[In vitro toxicity and mutagenicity of CoCrMo and TiAl wear particles]. / In-vitro-Toxizität und Mutagenität von CoCrMound TiAl-Abriebpartikeln.

Contact of wear particles with body fluids can result in widespread dissemination of extractable constituents from joint implants. The aim of this in vitro study is to clarify whether there is a mutagenic and/or carcinogenic risk from Co(28)Cr(6)Mo and Ti(6)Al(4)V wear particles. Particles of a representative size were produced by fretting; toxicity and mutagenicity were investigated using the Ames Salmonella/microsome test and the V79-HGPRT Test (Chinese hamster fibroblasts). To obtain the greatest possible elution of all constituents, the metallic wear particles were extracted with DMSO and water and the resulting eluates mixed together. After repeated test series under standardized conditions, neither the bacterial nor the mammalian cell assays produced evidence of toxic or mutagenic effects in the concentration range under study. It is therefore not to be expected that CoCrMo or TiAl alloys initiate carcinogenesis in the human organism.

In vitro toxicity and mutagenicity of CoCrMo and Ti6Al wear particles.

Contact of wear particles with body fluids can result in widespread dissemination of extractable constituents from joint implants. The aim of this in vitro study is to clarify whether there is a mutagenic and/or carcinogenic risk from CoCrMo and Ti6Al wear particles. Particles of a representative size were produced by fretting; toxicity and mutagenicity were investigated using the salmonella/microsome test according to AMES and the V79-HGPRT Test (Chinese Hamster Fibroblasts). To obtain the greatest possible elution of all constituents the metallic wear particles were extracted with dimethylsulfoxyd and water and the resulting eluates mixed together. Neither the bacterial assay nor the mammalian cell system after repeated test series under standardised conditions produced evidence of a toxic or mutagenic effect in the concentration range under study. It is therefore not to be expected that CoCrMo or Ti6Al alloys initiate carcinogenesis in the human organism.

Development of an allergy test model: activation of human mast cells with potentially allergenic substances.

Due to the permanent increase of newly developed and already existing allergies, simple, quick, and reliable test models for detecting potentially allergenic substances are still required. Here, we describe the development of a new in vitro allergy test based on isolated primary mast cells (MC) of non-allergic patients from lung tissue and foreskin specimens, respectively. To establish the specificity of the test model we used primary MC stimulated with immunoglobulin E (IgE), human recombinant stem cell factor (hrSCF), and anti-IgE antibodies to release significant amounts of histamine indicating the ability of MC to cause a hypersensitivity reaction of the immediate type. The general applicability of this test model for detecting allergenic substances could be confirmed by histamine release of primary MC stimulated with sera of patients suffering from house dust allergy, and the corresponding antigen Dermatophagoides pteronyssinus. The results of the present work suggest that this newly developed human in vitro model provides the opportunity of testing substances for their allergenic potential within days and at low costs. This could also be of particular interest for newly produced compounds.

Double-blind randomised placebo-controlled phase III study of an E. coli extract plus 5-fluorouracil versus 5-fluorouracil in patients with advanced colorectal cancer.

The primary aim of this study was to evaluate the toxicity (mucositis, diarrhea and leucopenia) of a therapy with 5-fluorouracil (CAS 51-21-8; 5-FU) plus an E. coli extract (LC-Extract, Laves coli extract, Colibiogen inject, cell-free soluble fraction from lysed E. coli, Laves strain) in comparison with 5-FU plus placebo. Secondary endpoints included general toxicity, response rate according to WHO, survival time and quality of life. 164 patients with advanced colorectal cancer were enrolled in this randomised, placebo-controlled, double-blind, multicenter phase III study. The treatment consisted of 0.167 ml/kg/d LC-Extract or placebo followed by 500-750 mg/m2/d 5-FU on five consecutive days, repeated every three weeks for up to six treatment cycles. 158 (77 verum, 81 placebo) patients were evaluable for toxicity, 144 (72 verum, 72 placebo) evaluable for response. The therapy with LC-Extract was well tolerated. Adverse events that occurred during the study were mainly judged as 5-FU- or tumor-related. Toxicity from treatment with 600 mg/m2/d 5-FU in both treatment groups was very low. After treatment with 750 mg/m2/d 5-FU patients in the placebo-group experienced a higher CTC toxicity than in the LC-Extract groups. Remission rate and survival time showed a slight trend in favour of LC-Extract. These results suggest a positive benefit-risk ratio of the additional application of LC-Extract to 5-FU in the treatment of advanced colorectal cancer especially for administration of high doses of 5-FU.

