Combined effect of liposomal and conventional amphotericin B in a mouse model of systemic infection with Candida albicans.

Amphotericin B may be employed as a potentially toxic deoxycholate complex or incorporated in liposomes and other particles which have an altered tissue distribution. A combination of both preparations could help to overcome the drawbacks of the individual preparations. We have employed a mouse model of systemic infection with Candida albicans to investigate whether this assumption was essentially true. We found that the combination of low dosages of both preparations (0.5 mg/kg of body weight/day of conventional and 0.5 mg kg of body weight/day liposomal amphotericin B) was more efficient than a similar dosage of conventional amphotericin B (1 mg/kg of body weight/day) in the liver and similar or higher dosages of liposomal amphotericin B (1 or 5 mg/kg of body weight/day) in the kidneys.

New molecular methods to study gene functions in Candida infections.

Candida albicans has become a model system for human pathogenic fungi in clinical research, mainly due to the increasing number of Candida infections. Molecular techniques to study C. albicans virulence properties have been improved over the last few years, despite difficulties in genetic manipulation of this fungus. Some of the recent achievements from our own laboratory or from other groups are described in this article. The molecular analysis of the recently identified ATP-dependent transporter Mlt1 using the green fluorescent protein (GFP) as reporter for protein localization and the dominant MPAR gene as a selection marker for gene inactivation provides an example for the study of gene functions in C. albicans.

Galactomannan enzyme immunoassay for monitoring systemic infection with Aspergillus fumigatus in mice.

Intravenous (i.v.) infection of immunocompetent mice with Aspergillus fumigatus was used to investigate the ability of a commercial galactomannan enzyme-linked immunosorbent assay (ELISA) to monitor the course of organ infection after dissemination. The test detected 100% of the fungemias which occurred for up to 5 days after infection. When blood-cultures became negative but there was a high load of fungi in the parenchymal organs and a positive culture from the brain, the ELISA was again positive in all animals. However, when blood cultures as well as brain cultures were negative and lower amounts of fungi demarcated by immune cells were present in the liver and kidneys which was the case between day 5 and 30 of infection, the test was negative in most of the animals. Therefore, the test was excellent for detection of early i.v. infection with Aspergillus fumigatus but not suited for detection of limited organ infection in immunocompetent mice.

Efficient treatment of murine systemic infection with Candida albicans using amphotericin B incorporated in nanosize range particles (emulsomes).

The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg(-1). In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans.

Influence of liposomal amphotericin B on CD8 T-cell function.

Liposomal amphotericin B was immunosuppressive on target cell lysis in vitro and on protection mediated by cytotoxic CD8 T cells in murine listeriosis. When dosages usually used for therapy in humans were compared, the immunosuppressive effect of 5 mg of liposomal amphotericin B/kg of body weight/day was similar to that of standard amphotericin B at 1 mg/kg/day, but a dosage of liposomal amphotericin B of 1 mg/kg/day was not suppressive in vivo.

Systemic and intracerebral infections of mice with Listeria monocytogenes successfully treated with linezolid.

Linezolid is an oxazolidinone derivative which is active mostly against Gram-positive bacteria. In this work its activity against the facultatively intracellular bacterium Listeria monocytogenes was examined in vitro, in tissue culture and in animal models of systemic and intracerebral infection and compared with ampicillin which is the antibiotic of choice for treatment of listeriosis. All strains of L. monocytogenes were susceptible to the substance, with minimal inhibitory concentrations (MICs) determined by E-test ranging from 0.38 to 1.5 mg/l which is below the preliminary breakpoint of this substance. Linezolid was bacteriostatic against L. monocytogenes since up to 64 times the MIC did not kill the bacteria in 24 hours. Linezolid was also bacteriostatic on L monocytogenes in infected tissue culture cells. In animal models of systemic and intracerebral infection, linezolid was able to inhibit bacterial growth but was clearly less effective than ampicillin. In conclusion, linezolid might be useful for the treatment of infections with L monocytogenes in humans when ampicillin may not be used.

Protein S-100B: a serum marker for ischemic and infectious injury of cerebral tissue.

