Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS.

In 2005, a new sibling species of Aspergillus fumigatus was discovered: Aspergillus lentulus. Both species can cause invasive fungal disease in immune-compromised patients. The species are morphologically very similar. Current techniques for identification are PCR-based or morphology-based. These techniques are labour-intense and not sufficiently discriminatory. Since A. lentulus is less susceptible to several antifungal agents, it is important to correctly identify the causative infectious agent in order to optimize antifungal therapy. In this study we determined whether Raman spectroscopy and/or MALDI-TOF MS were able to differentiate between A. lentulus and A. fumigatus. For 16 isolates of A. lentulus and 16 isolates of A. fumigatus, Raman spectra and peptide profiles were obtained using the Spectracell and MALDI-TOF MS (VITEK MS RUO, bioMérieux) respectively. In order to obtain reliable Raman spectra for A. fumigatus and A. lentulus, the culture medium needed to be adjusted to obtain colourless conidia. Only Raman spectra obtained from colourless conidia were reproducible and correctly identified 25 out of 32 (78 %) of the Aspergillus strains. For VITEK MS RUO, no medium adjustments were necessary. Pigmented conidia resulted in reproducible peptide profiles as well in this case. VITEK MS RUO correctly identified 100 % of the Aspergillus isolates, within a timeframe of approximately 54 h including culture. Of the two techniques studied here, VITEK MS RUO was superior to Raman spectroscopy in the discrimination of A. lentulus from A. fumigatus. VITEK MS RUO seems to be a successful technique in the daily identification of Aspergillus spp. within a limited timeframe.

Dynamics of interferon-gamma release assay and cytokine profiles in blood and respiratory tract specimens from mice with tuberculosis and the effect of therapy.

There are limitations on diagnostic methods to differentiate between active and latent tuberculosis (TB), and the prediction of latent progression to TB disease is yet complex. Traditionally, tuberculosis-specific host immune response was visualized using the tuberculin skin test. Nowadays, IFN-γ release assays (IGRA) provide a more specific and sensitive tool, by which exposure to Mtb could be determined. However, the merit of IGRA aids in diagnosing active TB is yet unclear. We adapted IGRA for use in mice, and quantifying bead-based flow cytometry techniques were used to assess cytokine profiles during the course of untreated infection and to investigate the value of IGRA and cytokines as biomarkers for therapy response. High variability of IGRA results during progression of active TB infection related to various phases of infection was obtained. However, a significant decrease in IGRA results and in levels of IFN-γ, IL-17, IP-10 or MIG was observed and appeared to be associated with successful therapy. This outcome does not support the value of IGRA to accurately diagnose active TB or to monitor infection progression. However, IGRA proved to be a useful biomarker to monitor therapy success. In addition, different cytokines might serve as biomarkers.

Course of murine tuberculosis and response to first-line therapy depends on route of infection and inoculum size.

BACKGROUND: In the search for new anti-tuberculosis drugs, numerous potential drugs are being screened in vitro. In animal models, promising new anti-tuberculosis drugs are assessed in terms of toxic side effects and comparative therapeutic efficacy. Mice are frequently used and experimental infections are established in different ways. OBJECTIVE: To investigate to what extent the route of Mycobacterium tuberculosis inoculation is a determinant in the pathogenesis of tuberculosis (TB) and the therapeutic outcome. Results will contribute to insight into the translational value of TB models used for preclinical studies. DESIGN: TB in mice was established through intratracheal or intravenous mycobacterial inoculation. The efficacy of a 26-week treatment regimen was evaluated, including assessment of relapse of infection 13 weeks post-treatment. RESULTS: It was shown that the course of TB and the therapeutic response, in terms of histopathological characteristics and mycobacterial load, in lungs and extra- pulmonary organs is substantially different and dependent on the route of infection applied and the inoculum size used. CONCLUSION: When evaluating the comparative therapeutic potential of novel anti-tuberculosis drugs or drug treatment schedules investigated in different studies, it should be noted that the route of infection applied and the inoculum size used influence the course of murine TB and the therapeutic response to the standard first- line anti-tuberculosis drug regimen.

Immunological parameters to define infection progression and therapy response in a well-defined tuberculosis model in mice.