Effects of crocidolite asbestos on human bronchoepithelial-dependent fibroblast stimulation in coculture: the role of IL-6 and GM-CSF.

Cocultures of human pulmonary epithelial cells (BEAS-2B) and lung fibroblasts (WISTAR-38), representing two cell types of central regulatory potential in (chronic) lung disease, were used as an in vitro model to study the role of interleukin 6 (IL-6) and of granulocyte macrophage-colony stimulating factor (GM-CSF) in early fibrogenesis. For this purpose, epithelial cells were pre-exposed to UICC crocidolite asbestos fibers or titanium dioxide (TiO2) particles for 96 h and subsequently cocultured with fibroblasts for additional 72 h. Gene expression of IL-6 or GM-CSF in both cell types as well as of alpha1 procollagens types I and III in fibroblasts was determined by RT-PCR. Synthesis of IL-6, GM-CSF or collagen I was quantified using IL-6 bioassay or ELISA tests, respectively. Both mediators were directly induced in bronchoepithelial cells by crocidolite but not by TiO2. Likewise, steady-state mRNA levels of procollagens as well as collagen synthesis were upregulated in cocultured fibroblasts. As a result of coculture, cytokine concentrations were synergistically enhanced and further increased by crocidolite in a dose-dependent manner. Suppression of cytokine induction by corresponding neutralizing antibodies consistently abrogated collagen enhancement. Direct stimulation of fibroblast monocultures with recombinant human IL-6 or GM-CSF significantly increased collagen synthesis and transcription in a dose-dependent manner. Thus, our results demonstrate that crocidolite selectively stimulated production of IL-6 and GM-CSF in bronchoepithelial cells. In epithelial-fibroblast interactions, these mediators appear to play a key role in regulating fibroblast activity, indicating a close correlation between these cytokines and the fibrogenic potential of particulates.

Combined liposomal immuno- and chemotherapy of visceral leishmaniasis.

Visceral leishmaniasis is a tropical disease caused by Leishmania donovani, an obligate intracellular parasite. Host cells of these parasites are macrophages. The conventional therapy of visceral leishmaniasis uses pentavalent antimony (Pentostam). To minimize the undesired side effects of antimony and to possibly reduce the therapeutical dose of antimony it was combined with interferon gamma (IFN gamma) and encapsulated both in multilamellar vesicles for treatment of murine visceral leishmaniasis. Using liposomes composed of one synthetic phospholipid and applying the freeze-thawing technique an encapsulation rate of about 30-70% of the offered antimony concentration was obtained. In the case of IFN gamma an entrapment of 20-30% was reached. Thus these liposomes were used to perform experiments in order to achieve pharmacological data about organ distribution of the encapsulated vs. free drug. For this purpose defined amounts of antimony were injected i.v. in B 10D2/n mice. At several time-points mice were sacrificed and lung, spleen, liver, kidneys and blood samples were assessed for antimony concentration. The results presented evidence that liposomal drugs were enriched in spleen and liver, organs mainly affected by visceral leishmaniasis. It has been shown previously that liposomes are phagocytozed by macrophages, the host cells of the parasites. Therefore treatment of L. donovani infected mice with liposomal antimony and IFN-gamma resulted in a nearly complete parasite reduction, while treatment with the free drug slightly reduced the parasite burden only in the liver. The aim of these studies was to minimize the undesired side effects of antimony and to possibly reduce the necessary dosage by 1 encapsulating it into multilamellar vesicles and 2, by combining the liposomal antimony with liposomal encapsulated IFN gamma, which is known to be a potent macrophage activating agent.

