Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

[Non-Antibiotic Strategies to Prevent the Recurrence of Uncomplicated Urinary Tract Infections in Women]. / Nicht antibiotische Strategien zur Rezidivprophylaxe von unkomplizierten Harnwegsinfektionen der Frau.

The aim of all medical treatment is "primum nihil nocere" ("First, do no harm").Restoring the integrity of intestinal microbiota and optimising the immune response in recurrent infections, especially in the urinary tract, are treatment alternatives which are closer to this target than the usual focus on antibiotic prevention of recurrence.In the future, antibiotics will continue to be recommended for the prevention of urinary tract infections on a case-by-case basis. However, the problems of an excessive use of antibiotics, e. g. resistance and long-term interference with intestinal microbiota, are forcing us to search for alternatives. The use of probiotics alone or in combination with immunotherapeutics, or the sole use of immunotherapeutics, are important treatment options, which are already routinely available in clinical practice. These therapies are focused on the pathomechanism of an infection and tackle the root cause of the problem. Phytotherapeutics or small molecules like mannose, which restricts the adherence of bacteria to the urothelium, are complementary approaches.The EAU guidelines recommend the following treatments for the long-term prevention of urinary tract infections:Oral and parenteral immunostimulants (StroVac(®)), local estrogen replacement and administration of Lactobacillus rhamnosus and Lactobacillus reuteri.

Conformational mapping of the cytosolic linker between domains III and IV of the cardiac Na+ channel protein and binding studies with a site-directed channel modifying antibody.

By combining antibody binding studies with conformational mapping using synthetic peptides, the structure of the cytosolic linker between domains III and IV of the cardiac Na+ channel alpha-subunit was analyzed. Inside-out patch clamp experiments with isolated cardiac Na+ channels from neonatal rat cardiocytes confirmed that a polyclonal antibody against amino acids 1490-1507 of the cardiac Na+ channel recognizes the linker in situ since Na+ inactivation became significantly retarded. Epitope fine mapping with a series of overlapping peptides identified the sequence YYNAMKKLG (corresponding to amino acids 1496-1504 of the cardiac sodium channel alpha-subunit) as the binding locus of the site directed antibody, an interesting result with respect to structure-function relationships because the functionally important hydrophobic amino-acid cluster in position 1487-1489 is not included. Circular dichroism measurements of synthetic 20-mer peptides in hydrophilic and lipophilic environments provided indications for a notable alpha-helical content only for segment GGQDIFMTEEQKKYYNAMKK. This sequence corresponds to amino acids 1483-1502 in the linker and adopts a highly ordered pattern of charge distribution due to this helical conformation. Ordered structure and helix dipole moment represent physical properties which may be important in a refined model for explaining the function of the linker in terminating the open channel configuration.

Separation of plasma membrane domains of calf thymocytes by affinity chromatography on ouabain-Sepharose.

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.

Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments.

The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-L-Cys-Ser and analogs with longer sequences, are polyclonal activators for B-lymphocytes. Triple-chain lipopeptides also constitute efficient low-molecular-weight carrier/adjuvant systems, which can be linked to antigens to yield immunogens for antibody production without further additives. This is the first report of monolayer experiments with chemically well defined, synthetic lipopeptide mitogens with the composition of the N-terminus of an important bacterial membrane protein. Various derivatives of the lipoprotein N-terminus were synthesized. These lipopeptides differed in the length of the peptide moiety, the number of fatty acid residues, and protective groups. In order to obtain the surface areas for the lipopeptides in isotherms and hysteresis isotherms, monolayer experiments with a computer-controlled film balance were performed. To get some information about the interaction of these compounds with typical membrane lipids mixed monolayers were formed from triple-chain lipopeptides with dipalmitoylphosphatidylcholine and cholesterol. A comparison of the mitogenic response of the compounds was made in an in vitro system with B-lymphocytes from Balb/c mice.

Interaction of immunologically-active lipopeptides with membranes.

Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Intracellular localization of a lipopeptide macrophage activator: immunocytochemical investigations and EELS analysis on ultrathin cryosections of bone marrow-derived macrophages.

Synthetic lipopeptides, structurally derived from the N-terminal part of bacterial lipoprotein, constitute macrophage and B-lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow-derived macrophages. The intracellular localization of the C-terminally biotinylated lipodipeptide Pam3Cys-Ser was investigated on ultrathin cryosections using the biotinstreptavidin-gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.

Induction of soluble antitumoral mediators by synthetic analogues of bacterial lipoprotein in bone marrow-derived macrophages from LPS-responder and -nonresponder mice.