To evaluate novel approaches for tuberculosis (TB) diagnostics and treatment, well-validated animal TB models are needed. Especially the emergence and spread of drug resistant TB requires innovative therapy and accurate parameters for monitoring success or failure of therapy. We developed a TB model in BALB/c mice, in which Mycobacterium tuberculosis (Mtb) infection was induced through the natural respiratory route, mimicking human TB infection. The lung showed a mild inflammatory infiltrate consisting of granulomas in the first phase of infection, followed by progressive increase of pneumonic lesions resulting in extensive lung consolidation in the chronic phase. Dissemination to the extra-pulmonary sites was observed. The model was validated in terms of therapeutic outcome. The 26-week standard therapy administered in human pharmacokinetic-equivalent doses, resulted in complete elimination of Mtb in all infected organs, without relapse of infection in the post-treatment period. However, a 13-week therapy, simulating patient non-adherence resulted in relapse of infection. In our quest to find biomarkers for monitoring success or failure of therapy, the concentrations of various cytokines in serum and lung, determined by cytometric bead array (CBA), were evaluated in relation to the in situ cytokine expression in the lung, assessed by immunohistochemistry. The level of IFN-gamma concentration in serum increased with infection progression, and decreased during effective therapy, and as such appeared to be an appropriate immunological parameter for success or failure of therapy. Relapse of infection, after inappropriate therapy, manifested as an increase in the serum IFN-gamma concentration.

Role of ceftazidime dose regimen on the selection of resistant Enterobacter cloacae in the intestinal flora of rats treated for an experimental pulmonary infection.

OBJECTIVES: The effect of ceftazidime dosing increments and frequency of dosing on the selection of ceftazidime-resistant Enterobacter cloacae in the intestine was studied in rats, during treatment of a pulmonary infection caused by Klebsiella pneumoniae. METHODS: Rats with pulmonary infection (n = 10 per group) received therapy with doses of ceftazidime at 3.1 to 400 mg/kg per day at a frequency of every 6,12 or 24 h for 18 days, starting 24 h after bacterial inoculation of the lung. Emergence of resistance in intestinal E. cloacae was monitored by culturing fresh stool specimens at days 0, 8, 15, 22, 29, 36 and 43 on agar plates with (6.4 mg/L) and without ceftazidime. Pharmacodynamic indices and time within the mutant selection window (MSW) were assessed in infected rats for each regimen. Ceftazidime-resistant E. cloacae mutants were characterized by determination of the beta-lactamase activity under cefoxitin-induced and non-induced conditions. RESULTS: A reduction of intestinal ceftazidime-susceptible E. cloacae was observed and showed a significant correlation with the fAUC/MIC at days 8, 15 and 22 and with the fCmax on days 8, 15, 22, 29 and 36. More rats treated with 12-25 and 50-100 mg/kg per day every 6 h were found colonized with ceftazidime-resistant E. cloacae mutants than animals treated every 12 h or every 24 h. The proportion of rats colonized with ceftazidime-resistant E. cloacae mutants at days 15, 36 and 43 correlated with the time during which ceftazidime plasma concentrations were within the boundaries of the MSW. Only at day 15 was a correlation demonstrated between the fCmax and significantly fewer rats colonized with ceftazidime-resistant E. cloacae. Ceftazidime-resistant E. cloacae mutants (MIC >or= 128 mg/L) were characterized as stable derepressed mutants. CONCLUSIONS: Colonization with stable derepressed ceftazidime-resistant E. cloacae mutants particularly occurred when rats were exposed to moderate doses of ceftazidime (12-25 or 50-100 mg/kg per day) administered every 6 h. Emergence of resistance was correlated with time within the MSW.

Host factors influencing the preferential localization of sterically stabilized liposomes in Klebsiella pneumoniae-infected rat lung tissue.

PURPOSE: To gain insight into the host factors influencing liposome localization at sites of bacterial infection. METHODS: In a unilateral Klebsiella pneumoniae pneumonia rat model, capillary permeability and number of circulating leukocytes was quantified and related to the degree of liposome target localization. RESULTS: Liposome localization was highest in the hemorrhagic zone of infection, a zone characterized by markedly increased capillary permeability and high bacterial numbers. Both liposome localization and capillary permeability correlated positively with severity of infection. Lung instillation of other inflammatory stimuli, such as lipopolysaccharide or 0.1 M HCl inducing increased capillary permeability, also promoted liposome localization. As liposomal target localization in leukopenic rats was similar to that in immunocompetent rats, contribution of circulating leukocytes seems limited. Intrapulmonary distribution of liposomes shows that leukocytes at the target site are involved in liposome uptake after extravasation. CONCLUSIONS: Increased capillary permeability plays a crucial role in liposome localization at the infected site, whereas contribution of leukocytes is limited. These results suggest inflammatory conditions that could benefit from liposomal drug delivery. The involvement of leukocytes in liposome uptake at the target site could be important information in the selection of appropriate drugs.