Augmentation of host defence against bacterial and fungal infections of mice pretreated with the non-pathogenic Escherichia coli strain Nissle 1917.

Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor) was investigated for its ability to enhance the immune response against bacterial or fungal infections in vivo. Mice were infected intravenously with either 6 x 10(3) colony forming units (cfu) of Listeria monocytogenes bacteria or 5 x 10(5) Candida albicans cells. One day prior to infection, mice were treated orally with four different concentrations of E. coli strain Nissle 1917 (10(6), 10(7), 10(8), and 10(9) viable cells). Three days after infection with L. monocytogenes or one day after infection with C. albicans, mice were sacrificed and the parasite burden of the main target organs of the respective infection model were examined. The protective effect of E. coli strain Nissle 1917, compared to placebo-treated controls and to mice treated with a dose of 10(4). Units interferon gamma, is shown as the reduction of viable bacteria in spleen and liver or viable fungi in the kidneys of infected animals, respectively. Orally administered E. coli strain Nissle 1917 reduced Listeria monocyto-genes and Candida albicans in a dose-dependent manner. Treatment with 10(9) cfu of E. coli bacteria led to a reduction of Listeria counts to 7.4% in spleen and 2.4% in liver. A more than 10-fold decrease of viable Candida albicans (residual parasitaemia 6.8%) in the kidneys of the infected animals was also achieved by this E. coli concentration. These results suggest that E. coli strain Nissle 1917 is a potent immunostimulator of bacterial origin with highly protective efficacy against pathogenic bacterial of fungal infections.

Treatment of an acute bacterial infection with a combination of acetylsalicylic acid/ibuprofen and interferon gamma.

The bacterial infection with Listeria monocytogenes is associated with an inhibition of the macrophage function, the first-line defense against bacterial infection. We studied the effect of acetylsalicylic acid (ASA, CAS 50-78-2) and ibuprofen (CAS 15687-21-1) alone and in combination with a suboptimal dose of recombinant interferon gamma. (IFN gamma) on the acute infection with Listeria monocytogenes in the Balb/c mouse. Animals were intravenously infected with a sublethal dose of Listeria monocytogenes. The therapy was carried out I) at the time of the infection, II) 30 min, III) 60 min, IV) 3 h and V) 24 h post infection. Six groups of mice were treated: i) untreated control, ii) 10(4) units IFN gamma, iii) 10 mg/kg ASA, i.v.) 10 mg/kg ASA + IFN gamma, i.v.) 12 mg/kg ibuprofen, and vi) 12 mg/kg ibuprofen + IFN gamma. The data shown that treatment with ibuprofen and ASA resulted in a significant reduction of viable bacteria in spleen and liver, the main organs of this infection. In combination with low dose interferon gamma, both non-steroidal anti-inflammatory drugs (NSAID) reduced the parasite burden in the examined organs by a factor of more than 10. The therapeutic efficacy showed its maximum 1 h after challenge with Listeria monocytogenes. These results suggest that ibuprofen and ASA possess antibacterial activity. In addition, IFN gamma significantly increases the antibacterial activity of ASA and ibuprofen. Presumably, these effects are due to an influence on the host immune system.

Influence of ibuprofen on the infection with Listeria monocytogenes.

