Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice.

Heated (20 min at 70 degrees C) amphotericin B-desoxycholate (hAMB-DOC) was further characterized, as was another formulation obtained after centrifugation (60 min, 3000 x g), hcAMB-DOC. Conventional AMB-DOC consisted of individual micelles (approximately 4 nm in diameter) and threadlike aggregated micelles, as revealed by cryo-transmission electron microscopy. For both hAMB-DOC and hcAMB-DOC, pleiomorphic cobweb structures were observed with a mean particle size of approximately 300 nm as determined by laser diffraction. The potent antifungal activity of AMB-DOC against Candida albicans is not reduced by heating. Effective killing of C. albicans (>99.9% within 6 h) was obtained at 0.1 mg/liter with each of the AMB formulations. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) from C. albicans of > or =50% was observed at 0.8, 0.4, and 0.4 mg/liter, respectively. After heating of AMB-DOC, toxicity was reduced 16-fold as determined by red blood cell (RBC) lysis. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, hemolysis of > or =50% was observed at 6.4, 102.4, and 102.4 mg/liter, respectively. In contrast, AMB-DOC and its derivates showed similar toxicities in terms of cation release from RBC. For AMB-DOC, hAMB-DOC, and hcAMB-DOC, cation release ((86)Rb(+)) of > or =50% was observed at 1.6, 0.8, and 0.8 mg/liter, respectively. In persistently leukopenic mice with severe invasive candidiasis, higher dosages of both hAMB-DOC and hcAMB-DOC were tolerated than those of conventional AMB-DOC (3 versus 0.8 mg/kg of body weight, respectively), resulting in significantly improved therapeutic efficacy. In conclusion, this new approach of heating AMB-DOC may be of great value for further optimizing the treatment of severe fungal infections.

Differential nitric oxide and TNF-alpha production of murine Kupffer cell subfractions upon priming with IFN-gamma and TNF-alpha.

AIMS/BACKGROUND: We have previously shown a striking heterogeneity of naive murine Kupffer cells (KC) that depends on cell size. METHODS: In the present study, we demonstrate a shift in response of KC fractions separated on cell size by countercurrent elutriation upon priming with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). RESULTS: Whereas unprimed large KC are most active in the production of TNF-alpha and nitric oxide (NO), after priming of KC with TNF-alpha predominantly small and intermediate sized KC produce TNF-alpha in response to bacteria. Priming with IFN-gamma enhanced NO production in all KC. A strong synergy, with respect to production of NO, was observed when KC subfractions were exposed to a combination of TNF-alpha and IFN-gamma. Concerning TNF-alpha production, priming of KC subfractions seemed to induce a shift of activity from large KC to smaller KC. CONCLUSIONS: The present data demonstrate a clear heterogeneity among murine KC with respect to immunologic response to stimuli. These results demonstrate that KC have different functions in immunologic reactions that seem to be related to size.

Bone marrow cellular composition in Listeria monocytogenes infected mice detected using ER-MP12 and ER-MP20 antibodies: a flow cytometric alternative to differential counting.

Detailed assessment of bone marrow cellular composition is essential in the evaluation of various experimental in vivo systems, such as expression of transgenes, null mutations and stimulation of host defence in infection. Traditional morphological analysis of mouse bone marrow is laborious, requires specific cytological expertise, and is somewhat subjective. As an alternative, we have examined whether double labelling of bone marrow with the anti-precursor monoclonal antibodies ER-MP12 and ER-MP20 could be used for differential analysis by flow cytometry, as these antibodies define six relatively homogeneous cell populations in mouse bone marrow. Following a sublethal infection of mice with Listeria monocytogenes, we monitored changes in cellular composition of the bone marrow at various time points in three ways: differential morphological count; single-color flow cytometric analysis using markers for the myeloid, erythroid and lymphoid lineages; and double labelling with ER-MP12 and ER-MP20. As expected, the bone marrow composition changed dramatically during infection, leading to an increase of myeloid cells which peaked after 1 week of infection. Data determined by ER-MP12/20 flow cytometric analysis appeared to be in close agreement with both morphology and lineage marker analysis. In addition, ER-MP12/20 analysis provided more detailed information with regards to the presence of early myeloid precursors compared to lineage marker analysis. These data show that flow cytometric analysis of bone marrow using ER-MP12 and ER-MP20 monoclonal antibodies provides a relatively simple, rapid and objective assay when evaluating cellular composition in the bone marrow of the mouse.