In vivo synergistic interaction of liposome-coencapsulated gentamicin and ceftazidime.

Antimicrobial agents may interact synergistically. But to ensure synergy in vivo, the drugs should both be present at the site of infection at sufficiently high concentrations for an adequate period of time. Coencapsulation of the drugs in a drug carrier may ensure parallel tissue distributions. Since liposomes localize preferentially at sites of infection, this mode of drug delivery could, in addition, increase drug concentrations at the focus of infection. The therapeutic efficacy of gentamicin and ceftazidime coencapsulated into liposomes was examined by monitoring survival in a rat model of an acute unilateral pneumonia caused by antibiotic-susceptible and antibiotic-resistant Klebsiella pneumoniae strains. It is shown that administration of gentamicin in combination with ceftazidime in the free form either as single dose or as 5-day treatment resulted in an additive effect on rat survival in both models. In contrast, targeted delivery of liposome-coencapsulated gentamicin and ceftazidime resulted in a synergistic interaction of the antibiotics in both models. Consequently, liposome coencapsulation of gentamicin and ceftazidime allowed both a shorter course of treatment at lower cumulative doses compared with administration of the antibiotics in the free form to obtain complete survival of rats. Liposomal coencapsulation of synergistic antibiotics may open new perspectives in the treatment of severe infections.

Therapeutic efficacy of liposomal gentamicin in clinically relevant rat models.

Sterically stabilized liposomes are able to localize selectively at sites of infection, potentially permitting targeted drug delivery. Up to now, the majority of studies investigating therapeutic efficacy of liposomes have been conducted in animals with an intact host defense infected with high antibiotic-susceptible bacteria. In the present study, the therapeutic efficacy of gentamicin encapsulated in sterically stabilized liposomes, alone or in combination with the free drug was studied in rats with intact host defense as well as leukopenic rats. Rats were inoculated with a high gentamicin-susceptible or low-gentamicin susceptible Klebsiella pneumoniae in the left lung, resulting in an acute unilateral pneumonia. Survival rates demonstrate the valuable therapeutic properties of the liposome-encapsulated drug in these clinically relevant animal models.

Improved efficacy of ciprofloxacin administered in polyethylene glycol-coated liposomes for treatment of Klebsiella pneumoniae pneumonia in rats.

Animal and clinical data show that high ratios of the area under the concentration-time curve and the peak concentration in blood to the MIC of fluoroquinolones for a given pathogen are associated with a favorable outcome. The present study investigated whether improvement of the therapeutic potential of ciprofloxacin could be achieved by encapsulation in polyethylene glycol (PEG)-coated long-circulating sustained-release liposomes. In a rat model of unilateral Klebsiella pneumoniae pneumonia (MIC = 0.1 microg/ml), antibiotic was administered at 12- or 24-h intervals at twofold-increasing doses. A treatment period of 3 days was started 24 h after inoculation of the left lung, when the bacterial count had increased 1,000-fold and some rats had positive blood cultures. The infection was fatal within 5 days in untreated rats. Administration of ciprofloxacin in the liposomal form resulted in delayed ciprofloxacin clearance and increased and prolonged ciprofloxacin concentrations in blood and tissues. The ED(50) (dosage that results in 50% survival) of liposomal ciprofloxacin was 3.3 mg/kg of body weight/day given once daily, and that of free ciprofloxacin was 18.9 mg/kg/day once daily or 5.1 mg/kg/day twice daily. The ED(90) of liposomal ciprofloxacin was 15.0 mg/kg/day once daily compared with 36.0 mg/kg/day twice daily for free ciprofloxacin; 90% survival could not be achieved with free ciprofloxacin given once daily. In summary, the therapeutic efficacy of liposomal ciprofloxacin was superior to that of ciprofloxacin in the free form. PEG-coated liposomal ciprofloxacin was well tolerated in relatively high doses, permitting once daily administration with relatively low ciprofloxacin clearance and without compromising therapeutic efficacy.

Therapeutic efficacy of liposome-encapsulated gentamicin in rat Klebsiella pneumoniae pneumonia in relation to impaired host defense and low bacterial susceptibility to gentamicin.