Listeria monocytogenes is a bacterial infection, which is facultatively localized in monocytes and macrophages. The influence of ibuprofen (CAS 15687-27-1), a nonsteroidal anti-inflammatory drug (NSAID), on this bacterial infection in balb/c mice was investigated. One day prior to sublethal infection, balb/c mice were treated intravenously with various therapeutic concentrations of ibuprofen alone or ibuprofen in combination with a suboptimal dosage of murine recombinant interferon gamma, a lymphokine produced by T-helper cells. Three days post-infection, parasite burdens of the mainly infected organs, spleen and liver, were determined by the colony-forming unit assay. It was shown that the prophylactic treatment with ibuprofen in a concentration of 4 mg/kg body weight resulted in a more than 10-fold reduction of viable Listeria monocytogenes in the spleen, whereas in liver 12 mg/kg Ibuprofen was necessary for a comparable kill of viable bacteria. A higher concentration of ibuprofen did not resulted in a higher antibacterial efficacy. In order to clarify the mechanism of ibuprofen action, molecular-biological experiments were performed to measure the messenger RNA (mRNA) induced by ibuprofen. It is presented here that therapeutic concentrations of ibuprofen induced significant higher amounts of mRNA for interleukin-1 in human monocytes compared to untreated cells. These findings support the hypothesis that ibuprofen influences the complex immune system to overcome a bacterial infection.

Interaction of alveolar macrophages and respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract illness in infants. However, the mechanisms leading to resolution of RSV infections are poorly understood. Since alveolar macrophages play an important role in defending the respiratory tract against infectious agents we investigated the interactions of RSV with these cells. Murine alveolar macrophages were challenged in vitro with RSV at different multiplicities of infection. The percentage of macrophages expressing viral antigen was determined by staining with monoclonal anti-RSV antibodies and evaluation by fluorescence microscopy or FACS analysis. The ability of macrophages to support virus replication was measured by a plaque forming assay on HEp-2 cells. Cell lysates of macrophages contained only small amounts of viable RSV in comparison to disrupted HEp-2 cells. The amount of viable RSV as well as the percentage of macrophages expressing viral antigen decreased rapidly over time. Activated macrophages had a reduced virus load in comparison to resting macrophages. RSV infected macrophages released biologically active tumor necrosis factor (TNF) in a virus dose dependent manner. In contrast, a high virus inoculum resulted in reduced microbicidal activity and oxygen radical production. Our results suggest that RSV infection influences different functions of alveolar macrophages in various ways. Since TNF is thought to restrict viral replication in several cell types it may play a role in limiting virus replication.

Acute effects of smoking and high experimental exposure to environmental tobacco smoke (ETS) on the immune system.

Controversial results have been published on the immune response to cigarette smoking while the effects of exposure to environmental tobacco smoke (ETS) have not yet been reported. In a controlled study, acute effects of smoking and of a high environmental exposure to ETS on immunological parameters have been investigated. The study consisted of four experimental days, two control and two exposure days. On control days, 1 and 3, smokers (n = 5) and nonsmokers (n = 5) sat in an unventilated 45 m3 room for 8 h. On the exposure days, 2 and 4, each of the smokers smoked 24 cigarettes in 8 h, while the nonsmokers were exposed to the ETS generated by the smoking volunteers. Blood was drawn before and after each exposure session on all four experimental days for dosimetry of tobacco smoke exposure and determination of the immune response. Flow cytometry using monoclonal antibodies was used to determine CD3+ cells (whole T cells), CD19+ cells (B lymphocytes), CD16+ and CD56+ cells (natural killer cells), CD4+ cells (T-helper cells), CD8+ cells (T-suppressor cells), the CD4+/CD8+ (helper/suppressor ratio), and Fc receptors on granulocytes. Serum was analyzed for soluble CD14 receptors (sCD14), interleukin 1, interleukin 6 and prostaglandin E2 (PGE2). Functional stimulation assays were performed to determine the basal and induced level of reactive oxygen intermediate (ROI) production by polymorphic neutrophils. Exposure to tobacco smoke in both groups was confirmed by dosimetry of carboxyhemoglobin, plasma nicotine, and cotinine levels. In comparison to nonsmokers, smokers had elevated granulocyte cell counts, increased CD16+ and CD56+ cell levels and decreased CD3+ and CD19+ levels. Acute smoking, but not exposure to ETS, resulted in a slight decrease in the number of CD19+ cells and an increase in the number of granulocytes; the latter was restricted to one subject. Acute smoking and exposure to high experimental concentrations of ETS resulted in a slight increase in CD16+ and CD56+ cells. None of the changes determined in immunological parameters after either acute smoking or exposure to ETS reached statistical significance. Serum sCD14, cytokine and PGE2, functional stimulation of in vitro ROI production, and changes in Fc receptors were not affected by acute smoking or exposure to ETS. Although no clear guidelines exist to assess immunotoxicity in man, our data do not favor immunosuppression and the possibility of increased risk of infection in nonsmokers exposed to ETS under real-life conditions.