The S-100B protein is released by injured astrocytes. After passage through a disintegrated blood-brain barrier (BBB) the molecule can be detected in the peripheral circulation. We investigated the association between the extent of brain injury and S-100B concentration in serum in cerebral injury caused by cerebral ischemia and cerebral fungal infection. Study I: The S-100B serum concentration was serially determined in 24 patients with ischemic stroke at 4, 8, 10, 24, 72 hours after the onset of symptoms. We observed that patients with brain lesions larger than 5 cm3 exhibited significantly increased serum levels of S-100B at 10, 24 and 72 hours compared to those with lesion volumes below 5 cm3. Furthermore, an association between S-100B serum concentration and neurological outcome was observed. Study II: In a mouse model of systemic fungal infection with Candida albicans we observed that serum levels of S-100B increased at day 1 after intravenous infection. At this time we could histologically demonstrate brain tissue injury by invading hyphae which had crossed the BBB. Furthermore, reactive astrogliosis was demonstrated by immunohistochemistry. On day 7 we found a significant decrease of S-100B serum level compared to day 1 and 4. This was associated with a demarcation of the fungi with leukocytes in brain tissue at this late phase of infection. No further invasion through the BBB was seen on day 7. In conclusion, serum levels of S-100B reflect the time course of tissue injury in cerebral ischemia and cerebral infection to a similar extent. Thus, S-100B may be a useful marker to assess cerebral tissue injury.

Differential activation of a Candida albicans virulence gene family during infection.

The yeast Candida albicans is a harmless commensal in most healthy people, but it causes superficial as well as life-threatening systemic infections in immunocompromised patients. C. albicans can colonize or infect virtually all body sites because of its high adaptability to different host niches, which involves the activation of appropriate sets of genes in response to complex environmental signals. We have used an in vivo expression technology that is based on genetic recombination as a reporter of gene expression to monitor the differential activation of individual members of a gene family encoding secreted aspartic proteinases (Saps), which have been implicated in C. albicans virulence, at various stages of the infection process. Our results demonstrate that SAP expression depends on the type of infection, with different SAP isogenes being activated during systemic disease as compared with mucosal infection. In addition, the activation of individual SAP genes depends on the progress of the infection, some members of the gene family being induced immediately after contact with the host, whereas others are expressed only after dissemination into deep organs. In the latter case, the number of invading organisms determines whether induction of a virulence gene is necessary for successful infection. The in vivo expression technology allows the elucidation of gene expression patterns at different stages of the fungus-host interaction, thereby revealing regulatory adaptation mechanisms that make C. albicans the most successful fungal pathogen of humans and, at the same time, identifying the stage of an infection at which certain virulence genes may play a role.

Inefficient T cell memory in the brain of mice infected with Candida albicans.

We compared the contribution of T cell memory to the clearance of the fungus Candida albicans from the liver, kidneys and brain of Balb/c mice in a model of secondary systemic infection. In secondary infection, the fungi were more rapidly eliminated from the liver and kidneys than during primary infection. This was most pronounced in the liver where the fungi were eliminated at day 14 of infection. In contrast, in the brain, cultivable yeasts were still detectable 35 days after infection. Although both CD4(+) and CD8(+) cells could be detected in the brain with immunohistology, these cells appeared later in infection and in lower numbers than in the liver, and there were no significant differences in the numbers of T cells detected in the brain between primary and secondary infection. In contrast to the liver and the kidneys where an effect of T cells on the fungal load could be demonstrated, depletion of neither CD4(+) nor CD8(+) nor Thy-1.2(+) cells resulted in a significant increase of the amount of fungi in the brain above levels measured in secondarily infected mice treated with irrelevant antibodies. We conclude that the contribution of CD4(+) and CD8(+) cells to the clearance of C. albicans in secondary infection is organ-dependent and that T cell memory is inefficient in the brain.

Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Suppression of acquired immunity against Listeria monocytogenes by amphotericin B-mediated inhibition of CD8 T cell function.

