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A F Kiderlen

Publications and source records attributed to A F Kiderlen.

34 records · Page 2Linked to original sources
PubMed1998

Detection of parasites with DNA-binding bisbenzimide H33258 in Pneumocystis carinii- and Leishmania-containing materials.

Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.

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PubMed1998

Activation and suppression of natural cellular immune functions by Pneumocystis carinii.

The regulatory role of soluble cytokines in innate cellular immune responses induced by Pneumocystis carinii was assessed in vitro in direct comparison to induction by Listeria monocytogenes. This report shows that P. carinii organisms, as well as L. monocytogenes, stimulated in whole spleen cell cultures of SCID mice the release of IFN-gamma, TNF-alpha/beta, IL-10, IL-12, and iNO. This response was independent of functional T cells. Both macrophages (M phi) and natural killer (NK) cells were necessary for either microorganism to induce release of these cytokines. Cocultures of purified M phi--including alveolar M phi--and purified NK cells indicated that no other cell population was necessarily involved. Microbial induction of NK cell-derived IFN-gamma has been reported to be mediated by the combined effects of TNF-alpha and IL-12 released by M phi upon adequate microbial stimulation. Interestingly, only L. monocytogenes, but not P. carinii organisms could directly induce detectable amounts of TNF-alpha/beta, IL-12, or iNO in purified M phi cultures. In dose-response experiments, release of IFN-gamma, TNF-alpha/beta, and iNO was reduced at high relative concentrations of either microorganism. This high-dose suppression was at least partially controlled by M phi-produced IL-10. Our data show that, P. carinii potently induces activating and inhibitory innate cellular immune response mechanisms and indicate that the initial step of macrophage-mediated NK cell activation might also involve other pathways than those described to date.

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PubMed1998

Leishmanicidal activity of aurones.

This is the first report on aurones as a new class of natural products with leishmanicidal activity. A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity test against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote L. donovani residing within murine macrophages. The compounds proved to be active at concentrations in the microgram range between 0.4 and 5.0 microg/ml. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds showed only moderate cytotoxicity (EC50 2.32-25.0 microg/ml).

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PubMed1997

A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines.

The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.

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PubMed1997

Leishmania major infection in C57BL/10 mice differing at the Lps locus: a new non-healing phenotype.

The course of cutaneous leishmaniasis was examined in mice from two genetically closely related strains, C57BL/10ScCr (Cr) and C57BL/10ScSn (Sn). Sn mice are able to heal Leishmania major infections, while Cr mice are unable to heal. The cutaneous lesions of the Cr mice progressed continuously and the increase in lesion size was paralleled by an unrestricted growth of the parasites in vivo. Cr mice, in contrast to their Sn counterparts, are highly resistant to all effects of lipopolysaccharide (LPS). The nonhealing L. major infection in Cr mice is in sharp contrast to the course of infection in another endotoxin-nonresponder mouse strain, C3H/HeJ, which heal infections with L. major. Cr mice exhibit, in addition to the defective LPS responsiveness, an impaired interferon-gamma (IFN-gamma) response after infection with a variety of microorganisms. The insufficient activation of parasitized macrophages to kill intracellular L. major could be due to the inability of splenocytes from infected Cr mice to secrete IFN-gamma upon restimulation with L. major. IFN-gamma is essential for the efficient activation of parasitized macrophages to kill intracellular L. major by producing nitric oxide (NO). Although bone marrow-derived Cr macrophage do not produce NO in response to LPS, both Sn and Cr macrophages release NO upon stimulation with IFN-gamma and tumor necrosis factor, indicating that they are responsive to activation by these cytokines.

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PubMed1991

Role of inorganic nitrogen oxides and tumor necrosis factor alpha in killing Leishmania donovani amastigotes in gamma interferon-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains.