Influence of acetylsalicylic acid on a Listeria monocytogenes infection.

The influence of acetylsalicylic acid (ASA, CAS 50-78-2) on the Listeria monocytogenes infection in balb/c mice was investigated. One day prior to lethal or sublethal infection, balb/c mice were treated intravenously with therapeutic concentrations of ASA alone or ASA in combination with murine recombinant interferon gamma, a lymphokine produced by T-helper cells. Three days post-infection, parasite burdens of spleen and liver were determined by the colony-forming unit assay. It was shown that the prophylactic application of ASA in a concentration of 5 mg/kg body weight resulted in a more than 10-fold reduction of viable Listeria monocytogenes in spleen and liver of balb/c mice. In addition, the combination of a suboptimal dosage of interferon gamma with ASA resulted in a significantly higher survival rate compared to the untreated controls.

Effect of acetylsalicylic acid, ascorbate and ibuprofen on the macrophage system.

The influence of ascorbic acid (CAS 50-81-7), acetylsalicylic acid (CAS 50-78-2) and ibuprofen (CAS 15687-27-1) on macrophages of C57BL/6 mice was investigated in vitro. It has been shown that ascorbic acid or acetylsalicylic acid alone did not stimulate or inhibit the production of interleukin-6, whereas a combination of both substances caused a significant stimulation. The viral replication in L929 fibroblasts was not affected by ascorbate and/or acetylsalicylic acid. In addition, the tumor-necrosis factor (TNF) synthesis of peritoneal macrophages was neither stimulated nor inhibited by both substances, alone or in combination. The oxygen radical production, however, was definitely inhibited by ascorbic acid, the effect of acetylsalicylic acid was far less marked, but at the high concentrations the inhibition was clearly discernible. Ibuprofen, a propionic acid derivate, was able to reduce the replication of vesicular stomatitis virus in L929 fibroblast cells. At the highest concentration of ibuprofen, 100 micrograms/ml, 34% of the fibroblast were able to survive. This protective effect declined as the ibuprofen concentration decreased. Ibuprofen could not stimulate peritoneal macrophages to secrete TNF, whereas the oxygen radical production was significantly reduced. In addition, ibuprofen activated mouse macrophages to produce interleukin-6 in a dose dependent way. The results of the in vitro experiments presented clearly show that ascorbic acid, acetylsalicylic acid in ibuprofen influenced the unspecific immune system.

Staphylococci surviving intracellularly in phagocytes from patients suffering from chronic granulomatous disease are killed in vitro by antibiotics encapsulated in liposomes.

