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.

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.

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.

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.

Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times.

One possibility for bringing drugs to their specific targets is to use the drug-laden liposomes that have been made target-specific by the attachment of appropriate proteins. Such 'directed' proteoliposomes and most other particles are rapidly removed from the bloodstream, however, by the mononuclear phagocytes in the liver and spleen. This causes suboptimal drug accumulation at the target site. Coating the liposome surface with poly(ethylene glycol) (PEG) may prolong the circulation time of liposomes. Using plasminogen as a homing device we have shown that the PEG-modified liposomes with such a homing device coupled to the ends of the long PEG chains may combine long vesicle circulation times in the blood with high target binding capability. The PEG-coated proteoliposomes with homing devices attached at the very bilayer surface, on the contrary, are longlived but have only little or no capability to bind to their targets.

Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue.

In an experimental model of unilateral pneumonia caused by Klebsiella pneumoniae in rats we investigated whether intravenous administration of liposomes with prolonged blood circulation time resulted in significant localization of liposomes in infected lung tissue. Liposomes (100 nm) composed of hydrogenated phosphatidylinositol:hydrogenated phosphatidylcholine:cholesterol (molar ratio, 1:10:5) radiolabeled with gallium-67-deferoxamine showed relatively long blood circulation time. The degree of localization of these long circulating liposomes in the infected left lung was significantly higher compared to that of localization of 110 nm egg phosphatidylglycerol:egg phosphatidylcholine:cholesterol (molar ratio, 1:10:5) liposomes which exhibited relatively short blood circulation time. At 16 h after administration of the long circulating liposomes (when 10% of the injected dose was still present in the bloodstream) localization of liposomes in the infected left lung was increased up to 10-fold compared to the left lung of uninfected rats, and appeared to be highly correlated with the intensity of the infection. In the uninfected right lung the localization of long circulating liposomes was not increased. The degree of localization of liposomes in the infected tissue is dependent on the residence time of liposomes in the blood compartment. The extent of localization of long circulating liposomes in infected tissue appeared to be dependent on the liposomal dose administered.

Doxorubicin entrapped in sterically stabilized liposomes: effects on bacterial blood clearance capacity of the mononuclear phagocyte system.

The introduction of long-circulating liposomes sterically stabilized by surface coating with polyethylene glycol has expanded the potential for drug targeting to tumors. In recent clinical studies, evidence of significant antitumor activity has been obtained with the industrially prepared formulation of long-circulating polyethylene glycol-coated liposomes containing doxorubicin, referred to as DOXIL. Previous studies performed in rats showed that doxorubicin-containing liposomes can exert major toxic effects on the liver macrophage population for a considerable period of time; a strong impairment of phagocytic function and even a substantial depletion of the liver macrophage populations were observed. In the present study, the phagocytic function of the mononuclear phagocyte system (MPS) after administration of DOXIL at a clinically relevant dosage schedule was evaluated in rats. Phagocytic function of the MPS was assessed by determining bacterial blood clearance capacity. The observations reported herein show that DOXIL is fairly well tolerated regarding bacterial blood clearance capacity of the MPS when administered in a regimen that resembles the clinical setting closely. This outcome has important implications with regard to the clinical utility of the liposomal drug, especially in the restricted context of immunocompromised cancer patients who easily develop systemic infections and should not be confronted with a therapy-induced reduction of the bacterial blood clearance capacity of the MPS.

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.

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.

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.

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.

Liposomal and lipid formulations of amphotericin B. Clinical pharmacokinetics.

Amphotericin B remains a very important drug for the treatment of fungal infections despite its toxicity. Encapsulation of amphotericin B into liposomes appears to reduce the toxic effects and to improve the clinical efficacy, allowing higher dosages to be given. The exact mechanism behind the reduced toxicity is not yet known. Amphotericin B is widely distributed after intravenous administration as the deoxycholate solubilisate. The highest concentrations are found in the liver, spleen and kidney. Protein binding and binding to the tissues is very high. The fate of the drug in the body is not known in detail. Renal and biliary excretion are both low and no metabolites have been identified. The drug is still detectable in the liver, spleen and kidney for as long as 1 year after stopping therapy. The pharmacokinetics of the different liposomal amphotericin B or lipid complexes of amphotericin B, which were recently developed, are quite diverse. A number of these preparations, such as amphotericin B lipid complex (ABLC), 'AmBisome' and amphotericin B colloidal dispersion (ABCD) are in clinical development. Their pharmacokinetics depend to a large extent on the composition and particle size of the liposomes or lipid complexes. Relatively large structures such as ABLC are rapidly taken up by the mononuclear phagocyte system, whereas smaller liposomes remain in the circulation for prolonged periods. In all studies only the total amphotericin B (both free and liposome- or lipid-associated) concentrations were determined. There is a need for studies correlating clinical efficacy and tolerability of liposomal amphotericin B with the pharmacokinetic properties of these formulations.

Impact of dosage schedule of antibiotics on the treatment of serious infections.

Experimental studies suggest that the importance of the antibiotic dosage schedule for therapeutic efficacy in severe infection and when host defences are impaired is related to the class of antibiotic. The efficacy of beta-lactams is mainly dependent on the maintenance of adequate antibiotic concentrations in plasma during the entire treatment interval, and not on high peak concentrations. The efficacy of aminoglycosides is related to the total dose administered, i.e., the area under the concentration-time curve, irrespective of the frequency of administration. This difference in efficacy between beta-lactams and aminoglycosides in relation to the dosage schedule correlate well with differences between both classes of antibiotics in kinetics of antibacterial activity in vitro and in vivo. Another factor relevant in this respect is the post-antibiotic effect (PAE) which means the suppression of bacterial regrowth at the end of the period of exposure to antibiotic.

Liposomes in the treatment of infections.

The use of liposomes in the treatment of severe infections is under investigation. Classical liposomes which localize in cells of the mononuclear phagocyte system (MPS) can be exploited in two ways. First for targeting of macrophage modulators such as muramyl peptides or IFN-gamma, to stimulate the cells of the MPS to maximal blood clearance capacity. This enhanced nonspecific anti-infectious resistance is important as in immunocompromised patients micro-organisms frequently appear in the blood from a local infection. Secondly, classical liposomes are successfully used as carriers of antibiotics in experimental intracellular parasitic-, viral-, fungal- or bacterial infections in MPS tissues. Based on these data extensive studies in patients with severe fungal infections have demonstrated successful treatment with liposomal or lipid-complexed amphotericin B. More recently, liposomal amphotericin B appeared to be effective in patients with drug-resistant visceral leishmaniasis. For the treatment of Mycobacterium avium complex infection in AIDS patients the efficacy of liposomal gentamicin is under investigation. With respect to infections in non-MPS tissues the applicability of Stealth liposomes characterized by long circulation half-lives is under investigation. Substantial localization of these liposomes in infected lung tissue of rats was demonstrated. Preliminary data in experimental bacterial lung infection showed superior efficacy of antibiotic encapsulated in Stealth liposomes.

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.

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.