Non-contact induction heating and SAAP-148 eliminate persisters within MRSA biofilms mimicking a metal implant infection.

Implant-associated infections are the primary cause of complications following orthopaedic surgery. Due to biofilm and persister formation, current treatments, i.e. surgical debridement followed by antibiotics, often fail. There is an urgent need for alternative strategies to combat such infections. Therefore, the present study investigated the effects of non-contact induction heating (NCIH), the antimicrobial peptide SAAP-148 and combinations thereof on bacterial counts in 7 d mature biofilms and in persister-enriched biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on titanium-aluminium-niobium (TAN) discs. Enrichment of persisters was achieved by daily exposure of mature biofilms to high doses of rifampicin and ciprofloxacin for 3 consecutive days. To heat up the TAN discs, a miniaturised induction heater was built and successfully validated. Using this apparatus, NCIH resulting in surface temperatures up to 85 °C eradicated all the bacteria in immature biofilms but not in mature biofilms, whereas persisters were already eliminated at surface temperatures ≥ 70 °C. SAAP-148 at concentrations > 25.6 µmol/L reduced the persister counts in antibiotics-exposed, mature biofilms. As surface temperatures > 60 °C can have detrimental effects on the surrounding tissues, the maximum temperature of NCIH used in combination with SAAP-148 on persisters was set to 60 °C. Results revealed that this combination was slightly more effective than the peptide or NCIH alone in eliminating biofilm-embedded persisters. NCIH and SAAP-148 can be applied both invasively and non-invasively in various treatment scenarios. Together, combinations of NCIH and SAAP-148 might be a promising treatment strategy to combat metal-implant-associated infections.

The licorice pentacyclic triterpenoid component 18ß-glycyrrhetinic acid enhances the activity of antibiotics against strains of methicillin-resistant Staphylococcus aureus.

This study aimed to identify compounds that enhance the activity of current antibiotics against multidrug-resistant bacteria. Screening of a 350+ compound proprietary small molecules library revealed that the Glycyrrhiza glabra (licorice)-derived triterpenoid 18ß-glycyrrhetinic acid (18ß-GA) potentiated the antibacterial activity of certain antibiotics against Staphylococcus aureus. Here, we evaluated the ability of pentacyclic triterpenoids to potentiate the activity of antibiotics against strains of methicillin-resistant S. aureus (MRSA). Checkerboard assays were used to assess the minimum inhibitory concentration (MIC) of tobramycin and ten pentacyclic triterpenoids against S. aureus. The effect of 18ß-GA on the MIC of different antibiotics against MRSA was also determined in an in vitro airway MRSA infection model. 18ß-GA enhanced the bactericidal activity of the aminoglycosides tobramycin, gentamicin and amikacin, and of polymyxin B against two MRSA strains, reducing the MIC of these antibiotics 32-64-fold [fractional inhibitory concentration index (FICI) of 0.12-0.13]. Other ß-amyrin triterpenoids and α-amyrin triterpenoids did not exert such synergistic effects. 18ß-GA did not enhance the activity of antibiotics from other structural classes against the MRSA strains. In an air-exposed airway epithelial cell culture, 18ß-GA enhanced the bactericidal activity of tobramycin and polymyxin B against the MRSA strain. These data demonstrate the potential of 18ß-GA to synergise with certain types of antibiotics to eliminate strains of MRSA.

Reduced filaggrin expression is accompanied by increased Staphylococcus aureus colonization of epidermal skin models.