Macrophage-dependent antitumoral activity is partly mediated by soluble factors including cytokines, reactive-oxygen intermediates (ROIs), and reactive-nitrogen intermediates (RNIs). Activation of macrophages for tumor cytotoxicity can be achieved with various bacterial compounds, such as lipopolysaccharides (LPSs), muramyl-dipeptides, and lipopeptides. We studied the production and release of oxygen radicals, nitric oxide, and tumor necrosis factor alpha (TNF-alpha) by bone marrow-derived macrophages (BMDMs) of different mouse inbred strains after they were stimulated with the lipopeptide P3CSK4, a water-soluble synthetic analogue of the lipidated N terminus of bacterial lipoprotein. The lipopeptide was able to induce a strong, long lasting release of oxygen radicals in BALB/c mouse macrophages. Furthermore, it induced nitric oxide release from BMDMs of several mouse strains (BALB/c, C57Bl/6, C57Bl/10ScSn, Sv129, NMRI, and LPS-nonresponder C57Bl/10ScCr). Stimulation with P3CSK4 also resulted in comparable production of TNF-alpha in LPS-responder and nonresponder BMDMs from C57Bl/10ScSn mice and C57Bl/10ScCr mice, respectively. All three antitumoral mediators reached functional levels or concentrations as shown by the strong cytostatic/cytotoxic activity of lipopeptide-activated macrophages for the cell lines Abelson 8-1, M12.5/P815, and L929, which are sensitive to ROIs, nitric oxide, and TNF-alpha, respectively. We found that synthetic lipopeptides can induce the secretion of effective levels of soluble tumor-cytotoxic/cytostatic mediators in BMDMs of LPS-responsive and, of particular interest, also of LPS-unresponsive mice. This result could indicate that the highly effective bacterial-macrophage activators P3CSK4 and LPS use different receptors and/or different intracellular signal transduction pathways.

Th1-orientated immunological properties of the bacterial extract OM-85-BV.

The bacterial extract OM-85-BV prepared from 21 pathogenic bacterial strains is administered orally to adults and children for the treatment and prevention of recurrent infections of the respiratory tract. We analyzed in vitro and in vivo the immunomodulatory effects of the extract. The lysate acted as a non specific macrophage activator, inducing NO production as well as the translocation of transcription factor NF-kappaB into the nucleus in murine bone marrow-derived macrophages. Besides stimulating unspecifically the immune system, a bacteria-specific humoral immune response was revealed. After oral application, a trend to increase bacteria-specific IgG and IgA in serum was observed. Also a marked increase of bacteria specific IgA in saliva as well as in supernatants of Peyer's patches and mesenteric lymph nodes-derived cell cultures was found. The immunomodulatory properties of the extract were also investigated with respect to shifting the Th1/Th2 bias in an in vivo allergy model. BALB/c mice were orally immunized with OM-85-BV and subsequently sensitized intraperitoneally with the allergen ovalbumin. The group pretreated with OM-85-BV showed a decrease of both total and ovalbumin specific IgE. Accordingly, in spleen cell supernatants the Th1-associated cytokine IFN-gamma was increased, and the Th2-associated cytokine IL-4 was downregulated. Our findings suggest that the immunoprotective effects of OM-85-BV observed in human beings may be correlated to its Th1 augmenting properties.

Generation of mouse polyclonal and human monoclonal antibodies against Bacillus anthracis toxin.

High titer antisera against the protective antigen (PA) from Bacillus anthracis were generated immunizing Balb/c mice two times intraperitoneally with PA in combination with lipopeptide adjuvant P3CSK4. The sera were able to protect the mouse macrophage cell line J774A.1 from an anthrax toxin challenge. We also tested the blood of anthrax vaccine-immunized persons for PA- and lethal factor (LF)-specific antibodies. An increased titer was found after three immunizations, and the sera were also able to protect the mouse macrophage cell line from a toxin challenge. For the preparation of human monoclonal antibodies, we used peripheral blood lymphocytes. After in vitro stimulation using PA or synthetic peptides derived from PA, B lymphocytes were immortalized by PEG fusion with the human mouse heteromyeloma cell line CB-F7. We obtained several clones producing high amounts of PA-specific immunoglobulin (Ig).

B cell activation by synthetic lipopeptide analogues of bacterial lipoprotein bypassing phosphatidylinositol metabolism and proteinkinase C translocation.

Synthetic analogues of bacterial lipoprotein induce proliferation of murine small resting B lymphocytes. We investigated the role of proteinkinase C (PKC) activation in lipopeptide-induced B cell stimulation. Using a standardized extraction procedure, there was no change in membrane bound and soluble PKC activity upon stimulation with lipopeptide. However, omitting Ca2+ chelators from the standard extraction medium resulted in a decrease of membrane bound PKC activity after stimulation. Lipopeptide failed to induce phosphoinositide degradation and the generation of the two second messengers cAMP and cGMP. To test whether guanosinetriphosphate-binding proteins are involved in lipopeptide-induced signal transfer we investigated the effect of LiCl, choleratoxin and pertussistoxin on B lymphocyte proliferation. LiCl and pertussistoxin did not inhibit cell activation, whereas choleratoxin reduced the proliferation rate at concentrations higher than 0.5 micrograms/ml. Similar results were observed when LPS was used as mitogen, whereas the anti-immunoglobulin-induced B cell activation was inhibited by all three compounds. Our results show, that B cell activation by bacterial lipopeptides bypasses phosphatidylinositol metabolism and PKC translocation.

Determination of second messengers and protein kinase C in bone marrow derived macrophages stimulated with a bacterial lipopeptide.

