Caspofungin modulates inflammatory responses to Aspergillus fumigatus through stage-specific effects on fungal beta-glucan exposure.

Echinocandins target fungal beta-1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of Aspergillus fumigatus, they do induce morphological changes in fungal hyphae. Because beta-1,3 glucans activate host antifungal pathways via the Dectin-1 receptor, we investigated the effect of echinocandins on inflammatory responses to A. fumigatus. Caspofungin- or micafungin-treated conidia and germlings induced less secretion of tumor necrosis factor (TNF) and CXCL2 by macrophages than did their untreated counterparts. Diminished secretion of TNF and CXCL2 correlated with diminished beta-glucan exposure on echinocandin-treated germ tubes. In contrast to treated conidia and germlings, echinocandin-treated hyphae stimulated increased release of TNF and CXCL2 by macrophages and demonstrated intense staining with a beta-glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin-induced morphological changes in A. fumigatus hyphae are accompanied by increased beta-glucan exposure, with consequent increases in Dectin-1-mediated inflammatory responses by macrophages.

Development of candidemia on caspofungin therapy: a case report.

Caspofungin, an echinocandin, is approved for use in invasive candidiasis. Few cases of break-through candidal infections during caspofungin therapy have been reported and none have involved Candida parapsilosis. Here, we report a patient who developed multiple post-operative complications after pancreaticoduodenectomy for a pancreatic mass, including fungemia due to C. parapsilosis, while on caspofungin for treatment of Candida glabrata peritonitis. The fungemia resolved after a central venous catheter was removed and therapy was switched from caspofungin to amphotericin B lipid complex. Studies of C. parapsilosis susceptibility and the pharmacodynamics and drug interactions of caspofungin that may contribute to breakthrough fungemia are discussed.

Both Th1 and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against Cryptococcus neoformans.

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) >> IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.

Intracellular parasitism of macrophages by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated fungal pathogen, causes meningoencephalitis in immunocompromised patients. Recent in vivo studies have demonstrated that C. neoformans is a facultative intracellular pathogen, as was previously suggested by in vitro studies. For survival in macrophages, C. neoformans utilizes a novel strategy for intracellular parasitism that includes the accumulation of intracellular polysaccharide in cytoplasmic vesicles. Confirmation of the fact that C. neoformans is a facultative intracellular pathogen could provide new insights into several poorly understood areas of cryptococcal pathogenesis, including mechanisms for latency and persistence and the lack of efficacy of humoral immunity. The finding that C. neoformans replicates inside macrophages in vitro in a manner similar to that observed in vivo provides an excellent system to dissect the molecular mechanisms responsible for this unique pathogenic strategy.

Intracellular crystal formation as a mechanism of cytotoxicity in murine pulmonary Cryptococcus neoformans infection.

Rod-like crystalline structures formed during eosinophilic Cryptococcus neoformans pneumonia in C57BL/6 mice. Crystals were found associated with yeast cells and free in host cell cytoplasm. The crystals apparently formed because of the interaction of a host protein with the cryptococcal polysaccharide. Crystal formation likely contributes to pathogenesis by causing cellular damage.

The effect of the echinocandin analogue caspofungin on cell wall glucan synthesis by Cryptococcus neoformans.

The echinocandin derivative caspofungin (MK-0991, L-743,872) inhibits 1,3-beta-d-glucan synthesis and is active against several medically important fungi but is relatively ineffective against Cryptococcus neoformans. To investigate the mechanism of C. neoformans resistance, the prevalence of 1,3- and 1,6-beta-d-glucan linkages was determined in cells grown with and without caspofungin, using affinity-purified antisera and gold particle immunoelectron microscopy. Cryptococcal strains ATCC 24067 (serotype D) and MY2061 (serotype A) were studied. Growth at 4 microg/mL of caspofungin reduced both glucan linkages in both strains. However, growth at 2 microg/mL resulted in reduced 1,6-beta-d-glucan linkage only for MY2061. Inhibition of 1,6-beta-d-glucan synthesis may be an additional mechanism of action for pneumocandins. The relatively low efficacy of caspofungin against C. neoformans may result from reduced activity against C. neoformans glucan synthase or from yet undiscovered mechanisms of action operative in other fungal pathogens but not in C. neoformans.

Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection.

To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungus Cryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformans occupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.

Antibody interactions with the capsule of Cryptococcus neoformans.

Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). Fixing C. neoformans cells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to the C. neoformans capsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to the C. neoformans polysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.

Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents.

The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformans cells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel(+)) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel(-)) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel(+) C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel(-) C. neoformans. A Mel(+) C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.

Melanization of Cryptococcus neoformans in murine infection.

Cryptococcus neoformans is a fungus that is pathogenic in humans and that can produce melanin in vitro. Melanization is associated with virulence, but there is no evidence that melanin is made during infection. Melanins are difficult to study because they are amorphous and insoluble. Melanin-binding peptides from a phage display library were used to demonstrate that C. neoformans makes melanin-like compounds in tissue. Melanin-binding peptides were characterized by a high proportion of positively charged and aromatic residues. Two other methods, demonstration of an antibody response to melanin in mice infected with C. neoformans and analysis of yeast cell walls in infected tissue by light microscopy, were used to support these findings. The demonstration that C. neoformans melanizes in tissue has important implications for pathogenesis and drug discovery.

Lactobacillus endocarditis: a case report of outpatient management.

A case of infective endocarditis due to Lactobacillus is presented. The diagnosis was established by positive blood cultures and transesophageal (but not transthoracic) echocardiography. The patient was cured with outpatient ceftriaxone therapy.