BACKGROUND: Atopic dermatitis is an inflammatory skin disease that is characterized by a reduced skin barrier function, reduced filaggrin (FLG) expression as well as increased colonization by Staphylococcus aureus. OBJECTIVE: This study focused on the possible involvement of FLG in epidermal colonization by S. aureus and/or whether it affects the epidermal defence mechanisms, including the expression of antimicrobial peptides (AMPs) and enzymes involved in stratum corneum barrier lipid synthesis. Furthermore, IL-31 has been shown to reduce FLG expression, but its effects on bacterial colonization and on the expression of AMPs and enzymes involved in the barrier lipid synthesis are not known. MATERIAL AND METHODS: We established N/TERT-based epidermal models (NEMs), after FLG knockdown (FLG-KD) and/or cultured with IL-31, that were colonized with S. aureus for 24 h. RESULTS: Both FLG-KD and IL-31 supplementation resulted in significantly increased epidermal S. aureus colonization, as well as in an up-regulation of S. aureus-induced IL-8 expression. IL-31, but not FLG-KD, prevented S. aureus-induced up-regulation of mRNA expression for the AMPs human ß-defensin 2 and -3 and RNAse7, whereas psoriasin expression remained unchanged. Furthermore, the S. aureus colonization induced changes in mRNA expression of ELOVL4 was not affected by FLG-KD, but was blocked by IL-31. Expression of SCD-1 and Gcase mRNA was reduced by IL-31, but not by FLG-KD. CONCLUSION: This study shows that NEMs, with FLG-KD and/or cultured in the presence of IL-31, mimic the skin of patients with atopic dermatitis in several aspects, including enhanced bacterial colonization, increased inflammatory and reduced protective responses.

Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.

The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

Rapid identification and antimicrobial susceptibility profiling of Gram-positive cocci in blood cultures with the Vitek 2 system.

Rapid identification and antimicrobial susceptibility profiling of the bacteria in blood cultures can result in clinical and financial benefits. Addition of saponin to the fluid from blood culture bottles promotes the recovery of the bacteria and thus may shorten the turnaround time of the microbiological analyses. In this study we compared the identification and susceptibility profiles of saponin-treated and untreated (standard method) blood cultures monomicrobial for Gram-positive cocci using Vitek 2. We concordantly identified 49 (89%) of 55 monobacterial cultures using the results with the standard method as reference. Complete categorical agreement between the susceptibility profiles with the new and the standard method was found for 26 (53%) of 49 isolates, while discrepancies were seen for 23 (47%) cultures. E-tests indicated that the new method resulted in a correct susceptibility profile for 8 (35%) of these 23 blood cultures. Therefore, 34 (69%) of 49 cultures showed a concordant/correct susceptibility profile for all antimicrobials with an overall error rate of 2.3%. Thus, addition of saponin to the fluid from blood culture bottles of the Bactec 9240 leads to the rapid (results available >or=12 hours earlier) and reliable identification and susceptibility profiling of Gram-positive cocci in blood cultures with Vitek 2.

Maggot secretions suppress pro-inflammatory responses of human monocytes through elevation of cyclic AMP.

AIMS/HYPOTHESIS: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As monocytes may contribute to the excessive inflammatory responses in such wounds, this study focussed on the effects of maggot secretions on the pro-inflammatory activities of these cells. METHODS: Freshly isolated monocytes were incubated with a range of secretions for 1 h and then stimulated with lipopolysaccharides (range 0-100 ng/ml) or lipoteichoic acid (range 0-5 microg/ml) for 18 h. The expression of cell surface molecules, cytokine and chemokine levels in culture supernatants, cell viability, chemotaxis, and phagocytosis and killing of Staphylococcus aureus were measured. RESULTS: Maggot secretions dose-dependently inhibited production of the pro-inflammatory cytokines TNF-alpha, IL-12p40 and macrophage migration inhibitory factor by lipopolysaccharides- and lipoteichoic acid-stimulated monocytes, while enhancing production of the anti-inflammatory cytokine IL-10. Expression of cell surface receptors involved in pathogen recognition remained unaffected by secretions. In addition, maggot secretions altered the chemokine profile of monocytes by downregulating macrophage inflammatory protein-1beta and upregulating monocyte chemoattractant protein-1 and IL-8. Nevertheless, chemotactic responses of monocytes were inhibited by secretions. Furthermore, maggot secretions did not affect phagocytosis and intracellular killing of S. aureus by human monocytes. Finally, secretions induced a transient rise in the intracellular cyclic AMP concentration in monocytes and Rp-cyclic AMPS inhibited the effects of secretions. CONCLUSIONS/INTERPRETATION: Maggot secretions inhibit the pro-inflammatory responses of human monocytes through a cyclic AMP-dependent mechanism. Regulation of the inflammatory processes by maggots contributes to their beneficial effects on chronic wounds.

