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2.  Potential Antifungal Effects of Human Platelets against Zygomycetes In Vitro 
The Journal of infectious diseases  2009;200(7):1176-1179.
Zygomycosis is increasingly recognized in immunocompromised hosts. We investigated whether platelets become activated after contact with Zygomycetes and adhere to conidial and hyphal structures using immunofluorescence. The platelets’ influence on fungal viability was evaluated by assessing hyphal elongation and hyphal damage. Platelets became activated and strongly adhered to conidia and hyphae of Zygomycetes. Platelets induced time dependent damage to hyphae and significantly reduced (P < .05) hyphal elongation. We found that platelets possess antifungal capacities against Zygomycetes based on granule dependent mechanisms and significantly reduce fungal growth and spread, both of which are of major importance in evolving invasive disease.
PMCID: PMC3017871  PMID: 19698079
3.  Interaction of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro 
This study examined the direct interaction of serotonin (5-hydroxytryptamine (5-HT)) with Aspergillus species. Accumulation of 5-HT in aspergilli was investigated by immunofluorescence staining and laser confocal scanning microscopy. The influence of 5-HT on fungal ergosterol content, cell membrane integrity, fungal growth and hyphal elongation was determined. 5-HT was localised in the cytoplasm of Aspergillus spp., as 5-HT fluorescent signals appeared after 30 min at 4°C and in the presence of inhibitors of oxidative phosphorylation. 5-HT treatment of Aspergillus spp. significantly affected ergosterol synthesis, fungal cell membrane integrity and hyphal elongation (P < 0.05). 5-HT treatment for 4 h resulted in a lag of re-growth (post-antifungal effect). In conclusion, our findings suggest that 5-HT affects hyphal growth and diminishes fungal cell membrane integrity.
PMCID: PMC3010239  PMID: 17276041
Serotonin; Aspergillus spp.; Ergosterol; Platelets
4.  Human Platelets Attenuate Aspergillus Species via Granule-Dependent Mechanisms 
The Journal of infectious diseases  2008;198(8):1243-1246.
Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [3H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (P<.05), hyphal elongation (P<.001), colony size, pig-mentation, and 5-HT release (P<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.
PMCID: PMC2980866  PMID: 18752432
5.  Complement as an Endogenous Adjuvant for Dendritic Cell-Mediated Induction of Retrovirus-Specific CTLs 
PLoS Pathogens  2010;6(4):e1000891.
Previous studies have demonstrated the involvement of complement (C) in induction of efficient CTL responses against different viral infections, but the exact role of complement in this process has not been determined. We now show that C opsonization of retroviral particles enhances the ability of dendritic cells (DCs) to induce CTL responses both in vitro and in vivo. DCs exposed to C-opsonized HIV in vitro were able to stimulate CTLs to elicit antiviral activity significantly better than non-opsonized HIV. Furthermore, experiments using the Friend virus (FV) mouse model illustrated that the enhancing role of complement on DC-mediated CTL induction also occurred in vivo. Our results indicate that complement serves as natural adjuvant for DC-induced expansion and differentiation of specific CTLs against retroviruses.
Author Summary
Upon entering the body, HIV initiates immediate responses of the immune system. The complement system constitutes a first line of defense against HIV and bridges innate and adaptive immunity. Thus, in the acute phase of infection, HIV is coated with complement fragments. Following seroconversion, when HIV-specific antibodies appear, antibodies and complement are deposited onto HIV. Dendritic cells (DCs), the most potent antigen-presenting cells, interact with complement- and Ig-opsonized HIV due to expression of the appropriate receptors. Recently, we found differences with respect to infection of DCs with complement- and Ig-opsonized HIV. We have now investigated whether these differently opsonized HIV preparations also modulated the antigen-presenting capacity of DCs. Since it is extremely difficult to investigate the role of HIV-complement interactions on the induction of virus-specific cytotoxic T lymphocytes (CTLs) in vivo, we used the well-characterized Friend virus (FV) mouse model for our in vivo studies. We now show that complement opsonization of retroviral particles enhanced the ability of DCs to induce CTL responses against HIV or FV. Thus, our results indicate that complement serves as natural adjuvant for DC-induced expansion and differentiation of specific CTLs against retroviruses.
PMCID: PMC2861708  PMID: 20442876
6.  Posaconazole Enhances the Activity of Amphotericin B against Hyphae of Zygomycetes In Vitro▿  
The in vitro activity of posaconazole plus amphotericin B against conidia and hyphae of 30 clinical zygomycetes was investigated. The combination of posaconazole with amphotericin B was found to be significantly more synergistic (40%) against hyphae (P < 0.05) than against conidia (10%). Antagonism was not observed.
PMCID: PMC2443899  PMID: 18458135
7.  Susceptibility Testing of Anidulafungin and Voriconazole Alone and in Combination against Conidia and Hyphae of Aspergillus spp. under Hypoxic Conditions▿  
MICs and fractional inhibitory concentrations were evaluated for anidulafungin and voriconazole alone and in combination against conidia and hyphae under hypoxic (1% oxygen-5% CO2-94% nitrogen) conditions against 31 Aspergillus isolates. Anidulafungin exhibited excellent activity against conidia and hyphae of Aspergillus spp. The visual reading of the MIC for anidulafungin was optimal under hypoxic conditions.