Granulocytes and monocytes/macrophages from patients suffering from chronic granulomatous disease (CGD) are ineffective in killing specific kinds of phagocytized bacteria, e.g., Staphylococcus aureus, due to decreased or lacking ability to produce reactive oxygen intermediates. Commonly used antibiotics like flucloxacillin are of limited therapeutic value, because the staphylococci are protected against their action in the interior of phagocytes. However, encapsulation of flucloxacillin into liposomes could enable its entrance into the interior of neutrophils from two CGD patients to kill phagocytized bacteria there. The effect of rifampicin against intracellular staphylococci could be similarly enhanced by liposome encapsulation. Dose-response relations and kinetics of killing of intracellular bacteria by antibiotics in the free and encapsulated form were studied under different conditions using J 774 mouse macrophages, because phagocytes from CGD patients are not available in great amounts. Preincubation of phagocytes with either antibiotic in liposomes subsequently endowed the cells with a strongly enhanced ability to kill phagocytized bacteria. Our data show that a drug which normally will not reach a phagosome can be delivered to this intracellular compartment by a liposome. A possible clinical use is discussed.

[Immunomodulating effect of killed, apathogenic Escherichia coli, strain Nissle 1917, on the macrophage system]. / Immunmodulierende Wirkung von abgetöteten apathogenen Escherichia coli, Stamm Nissle 1917, auf das Makrophagensystem.

The influence of formaldehyde-killed Escherichia coli strain Nissle 1917 (SK 22) on macrophages of C57BL/6 mice was investigated in vitro. It has been shown that SK 22 activated macrophages derived from bone marrow produced Interleukin-6 with high efficiency. In addition, SK 22 stimulated macrophages to secrete tumor necrosis factor, as measured by a bioassay. Furthermore, macrophages were activated by SK 22 to produce a 3 fold amount of oxygen radicals compared to the spontaneous oxygen radical production. In contrast to this finding, the phagocytic capacity of these macrophages was only slightly increased. The specific lysis of P 815 tumor cells by peritoneal macrophages after coincubation with SK 22 was measured using tumor cells prelabelled with radioactive 51Cr. The results of the in vitro experiments presented clearly show that the E. coli preparation SK 22 is an efficient immunomodulator of the unspecific immune system.

Immunotherapy of murine visceral leishmaniasis with murine recombinant interferon-gamma and MTP-PE encapsulated in liposomes.

The efficiency of immunotherapy with murine recombinant interferon-gamma (rIFN-gamma) in mouse visceral leishmaniasis caused by Leishmania donovani was examined. To avoid the side effects encountered after the in vivo administration of high dosages of free IFN-gamma, this lymphokine and muramyltripeptide (MTP-PE) were encapsulated into multilamellar liposomes. We established that a combination of 5 X 10(3) U of IFN-gamma and 6 micrograms of MTP-PE, encapsulated in liposomes and given i.v. in C56BL/6 and BALB/c mice activates macrophages from spleen and liver in vivo to kill L. donovani in vitro. Neither empty liposomes nor the same concentration of free IFN-gamma and/or MTP-PE injected i.v. resulted in a leishmanicidal activity of these macrophage populations. For verification of these results in an in vivo infection model, susceptible mice were infected with L. donovani and were treated with IFN-gamma and MTP-PE encapsulated in multilamellar vesicles. Treatment consisted of multiple i.v. injections beginning 4 and 2 days before infection (prophylactic), either simultaneously with the infection or at various times of the exacerbation and remission phases of visceral leishmaniasis. These mouse strains treated with IFN-gamma and MTP-PE in liposomes had significantly fewer splenic parasites than untreated mice or control animals treated with free drugs or empty liposomes. The targetting of multilamellar vesicles to liver and spleen make them particularly suited for the delivery of macrophage-activating substances used for treatment of visceral L. donovani infection.

Involvement of membrane-associated TNF in the killing of Leishmania donovani parasites by macrophages.