IL-10 promotor haplotypes associated with susceptibility to and severity of bacterial corneal ulcers.

Interleukin-10 plays an important role in modulating inflammation and antimicrobial defences. In animal models for bacterial corneal ulcers, high IL-10 levels were associated with a better clinical outcome. We investigated whether IL-10 promotor haplotypes, known to determine IL-10 expression in vitro, are associated with susceptibility to and/or clinical outcome of bacterial corneal ulcers in patients. IL-10 promotor polymorphisms C-819T, G-1082A, A-2763C, and A-2849G for 83 patients with bacterial corneal ulcers and 115 healthy controls were determined by restriction fragment length PCR analysis. For 63 patients and all healthy controls the most frequently occurring IL-10 promotor haplotypes were inferred from these data using the program SNPHAP. A significant underrepresentation of the A-2849A genotype was observed in patients as compared to healthy controls. Both the -2763A allele and the IL-10.1 promotor haplotype were associated with a poor clinical outcome, whereas a favourable clinical outcome was seen in patients carrying the IL-10.2 promotor haplotype. Together, IL-10 promotor haplotypes associated with low IL-10 levels seem to protect against the onset of bacterial corneal ulcers. Once a corneal ulcer has developed, patients carrying IL-10 haplotypes associated with a high IL-10 expression may have a favourable outcome.

Human antimicrobial peptides' antifungal activity against Aspergillus fumigatus.

In light of the need for new antifungals, we compared the in vitro antifungal activity of two peptides derived from human lactoferrin (hLF), i.e., hLF(1-11) and hLF(21-31), two analogs of histatin 5, further referred to as dhvar4 and dhvar5, and two ubiquicidin (UBI)-derived peptides, i.e., UBI 18-35 and UBI 29-41, with that of amphotericin B against Aspergillus fumigatus hyphae using the MTT assay. The results revealed a dose-dependent antifungal activity for all peptides, with dhvar5 being the most potent peptide. In addition, hLF(1-11), dhvar5, and UBI 18-35 were effective against A. fumigatus conidia. Furthermore, hLF(1-11) did not lyze human erythrocytes, whereas dhvar5 (>or=16 microM) and UBI 18-35 (>or=20 microM) were hemolytic. Based on these in vitro results and their effectiveness against infections in mice, we concluded that hLF(1-11) and dhvar5 are promising candidates for the development of new agents against A. fumigatus infections.

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.

Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation.

Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation.

Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145.

The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Detection of fungal infections using radiolabeled antifungal agents.

The outcome of antifungal therapy depends on the progression of the infection at the start of therapy. Unfortunately, most patients are diagnosed once the fungal infection has progressed considerably as a result of the non-specific clinical signs of fungal infections in immunocompromised patients and the poor sensitivity of current mycological diagnostic tests. This review will highlight current fungal diagnostic techniques and will focus on scintigraphic methods for the specific detection of fungal infections in mice. For this purpose, antifungal components (e.g. fluconazole and antifungal peptides) are radiolabeled e.g. with technetium-99m ((99m)Tc) and their in vivo distribution is monitored in infected mice. It has been demonstrated that (99m)Tc-fluconazole is an excellent tracer to detect Candida albicans infections in mice as it distinguishes these infections from bacterial infections and sterile inflammations. However, this radiopharmaceutical only poorly detects infections with Aspergillus fumigatus in mice. (99m)Tc-peptides derived from antifungal peptides/proteins, such as human ubiquicidin and lactoferrin, can distinguish C. albicans and A. fumigatus infections from sterile inflammations, but not from bacterial infections, in mice. Furthermore, the efficacy of fluconazole in C. albicans-infected mice could be successfully monitored using (99m)Tc-ubiquicidin. In conclusion, neither (99m)Tc-fluconazole nor the (99m)Tc-peptides tested are optimal tracers for fungal infections. Nonetheless, since early initiation of antifungal therapy for candidemia reduces its high mortality rate, a positive result with (99m)Tc-fluconazole scintigraphy is of clinical relevance. Finally, the possibility that other (radiolabeled) antifungal agents, e.g. voriconazole, caspofungin, antifungal plant or insect defensins, can be useful for detection of fungal infections should be considered.