Long-circulating liposomes (LCL) may be used as targeted antimicrobial drug carriers as they localize at sites of infection. As a result, LCL-encapsulated gentamicin (LE-GEN) has demonstrated superior antibacterial activity over the free drug in a single-dose study of immunocompetent rats with Klebsiella pneumoniae pneumonia. In the present study, the therapeutic efficacy of LE-GEN was evaluated by monitoring rat survival and bacterial counts in blood and lung tissue in clinically relevant models, addressing the issue of impaired host defense and low bacterial antibiotic susceptibility. The results show that in immunocompetent rats infected with the high-GEN-susceptibility K. pneumoniae strain, a single dose of LE-GEN is clearly superior to an equivalent dose of free GEN. Yet complete survival can also be obtained with multiple doses of free GEN. In leukopenic rats infected with the high-GEN-susceptible K. pneumoniae strain, free GEN at the maximum tolerated dose (MTD) was needed to obtain survival. However, with the addition of a single dose of LE-GEN to free-GEN treatment, complete survival can be obtained using a sevenfold-lower cumulative amount of GEN than with free-GEN treatment alone. In leukopenic rats infected with low-GEN-susceptible K. pneumoniae cells, free GEN at the MTD did not result in survival. The use of LE-GEN is needed for therapeutic success. Increasing LE-GEN bilayer fluidity resulted in an increased GEN release from the liposomes and hence improved rat survival, thus showing the importance of the liposome lipid composition for therapeutic efficacy. These results warrant further clinical studies of liposomal formulations of aminoglycosides in immunocompromised patients with severe infections.

In vitro activity of trovafloxacin against Bacteroides fragilis in mixed culture with either Escherichia coli or a vancomycin- resistant strain of Enterococcus faecium determined by an anaerobic time-kill technique.

To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of Bacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant strain of Enterococcus faecium (VREF). With inocula of 5 x 10(5) CFU/ml and trovafloxacin concentrations of /=6.1 (log(10) CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10(8) CFU/ml, a similar E(max) and a similar concentration to produce 50% of E(max) (EC(50)) for B. fragilis were found in both pure cultures and mixed cultures with E. coli. However, to produce a similar killing of B. fragilis in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar "protective" effect on B. fragilis against the activity of trovafloxacin. Using inocula of 10(9) CFU/ml, the activity of trovafloxacin was retained for E. coli and B. fragilis and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of B. fragilis in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of B. fragilis and E. coli, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.

Localization of sterically stabilized liposomes in experimental rat Klebsiella pneumoniae pneumonia: dependence on circulation kinetics and presence of poly(ethylene)glycol coating.

Preferential localization of liposomes at sites of infection or inflammation has been demonstrated in a variety of experimental models. Most studies report enhanced localization at the target site of poly(ethyelene) glycol (PEG)-coated liposomes as compared to conventional non-coated liposomes. It is generally accepted that the prolonged circulation time of PEG-coated liposomes increases target site exposure, which results in increased target localization. A quantitative relationship between circulation kinetics and localization at the pathological site has not been defined as yet. Besides, an effect of the PEG coating itself has been suggested, as theoretically the PEG coating may facilitate liposome extravasation. In the present study, in a rat model of an acute unilateral Klebsiella pneumoniae pneumonia, circulation kinetics of PEG-coated liposomes were manipulated by incorporation of different amounts of phosphatidylserine (PS) and variation of lipid dose, additionally allowing evaluation of the saturability of the localization process. In addition, this paper addresses the effect of the PEG coating, by comparing the circulation kinetics and target localization of long-circulating 'PEG-free' and PEG-coated liposomes. It is shown that the degree of liposome localization at the target site is positively linearly related to the area under the blood concentration time curve (AUC) of the liposome formulations, irrespective of PEG coating. This finding is discussed in relation to the equation of Kedem and Katchalsky, which describes protein influx into sites of infection or inflammation.

Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model.

Two diagnostic tests, an Aspergillus-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (P < 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis.

Efficacy of liposomal amphotericin B with prolonged circulation in blood in treatment of severe pulmonary aspergillosis in leukopenic rats.