Superior efficacy of liposomal amphotericin B with prolonged circulation in blood in the treatment of severe candidiasis in leukopenic mice.

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg . day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.

Activity of liposomal amphotericin B with prolonged circulation in blood versus those of AmBisome and fungizone against intracellular Candida albicans in murine peritoneal macrophages.

Activity against intracellular Candida albicans was assessed in C. albicans-infected murine peritoneal macrophages exposed to long-circulating pegylated amphotericin B liposomes (PEG-AMB-LIP), AmBisome, or Fungizone. The level of antifungal activity of Fungizone is much higher than that of AmBisome or PEG-AMB-LIP, while PEG-AMB-LIP and AmBisome show equivalent activity levels. Previous exposure of uninfected macrophages to PEG-AMB-LIP or AmBisome is advantageous for intracellular antifungal activity.

Involvement of T cells in enhanced resistance to Klebsiella pneumoniae septicemia in mice treated with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine or gamma interferon.

We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-gamma) results in strongly increased survival of mice from a normally lethal septicemia with Klebsiella pneumoniae. It was anticipated that the treatment acts on macrophages and nonspecifically augments host resistance to various infections. In the present study, we provide evidence for a key role for T cells in host defense potentiation by the liposomal immunomodulators toward K. pneumoniae septicemia. It is shown that both CD4 and CD8 cells are important in immunomodulation, most likely due to production of IFN-gamma. Depletion of circulating IFN-gamma resulted in strong reduction of the antimicrobial host defense activation. Administration of interleukin-10 resulted in decreased antimicrobial host defense activation by liposomal immunomodulators. Moreover, administration of liposomal immunomodulators was shown to induce predominantly T-helper type 1 (Th1) cell populations in the spleen. These findings indicate that immunomodulation with liposomal MTPPE and IFN-gamma favors Th1 and NK cell activation.

Isolation and characterization of murine Kupffer cells and splenic macrophages.

A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 x 10(6) /g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages based on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-alpha (TNF-alpha) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.

Enhancement of nonspecific resistance by liposome-encapsulated immunomodulators does not affect skin graft rejection in mice.

Administration of liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidyl ethanolamine (LE-MTPPE) or interferon-gamma (LE-IFN-gamma), or co-encapsulated MTPPE and IFN-gamma (LE-(MTPPE/IFN-gamma)) resulted in a dramatic increase of the nonspecific antimicrobial resistance in mice, as shown before. This kind of treatment is especially of use in immunocompromised hosts who are prone to severe infections. Application of these immunomodulators might protect these patients, e.g., transplant recipients, from opportunistic infections. However, accelerated rejection of the graft, resulting from augmentation of the antimicrobial defense in a nonspecific way, has to be avoided. In this study, the effect of treatment with LE-MT-PPE, LE-IFN-gamma, or LE-(MTPPE/IFN-gamma) on skin graft rejection in mice was investigated. It was found that prophylactic treatment of skin-grafted mice with immunomodulating formulations did not influence rejection of the graft. Moreover, in T cell-depleted mice, which showed a prolonged graft survival compared with immunocompetent recipients, the administration of immunomodulators did not change the survival time of the grafts compared with T cell-depleted mice that did not receive immunomodulators. The results clearly show that, in this experimental setting, application of the antimicrobial resistance-enhancing formulations (LE-MTPPE, LE-IFN-gamma, and LE-(MTPPE/IFN-gamma)) is allowed in graft-bearing recipients, without influencing graft survival.