Monocytes incubated with surfactant: a model for human alveolar macrophages?

Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfactant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macrophages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of surfactant, efficient phagocytosis of opsonized Staphylococcus aureus, and poor intracellular killing of ingested bacteria. The suppressive effect of surfactant on bactericidal activities of monocytes could not be attributed to either the surfactant lipid fraction or surfactant protein A. Monocytes incubated with surfactant differed from alveolar macrophages with respect to expression of various Fc and complement receptors involved in intracellular killing of bacteria. Surfactant-incubated monocytes produced significantly more H2O2 upon stimulation with phorbol ester than alveolar macrophages, but significantly less than control monocytes. Together, monocytes and MDM incubated with surfactant, although similar to alveolar macrophages in some aspects, are not an adequate model for alveolar macrophages. Most likely, factors other than surfactant in the microenvironment of the alveoli, such as oxygen tension, play a role in the differentiation of monocytes to alveolar macrophages as well.

Ubiquicidin, a novel murine microbicidal protein present in the cytosolic fraction of macrophages.

Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon-gamma (IFN-gamma). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (Mr 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Aminoterminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death.

The characterization, origin, and kinetics of skin macrophages during inflammation.

This report deals with the characterization, origin, and kinetics of exudate skin macrophages. The inflammatory stimulus used was a subcutaneously inserted glass coverslip. The macrophages adhering to the glass surface have many characteristics in common with circulating monocytes. During this kind of inflammation there is little differentiation into a more mature or activated type of mononuclear phagocyte. The kinetic studies with [3H]thymidine as cell marker and calculation of local production at the site of inflammation as well as the influx of cells to that site led to the conclusion that greater than or equal to 99% of the exudate skin macrophages were monocyte derived and less than or equal to 1% originated by local division of macrophages.

Effects of monomethylfumarate on human granulocytes.

Monomethylfumarate (MMF) is the most active metabolite of the new antipsoriasis drug Fumaderm. Because granulocytes play an important role in the pathophysiology of psoriasis, the effects of this drug on the functional activities of these cells were investigated. MMF stimulated polarization and elastase release, and enhanced the intracellular killing of bacteria by granulocytes. This compound suppressed the formyl-Met-Nle-Phe (FMLP)-stimulated respiratory burst in these cells. MMF and dimethylfumarate but not its stereoisomer dimethylmaleate, fumaric acid, or dimethylmalate stimulated polarization of and elastase release by granulocytes, indicating that methylated fumarate derivatives interact with granulocytes in a specific fashion. MMF did not affect the binding of formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein isothiocyanate to the FMLP receptor on granulocytes. This compound induced an increase in the intracellular Ca++ ([Ca++]i) and cyclic adenosine monophsphate concentration. The agonistic effects of MMF on granulocytes are thought to be mediated by the rise in the [Ca++]i and the antagonistic effects by the increase in the cyclic adenosine monophosphate concentration. These effects of MMF on granulocytes may in part explain the beneficial action of methylated fumarate derivatives on psoriatic skin lesions.

Phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus by mouse fibroblasts expressing human Fcgamma receptor type IIa (CD32).

