Cornelia Speth

Activities of Antifungal Agents against Yeasts and Filamentous Fungi: Assessment according to the Methodology of the European Committee on Antimicrobial Susceptibility Testing

ABSTRACT We compared the activities of antifungal agents against a wide range of yeasts and filamentous fungi. The methodology of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for yeasts and spore-forming molds was applied; and a total of 349 clinical isolates of Candida spp., other yeast species, Aspergillus spp., and nondermatophyte non-Aspergillus spp. were investigated. The average geometric mean (GM) of the MICs of the various drugs for Candida spp. were as follows: amphotericin B (AMB), 0.55 μg/ml; liposomal amphotericin B (l-AMB); 0.35 μg/ml; itraconazole (ITC), 0.56 μg/ml; voriconazole (VRC), 0.45 μg/ml; posaconazole (POS), 0.44 μg/ml; and caspofungin (CPF), 0.45 μg/ml. The data indicated that the majority of Candida spp. were susceptible to the traditional and new antifungal drugs. For Aspergillus spp., the average GM MICs of AMB, l-AMB, ITC, VRC, POS, and CPF were 1.49 μg/ml, 1.44 μg/ml, 0.65 μg/ml, 0.34 μg/ml, 0.25 μg/ml, and 0.32 μg/ml, respectively. For the various zygomycetes, the average GM MICs of AMB, l-AMB, ITC, and POS were 1.36 μg/ml, 1.42 μg/ml, 4.37 μg/ml, and 1.65 μg/ml, respectively. Other yeastlike fungi and molds displayed various patterns of susceptibility. In general, the minimal fungicidal concentrations were 1 to 3 dilutions higher than the corresponding MICs. POS, AMB, and l-AMB showed activities against a broader range of fungi than ITC, VRC, and CPF did. Emerging pathogens such as Saccharomyces cerevisiae and Fusarium solani were not killed by any drug. In summary, the EUCAST data showed that the in vitro susceptibilities of yeasts and filamentous fungi are variable, that susceptibility occurs among and within various genera and species, and that susceptibility depends on the antifungal drug tested. AMB, l-AMB, and POS were active against the majority of pathogens, including species that cause rare and difficult-to-treat infections.

Induction of Maturation and Cytokine Release of Human Dendritic Cells by Helicobacter pylori

ABSTRACT Helicobacter pylori causes a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease. Despite an intensive proinflammatory response, the immune system is not able to clear the organism. However, the immune escape mechanisms of this common bacterium are not well understood. We investigated the interaction between H. pylori and human dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system. Stimulation of DCs with different concentrations of H. pylori for 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production. Lipopolysaccharide (LPS) from Escherichia coli, a known DC maturation agent, was used as a positive control. The cytokine release after stimulation with LPS was comparable to that induced by H. pylori except for IL-12. After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced by H. pylori. We further investigated the potential of H. pylori to induce maturation of DCs. Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation with H. pylori or LPS. We found no significant differences between H. pylori seropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules. These data clearly demonstrate that H. pylori induces a strong activation and maturation of human immature DCs.

Cutting Edge: Productive HIV-1 Infection of Dendritic Cells via Complement Receptor Type 3 (CR3, CD11b/CD18)

In the present study, we demonstrate that macrophage-tropic HIV-1 opsonized by complement and limited amounts of anti-HIV-IgG causes up to 10-fold higher productive infection of human monocyte-derived dendritic cells than HIV treated with medium or HIV opsonized by Ab only. Enhanced infection is completely abolished by a mAb specific for the ligand-binding site of CD11b (i.e., α-chain of complement receptor 3, receptor for iC3b), proving the importance of complement receptor 3 in this process. Inhibition of complement activation by EDTA also prevents enhanced infection, further demonstrating the role of complement in virus uptake and productive infection. Since HIV is, even in the absence of Abs, regularly opsonized by complement, most probably the above-described mechanism plays a role during in vivo primary infection.

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

The supportive role of complement in HIV pathogenesis.

This review focuses on interactions of HIV with the first‐line defence of native immunity, the complement system. In all body compartments tested so far, HIV meets complement. Activation of the complement system results in deposition of C3 fragments on the viral surface, but in contrast to other pathogens, most of HIV is not or is only poorly lysed by membrane attack complexes. To survive complement‐mediated lysis, HIV has not only developed resistance mechanisms, but uses opsonisation with complement fragments for its own advantage. Opsonised virions interact with complement receptor‐expressing cells, which are either subsequently infected with high efficiency or retain viral particles on their surface, which promotes transmission of virus to other permissive cells. Our knowledge of these mechanisms has increased enormously over the past few years. A complete understanding of these complex interactions of HIV with the complement system opens new perspectives for development of alternative therapeutic strategies.