Organ-dependent variation of capsule thickness in Cryptococcus neoformans during experimental murine infection.

In studies of murine infection, the capsule thickness of Cryptococcus neoformans varied depending on the organ. The relative order of thickness was as follows: lung > brain > in vitro isolates. The differences in capsule thickness suggest that there are organ-related differences in the expression of genes responsible for capsule thickness.

Effect of antibody to capsular polysaccharide on eosinophilic pneumonia in murine infection with Cryptococcus neoformans.

The effect of the murine IgG1 monoclonal antibody (MAb) 2H1, which binds to Cryptococcus neoformans glucuronoxylomannan (GXM), on pulmonary infection in immunocompetent C57Bl/6 mice was examined. C57Bl/6 mice develop eosinophilic pneumonia in response to pulmonary cryptococcal infection. Survival, organ fungus burden, serum anticapsular antibody levels, and histopathology by light and electron microscopy were studied. MAb administration prior to infection prolonged survival without reducing the number of yeast in the lung or extrapulmonary sites. Compared with uninfected mice, occasional control and MAb-treated mice produced more IgM antibody to GXM or low levels of GXM-binding IgG1, IgG2b, or IgG3 antibodies. MAb-treated mice had fewer granules per eosinophil, indicating alteration in eosinophil physiology or degranulation (or both). Our results provide additional evidence that antibody administration can produce quantitative and qualitative changes in the inflammatory response to a pathogen.

Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies.

The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(kappa)] is in preclinical development for treatment of Cryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.

Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein.

We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (Kd approximately 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP.

Mechanism of action of antibody to capsular polysaccharide in Cryptococcus neoformans infection.

Cryptococcus neoformans is an encapsulated fungus that causes meningoencephalitis in 5-10% of patients with AIDS. While the immune response that controls infection is predominantly cell-mediated, Ab-mediated immunity is being studied for therapeutic use. mAbs to glucuronoxylomannan (GXM), the predominant constituent of the polysaccharide capsule are protective in a variety of murine infection models. However, the mechanism of Ab action in this infection is unknown. We review the literature on the effect of Ab in cryptococcal infection and potential mechanisms of action. The mechanism is likely multifactorial, involving enhancement at several branches of the immune response, including opsonization, antigen presentation and altered effector cell function. Removal of the toxic and immunosuppressive effects of GXM may be an important component of the mechanism of Ab action. Changes in pathology in response to monoclonal antibody (mAb) administration suggest that alterations in cytokine production may mediate mAb effects. In summary, specific Ab can modulate the course of cryptococcal infection to the benefit or detriment of the host, but significant questions remain concerning the mechanism of action and the relative importance of antibody-mediated immunity in normal and immunocompromised hosts.

Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro.

Eosinophils are components of inflammatory responses to a variety of pathogens. Although a variety of beneficial and harmful functions have been ascribed to these cells, their role in protection against infectious agents remains uncertain. Previous studies have reported eosinophilic pneumonia in mice infected intratracheally with Cryptococcus neoformans. We confirmed this observation and studied the inflammatory response in the lung at day 14 by light and electron microscopy. Immunostaining for glucuronoxylomannan showed isolated cryptococci inside the eosinophilic cuffs. Eosinophils were found to be in close association with C. neoformans in vivo. Cryptococci were associated with eosinophils within eosinophilic perivascular cuffs, within granulomas, and lining the alveolar space. To further investigate this phenomenon in vitro, we isolated rat peritoneal eosinophils and studied cryptococcus-eosinophil interactions in the presence and absence of anti-capsular immunoglobulin G1 (IgG1) and IgE monoclonal antibody (MAb). Eosinophils phagocytosed C. neoformans only in the presence of specific antibody. Phagocytosis was rapid, and dense rings that appeared to consist of granule contents were formed around the organisms. Mast cells were observed to occasionally phagocytose C. neoformans in vitro in the presence of IgE MAb. Our observations suggest that eosinophils may be effector cells against C. neoformans.

Effect of serum IgG1 to Cryptococcus neoformans glucuronoxylomannan on murine pulmonary infection.

Little is known about the role of Ab in protection against pulmonary fungal pathogens. The ability of murine IgG1 mAb 2H1 to modify pulmonary Cryptococcus neoformans infection was investigated in intratracheal infection. mAb 2H1 binds C. neoformans glucuronoxylomannan. mAb 2H1 was given to A/JCr mice 24 h before infection. Two C. neoformans strains were studied: ATCC 24067 (serotype D) and 62070 (serotype A). Fungal burden was determined by CFU 14 days after infection for both strains, and at 2 h, 24 h, 48 h, 7 days, and 28 days after infection for strain 24067. On day 14, mAb 2H1 treatment reduced CFU in the lung, brain, and liver for strain 62070 infection. Minor reductions in lung CFU followed infection with strain 24067 in mAb 2H1-treated mice, despite prolonged survival. The limited ability of mAb 2H1 to reduce lung CFU may reflect rapid phagocytosis of yeast by alveolar macrophages, seen by electron microscopy 2 h after infection, regardless of whether mice had received mAb. Alveolar macrophages phagocytosed and reduced C. neoformans CFU in vitro only in the presence of mAb 2H1. Differences were apparent in phagocytosis and in vitro killing between strains 24067 and 62070. Serum IgG1 modified the course of pulmonary infection in mice by prolonging survival, reducing CFU, and reducing tissue glucuronoxylomannan Ag. mAb administration was associated with enhanced granulomatous inflammation, but did not prevent infection or dissemination. Despite incomplete protection by serum Ab against pulmonary infection, our results provide encouragement for continued vaccine development.