Biodistribution of liposomal amphotericin B (AmBisome) and amphotericin B-desoxycholate (Fungizone) in uninfected immunocompetent mice and leucopenic mice infected with Candida albicans.

The biodistribution of liposomal amphotericin B (L-AmB; AmBisome) and amphotericin B-desoxycholate were compared after a single injection of drug in uninfected immunocompetent mice and in leucopenic mice 6 h after inoculation with Candida albicans. Amphotericin B-desoxycholate was administered at the maximum tolerated dose (MTD) of 0.3 mg/kg whereas L-AmB was given at either 0.3 mg/kg or the MTD of 7 mg/kg. Amphotericin B (AmB) concentrations in the blood, liver, spleen, lungs and kidneys were determined by HPLC analysis at various intervals during the 48 h after administration. The biodistribution of both preparations of AmB followed similar patterns in both uninfected immunocompetent mice as well as those that were leucopenic and infected with C. albicans. Administration of L-AmB resulted in increased concentrations of drug in the blood, liver, and spleen but decreased concentrations in the kidney and lung. Hepatosplenic uptake of L-AmB was highly dose dependent with 7 mg/kg resulting in a relatively prolonged blood circulation. Blood and tissues retained high AmB concentrations after administration of L-AmB at the MTD. By using radiolabelled L-AmB, it was found that the high AmB concentrations in blood represented liposome-associated AmB and that during circulation in blood slow release of AmB occurred.

Modulation of nonspecific antimicrobial resistance of mice to Klebsiella pneumoniae septicemia by liposome-encapsulated muramyl tripeptide phosphatidylethanolamine and interferon-gamma alone or combined.

Activation of the host defense system in a nonspecific way might provide tools to support failing antibiotic treatment in certain infectious diseases. The antimicrobial effect was investigated of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (MTPPE) and interferon (IFN)-gamma and liposome-coencapsulated MTPPE and IFN-gamma on Klebsiella pneumoniae septicemia in mice. Prophylactic treatment of mice with five doses of liposomal MTPPE or IFN-gamma increased survival from 0 to 65%. Administration of MTPPE and IFN-gamma coencapsulated in liposome resulted in 100% survival. In vitro, peritoneal macrophages by themselves were stimulated by these agents but were unable to kill K. pneumoniae. However, production of both oxygen and nitrogen intermediates increased when immunomodulators were added to macrophages. These results indicate that effective prophylactic treatment of septicemia due to K. pneumoniae with coencapsulated MTPPE and IFN-gamma is not solely due to activation of the resident macrophages.

Treatment of Klebsiella pneumoniae septicemia in normal and leukopenic mice by liposome-encapsulated muramyl tripeptide phosphatidylethanolamide.

The effect of free muramyl tripeptide phosphatidylethanolamide (MTPPE) and liposome-encapsulated MTPPE (LE-MTPPE) on Klebsiella pneumoniae septicemia resulting from intraperitoneal bacterial inoculation was investigated in mice. When administering a single prophylactic dose at 24 h before bacterial inoculation, the percentage survival was 55% (MTPPE) or 40% (LE-MTPPE), whereas untreated control mice died. Only repeated prophylactic treatment with LE-MTPPE could further increase survival up to 85%.

Free versus liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) and interferon-y (IFN-y) in experimental infection with Listeria monocytogenes.