Amphotericin B is frequently used for the treatment of fungal infections of immunocompromised individuals. Whereas immunomodulatory side effects of this agent are known, the influence of amphotericin B was studied in the model of murine Listeria monocytogenes infection. Treatment of L. monocytogenes-immune mice with a nontoxic dose of amphotericin B (0.75 mg/kg) reduced antilisterial protection by 4-5 orders of magnitude, while it had no significant effect on natural immunity against L. monocytogenes in naive mice. Treatment of mice with amphotericin B also abolished the protection mediated by transfer of an L. monocytogenes-specific CD8 T cell line. Furthermore, in vitro analysis showed that amphotericin B impaired target cell lysis and interferon-gamma production by peptide-specific CD8 T cell lines and antigen presentation by L. monocytogenes-infected macrophagelike cells. These data indicate that amphotericin B has a strong suppressive effect on the function of CD8 T cells in vitro and in vivo.

The ketolide antibiotic HMR 3647, a candidate substance for the treatment of systemic and intracerebral infections with Listeria monocytogenes.

HMR 3647 is a novel macrolide derivative with a broad spectrum of activity against grampositive bacteria and some fastiduous gramnegative bacteria, anaerobes and Toxoplasma gondii. In this work, its activity against the facultatively intracellular bacterium, Listeria monocytogenes, was examined in vitro, in tissue culture and in animal models of systemic and intracerebral infection and compared with that of erythromycin. All strains of L. monocytogenes were susceptible to the substance, with minimal inhibitory concentrations (MICs) that were consistently lower than the MICs of erythromycin. HMR 3647 was bacteriostatic against L. monocytogenes since concentrations of up to 64 times the MIC did not kill the bacteria within 24 hours. HMR 3647 produced a pronounced postantibiotic effect (PAE) and was bacteriostatic in tissue culture cells infected with L. monocytogenes. In animal models of systemic and intracerebral infection, HMR 3647 was slightly more effective than erythromycin in the livers and spleens and comparably effective in the brains when given in the same dosage. In conclusion, HMR 3647 is a candidate substance for the treatment of infections with L. monocytogenes in immunocompetent subjects.

Quinupristin/dalfopristin attenuates the inflammatory response and reduces the concentration of neuron-specific enolase in the cerebrospinal fluid of rabbits with experimental Streptococcus pneumoniae meningitis.

The inflammatory response following initiation of antibiotic therapy and parameters of neuronal damage were compared during intravenous treatment with quinupristin/dalfopristin (100 mg/kg as either a short or a continuous infusion) and ceftriaxone (10 mg/kg/h) in a rabbit model of Streptococcus pneumoniae meningitis. With both modes of administration, quinupristin/dalfopristin was less bactericidal than ceftriaxone. However, the concentration of proinflammatory cell wall components (lipoteichoic acid (LTA) and teichoic acid (TA)) and the activity of tumour necrosis factor (TNF) in cerebrospinal fluid (CSF) were significantly lower in the two quinupristin/dalfopristin groups than in ceftriaxone-treated rabbits. The median LTA/TA concentrations (25th/75th percentiles) were as follows: (i) 14 h after infection: 133 (72/155) ng/mL for continuous infusion of quinupristin/dalfopristin and 193 (91/308) ng/mL for short duration infusion, compared with 455 (274/2042) ng/mL for ceftriaxone (P = 0.002 and 0.02 respectively); (ii) 17 h after infection: 116 (60/368) ng/mL for continuous infusion of quinupristin/dalfopristin and 117 (41/247) ng/mL for short duration infusion, compared with 694 (156/2173) ng/mL for ceftriaxone (P = 0.04 and 0.03 respectively). Fourteen hours after infection the median TNF activity (25th/75th percentiles) was 0.2 (0.1/1.9) U/mL for continuous infusion of quinupristin/dalfopristin and 0.1 (0.01/3.5) U/mL for short duration infusion, compared with 30 (4.6/180) U/mL for ceftriaxone (P = 0.02 for each comparison); 17 h after infection the TNF activity was 2.8 (0.2/11) U/mL (continuous infusion of quinupristin/dalfopristin) and 0.1 (0.04/6.1) U/mL (short duration infusion), compared with 48.6 (18/169) U/mL for ceftriaxone (P = 0.002 and 0.001). The concentration of neuron-specific enolase (NSE) 24 h after infection was significantly lower in animals treated with quinupristin/dalfopristin: 4.6 (3.3/5.7) microg/L (continuous infusion) and 3.6 (2.9/4.7) microg/L (short duration infusion) than in those treated with ceftriaxone (17.7 (8.8/78.2) microg/L) (P = 0.03 and 0.009 respectively). In conclusion, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space after initiation of antibiotic therapy. The concentration of NSE in the CSF, taken as a measure of neuronal damage, was lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits.