The synthetic lipopeptide Pam3Cys-Ala-Gly, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage activator. The role of protein kinase C (PKC) in lipopeptide induced signal transduction was investigated. As determined by enzymatic and immunochemical methods, translocation of PKC could not be observed in lipopeptide stimulated bone marrow derived macrophages. Our studies showed that the membrane-associated form of PKC displayed different characteristics than the cytosolic form. The second messengers, inositoltrisphosphate, cAMP and cGMP, did not seem to be involved in signal transduction. Unlike LPS, Pam3Cys-Ala-Gly induced a rapid rise in cytosolic Ca2+, which was due to an influx of extracellular calcium as well as to a redistribution of intracellular calcium. The data suggest that one major intracellular signal transduction mechanism initiated by lipopeptide consists of altering internal Ca2+ concns.

Role of proteinkinase C and phosphatidylinositol metabolism in lipopeptide-induced leukocyte activation as signal transducing mechanism.

Synthetic lipopeptides are potent B-lymphocyte and macrophage activators. The role of phosphatidylinositol metabolism and proteinkinase C in lipopeptide induced leukocyte activation were investigated. In murine B-lymphocytes and in bone marrow derived macrophages, lipopeptide failed to induce phosphatidylinositol breakdown, whereas in the macrophage cell line P388D1 formation of inositolphosphates was increased. Translocation of proteinkinase C from a cytosolic to a membrane compartment was only observed in the cell line P388D1 indicating that in the other cells tested lipopeptide acts via different signal transduction pathways.

Rapid alterations in the plasma membrane structure of macrophages stimulated with bacterial lipopeptides.

Synthetic lipopeptide analogues of the N-terminal region of bacterial lipoprotein are potent activators of macrophages. In a previous study we showed that within minutes after their addition to macrophage cultures, lipopeptides were found attached to the plasma membranes and within different compartments of the cells. Their rapid interaction with the plasma membrane is thought to occur via the insertion of their three fatty acids. We used the freeze-fracture technique to study the influence of lipopeptides on the architecture of plasma membranes. Fifteen to thirty seconds after addition of the lipopeptides, the freeze-fractured plasma membranes show a rapid decrease in the particle density. This effect is not due to a loss of proteins, but is caused by lateral diffusion of single particles, which subsequently aggregate. These alterations are transient, temperature-sensitive and disappear 20 min after stimulation. At 4 degrees C, no change is found in the architecture of the plasma membranes. Using electron energy loss spectroscopy (EELS), lipopeptides can neither be detected on the membrane nor within the cells when incubated at this temperature. Our findings suggest that membrane protein aggregation is involved in the rapid uptake of lipopeptides into macrophages after their interaction with the plasma membranes.

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages.

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-14C]oleate and [3H]inositol, increased the production of inositol phosphates and the release of 1,2-[14C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.

Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas.

Murine bone marrow-derived macrophages (BMDM), a homogeneous cell population easily obtainable in large quantities and at reproducible quality by in vitro differentiation, were used as feeder cells for human B cell hybridomas after fusion or during recloning. We used as antigens for the in vitro immunization of human B lymphocytes from peripheral blood as well as from tonsils: (i) synthetic peptides representing immunogenic sequences of gp160 and Nef of HIV-1, coupled to the lipopeptide carrier N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2 RS)-propyl]-(R)-cysteinyl(-seryl-seryl) (P3 CSS-[gp160(303-329)] and P3C-nef24), (ii) the toxins saxitoxin and microcystin, coupled to BSA (BSA-STX and BSA-MCYST). After fusion with the mouse-human heteromyeloma CB-F7, we could demonstrate that BMDM exert a strong growth supporting effect on post-fusion cultures, resulting in 81.6% versus 23.6% growth-positive wells for P3C-nef24, and 100% versus 71.2% growth-positive wells for BSA-STX stimulated cells in cultures with and without BMDM, respectively. Furthermore, clones in wells with BMDM grew much more rapidly, resulting in 24.3% versus 3.6%, 98.1% versus 42.2% and 56.7% versus 6.7% of cultures ready for screening 2 weeks after fusion of P3C-nef24, P3CSS-[gp160(303-329)], and BSA-STX stimulated lymphocytes, respectively. Apart from their effect on cell growth, murine BMDM also increased the percentage of immunoglobulin (Ig)-producing cultures after fusion, as shown for BSA-STX stimulated lymphocytes (47.8% versus 6.7%), as well as the percentage of cultures producing specific antibodies, as demonstrated with BSA-MCYST activated cells (42% versus 10%). Finally, recloning efficiencies of two human B cell hybridomas (E 10 and F 2) were raised profoundly by BMDM, resulting in 100% versus 64.2% and 90.9% versus 44.2% growth-positive wells after recloning on a ten cells/well level. As murine BMDM can also be stored in liquid nitrogen without loss of activity, they constitute ideal feeder cells for the establishment of human B cell hybridomas.

Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages, and adjuvanticity in vivo and in vitro.

Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution. 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom). The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 [2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes. The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity. (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants. A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL. Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone. Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages.