Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes.

BACKGROUND: Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8(+) T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8(+) T-cell responses. OBJECTIVE: We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)-mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. METHODS: Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8(+) T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4(+) T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. RESULTS: We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8(+) T-cell response compared with the earlier described efficient CD8(+) T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8(+) T-cell clones. CONCLUSION: Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs.

Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific CTLs.

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.

Candida albicans Hgt1p, a multifunctional evasion molecule: complement inhibitor, CR3 analogue, and human immunodeficiency virus-binding molecule.

BACKGROUND: The complement system is tightly controlled by several regulators. Two of these, factor H (FH) and C4b-binding protein (C4BP), can be acquired by pathogens conveying resistance to complement attack. The aim of the study was to characterize the FH binding molecule of Candida albicans, a potentially life-threatening yeast. METHODS: The gene coding for this molecule was identified by probing an expression library and homozygous deletion mutants of the respective gene were constructed. Binding and functional assays were undertaken to compare wild-type and knockout strains. RESULTS: The high-affinity glucose transporter 1 (CaHgt1p) was identified as an FH-binding molecule. Homozygous hgt1Δ/Δ deletion mutants, but not the restored strain in which HGT1 was reintegrated, showed a decreased binding of FH and even of C4BP, demonstrating its function as an FH- and C4BP-binding protein. This led to an enhanced terminal complement complex deposition after incubation with human serum; CaHgt1p thus functions as complement inhibitor. hgt1Δ/Δ mutants failed to form rosettes with complement-coated sheep erythrocytes, and show reduced binding to HIV-gp160, implying that a complement receptor 3 (CR3) moiety, known as fungal HIV binding molecule is lacking. CONCLUSIONS: CaHgt1p is a multifunctional evasion molecule, as complement inhibitor, CR3 analogue and HIV receptor.

Shiga toxin activates complement and binds factor H: evidence for an active role of complement in hemolytic uremic syndrome.

Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6-8 and SCRs18-20, but not to SCRs16-17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Deltanef and challenged with pathogenic SIVmac251.

BACKGROUND: To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live-attenuated SIVmac239Deltanef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. RESULTS: Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post-challenge. Concomitantly, viremia correlated inversely with SIV-specific IgG, complement-mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10(6) PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). CONCLUSIONS: As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.

Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups.

Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups--considered to be highly pathogenic--were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype alpha was found, whereas in most of the other non-O157 serogroups, subtypes beta and gamma were found. Subtype delta was not detected in our strain collection. Regarding the espP subtypes, only subtype alpha, but not beta and gamma, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype alpha, is associated with highly pathogenic serogroups.

Complement induction and complement evasion in patients with cerebral aspergillosis.

Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the "sealing off" in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches.

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% CO(2)-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.

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.

Basidiomycete metabolites attenuate virulence properties of Candida albicans in vitro.

Secreted aspartic proteases (Saps) represent an important virulence factor facilitating fungal adherence. Several protease inhibitors (PIs), including HIV PIs, have been shown to reduce Candida adhesion. The aim of this study was to ascertain whether or not the recently discovered PIs Aureoquinone and Laccaridiones A and B, isolated from Basidiomycete cultures, or Bestatin, act as Sap-inhibitors and/or inhibitors of fungal adhesion. Drug effects on candidial Sap-production were determined by Sap-ELISA. Control tubes, in the absence of drug, served as positive controls, while tubes excluding both drug and proteinase induction medium were used as negative controls. Aureoquinone as well as Laccaridiones A and B, but not Bestatin, significantly inhibited Candida albicans adhesion to both epithelial and endothelial cells in a dose dependent manner and also reduced Sap-release (effects were not because of a direct interaction of the Basidiomycete metabolites with secreted Saps). Laccaridione B was consistently found to be the most effective PI tested. Interestingly, these drugs are neither fungistatic nor fungicidal at the concentrations applied. Laccaridione B may represent a promising novel type of antimycotic drug--targeting virulence factors without killing the yeast.

Prevalence of Shiga toxin-, intimin- and haemolysin genes in Escherichia coli isolates from drinking water supplies in a rural area of Austria.

Literature harbours several reports of potable water-associated outbreaks. We studied the prevalence of Shiga toxin- (stx1/2), intimin- (eae) and haemolysin (hlyA) genes in Escherichia coli isolates from drinking water of private and public water supplies in a rural area of Upper Austria; 2633 water samples were gained between November 2000 and December 2003. Two hundred and eighty of these water samples were positive for E. coli (10.6%). Of these, 101 samples were drawn from drilled wells (36%), 96 from dug wells (34%), 61 from springs (22%) and 22 from water supplies without available information on technical details (8%); 141 of the samples were from public water supplies, 139 from private water supplies. Eleven of the E. coli isolates were found to be positive for one of the investigated virulence genes (3.9%): one isolate yielded stx2, seven eae, and three isolates had hlyA. The presence of these genes underlines the importance of control of water quality in public and also private water supplies.

Immune response to retroviral infections of the brain.

Various neurological manifestations of retroviral infections have been reported, including peripheral neuropathy, encephalopathy and neuronal degeneration. After penetration into the central nervous system (CNS) the invading retroviruses meet a unique immunological situation that differs significantly from that in the periphery. Due to the blood-brain barrier with its general access restrictions peripheral T-cells, monocytes and B-cells are only "guests" in the brain; instead the immune balance is shifted in favour of the local innate immunity with microglia, astrocytes, cytokines/chemokines and complement forming the dominating defence network. The present article focuses on the most important retroviral infections and highlights the immunological aspects of the neuropathogenesis induced by selected retroviruses. These aspects include: (i) local and infiltrated immune cells as targets of retroviral infection; (ii) stimulation of the cerebral immunity network by retroviruses and subsequent steps of antiviral defence; and (iii) immune activation products as potential contributors to neural damage in the sensitive brain tissue.

A single vaccination with attenuated SIVmac 239 via the tonsillar route confers partial protection against challenge with SIVmac 251 at a distant mucosal site, the rectum.

Elucidating the mechanisms that protect monkeys previously immunized with attenuated SIV (SIVDeltanef) against challenge infection with pathogenic virus may reveal new strategies for the development of an effective HIV vaccine. Here we show that a single atraumatic application of SIVDeltanef to the tonsils of four rhesus macaques conferred protection against SIVmac251 applied intrarectally 26 weeks later. While this protection was not complete, i.e., challenge virus could be isolated from all immunized animals, it was reflected by significantly lower viral loads in the blood (weeks 2-16 after challenge, p < 0.01) and considerably lower loads in lymphoid organs, and more stable peripheral CD4 counts in a proportion of the immunized animals as compared to four non-immunized, SIVmac251-infected control monkeys. SIV-specific humoral as well as systemic and mucosal T cell responses were detected in the immunized animals, but there was no correlation between their magnitude of expression and the level of protection. Analyses of leukocyte subsets in these animals at necropsy (24 weeks after challenge) did not reveal a significantly enhanced proportion of gamma/delta T cells in the tissues of protected monkeys. Therefore, tonsillar application of attenuated SIV induces protection in some animals against a superinfection with wild-type SIV distant at a distant mucosal site.

Variability in tellurite resistance and the ter gene cluster among Shiga toxin-producing Escherichia coli isolated from humans, animals and food.

Tellurite-containing media are widely used for the screening and isolation of Shiga toxin-producing Escherichia coli (STEC) O157:H7, but tellurite resistance among non-O157 STEC is poorly characterized. Therefore, we investigated 202 STEC strains representing 61 different serotypes from humans, animals or food for the presence of ter genes by PCR and their correlation with tellurite resistance, by assessing growth on cefixime-tellurite sorbitol MacConkey agar. All strains were screened for terC, terE and terF as markers for the ter gene cluster. Of the 202 strains, 127 contained terC and terE and were tellurite-resistant, but only 121 of these also contained terF. All 72 non-sorbitol-fermenting O157:H7 and O157:NM (non-motile) strains contained terC, terE and terF and expressed tellurite resistance. In contrast, all eight sorbitol-fermenting STEC O157:NM were terC-, terE- and terF-negative and tellurite-sensitive. Among non-O157 STEC, terC, terE and terF were found in all seven O145:NM, four O111:H8/NM, 17 of 18 O26:H11/NM and in 21 strains of 14 other serotypes. The strong correlation between the presence of ter genes and the ability to grow on tellurite-containing media suggest that the ter genes encode tellurite resistance in the vast majority of these strains. The presence of the ter gene cluster was significantly (P<0.00001) associated with the presence of eae genes. We conclude that the use of tellurite-containing media in screening for STEC will allow the detection of STEC O26, O111, O145 and non-sorbitol-fermenting O157, but most strains (in this study 74.3%) from other serotypes will be missed.

Comparison of an immunochromatographic rapid test with enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Shiga toxins from human stool samples.

