Induction of tumouricidal macrophages by MTP-PE: its enhancement by a factor produced spontaneously by tumour cells

Tumouricidal activity of rat alveolar macrophages is induced by MTP-PE in vitro. This tumouricidal activity is enhanced by a factor (tumour cell derived immunostimulating factor, TCIF) contained in tumour cell culture supernatants. TCIF is not species specific, since culture supernatants of rat MADB-200 as well as mouse B16 or Meth A tumour cells showed similar effects on rat alveolar macrophages. TCIF is not produced in cultures of normal cells, e.g. rat embryo cells. TCIF produced by MADB-200 tumour cells is relatively heat-stable and dialyzable. It is destroyed by treatment at pH 2 for 24 hrs. These results suggest that TCIF can participate in macrophage activation and could be of potential therapeutic value.     

Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Inhibition of murine hepatic tumor growth by liposomes containing a lipophilic muramyl dipeptide

Summary We have investigated the ability of liposomes containing a lipophilic muramyl dipeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine glycerol dipalmitate (MDP-GDP) to activate Kupffer cell tumoricidal activity in situ and to inhibit the growth of experimental hepatic micrometastases of tumor cell line H-59, a liver-homing variant of the Lewis lung carcinoma. Liposomes prepared from distearoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DSPC/DMPG) and containing MDP-GDP (1 mol and 2 g, respectively) were efficiently taken up by the liver after i.v. administration. A single i.v. injection of DSPC/DMPG liposomes containing MDP-GDP was capable of inducing Kupffer cell tumoricidal activity against H-59 tumor cells as measured in vitro. Control liposomes or 100 g free MDP were ineffective in inducing Kupffer cell tumoricidal activity in situ. Two treatment regimens were evaluated in vivo: firstly, C57BL/6 mice were injected with tumor cell line H-59 and subsequently treated with multiple injections of liposomal MDP-GDP. Secondly, treatment with liposomal MDP-GDP was initiated prior to tumor cell injection and continued after tumor cell injection. The ability of liposomes containing MDP-GDP to reduce the number of hepatic micrometastases using the first protocol was related to the tumor cell inoculum, significant inhibition being observed at lower liver tumor burdens (<25 tumor nodules). Pretreatment of the mice prior to tumor cell challenge followed by treatment afterwards greatly enhanced the efficacy of liposomal MDP-GDP and brought about a highly significant inhibition of the growth of experimental metastases even at high liver tumor burdens (>50 nodules).     

Enhancement of dengue virus infection in cultured mouse macrophages by lipophilic derivatives of muramyl peptides

Dengue virus multiplication in cultures of a murine myelomonocytic cell line (WEHI-3) as well as mouse peritoneal macrophages was enhanced by treatment of the cells with lipophilic derivatives of muramyl peptides for 2 or 3 days before virus inoculation, but not for 2 hr before virus inoculation or during the adsorption period. The infection-enhancing activity of the materials was dependent on their chemical structure, correlating with their immunoadjuvanticity. The infection enhancement in WEHI-3 cells was due primarily to an increase in the number of virus-infected cells which was accompanied by an increased cellular capacity to bind latex particles to their cell surfaces.     

Induction of interleukin 1 by synthetic and naturally occurring muramyl peptides

Like bacterial lipopolysaccharides (endotoxins), synthetic muramyl peptides (MPs) are thought to exert many of their biological effects by inducing the production of various mediators from host cells. Both synthetic muramyl dipeptide (MDP) and naturally occurring sleep factor (SF), which contains an MP structure, stimulate human monocytes to produce interleukin 1 (IL 1). IL 1 is a family of unique polypeptides that mediate a variety of host defense functions and possess several biological properties, many of which are shared with MPs. Endotoxins are potent inducers of IL 1, but polymyxin B, which blocks endotoxin's biological activities, has no effect on MP-induced IL 1 production. SF purified from human urine and SF isolated from the peritoneal fluid of patients undergoing chronic ambulatory peritoneal dialysis (CAPD) induce IL 1 when incubated with human mononuclear cells in vitro. SF from urine or CAPD fluid induces IL 1 production in the picrogram per milliliter range whereas synthetic MDP requires microgram per milliliter concentrations. Thus, both synthetic and naturally occurring MPs exert their biological effects, in part, by inducing IL 1.     

ImmTher, a lipophilic disaccharide derivative of muramyl dipeptide, up-regulates specific monocyte cytokine genes and activates monocyte-mediated tumoricidal activity

ImmTher, a liposome-encapsulated lipophilic disaccharide tripeptide derivative of muramyl dipeptide, previously showed activity against liver and lung colorectal metastases in a phase I trial. The purpose of the current studies was to investigate whether ImmTher could up-regulate specific cytokine gene expression and protein production, as well as activate the tumoricidal or cytostatic activity of human monocytes. ImmTher induced the expression and production of interleukin(IL)-1α IL-1β, IL-6, IL-8, IL-12, macrophage chemotactic and activating factor, and tumor necrosis factor α but not IL-2 or IL-10. Cytostatic or cytotoxic monocyte activity was stimulated against human Ewing's sarcoma, osteosarcoma, and melanoma cells but not breast cancer cells. Production and secretion of these cytokine proteins may play a role in the antitumor activity of ImmTher. Received: 15 December 1998 / Accepted: 18 March 1999     