PMCID: PMC2346651  PMID: 18347112
8.  Potential Basis for Amphotericin B Resistance in Aspergillus terreus▿  
This study investigated the basis for intrinsic amphotericin B (AMB) resistance in Aspergillus terreus. The ergosterol content, cell wall composition, and lipid peroxidation level had no influence on AMB resistance. The level of catalase production in A. terreus was significantly higher than that in A. fumigatus (P < 0.05). This higher-level production may contribute to AMB resistance in A. terreus since oxidative damage has been implicated in AMB action.
PMCID: PMC2292529  PMID: 18268082
9.  Posaconazole Enhances the Activity of Amphotericin B against Aspergillus Hyphae In Vitro▿  
The MICs and fractional inhibitory concentrations of posaconazole (POS) and voriconazole (VRZ), alone and in combination with amphotericin B (AMB), for the conidia and hyphae of 100 Aspergillus isolates were evaluated. POS-AMB had more synergistic activity against hyphae (75% of isolates) than VRZ-AMB (37%) and significantly more synergistic activity against hyphae than against conidia (12%).
PMCID: PMC1797736  PMID: 17116665
11.  In Vitro Activities of Amphotericin B and Voriconazole against Aleurioconidia from Aspergillus terreus 
This study used aleurioconidia as inoculum and compared the MICs of amphotericin B and voriconazole to those obtained for conidia of 31 Aspergillus terreus strains. For conidia and aleurioconidia, the MIC at which 90% of strains were inhibited was 2.5 μg/ml and 5 μg/ml with amphotericin B and 1 μg/ml and 2 μg/ml with voriconazole.
PMCID: PMC1140531  PMID: 15917567
12.  Diagnosing Invasive Aspergillosis during Antifungal Therapy by PCR Analysis of Blood Samples 
Journal of Clinical Microbiology  2004;42(9):4154-4157.
We evaluated the value of Aspergillus PCR as a tool for diagnosing invasive aspergillosis from whole-blood samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens. A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoalveolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had probable, and 12 had possible invasive Aspergillus infection according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive PCR results for Aspergillus were associated with fatal outcome, as three of six patients died. For patients with probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has been started. Performance of Aspergillus PCR using tissue samples is recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.
PMCID: PMC516315  PMID: 15365004
13.  Chlamydophila abortus Pelvic Inflammatory Disease 
Emerging Infectious Diseases  2003;9(12):1642-1644.
We report the first documented case of an extragestational infection with Chlamydophila abortus in humans. The pathogen was identified in a patient with severe pelvic inflammatory disease (PID) by sequence analysis of the ompA gene. Our findings raise the possibility that Chlamydiaceae other than Chlamydia trachomatis are involved in PID.
PMCID: PMC3034334  PMID: 14720414
Chlamydophila abortus; pelvic inflammatory disease; ompA-gene; stress-response protein; hsp60; intrauterine device; copper
14.  Phagocytosis and Killing of Bacteria by Professional Phagocytes and Dendritic Cells 
Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroy Staphylococcus aureus and Escherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.
PMCID: PMC130096  PMID: 12414745
15.  Mechanism of Human Immunodeficiency Virus-Induced Complement Expression in Astrocytes and Neurons 
Journal of Virology  2002;76(7):3179-3188.
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.
PMCID: PMC136041  PMID: 11884542
16.  Human Immunodeficiency Virus Type 1 Induces Expression of Complement Factors in Human Astrocytes 
Journal of Virology  2001;75(6):2604-2615.
Since the brain is separated from the blood immune system by a tight barrier, the brain-resident complement system may represent a central player in the immune defense of this compartment against human immunodeficiency virus (HIV). Chronic complement activation, however, may participate in HIV-associated neurodegeneration. Since the level of complement factors in the cerebrospinal fluid is known to be elevated in AIDS-associated neurological disorders, we evaluated the effect of HIV type 1 (HIV-1) on the complement synthesis of brain astrocytes. Incubation of different astrocytic cell lines and primary astrocytes with HIV-1 induced a marked upregulation of the expression of the complement factors C2 and C3. The synthesis of other secreted or membrane-bound complement proteins was not found to be altered. The enhancement of C3 production was measured both on the mRNA level and as secreted protein in the culture supernatants. HIV-1 laboratory strains as well as primary isolates were capable of inducing C3 production with varied effectiveness. The usage of viral coreceptors by HIV-1 was proved to be a prerequisite for the upregulation of C3 synthesis, which was modulated by the simultaneous addition of cytokines. The C3 protein which is secreted after incubation of the cells with HIV was shown to be biologically active as it can participate in the complement cascade.
PMCID: PMC115884  PMID: 11222683
17.  Studies of In Vitro Activities of Voriconazole and Itraconazole against Aspergillus Hyphae Using Viability Staining 
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.
PMCID: PMC90249  PMID: 11120954
18.  Detachment of Human Immunodeficiency Virus Type 1 from Germinal Centers by Blocking Complement Receptor Type 2 
Journal of Virology  2000;74(17):7997-8002.
