis, and thus it could be speculated thatC. pyrenoidosa modulates the immune response at the macrophage level.Acknowledgments. This work was supported by the Secrétariat d'Etat aux Universités and by the DGRST, grant No. 77.71347.     

Cardenolide glycosides from the adults and eggs ofChrysolina fuliginosa (Coleoptera: Chrysomelidae)

Besides the known sarmentogenin 3-O--D-allopyranoside (1) and digitoxigenin-3-O-[-D-xylopyranosyl-(14)-2, 3-di-O-acetyl--D-allopyranoside] (4), four new cardiac glycosides (2, 3, 5 and6) have been isolated from the defensive glands of adults ofChrysolina fuliginosa. The structures of the new compounds were determined by¹H NMR at 600 MHz and FABMS. Compounds1–6, present in the adults ofC. fuliginosa, have also been identified in the eggs.     

At least two toxins are involved inEscherichia coli mastitis

Summary Culture filtrate ofEscherichia coli produces changes in the bovine udder identical to those seen in experimental infections with the same organism.E. coli endotoxin produces an acute inflammatory response but none of the cell damage induced by culture filtrate. It appears therefore that at least 2 toxins are involved in the disease.Acknowledgments. We are grateful to Mrs K. Wells, Miss H. Meads and Miss A. Ashford for technical assistance.     

Bacterial lipopolysaccharide (LPS) enhances concanavalin A reactivity of thymocytes from the low-LPS-responder mouse strain C3H/Hej

Summary The capacity of LPS to enhance Con A reactivity of thymocytes was studied comparatively in the low-LPS-responder C3H/Hej mice and the high-LPS-responder CBA mice. The extent of synergism LPS+Con A was found similar in both strains.This work was supported by a grant from the INSERM (CRL: 76-5-101-1).     

Course of fever response to repeated administration of sublethal doses of lipopolysaccharides,polyinosinic: Polycytidylic acid and muramyl dipeptide to rabbits

Summary The purpose of the present study was to examine the development of tolerance to three structurally dissimilar pyrogens, i.e., lipopolysaccharide (LPS), muramyl dipeptide (MDP) and polyinosinic: polycytidylic acid (poly I:C) in rabbits. The possibility of pyrogenic cross-tolerance among these agents has also been studied. It was observed that repeated injection of sublethal doses of LPS and MDP was connected with the changing of biphasic fever to monophasic. The consequence of this was a drop in the fever index. In contrast to LPS and MDP, the repeated administration of poly I:C did not result in such changes. Successive injections of this pyrogen always evoked biphasic fever. We also demonstrated that pyrogenic cross-tolerance between LPS and MDP did not occur. The cross-tolerance between LPS and MDP did not occur. The cross-tolerance among pyrogens was possible if they originated from the same class, for example endotoxin fromSalmonella abortus eq. and endotoxin fromEscherichia coli.