The effect of free and liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) and interferon-y (IFN-y) on the resistance against Listeria monocytogenes infection in mice was investigated. It was shown that administration of MTPPE or IFN-y at 24 h before bacterial inoculation led to increased resistance against L. monocytogenes infection in terms of a decrease in bacterial numbers in liver and spleen. Encapsulation of MTPPE and IFN-y in liposomes increased their efficacy 33- or 66-fold, respectively. In addition, liposomal encapsulation led to a more rapid decrease in bacterial numbers. The immunomodulator to lipid ratio appeared to be very important in the antibacterial effect of LE-MTPPE and LE-IFN-y. When nontherapeutic doses of liposome-encapsulated MTPPE or IFN-y were administered in a larger amount of lipid (so at higher lipid: immunomodulator ratio), these doses became effective. Exposure of macrophages in monolayer infected with L. monocytogenes in vitro to MTPPE had no effect, whereas exposure to IFN-y only led to growth inhibition of the intracellular bacteria. However, incubation of macrophages with a combination of MTPPE and IFN-y resulted in killing of the intracellular bacteria. Exposure of macrophages in vivo to both immunomodulators in combination can be effected by using liposomes as carriers. It was observed that administration of MTPPE and IFN-y co-encapsulated in liposomes resulted in a synergistic enhanced antibacterial resistance against L. monocytogenes. Both reactive oxygen and nitrogen intermediates seemed to play a role in the killing of L. monocytogenes by macrophages activated with a combination of MTPPE and IFN-y.

Tissue distribution and cellular distribution of liposomes encapsulating muramyltripeptide phosphatidyl ethanolamide. Tissue and cellular distribution of LE-MTPPE.

In previous studies it was shown that administration of liposome-encapsulated MTPPE (LE-MTPPE) led to resistance against Klebsiella pneumoniae infection. To get more insight in the cell types that are involved in this by LE-MTPPE induced antibacterial resistance, the tissue distribution of liposomes encapsulating MTPPE and the distribution over the cells in the main target organs were investigated. After intravenous injection of the liposomes in mice a substantial amount was recovered from liver and spleen and a smaller amount from the lung. In the liver 83% of the liposomes was taken up by the macrophages. In the spleen also most liposomes were taken up by macrophages of the red and white pulp as well as by dendrocytes. The liver and spleen were also the organs in which, after intravenous inoculation, K. pneumoniae was trapped. It was observed that cells containing LE-MTPPE often had not taken up bacteria. Most bacteria, about 73%, were found in cells not containing liposomes. The capacity of the liposome-containing cells to take up bacteria did not change with time. This suggests that the by LE-MTPPE immunostimulating effect is due to the production of cytokines by the cells that take up LE-MTPPE. These cytokines might stimulate other cells to the killing of bacteria.

Liposomes as carriers of antimicrobial agents or immunomodulatory agents in the treatment of infections.

Targeting of antimicrobial agents by means of liposomes is under investigation and may be of importance in the treatment of infections that prove refractory to conventional forms of antimicrobial treatment. The ability to achieve a significantly longer residence time of liposomes in plasma and limited uptake of liposomes by the mononuclear phagocyte system opens up new areas of investigation and potential therapeutic application. By manipulating the liposomal composition, rates of uptake and intracellular degradation can be influenced and thereby the rates at which liposome-encapsulated agents are released and become available to exert their therapeutic action. With respect to the targeting of macrophage modulators at the mononuclear phagocyte system by means of liposomes for maximal stimulation of the nonspecific antimicrobial resistance, experimental evidence is now available of the potential usefulness of liposomes as carriers of these agents. This approach may also be of importance for the potentiation of treatment of severe infections.

Roles of peripheral leukocytes and tissue macrophages in antibacterial resistance induced by free or liposome-encapsulated muramyl tripeptide phosphatidylethanolamide.