The capacity of mature bone-marrow-derived macrophages and resident peritoneal macrophages from Lshr versus Lshs congenic mice to kill intracellular Leishmania donovani amastigotes when activated by recombinant gamma interferon-lipopolysaccharide (rIFN-gamma-LPS) was examined. IFN-gamma alone in doses up to 100 U/ml was unable to activate macrophages to kill L. donovani amastigotes in vitro; LPS was a necessary secondary stimulus. Similarly, LPS alone in doses up to 100 ng/ml produced no leishmanicidal activity. In bone marrow macrophages, a dose-dependent increase in leishmanicidal activity was observed as increasing rIFN-gamma-LPS dose combinations were introduced, with Lshr macrophages maintaining a significant but not dramatic advantage within any particular dose combination. For peritoneal macrophages, the reverse was true, with macrophages from Lshs mice being more efficient at killing for doses of LPS up to 10 ng/ml with doses of rIFN-gamma in the range of 11 to 33 U/ml. The degree of killing in both bone marrow and peritoneal macrophages correlated well with the levels of nitrites measured in the supernatants at 72 h, and a highly significant correlation was observed between 4-, 24-, or 72-h tumor necrosis factor alpha (TNF-alpha) release and nitrite production measured at 72 h. Inclusion of 200 microM NG-monomethyl-L-arginine, a competitive inhibitor of the L-arginine-dependent pathway for the synthesis of inorganic nitrogen oxides, inhibited the killing, as did the addition of neutralizing anti-TNF-alpha antibody. These results are consistent with previous data showing an important autocrine role for TNF-alpha in enhancing production of inorganic nitrogen oxides by primed or activated macrophages. In addition, our results suggest that production of TNF-alpha and nitrites after priming or activation signals may be under a different regulatory control in mature bone marrow macrophages than in the resident peritoneal macrophage population.

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PubMed1990

A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites.

An in vitro method is described which colorimetrically assesses the activation of macrophages for intracellular cytotoxicity against the obligate intracellular parasite Leishmania donovani. The assay system uses a highly purified macrophage population derived from 10-day murine bone marrow cultures. These were infected in vitro as a suspension culture with viable L. donovani amastigotes and then exposed to activating agents. After 48 h the intracellular parasites were released by SDS lysis of the macrophages. Surviving Leishmania organisms were quantitated by their conversion of the chromophore MTT. The sensitivity of this method was comparable with the established method of [3H]dThd incorporation. This assay system has been used to show that there is a dual signal requirement (recombinant interferon-gamma and bacterial endotoxin (LPS] for effective activation of macrophages for leishmanicidal activity.

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PubMed1988

Extracellular killing of Leishmania promastigotes and amastigotes by macrophage precursors derived from bone marrow cultures.

Flagellates of the genus Leishmania are obligate intracellular parasites of vertebrates including man. The microorganisms reside and multiply inside the phagolysosomes of cells of the mononuclear phagocyte lineage. We here report on the spontaneous leishmanicidal activity exerted extracellularly by immature cells of the mononuclear phagocyte lineage. Highly purified, bone marrow-derived macrophage precursor cells displayed a strong spontaneous leishmanicidal activity already at very low effector/target rations (3:1, 6:1). This leishmanicidal activity was effective against both promastigotes and amastigotes as targets. The cytotoxic effect was evident within 4 h and maximal after 12 h of effector-target organism cocultivation, as determined by a radiolabel-release assay. An intimate cell-cell contact seemed necessary for the parasites to be killed.

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PubMed1986

A fast and objective assay for cell mediated intra- and extracellular killing of Leishmania promastigotes.

An in vitro radiometric assay is described for the detection of cytolytic activity against the obligate intracellular parasite Leishmania enriettii. The assay system can be equally well applied to non-cellular (humoral), cellular non-phagocytic, and cellular phagocytic effector situations. Leishmania promastigote organisms are DNA-labeled with [methyl-3H]thymidine [( 3H]dThd) with high efficiency. Spontaneous label release remains very low even in non-ideal culture conditions. We have modeled a leishmanicidal situation by co-cultivating L. enriettii with starch-elicited murine peritoneal macrophages for different time periods. At an effector-to-target ratio of 1:6 a highly significant [3H]dThd release can be observed after 4 h of co-cultivation, reaching 60% after 18 h. The principle advantages of this assay system are speed, high sensitivity and objectivity. This makes it suitable for mass screening of, e.g., immunomodulatory or parasiticidal agents and equally useful in both phagocytic and non-phagocytic situations.

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PubMed1986

Functional heterogeneity of murine macrophage precursor cells from spleen and bone marrow.