Synthesis, mass spectral characterization and immunoadjuvant activity of some novel lipophilic derivatives of muramyl dipeptides

Some novel lipophilic derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) have been prepared and rigorously evaluated by spectroscopic means. Fast atom bombardment and field desorption mass spectrometry provided information about both molecular weight and structural detail. The new MDP derivatives have been tested in guinea pigs for immunoadjuvant activity using egg albumin as the model antigen. Amongst these derivatives, 6-O-[3-(5-cholesten-3 beta-yloxycarbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (CSMDP), 6-O-[3-1,2-dipalmitoyl-sn-glycero-3-carbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (GSMDP) and N-palmitoyl muramyl-L-alanyl-D-isoglutamine (PMDP) possessed significantly better activity than MDP, as judged by the antigen-specific antibody and delayed hypersensitivity responses in the immunized animals. In addition, CSMDP was found to induce strong delayed hypersensitivity response even in saline. These three active compounds were also tested for their pyrogenic response in rabbits, and were found to be lesser pyrogenic than MDP. Some of these MDP derivatives hold promise as adjuvants in immunization.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Induction of chromosomal breakage in cultured human leucocytes by luteoskyrin

Summary Luteoskyrin, a Polyhydroxy-Bis-Dihydroanthrachinon isolated from Penicillium islandicum Sopp, is known to have strong cytotoxic effects. In long-term cultures of Ehrilich-Ascites-Tumour cells it causes the formation of very long abnormal chromosomes (Schachtschabel). Experiments with short-term cultures of human leucocytes are reported on which have shown Luteoskyrin to induce chromosomal breaks and interchromosomal bridges.

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Zusammenfassung Das Polyhydroxy-Bis-Dihydroanthrachinon Luteoskyrin, ein Stoffwechselprodukt des Schimmelpilzes Penicillium islandicum Sopp, hat eine starke cytotoxische Wirkung. In Langzeitkulturen von Ehrlich-Ascites-Tumorzellen verursacht es die Bildung von abnormal langen Chromosomen (Schachtschabel). Es wird über Versuche mit Kurzzeitkulturen von menschlichen Leukocyten berichtet, in denen durch Luteoskyrin Chromosomenbrüche und interchromosomale Brücken entstehen.

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Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 128/1).     

Induction of salivary kallikreins by the diet containing a sweet-suppressive peptide, gurmarin, in the rat

Gymnema sylvestre (gymnema) contains gurmarin that selectively inhibits responses to sweet substances in rodents. The present study investigated possible interaction between gurmarin and the submandibular saliva in rats fed diet containing gymnema. Electrophoretic analyses demonstrated that relative amounts of two proteins in the saliva clearly increased in rats fed the gymnema diet. However, rats previously given section of the bilateral glossopharyngeal nerve showed no such salivary protein induction. Analyses of amino acid sequence indicate that two proteins are rat kallikrein 2 (rK2) and rat kallikrein 9 (rK9). rK2 and rK9, a family of serine proteases, have a striking resemblance of cleavage site in the protein substrates. Interestingly, gurmarin possesses comparable residues with those rK2 and rK9 prefer. The kallikreins significantly inhibited immunoreaction between gurmarin and antigurmarin antiserum. These results suggest that rK2 and rK9 increased by chemosensory information for the gymnema diet via the glossopharyngeal nerve might cleave gurmarin or at least cause specific binding with it.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative

The lipophilic amino acid, (S)-2-aminoundecanoic acid, was synthesized and incorporated at a number of specific positions within the peptide sequence of anoplin. These lipophilic anoplin analogs showed to be more active against Escherichia coli and Staphylococcus aureus compared to native anoplin, while the EC₅₀-value of hemolysis was at least one order of magnitude lower than the MIC values. This was in sharp contrast to the N-acylated anoplin derivative, where a gain in activity also led to a complete loss of selectivity. Thus, the incorporation of a lipophilic amino acid residue into anoplin enhanced the antimicrobial activity, while selectivity towards microbial membranes was retained.     

Molecular mechanisms of induction of antigen-specific allograft tolerance by intranasal peptide administration

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.     

Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation

Peptide nucleic acids (PNAs) are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to cells. To overcome this problem we made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond. The TPP cation led to efficient PNA uptake into the cytoplasm where the disulphide bond was reduced, releasing the antisense PNA to block expression of its target gene. This method of directing PNAs into cells is a significant improvement on current procedures and will facilitate in vitro and pharmacological applications of PNAs.     

Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide.

Comparative surface feature analyses of the VP1 sequences of hepatitis A virus (HAV) and poliovirus type 1 allowed an alignment of the two sequences and an identification of probable HAV neutralization antigenic sites. A synthetic peptide containing the HAV-specific amino acid sequence of one of these sites induced anti-HAV-neutralizing antibodies. It is concluded that a structural homology exists between the two viruses, despite minimal primary sequence conservation.     

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.