Rapid detection of enterohemorrhagic Escherichia coli is important for its successful treatment. We have evaluated the immunochromatographic Duopath Verotoxin-test for detection of Shiga toxins, in comparison with enzyme-linked immunosorbent assay and polymerase chain reaction, on 240 clinical human stool samples. The Duopath-test showed a lower sensitivity and specificity.

The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome.

Shiga toxins (Stx) are believed to play a key role in the pathogenesis of diseases caused by Stx-producing Escherichia coli (STEC), including the potentially life-threatening hemolytic uremic syndrome (HUS). In this study, 201 STEC strains collected from patients and environmental sources were investigated with regard to the stx genotypes and pathogenicity. The stx(2) and stx(2c) alleles were associated with high virulence and the ability to cause HUS, whereas stx(2d), stx(2e,)stx(1), and stx(1c) occurred in milder or asymptomatic infections. Quantification of Stx using an enzyme immunoassay and the Vero cell cytotoxicity assay showed no significant differences between the strains associated with HUS and those causing milder diseases. We hypothesize that the stx genotype and perhaps other yet unknown virulence factors rather than the amount of Stx or the in vitro cytotoxicity correlate with the development of HUS.

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 30min at 4 degrees 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 4h 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.

Subtype and genotypic resistance analysis of HIV-1 infected patients in Austria.

BACKGROUND: Analysis of HIV-1 subtypes and genotypic resistance have been shown to be relevant for epidemiologic and therapeutic studies or for vaccine development. In Europe, the majority of HIV-1 isolates belong to subtype B. Due to migration an increasing incidence for additional subtypes and complex recombinant forms are expected. OBJECTIVES AND STUDY DESIGN: To evaluate the prevalence of HIV-1 subtypes in Austria, 188 plasma samples of treatment experienced patients were investigated. For phylogenetic analysis protease and reverse transcriptase genes were amplified and sequenced. Subtypes were determined by comparing reference sequences. For genotypic resistance determination, the Resistance-Algorithm-Comparison from Stanford University was used. RESULTS: Non-B subtypes were found in 20.2% of all patients with a dominant prevalence (50%) in the Southern provinces of Austria. With 85% CRF01_AE and CRF02_AG are the predominant circulating recombinant forms in Austria. When resistance mutations were analyzed, 57.4% of all patients were susceptible to all three groups of antiretroviral drugs, whereas in 12.2% resistance against all three classes of antiretroviral drugs was found. CONCLUSION: HIV-1 subtype B is still dominant in major parts of Austria. However, a significantly increasing percentage of non-B subtypes and recombinant forms are observed in the Southern provinces.

Cytolethal distending toxins in Shiga toxin-producing Escherichia coli: alleles, serotype distribution and biological effects.

To assess the prevalence of cytolethal distending toxin (CDT) among Shiga toxin-producing Escherichia coli (STEC), 202 STEC strains were investigated using PCRs targeting various cdt alleles (cdt-I to cdt-V). Seven of the 202 strains contained cdt-III and an additional seven contained cdt-V. All 14 cdt-positive strains produced biologically active CDT, as demonstrated by a progressive distension of cultured Chinese hamster ovary cells. The CDT-positive STEC belonged to eight different serotypes, including sorbitol-fermenting O157 : NM (non-motile). The data demonstrate that CDT is present in some STEC serotypes only. However, more studies are required to evaluate whether CDT presence is associated with severe disease.

Prevalence, structure and expression of urease genes in Shiga toxin-producing Escherichia coli from humans and the environment.

A component of the ure gene cluster in E. coli, ureC, encodes a subunit of urease. We have investigated the distribution of ureC in 202 Shiga toxin-producing E. coli (STEC) strains from Austria belonging to 61 different serotypes. These strains were of human (n=150), animal (n=38), and food (n=14) origin. ureC was present in all 72 E. coli O157:H7 and O157:NM (non-motile) strains, as well as in all 29 strains of serotypes O26:H11/NM, O111:H8/NM and O145:NM. In contrast, none of eight sorbitol-fermenting E. coli O157:NM were ureC-positive. ureC occurred significantly more frequently among STEC that carry eae (113 of 132; 85.6%) than among eae-negative STEC strains (four of 70; 5.7%; p<0.0001). However, only 4 (2%) of the 202 strains (3.4% of ureC positive strains) expressed urease activity. There was no significant association (p=0.56) between urease expression and the source of the isolates (humans vs. animals). Nucleotide sequence analysis of PCR amplicons derived from all seven genes of the ure cluster in STEC of 10 different serotypes demonstrated a high degree of homology (>or=99%), indicating a recent acquisition of not necessarily expressed ure genes.