Candida: Platelet Interaction and Platelet Activity in vitro.

Over the last 2 decades, platelets have been recognized as versatile players of innate immunity. The interaction of platelets with fungal pathogens and subsequent processes may critically influence the clinical outcome of invasive mycoses. Since the role of platelets in Candida infections is poorly characterized and controversially discussed, we studied interactions of human platelets with yeast cells, (pseudo-)hyphae, biofilms and secretory products of human pathogenic Candida species applying platelet rich plasma and a whole blood model. Incubation of Candida with platelets resulted in moderate mutual interaction with some variation between different species. The rate of platelets binding to -Candida (pseudo-) hyphae and candidal biofilm was comparably low as that to the yeast form. Candida-derived secretory products did not affect platelet activity - neither stimulatory nor inhibitory. The small subset of platelets that bound to Candida morphotypes was consequently activated. However, this did not result in reduced growth or viability of the different Candida species. A whole blood model simulating in vivo conditions confirmed platelet activation in the subpopulation of Candida-bound platelets. Thus, the inability of platelets to efficiently react on Candida presence might favor fungal survival in the blood and contribute to high morbidity of Candida sepsis.

Tolerance towards gentamicin is a function of nutrient concentration in biofilms of patient-isolated Staphylococcus epidermidis.

Staphylococcus epidermidis is a biofilm-forming bacterial strain that can cause major problems as an agent of nosocomial infections. Bacteria in biofilms are shielded from the environment and can survive high doses of antibiotics. We here test the antibiotic susceptibility of Staphylococcus epidermidis to rising gentamicin concentrations in optimal growth conditions as used in routine bacteriology laboratories with low nutrient situations as suggested to be found in clinical situations. We found that gentamicin-resistant Staphylococcus epidermidis biofilms survived in the absence of external nutrient supply in PBS. While addition of gentamicin sulfate significantly reduced the pH value of all used media and solutions, this acidification did not alter survival of bacteria in the biofilm. We found a statistically significant and dose-dependent reduction of survival in low nutrient situations using gentamicin sulfate in three out of four patient isolates of Staphylococcus epidermidis which have been tested to be gentamicin-resistant under optimal growth conditions. Supporting the original profiling, survival in full media under the same antibiotic dosages was not significantly reduced. Our data here show that antibiotic resistance is a function of the provided nutrient concentration. Antibiotic resistance profiling should consider variations in nutrient availability.

Lyophilized allogeneic bone tissue as an antibiotic carrier.

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.

Changes in the Chemical Quality of Bone Grafts During Clinical Preparation Detected by Raman Spectroscopy.

METHODOLOGY: We determined the content of amide I, amide III, PO4, CO3, and CH2 in samples of fresh bone, bone frozen at -80°C thawed once, bone after two freeze-thaw cycles, and chemically cleaned bone chips. A total of 750 Raman spectra were collected per sample group and the derived quantitative values compared statistically by one-way ANOVA. RESULTS: We found statistically significant differences between the investigated sample groups differing in their treatment already after one freeze-thaw cycle and as well after multiple freeze-thaw cycles, and/or chemical cleaning. Chemical cleaning decreased the content of all measured components compared to the fresh sample as detected by Raman spectroscopy. We further used the derived data to calculate the mineral to matrix ratios for each sample group. DISCUSSION: Our data indicate that significant changes of the chemical quality and mineral to matrix ratio occur during freeze-thawing and chemical cleaning. At the same time, this study highlights the importance of sampling and testing at multiple locations for reliable predictions of the chemical composition. We think that it is very desirable to test the quality of bone graft material before transfer to a recipient; this might ultimately help define parameters to choose the best graft for the patient. It is also important to highlight that this is a preliminary study, which shows the importance of detecting changes in the chemical quality of bone grafts before transfer to the patient.

Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis.

N-chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT.

Effect of storage temperature and antibiotic impregnation on the quantity of bone morphogenetic protein seven in human bone grafts.