A killing process is described where macrophages destroy 3H Thymidine prelabeled Leishmania parasites during the process of phagocytosis. This phagocytosis associated killing may represent a first line of defense and reduces drastically the amount of parasites homing in the macrophages. Treatment of the macrophages with anti-TNF antibody inhibited the phagocytosis associated killing. Soluble TNF had no effect on Leishmania parasites. Macrophages performed the killing process in the complete absence of any secreted TNF. Experiments using fixed macrophages and macrophage membranes revealed the existence of a 26 Kd membrane-TNF molecule. This membrane TNF was active against a TNF sensitive tumor cell line whereas the fixed cells or the isolated membranes had no cytotoxic effect on Leishmania parasites. This data indicates, that the Leishmanicidal effect was mediated through TNF but that apparently TNF itself was not the cytotoxic principle. Experiments using organ macrophages from Leishmania infected susceptible C 57/Bl 6 healer mice demonstrated that during the first 4 weeks of the infection the organ macrophages were totally unable to destroy Leishmania parasites independent of additional activation whereas the mechanism to destroy P815 tumor cells was unaffected. Similar data were obtained in therapy protocols using interferon gamma encapsulated in liposomes in order to target the substance to the macrophages. Also under these in vivo conditions the organ macrophages could not be activated and no beneficial effect was obtained by this activation treatment. In contrast, when therapy was started early (day 0 or 1) or late (5 weeks) after infection a highly significant reduction of the parasite burden was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunomodulating effect of lysed immunoactive fractions of selected Escherichia coli strains on the macrophage system. An in vitro study]. / Immunmodulierende Wirkung von lysierten, immunaktiven Fraktionen aus ausgewählten Escherichia-coli-Stämmen auf das Makrophagensystem. Eine In-vitro-Studie.

The immunomodulator Uro-Vaxom (an immunoactive fraction of E. coli, FEC) is a therapeutic agent used to control bacterial infections. FEC was applied to macrophages of C57BL/6 mice to investigate the in vitro activation of these cells of the unspecific immune system. Experiments were designed to test the secretory, immuno-regulatory and cytotoxic function of macrophages after application of FEC. As presented here, production of Interleukin-6 and tumor-necrosis-factor were significantly and dose-dependent way increased, whereas it was not possible to induce a secretion of Interleukin-1. In addition, FEC activated macrophages kill Leishmania donovani promastigotes, Candida albicans and Staphylococcus aureus. In comparison to a pressed echinaceae-preparation, FEC activated mouse macrophages secrete Interleukin-6 and tumor-necrosis-factor and kill protozoa, fungi and bacteria, with higher efficiency.

Mouse interferon-gamma in liposomes: pharmacokinetics, organ-distribution, and activation of spleen and liver macrophages in vivo.

Recombinant mouse interferon-gamma (IFN-gamma) was encapsulated into multilamellar vesicles and the proportion of encapsulated IFN-gamma determined by biological activity was 19%. The distribution of 125I-labeled IFN-gamma liposomes in C57BL/6 mice was analyzed. After an initial enrichment of liposomes in lung, more than 60% of total 125I-labeled IFN-gamma was accumulated in spleen and liver. Furthermore, it was observed if the encapsulation of IFN-gamma in liposomes prevented the rapid decay of IFN-gamma in serum of C57BL/6 mice after intravenous injection. We compared the serum decay curve of liposomal and free IFN-gamma, and showed that IFN-gamma encapsulated in liposomes has an elongated availability in the serum. In addition, we established that a combination of 10(2) U/ml IFN-gamma and 1 microgram/ml MTP-PE, encapsulated in liposomes, activates splenic and starch-elicited peritoneal macrophages in vitro synergistically to kill Leishmania donovani promastigotes. After intravenous injection of liposomal IFN-gamma (5 X 10(3) U) and muramyltripeptide (MTP-PE) (6 micrograms) in C57BL/6 mice, splenic and liver macrophages were activated in vivo to kill Leishmania species in vitro. Neither an injection of the same amount of free substances nor injection of empty liposomes resulted in an increased leishmanicidal activity.

Susceptibility of 3H-thymidine-prelabelled Leishmania donovani amastigotes and promastigotes to the cytotoxic activity of peritoneal, splenic and liver macrophages.

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.