Neutrophils Turn Plasma Proteins into Weapons against HIV-1.

As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms.

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.

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.

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.

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.

Potential antifungal effects of human platelets against zygomycetes in vitro.

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.

Induction of complement-mediated lysis of HIV-1 by a combination of HIV-specific and HLA allotype-specific antibodies.

HLA-specific antibodies generated by allo-immunization are supposed to be involved in the control of HIV infections by both the neutralizing capacity of HLA-specific antibodies (Abs) and HLA-specific Ab-dependent complement-mediated lysis (CML). We further characterized CML of HIV primary isolates induced by HLA-specific Abs. Although HIV-specific and HLA allo-type specific Abs induced only weak CML of HIV primary isolates, several combinations of HLA allo-type specific Abs with HIV-specific Abs could enhance CML significantly. Nevertheless, certain HLA-specific Abs did not improve but even inhibit CML of HIV, although the corresponding HLA molecules were present. Thus, our results emphasize a possible limitation of allo-immunization as a potential approach to induce protective immunity against HIV.

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.

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.

Human immunodeficiency virus type-1 (HIV-1) Pr55gag virus-like particles are potent activators of human monocytes.

Human immunodeficiency virus type-1 (HIV-1) Pr55(Gag) virus-like particles (VLP) represent an interesting HIV vaccine component since they stimulate strong humoral and cellular immune responses. We demonstrated that VLP expressed by recombinant baculoviruses activate human PBMC to release pro-inflammatory (lL-6, TNF-alpha), anti-inflammatory (IL-10) and Th1-polarizing (IFN-gamma) cytokines as well as GM-CSF and MIP-1alpha in a dose-and time-dependent manner. Herein, residual baculoviruses within the VLP preparations showed no or minor effects. Monocytes could be identified as a main target for VLP to induce cytokine production. Furthermore, VLP-induced monocyte activation was shown by upregulation of molecules involved in antigen presentation (MHC II, CD80, CD86) and cell adhesion (CD54). Exposure of VLP to serum inactivates its capacity to stimulate cytokine production. In summary, these investigations establish VLP as strong activators of PBMC and monocytes therein, potently enhancing their functionality and potency to promote an efficient immune response. This capacity makes VLP an interesting component of combination vaccines.

Human platelets attenuate Aspergillus species via granule-dependent mechanisms.

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 [(3)H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (p<.05), hyphal elongation (p<.001), colony size, pigmentation, 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.

[WHO Polio Eradication Programme:Status quo and implementation in Austria]. / WHO-Eradikationsprogramm für Poliomyelitis: Status quo und Umsetzung in Osterreich.

In 1988 the 41(th) World Health Assembly declared polio to be worldwide eradicated until the year 2000. Although this ambitious aim could not be reached completely, the yearly worldwide incidence was reduced by 99% and three WHO-Regions were declared polio-free (Americans, West Pacific, Europe). To maintain this status the following measures have to be carried out: polio vaccination, enterovirus surveillance, AFP-surveillance, quality control of laboratories and notification of labs keeping stocks of polio wildvirus. Especially after the Second World War Austria faced severe polio epidemics and thus general and free of charge polio vaccination for children and young adults up to 21 years was started in winter 1961/62 by the Austrian Ministry of Health (MoH). Immediately the yearly incidence dropped from 3.65/100.000 (n = 292) in 1961 to 0.1/100.000 (n = 8) in 1962. Since 1998 all mandatory national measures according the WHO polio eradication programme have been performed. Despite the worldwide success of the programme there are currently still four countries with endemic polio and since November 2006 eleven further countries have faced epidemics due to imported cases. Therefore the 60(th) World Health Assembly in 2007 again pointed to the threat of failing worldwide polio eradication. Currently the Global Polio Eradication Initiative (GPEI) works intensively with the concerned countries to fight against this development.

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.

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.

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.