After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC.
PMCID: PMC112331  PMID: 10933708
19.  Simultaneous Infection with Two Drug-Susceptible Mycobacterium tuberculosis Strains in an Immunocompetent Host 
Journal of Clinical Microbiology  1999;37(12):4156-4157.
An important assumption for DNA fingerprinting of Mycobacterium tuberculosis is that patients are infected with only one strain at a time. Nonetheless, we demonstrate a case of simultaneous infection with two drug-susceptible strains of M. tuberculosis in an immunocompetent patient by IS6110 restriction fragment length polymorphism and spoligotyping. Epidemiological data prove the patient's involvement in two independent clusters. Thus, double infections should be suspected with fingerprints showing divergent band intensities.
PMCID: PMC85908  PMID: 10565951
20.  Epstein-Barr Virus Infection of Human Astrocyte Cell Lines 
Journal of Virology  1999;73(9):7722-7733.
Epstein-Barr virus (EBV) is implicated in different central nervous system syndromes. The major cellular receptor for EBV, complement receptor type 2 (CR2) (CD21), is expressed by different astrocyte cell lines and human fetal astrocytes, suggesting their susceptibility to EBV infection. We demonstrated the infection of two astrocyte cell lines, T98 and CB193, at low levels. As infection was mediated by CR2, we used two stable CR2 transfectant astrocyte cell lines (T98CR2 and CB193CR2) to achieve a more efficient infection. We have monitored EBV gene expression for 2 months and observed the transient infection of T98 and T98CR2 cells and persistent infection of CB193 and CB193CR2 cells. The detection of BZLF1, BALF2, and BcLF1 mRNA expression suggests that the lytic cycle is initiated at early time points postinfection. At later time points the pattern of mRNA expressed (EBER1, EBNA1, EBNA2, and LMP1) differs from latency type III in the absence of LMP2A transcription and in the expression of BALF2 and BcLF1 but not BZLF1. A reactivation of the lytic cycle was achieved in CB193CR2 cells by the addition of phorbol esters. These studies identify astrocyte cell lines as targets for EBV infection and suggest that this infection might play a role in the pathology of EBV in the brain.
PMCID: PMC104299  PMID: 10438862
21.  Human Immunodeficiency Virus Type 1 Derived from Cocultures of Immature Dendritic Cells with Autologous T Cells Carries T-Cell-Specific Molecules on Its Surface and Is Highly Infectious 
Journal of Virology  1999;73(4):3449-3454.
During the budding process, human immunodeficiency virus type 1 (HIV-1) acquires cell surface molecules; thus, the viral surface of HIV-1 reflects the antigenic pattern of the host cell. To determine the source of HIV-1 released from cocultures of dendritic cells (DC) with T cells, immature DC (imDC), mature DC (mDC), T cells, and their cocultures were infected with different HIV-1 isolates. The macrophage-tropic HIV-1 isolate Ba-L allowed viral replication in both imDC and mDC, whereas the T-cell-line-tropic primary isolate PI21 replicated in mDC only. By a virus capture assay, HIV-1 was shown to carry a T-cell- or DC-specific cell surface pattern after production by T cells or DC, respectively. Upon cocultivation of HIV-1-pulsed DC with T cells, HIV-1 exclusively displayed a typical T-cell pattern. Additionally, functional analysis revealed that HIV-1 released from imDC–T-cell cocultures was more infectious than HIV-1 derived from mDC–T-cell cocultures and from cultures of DC, T cells, or peripheral blood mononuclear cells alone. Therefore, we conclude that the interaction of HIV-1-pulsed imDC with T cells in vivo might generate highly infectious virus which primarily originates from T cells.
PMCID: PMC104111  PMID: 10074201
22.  Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages 
Journal of Virology  1998;72(8):6671-6677.
Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4+ T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4+ cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83− DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83+ DC (maDC); this finding is in contrast to data previously obtained with CD4+ T cells. Third, maturation from imDC to maDC efficiently silenced expression of β2-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.
PMCID: PMC109863  PMID: 9658114
Cultured human lymphoid cells RPMI 8866 at different stages of their growth cycle vary in their susceptibility to lysis by rabbit, human, and guinea pig complement activated by HL-A antibodies or heterologous antibodies directed to membrane antigens; cells in G1 phase are the least sensitive to lysis. To investigate the cause of differential susceptibility of cells RPMI 8866 to lysis, the expression of HL-A determinants and the ability of cells to react with complement were investigated. No change was detected in the density of HL-A antigens on RPMI 8866 cells in synchronous growth as determined by quantitative microabsorption assays, isotopic antiglobulin tests and yields of soluble HL-A antigens. Cells did not vary during the growth cycle in their ability to interact with complement components and in their capacity to activate the complement system through the classical or alternate pathway. These data suggest that variability in lytic susceptibility is due to changes in the structure of the cell membrane or in its ability to repair complement induced damage at certain intervals during the cell cycle. Therefore, this cell line constitutes a useful model to investigate the final steps of the cytolytic reaction.
PMCID: PMC2139582  PMID: 4527075

Results 1-23 (23)