Administration of free muramyl tripeptide phosphatidylethanolamide (MTPPE) or liposome-encapsulated MTPPE (LE-MTPPE) in a twofold-lower dose at 24 h before bacterial inoculation resulted in clearance of intravenously inoculated Klebsiella pneumoniae by tissue macrophages, whereas in control mice, bacteria were not effectively cleared from the blood. In addition, MTPPE and LE-MTPPE led to increased numbers of leukocytes in the blood, which could compensate for the leukopenia in mice resulting from infection with K. pneumoniae. In an attempt to elucidate the relative contributions of the activation of tissue macrophages and the recruitment of leukocytes to the antibacterial resistance induced by MTPPE and LE-MTPPE, mice were infected intraperitoneally with K. pneumoniae. In these MTPPE- and LE-MTPPE treated mice, intraperitoneal influx of leukocytes and the phagocytic capacity of leukocytes were not higher than in untreated control mice. However, MTPPE- and LE-MTPPE-treated mice survived much longer; eventually 33% of the LE-MTPPE-treated mice survived, whereas all untreated control mice died as a result of bacterial septicemia. This prevention of early death appeared to be the result of an increased clearance of bacteria from the blood by activated tissue macrophages. It was observed that depletion of these tissue macrophages in liver and spleen abrogates the effect of LE-MTPPE treatment, indicating that tissue macrophages are of major importance in the LE-MTPPE-induced resistance against K. pneumoniae infection.

Free versus liposome-encapsulated muramyl tripeptide phosphatidylethanolamide in treatment of experimental Klebsiella pneumoniae infection.

The effect of free and liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) on resistance to Klebsiella pneumoniae infection in mice was investigated. It was shown that administration of MTPPE, at 24 h before bacterial inoculation, led to a dose-dependent antibacterial resistance in terms of increased clearance of bacteria from the blood and bacterial killing in various organs. The lowest effective dose of MTPPE was 50 micrograms per mouse. Administration of liposome-encapsulated MTPPE was also effective at a dose of 25 micrograms per mouse. The time of administration of both free and liposome-encapsulated MTPPE, with respect to the appearance of bacteria in the blood, was very important and indicated that repeated administration is necessary to obtain protection for a prolonged period. In view of the toxicity of MTPPE, it was an important observation that repeated administration of MTPPE in the liposome-encapsulated form also produced antibacterial resistance. Administration of free and liposome-encapsulated MTPPE resulted in increased numbers of granulocytes, monocytes, and lymphocytes in the blood of uninfected mice and prevented leukopenia in infected mice.

Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in The Netherlands.

During a two-year period data were collected nationwide in The Netherlands on 438 episodes of bacterial endocarditis (BE) in 432 patients. Of the strains isolated in these patients 419 were available for analysis. Of these, 326 were isolated in native valve endocarditis (NVE) and 93 in prosthetic valve endocarditis (PVE). Viridans streptococci, staphylococci and enterococci together constituted 87% of the isolates. More than 46% of the viridans streptococci consisted of Streptococcus sanguis. Enterococcus faecalis and Staphylococcus aureus were the predominant species in the late form of PVE. The majority of the viridans streptococci and haemolytic streptococci were highly susceptible to penicillin. Five of 35 strains of coagulase negative staphylococci were resistant to methicillin. Eleven percent of a random sample of the streptococci collected were tolerant to penicillin. After repeated exposure to a concentration gradient of an appropriate beta-lactam antibiotic, this figure increased to 49%. Of the staphylococci, 5-6% of the strains were tolerant before induction and 16-20% after induction. Of the Enterococcus strains (n = 40), 12.5% showed high-level resistance to one or more aminoglycoside.

Typing of Acinetobacter calcoaceticus strains isolated from hospital patients by cell envelope protein profiles.

The usefulness of sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of cell envelope proteins for classifying strains of Acinetobacter calcoaceticus was studied using 129 isolates from 16 in-patients in a teaching hospital. In 11 patients, all of the isolates from each patient exhibited the same pattern irrespective of the body site or time of isolation. The patterns of the isolates from four other patients were indistinguishable, with the exception of one isolate per patient. In the isolates from one patient five patterns were observed. In several cases isolates from different patients exhibited the same pattern. The relative frequency of some of these patterns was low. Epidemiological data were compatible with the assumption that the concurrent presence of bacteria of these patterns in the patients was the result of cross-infection. For one pattern, which was seen in seven patients, cross-infection could not be substantiated. On the basis of analysis of electrophoretic patterns in combination with epidemiological data on a number of strains it is concluded that cell-envelope protein profiles appear to be a useful aid in studying the dissemination of Acinetobacter in the hospital environment.