A novel in vitro model for haematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism.

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the process. Using an in vitro catheter infection model, we studied S. aureus biofilm growth, late-stage dispersal, and reattachment to downstream endothelial cell layers. The ability of the released biofilm material to resist host response and disseminate in vivo was furthermore studied in whole blood and phagocyte survival assays and in a short-term murine infection model. We found that S. aureus biofilms formed in flow of human plasma release biofilm thromboemboli with embedded bacteria and bacteria-secreted polysaccharides. The emboli disseminate as antibiotic and immune resistant vehicles that hold the ability to adhere to and initiate colonisation of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth-grown bacteria but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S. aureus from in vivo-like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections.

In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). MATERIAL AND METHODS: Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. RESULTS: The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1ß, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). CONCLUSION: Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

Calibrated kallikrein generation in human plasma.

OBJECTIVES: The physiological role of the contact system remains inconclusive. No obvious clinical complications have been observed for factor XII (FXII), prekallikrein (PK), or high molecular weight kininogen deficiencies even though the contact system in vitro is associated with coagulation, fibrinolysis, and inflammation. A global generation assay measuring the initial phase of the contact system could be a valuable tool for studies of its physiological role. DESIGN AND METHODS: We investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s-1), and kallikrein generation could be measured in undiluted plasma when silica was added as activator. Disturbing effects, including substrate depletion and the inner-filter effect, however, affected the signal. These problems were corrected for by external calibration with α2-macroglobulin-kallikrein complexes. Selectivity studies of the substrate, experiments with FXII and PK depleted plasmas, and plasma with high or low complement C1-esterase inhibitor activity indicated that the obtained and calibrated signal predominantly was related to FXII-dependent kallikrein activity. CONCLUSIONS: The findings described show that establishment of a kallikrein generation method is possible. Potentially, this setup could be used for clinical studies of the contact system.

Pre-existing IgG antibodies cross-reacting with the Fab region of infliximab predict efficacy and safety of infliximab therapy in inflammatory bowel disease.

BACKGROUND: Infliximab (IFX) is a chimeric murine/human anti-TNF antibody (Ab) used for the treatment of Crohn's disease (CD) and ulcerative colitis (UC). Loss of response is common and associated with development of anti-IFX Abs during ongoing therapy. However, human anti-murine immunoglobulin Abs are common and may cross-react with the murine part of IFX. AIM: To investigate if Abs binding to IFX's Fab region (IFX-Fab) are present in IBD patients before exposure to IFX, and whether they predict efficacy and safety of IFX therapy. METHODS: Observational, retrospective cohort study of patients with CD (n = 29) and UC (n = 22). RESULTS: Pre-treatment levels of IFX-Fab reactive IgG Abs were significantly lower in CD patients in remission after 1 year of maintenance IFX (median 91 mU/L, n = 8) than in the rest of the patients (639 mU/L, n = 21; P < 0.01), and lower than in patients with secondary loss of response in particular (692 mU/L, n = 7; P < 0.01). A cut-off concentration of <439 mU IFX-Fab reactive IgG Ab per litre comprised all patients who later obtained long-term sustained remission on IFX (sensitivity 100%, specificity 67%). Similar trends were observed in UC. The pre-treatment levels of IFX-Fab reactive IgG Abs were markedly higher in patients developing infusion reactions to IFX (1037 mU/L, n = 7) than in the remaining patients (349 mU/L, n = 44; P = 0.036). CONCLUSIONS: IFX-Fab reactive IgG antibodies present in serum from IBD patients before infliximab therapy associate with lack of long-term efficacy and safety. Assessments of such antibodies may help clinicians to choose between treatment with infliximab and more humanised agents.

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1).

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.

Novel assays to assess the functional capacity of the classical, the alternative and the lectin pathways of the complement system.

Deficiencies in many of the complement proteins and their regulatory molecules have been described and a variety of diseases, such as recurrent infections, systemic lupus erythematosus (SLE) and renal diseases, may be linked to deficiency in the complement system. Screening for complement defects is therefore of great importance. In this study, we present novel improved enzyme-linked immunosorbent assays for the functional assessment of the three individual pathways of the complement system. The method is applicable at high serum concentrations and we demonstrate that it minimizes both false negative as well as false positive results. In particular, for the functional mannose-binding lectin activity it represents an improvement on the existing assays. In this respect, the present assays represent novel improved diagnostic protocols for patients with suspected immunodeficiencies related to the complement system.