Phagocytes bear more than one class of receptors for the Fc domain of IgG (FcgammaR). In addition the same ligand can interact with different classes of FcgammaR. This complexity makes it difficult to study the contribution of the various classes of FcgammaR to antimicrobial functions. To circumvent this difficulty, in the present study mouse 3T6 fibroblasts transfected with cDNA encoding for human FcgammaR type IIa (FcgammaRIIa-expressing cells) were used to determine the role of this receptor in phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus. Experiments using microbiological and fluorescent techniques to discriminate between cell-adherent and intracellular bacteria revealed that serum-opsonized bacteria are phagocytized by FcgammaRIIa-expressing cells, but not by parental fibroblasts. Non-opsonized bacteria were poorly internalized by FcgammaRIIa-expressing as well as parental fibroblasts. Furthermore, incubation of FcgammaRIIa-expressing cells with opsonized bacteria at 4oC and incubation of FcgammaRIIa-expressing cells with cytochalasin E prior to addition of opsonized bacteria inhibited the phagocytosis of these bacteria almost completely. Phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells was partly inhibited by selective inhibition of protein tyrosine kinases (PTK). FcgammaRIIa cross-linking initiated transient tyrosine phosphorylation of various proteins in FcgammaRIIa-expressing cells. These data indicate that activation of PTK is involved in the FcgammaRIIa-mediated phagocytosis of opsonized S. aureus by transfected fibroblasts. Human serum from normal individuals and agammaglobulinemic patients triggered the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts. Surprisingly, heat-inactivated human serum, IgG and incubation with anti-FcgammaRII antibodies followed by a bridging secondary antibody did not stimulate the killing process. The possibility that these ligands did not interact with FcgammaRIIa on the cells can be excluded since they induced tyrosine phosphorylation of cellular proteins. The serum factor that stimulates the intracellular killing of bacteria by FcgammaRIIa-expressing cells is not yet identified. Oxygen-independent mechanisms are thought to be responsible for the killing of intracellular bacteria by these cells since the NADPH oxidase inhibitor diphenylene iodonium did not affect the serum-stimulated intracellular killing of S. aureus and no reactive oxygen and nitrogen intermediates were produced by FcgammaRIIa-expressing cells after appropiate stimulation. Taken together, these data show that phagocytosis but not intracellular killing of S. aureus is mediated via FcgammaRIIa on cells expressing this receptor.

A cell-ELISA for the quantification of adherent murine macrophages and the surface expression of antigens.

The present study was performed in order to establish whether a cell-ELISA could be used to determine the expression of antigens by adherent murine peritoneal macrophages and also quantify the numbers of such macrophages. Accurate determination of the number of adherent macrophages proved to be possible with a cell-ELISA designed to assess complement receptor type III (CRIII) expression. Expression of CRIII was considerably more sensitive than determination of the cell-protein or DNA content as a measure of the number of adherent macrophages. For the calculation of the expression of CRIII, Ia antigen, and antigen F4/80 by resident and activated macrophages, use was made of the linear part of the curve obtained when the numbers of macrophages were plotted against the absorbance values for each of the antigens. The values for CRIII expression did not differ significantly between resident macrophages, macrophages activated with recombinant interferon-gamma (rIFN-gamma) and macrophages activated with BCG/PPD. IFN-gamma-activated and BCG/PPD-activated macrophages expressed Ia antigen significantly more intensely than did resident peritoneal macrophages. In contrast the activated macrophages expressed F4/80 significantly less intensely than resident peritoneal macrophages.

Mean cell volume of human blood leucocytes and resident and activated murine macrophages.

The mean cell volume (MCV) of human blood leucocytes and resident and activated murine macrophages was measured with a Coulter counter connected to a 256 channelyzer. The values found for human blood monocytes, granulocytes, and lymphocytes were 421 +/- 24 femtolitre (fl), 334 +/- 32 fl, and 204 +/- 19 fl, respectively. Resident murine peritoneal macrophages were significantly smaller than rIFN-gamma-activated and BCG/PPD-activated peritoneal macrophages and resident alveolar macrophages.