Host-induced, stage-specific virulence gene activation in Candida albicans during infection.

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.

Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua.

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.

Parameters for determination of Candida albicans virulence in murine peritonitis.

Using intraperitoneal (i.p.) infection of mice with Candida albicans we determined which parameters might be useful for characterization of virulence in this model. Upon i.p. infection of mice with two reference strains striking differences in lethality were detected. These differences in virulence corresponded with invasion of the liver and pancreas by the virulent strain and with a lack of invasion by the avirulent strain. The virulent strain was able to release high amounts of the enzymes alanine aminotransferase (ALT) and alpha-amylase (AM) from liver and pancreas into the blood plasma. Most likely, these enzymes were released by penetration of hyphae into the cytoplasm which was shown with electron microscopy. When invasion slowed down, there was also a drop in the activities of ALT and AM measured in the blood of infected mice. As both strains disseminated to the heart, kidneys, and lungs, dissemination into these organs was no reliable parameter for virulence in this model. However, only the virulent strain was able to reach the brain and to germinate in the kidneys and brain. In contrast to invasion and enzyme activities, the fungal load in the peritoneal cavity and in the neighbouring organs appeared not to be related with virulence. This may be concluded from the fact that there were no differences in the absolute colony forming units (cfu) and the length of persistence of both strains when similar inocula were used. We conclude that the ability of a given strain of C. albicans to invade neighbouring organs, to reach the brain upon dissemination and germination in the brain and kidneys may be used for measurement of virulence in this model when virulence is defined as lethality.

Parameters for determination of Candida albicans virulence in murine peritonitis.

Using intraperitoneal (i. p.) infection of mice with Candida albicans we determined which parameters might be useful for characterization of virulence in this model. Upon i. p. infection of mice with two reference strains striking differences in lethality were detected. These differences in virulence corresponded with invasion of the liver and pancreas by the virulent strain and with a lack of invasion by the avirulent strain. The virulent strain was able to release high amounts of the enzymes alanine aminotransferase (ALT) and α-amylase (AM) from liver and pancreas into the blood plasma. Most likely, these enzymes were released by penetration of hyphae into the cytoplasm which was shown with electron microscopy. When invasion slowed down, there was also a drop in the activities of ALT and AM measured in the blood of infected mice. As both strains disseminated to the heart, kidneys, and lungs, dissemination into these organs was no reliable parameter for virulence in this model. However, only the virulent strain was able to reach the brain and to germinate in the kidneys and brain. In contrast to invasion and enzyme activities, the fungal load in the peritoneal cavity and in the neighbouring organs appeared not to be related with virulence. This may be concluded from the fact that there were no differences in the absolute colony forming units (cfu) and the length of persistence of both strains when similar inocula were used. We conclude that the ability of a given strain of C. albicans to invade neighbouring organs, to reach the brain upon dissemination and germination in the brain and kidneys may be used for measurement of virulence in this model when virulence is defined as lethality.

A two-step model for the induction of organ-specific autoimmunity.

Peripheral tolerance is considered to be a safeguard against autoimmunity but the mere existence of anergic T cells renders them potentially dangerous. Using transgenic mice that were tolerant to a foreign MHC class I antigen (Kb) exclusively expressed in the liver, we investigated whether reversal of tolerance in vivo would directly result in autoimmunity. Breaking of tolerance was achieved by application of tumour cells expressing both Kb and interleukin 2. Despite the fact that the respective mice were now able to reject Kb-positive grafts, the reversed T cells did not infiltrate and attack the Kb-positive liver. However, when the liver was 'conditioned' through an inflammatory reaction either by irradiation or by infection with Listeria, massive T cell infiltration and liver damage were observed in the reversed mice. The results show that at least two steps are required for autoimmunity: (1) activation of antigen-specific T cells, and (2) conditioning of the target organ. It will be important to determine the factors leading to conditioning but it is likely that adhesion molecules are involved. These experiments are not only of relevance for treatment of autoimmune disease but also for tumour therapy.