The therapeutic efficacy of long-circulating polyethylene glycol-coated liposomal amphotericin B (AMB) (PEG-AMB-LIP) was compared with that of AMB desoxycholate (Fungizone) in a model of severe invasive pulmonary aspergillosis in persistently leukopenic rats as well as in temporarily leukopenic rats. PEG-AMB-LIP treatment (intravenous administration) consisted of a single, or double (every 72 h), or triple (every 72 h) dose of 10 mg of AMB/kg of body weight, a double dose (every 72 h) of 14 mg of AMB/kg, or a 5-day treatment (every 24 h) with 6 mg/kg/dose. AMB desoxycholate was administered for 10 consecutive days at 1 mg of AMB/kg/dose. Treatment was started 30 h after fungal inoculation, at which time mycelial growth was firmly established. Both persistently and temporarily leukopenic rats died between 4 and 9 days after Aspergillus fumigatus inoculation when they were left untreated or after treatment with a placebo. In persistently leukopenic rats, a single dose of PEG-AMB-LIP (10 mg/kg) was as effective as the 10-day treatment with AMB desoxycholate (at 1 mg/kg/dose) in significantly prolonging the survival of rats infected with A. fumigatus and in reducing the dissemination of A. fumigatus to the liver. Prolongation of PEG-AMB-LIP treatment (double or triple dose or 5-day treatment) did not further improve efficacy. For temporarily leukopenic rats no major advances in efficacy were achieved compared to those for persistently leukopenic rats, probably because the leukocyte numbers in blood were restored too late in the course of infection.

Localization of sterically stabilized liposomes in Klebsiella pneumoniae-infected rat lung tissue: influence of liposome characteristics.

Sterically stabilized liposomes are able to localize at sites of infection and could serve as carriers of antimicrobial agents. For a rational optimization of liposome localization, the blood clearance kinetics and biodistribution of liposomes differing in poly(ethylene glycol) (PEG) density, particle size, bilayer fluidity or surface charge were studied in a rat model of a unilateral pneumonia caused by Klebsiella pneumoniae. It is shown that all liposome preparations studied localize preferentially in the infected lung compared to the contralateral non-infected lung. A reduction of the PEG density or rise in particle size resulted in a higher uptake by the mononuclear phagocyte system, lower blood circulation time and lower infected lung localization. Differences in bilayer fluidity did not affect blood clearance kinetics or localization in the infected lung. Increasing the amount of negatively charged phospholipids in the liposome bilayer did not affect blood clearance kinetics, but did reduce localization of this liposome preparation at the site of lung infection. In conclusion, the degree of localization at the infected site is remarkably independent of the physicochemical characteristics of the PEG liposomes. Substantial selective liposome localization can be achieved provided that certain criteria regarding PEG density, size and inclusion of charged phospholipids are met. These properties seem to be a direct consequence of the presence of the polymer coating operating as a repulsive steric barrier opposing interactions with biological components.

Scintigraphic imaging of bacterial and fungal infection in granulocytopenic rats.

UNLABELLED: Scintigraphic imaging in granulocytopenic patients can be very useful to detect and localize infections, which often do not show localizing signs and symptoms. We studied the potential of 99mTc-labeled polyethylene glycol (PEG)-coated liposomes and 99mTc-labeled IgG to image bacterial and fungal infection in a granulocytopenic rat model. 67Ga-citrate was used as a reference agent. METHODS: 99mTc-PEG-liposomes, 99mTc-hydrazinonicotinate (HYNIC)-IgG or 67Ga-citrate was administered to granulocytopenic rats with a Staphylococcus aureus abscess or with unilateral invasive pulmonary aspergillosis. Imaging and biodistribution studies were performed. RESULTS: All agents visualized the S. aureus infection from 1 h after injection onward. However, only with 99mTc-PEG-liposomes and with 99mTc-HYNIC-IgG did activity in the infectious foci increase with time up to 24 h. 99mTc-PEG-liposomes and 99mTc-HYNIC-IgG showed significantly higher accumulation in the infectious focus compared with 67Ga-citrate (1.33+/-0.31 and 1.40+/-0.16 percentage injected dose per gram [%ID/g], respectively, versus 0.31+/-0.04 %ID/g 24 h after injection; P<0.05). At 24 h after injection, abscess-to-muscle ratios were highest for 99mTc-liposomes (72.1+/-19.1), followed by 99mTc-HYNIC-IgG (18.3+/-3.3) and 67Ga-citrate (4.4+/-0.7). In pulmonary aspergillosis, both 99mTc-PEG-liposomes and 99mTC-HYNIC-IgG showed significantly higher uptake in the infected lung than did 67Ga-citrate (3.6+/-0.4 and 8.3+/-0.8 %ID/g, respectively, versus 1.3 %ID/g at 24 h after injection; P<0.05). CONCLUSION: 99mTc-PEG-liposomes and 99mTc-HYNIC-IgG performed better than did 67Ga-citrate in the localization of peripheral bacterial infection and fungal infection in the lung in granulocytopenic rats. The high focal uptake and high target-to-nontarget ratios of 99mTc-PEG-liposomes and 99mTc-HYNIC-IgG indicate that both radiopharmaceuticals may become valuable agents to image infection in granulocytopenic patients.

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.