PURPOSE: The aim of this study was to quantify the amount of bone morphogenic protein 7 (BMP-7) in bone samples in different storage and treatment conditions used in bone banks and thereby evaluate the benefit of this test as a routine measure before bone grafting. METHODS: Fresh as well as frozen bone chips, each with and without antibiotic impregnation, were screened for their BMP-7 content. Human bone chips were produced from femoral heads of two female donors who had undergone total hip replacement surgery. The amount of BMP-7 was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) test. RESULTS: There were no significant differences between groups in samples obtained from the first femoral head. Bone-chip samples derived from the second femoral head showed significant differences between groups. The actual amount of these differences was small and most likely biologically irrelevant. It is important to note that there was a significant difference between groups when comparing both femoral heads, reflecting donor-to-donor variability. CONCLUSION: ELISA testing for BMP-7 as a qualitative measurement of bone grafts should be considered a routine quality-control test for bone banks.

Bactericidal activity of N-chlorotaurine against biofilm-forming bacteria grown on metal disks.

Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.

Inflammatory status of transmigrating primary rat monocytes in a novel perfusion model simulating blood flow.

It remains unclear whether monocyte infiltration plays a protective or detrimental role in neurodegenerative disease. The present study characterizes the inflammatory status of primary monocytes in a novel in vitro perfusion model. Monocytes under perfusion do not undergo elevated cell death. However, perfusion does lead to altered morphology, which can be counteracted by anti-inflammatory drugs. Functional studies indicate that cytokine levels are significantly reduced in perfusion compared to stationary conditions and enhanced with brain slices or capillary endothelial cells. Understanding monocyte properties could lead to refined treatment and new ways to interfere with inflammation in diseased brains.

Targeting viral antigens to CD11c on dendritic cells induces retrovirus-specific T cell responses.

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.

Negative impact of IFN-γ on early host immune responses to retroviral infection.

The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8+ T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response.

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.

Effects of acute and chronic murine norovirus infections on immune responses and recovery from Friend retrovirus infection.

Murine norovirus (MNV) is a highly infectious but generally nonpathogenic agent that is commonly found in research mouse colonies in both North America and Europe. In the present study, the effects of acute and chronic infections with MNV on immune responses and recovery from concurrent Friend virus (FV) infections were investigated. No significant differences in T-cell or NK-cell responses, FV-neutralizing antibody responses, or long-term recovery from FV infection were observed. We conclude that concurrent MNV infections had no major impacts on FV infections.

Lactate dehydrogenase-elevating virus induces systemic lymphocyte activation via TLR7-dependent IFNalpha responses by plasmacytoid dendritic cells.

BACKGROUND: Lactate dehydrogenase-elevating virus (LDV) is a natural infectious agent of mice. Like several other viruses, LDV causes widespread and very rapid but transient activation of both B cells and T cells in lymphoid tissues and the blood. The mechanism of this activation has not been fully described and is the focus of the current studies. PRINCIPAL FINDINGS: A known inducer of early lymphocyte activation is IFNalpha, a cytokine strongly induced by LDV infection. Neutralization of IFNalpha in the plasma from infected mice ablated its ability to activate lymphocytes in vitro. Since the primary source of virus-induced IFNalpha in vivo is often plasmacytoid dendritic cells (pDC's), we depleted these cells prior to LDV infection and tested for lymphocyte activation. Depletion of pDC's in vivo eradicated both the LDV-induced IFNalpha response and lymphocyte activation. A primary receptor in pDC's for single stranded RNA viruses such as LDV is the toll-like receptor 7 (TLR7) pattern recognition receptor. Infection of TLR7-knockout mice revealed that both the IFNalpha response and lymphocyte activation were dependent on TLR7 signaling in vivo. Interestingly, virus levels in both TLR7 knockout mice and pDC-depleted mice were indistinguishable from controls indicating that LDV is largely resistant to the systemic IFNalpha response. CONCLUSION: Results indicate that LDV-induced activation of lymphocytes is due to recognition of LDV nucleic acid by TLR7 pattern recognition receptors in pDC's that respond with a lymphocyte-inducing IFNalpha response.

Potent human immunodeficiency virus-neutralizing and complement lysis activities of antibodies are not obligatorily linked.

To evaluate the contribution of complement-mediated lysis to the in vivo activities of neutralizing antibodies, we analyzed the influence of complement activation on treatment success in a recent passive immunization trial with the neutralizing monoclonal antibodies 2G12, 2F5, and 4E10. Administration of monoclonal antibodies led to an immediate, high activation of the complement system even in the absence of viremia in the 14 participating human immunodeficiency virus-infected individuals. Lysis activity measured in patient plasma increased during passive immunization; however, the increases were modest and only partially attributable to the administration of antibodies. We found that unlike neutralization activity, lysis activity was not associated with treatment success in this trial. Compared to complement lysis mounted by the polyclonal antibody response in vivo, monoclonal antibodies were weak inducers of this activity, suggesting that polyclonal responses are more effective in reaching the required threshold of complement activation. Importantly, strong neutralization activity of the monoclonal antibodies did not predict complement lysis activity against patient and reference viruses, suggesting that these activities are not linked. In summary, our data support the notion that the in vivo activities of 2G12, 2F5, and 4E10 are likely due to direct neutralization or Fc receptor-mediated mechanisms such as phagocytosis and antibody-dependent cellular cytotoxicity.

Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells.

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells.

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.

C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells.

HIV directly activates the complement cascade and is, therefore, opsonized with C3-cleavage products in vivo. This cloud of C3 fragments on the viral surface may impair the interaction of the HIV envelope glycoproteins gp120/gp41 with C-type lectins expressed on immature dendritic cells (iDC). Therefore, we determined the accessibility of gp120 after opsonization and compared the interaction of DC with non-opsonized or complement-opsonized HIV. The recognition of native gp120 was drastically impaired when the virus was covered by complement. Independent of opsonization, similar amounts of HIV bound to DC. The interaction of iDC and the infection of DC-PBL co-cultures with non-opsonized virus was significantly reduced by mannan and antibodies which inhibit the ICAM-1-CR3 interaction. The binding of opsonized virus to iDC was reduced by an anti-CR3-antibody, which interferes with the binding of C3 fragments, but was not affected by mannan. Complement enhanced the HIV infection of DC and DC-PBL co-cultures significantly. Mannan did not inhibit the complement-dependent enhancement of infection. Thus, non-opsonized and opsonized HIV interacted with iDC, although the binding mechanisms seemed to differ. As HIV is opsonized in vivo, the C-type lectin-independent interaction of opsonized viruses with iDC has to be taken into account.

Complement-opsonized HIV: the free rider on its way to infection.

The complement system (C) is one of the main humoral components of innate immunity. Three major tasks of C against invading pathogens are: (i) lysis of pathogens by the formation of the membrane attack complex (MAC); (ii) opsonization of pathogens with complement fragments to favor phagocytosis; and (iii) attraction of inflammatory cells by chemotaxis. Like other particles, HIV activates C and becomes opsonized. To escape complement-mediated lysis, HIV has adopted various properties, which include the acquisition of HIV-associated molecules (HAMs) belonging to the family of complement regulators, such as CD46, CD55, CD59, and the interaction with humoral regulatory factors like factor H (fH). Opsonized virus may bind to complement receptor positive cells to infect them more efficiently or to remain bound on the surface of such cells. In the latter case HIV can be transmitted to cells susceptible for infection. This review discusses several aspects of C-HIV interactions and provides a model for the dynamics of this process.

Passive immunization with the anti-HIV-1 human monoclonal antibody (hMAb) 4E10 and the hMAb combination 4E10/2F5/2G12.

OBJECTIVES: To study the safety, immunogenicity and pharmacokinetics of the human monoclonal antibody (hMAb) 4E10 alone and in combination with the hMAbs 2F5 and 2G12 in HIV-1-infected persons. MATERIALS AND METHODS: Eight healthy volunteers with > or =350 CD4 cells/mm3 and < or =100 000 HIV-1 RNA copies/mL were enrolled, seven finished the study. A single 4E10 infusion was administered on day 0, followed by three doses of the hMAb combination 4E10/2F5/2G12 on days 7, 14 and 21 (total amount 8.5 g). Safety was assessed by physical examination, blood chemistry, complete blood cell count and recording of adverse events. 4E10, 2F5 and 2G12 plasma levels were determined before and at the end of each infusion and during the 7 week follow-up. RESULTS: No drug-related adverse events were observed throughout the study. The median plasma concentrations immediately after the first infusion were 371, 253 and 139 microg/mL for 4E10, 2F5 and 2G12. Multiple infusions resulted in maximum plasma concentrations of 407, 294 and 210 microg/mL for 4E10, 2F5, and 2G12, respectively. The median elimination half-lives (t1/2beta) were 6.6, 3.2 and 14.1 days for 4E10, 2F5 and 2G12. A low level antibody response against 2G12 was found in two patients. CONCLUSION: This Phase I trial showed that the hMAb 4E10 can be safely administered, both alone and in combination with 2F5 and 2G12 to HIV-1-infected patients.