Mechanism of Human Immunodeficiency Virus-Induced Complement Expression in Astrocytes and Neurons

ABSTRACT The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.

Studies of In Vitro Activities of Voriconazole and Itraconazole against Aspergillus Hyphae Using Viability Staining

ABSTRACT The minimal fungicidal concentrations (MFCs) of voriconazole and itraconazole for five clinical isolates each of Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were determined by a broth macrodilution method. Conidial suspensions as inocula were compared to hyphae as inocula since the invasive form of aspergillosis is manifested by the appearance of hyphal structures. In addition, cell viability staining with the dye FUN-1 was performed to assess time-dependent damage of hyphae exposed to various concentrations of the antifungal agents. With conidial inocula the MFC ranges of voriconazole were 0.5 to 4 μg/ml and those of itraconazole were 0.25 to 2 μg/ml, whereas the MFCs (2 to >16 μg/ml) with hyphal inocula were substantially higher (P < 0.01) for both itraconazole and voriconazole. Only minor differences between the tested antifungals were observed since 16 of 20 and 17 of 20 of the isolates of Aspergillus spp. tested appeared to be killed by voriconazole and itraconazole, respectively. The results of FUN-1 viability staining correlated closely to colony counts, but various time- and dose-dependent levels of viability of hyphae were also observed. In conclusion, our study demonstrates the importance of the type of inoculum used to test antifungals and the applicability of FUN-1 staining as a rapid and sensitive method for assaying the viability of hyphae.

Human immunodeficiency virus type 1 protease inhibitor attenuates Candida albicans virulence properties in vitro.

The putative virulence factor secreted aspartyl proteinase (SAP) of Candida albicans and the human immunodeficiency virus type 1 (HIV-1) protease both belong to the aspartyl proteinase family. The present study demonstrates that the HIV-1 protease inhibitor Indinavir is a weak but specific inhibitor of SAP. In addition, Indinavir reduces the amount of cell bound as well as released SAP antigen from C. albicans. Furthermore, viability and growth of C. albicans are markedly reduced by Indinavir. These findings indicate that HIV-1 protease inhibitors may possess antifungal activity and we speculate that in vivo SAP inhibition may add to the resolution of mucosal candidiasis in HIV-1 infected subjects.

Assessment of retroviral activity using a universal retrovirus chip.

A DNA chip-based assay is described for parallel detection and identification of a wide variety of human and mammalian exogenous and endogenous retroviruses. The assay combines multiplex polymerase chain reaction (PCR) using fluorochrome-modified primer mixtures and chip hybridization. The microarray is composed of retrovirus-specific synthetic oligonucleotides as capture probes deposited on glass slides. The retrovirus chip can be used to assess the occurrence of reverse transcriptase (RT)-related transcripts in biological samples of human and mammalian origin. For example, distinct expression profiles of human endogenous retroviruses (HERV) were established reproducibly in human white blood cells, mammary gland and other human tissues. In particles released by human cells, packaging of specific HERV transcripts could be observed. Monitoring of human exogenous retroviruses (HIV, HTLV) and detection of putative cross-species transmissions (MLV, PERV) in human samples was efficient and reliable. The DNA chip should be an excellent tool for the detection of most relevant retroviruses and offers insights into differential retroviral activities and replication strategies. Furthermore, it could improve significantly the safety of gene therapy, tissue engineering, xenotransplantation and production of therapeutic polypeptides in cell culture.

gp41 Envelope Protein of Human Immunodeficiency Virus Induces Interleukin (IL)-10 in Monocytes, but Not in B, T, or NK Cells, Leading to Reduced IL-2 and Interferon-γ Production

The effect of extracellular domain of human immunodeficiency virus (HIV-1) transmembrane glycoprotein gp41 on interleukin (IL)-10, IL-2, interferon (IFN)-y, IL-4, and tumor necrosis factor-alpha production by human peripheral blood mononuclear cells (PBMC) was assessed by ELISA. Rapid gp41-induced increase of IL-10 production was detected in resting PBMC and isolated monocytes but not in B, T, or NK cells. Furthermore, gp41 also enhanced IL-10 production in staphylococcal enterotoxin B-stimulated PBMC, while synthesis of IL-2, IFN-gamma, and IL-4 in these cells was down-modulated. Kinetic studies revealed that increased IL-10 production preceded reduction of IL-2, indicating the possible IL-10 regulatory role in the gp41-induced down-modulation of this cytokine. Anti-IL-10 antibody reversed almost completely the gp41 inhibitory effect on IL-2 production. In this study, HIV-1 gp41 was a potent modulator of cytokine production by PBMC, in particular by increasing IL-10 secretion from normal monocytes/macrophages and consequently down-regulating IL-2 and IFN-gamma.