We previously reported that highly purified bone marrow-derived macrophage precursors can exert strong spontaneous cytotoxicity against YAC-1 tumor cells, Candida albicans, and protozoa of the genus Leishmania. In the present paper, evidence is shown that macrophage precursors in normal untreated mice are not confined to the bone marrow compartment but can also be found in the spleen. These organ-associated cells, which have the same buoyant density as large granular lymphocytes, have been positively sorted by means of an indirect rosetting technique employing the macrophage-specific monoclonal antibodies F4/80 and M143. The rosetting fractions represented an extremely homogeneous population of macrophage precursors characterized by high candidacidal and natural killer activity and by a strong proliferative response to the macrophage-specific colony-stimulating factor CSF-1. Spleen- and bone marrow-derived macrophage precursors differed in their target selectivity. In addition, the mature macrophages derived in vitro from these two precursor populations displayed striking differences in their candidacidal activity. The implications of these findings in relationship to heterogeneity in the macrophage differentiation line are discussed.

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PubMed1985

Liver macrophages (Kupffer cells) as cytotoxic effector cells in extracellular and intracellular cytotoxicity.

The potential of the resident murine Kupffer cell to be cytotoxic in extra- and intracellular killing systems in vitro was investigated. Kupffer cells exerted no spontaneous cytotoxicity but were readily susceptible to activation with lymphokines. Such activated Kupffer cells very efficiently killed extracellular P815 cells and intracellular Leishmania spp. parasites. Kupffer cells could be induced to proliferate in vitro under the influence of colony-stimulating factor (D.-M. Chen, H.-S. Lin, P. Stahl, and R. Stanley, Exp. Cell Res. 121:103-109, 1979). Kupffer cell-derived macrophages cultured in vitro were identical to their liver-derived progenitors in terms of macrophage-specific surface antigens and with respect to extra- and intracellular cytotoxicity. These results demonstrated that Kupffer cells have a strong self-renewing potential and that essential Kupffer cell properties like antigenic determinants and cytotoxic potential remained stable throughout the replicative process. The possibility of Kupffer cell self-renewal in the intact organism is also discussed.

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PubMed1984

Protection of mice against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon.

Immune interferon, available at high specific activity through recombinant DNA technology, is known to activate macrophages to intra- and extracellular cytotoxicity. We now report that murine recombinant IFN-gamma activates macrophages to cytotoxicity also when applied in vivo. Furthermore, recombinant IFN-gamma can protect mice in vivo against the intracellular bacterial pathogen Listeria monocytogenes in a local as well as in a systemic infection model. The role of T lymphocyte-produced lymphokines in acquired resistance to facultative intracellular pathogens and their possible involvement in novel immunotherapy are discussed.

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PubMedDate not supplied

Tannins and related compounds: killing of amastigotes of Leishmania donovani and release of nitric oxide and tumour necrosis factor alpha in macrophages in vitro.

The antileishmanial and immunomodulatory potencies of a series of 28 polyphenols were evaluated in terms of extra- and intracellular leishmanicidal activity and macrophage activation for release of nitric oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)-like properties. For this, several functional bioassays were employed including an in vitro model for leishmaniasis in which murine bone marrow-derived macrophages (BMMphi) were infected with the obligate intracellular parasite Leishmania donovani, an extracellular Leishmania proliferation assay, a fibroblast-lysis assay (TNF-activity), and a biochemical assay for NO. Except for gallic acid, its methyl ester, shikimic acid and catechin (EC50 25.8-67.9 nM) all polyphenols tested significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.4-13.9 nM) when compared with the clinically used agent, sodium stibogluconate (EC50 10.6 nM). In contrast, none of the samples proved to be directly toxic for the extracellular promastigote form of the parasite. Noteworthy, the phenolic samples showed only moderate or no cytotoxicity against the murine host cells (EC50 10 to >144 nM). Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate ranging from 7-54 microM (IFN-gamma/LPS 119 microM). On the other hand, inhibition of NO production had no apparent effect on intracellular leishmanicidal activity of polyphenols. Their in vitro TNF-inducing potential producing 50% lysis in murine L929 cells increased in the order of simple phenols and flavanols (34-48 U/ml) < A-type proanthocyanidins (53-80 U/ml) < B-type proanthocyanidins (64-200 U/ml) < hydrolyzable tannins (287-350 U/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, gallic acid and some hydrolyzable tannins showed appreciable IFN-like activities (14-23 U/ml) as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L 929 cells. The results provide a rational basis for the recorded anti-infectious efficacy of traditionally used herbal medicines containing tannins in vivo, in the light of both only moderate direct antimicrobial activities of distinct polyphenols in vitro and the limited knowledge on their uptake in humans.

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