Natural Killer Cell Activity Assessment in a Whole Blood-Based Culture System Using Multiplex Cytokine and Flow Cytometry Measurements

Natural killer (NK) cells represent crucial players of the innate immune system and fulfil their main function in first line of defense by recognizing and eliminating tumor degenerated and virus infected cells. To analyze and influence NK cell behavior it is necessary to be able to specifically activate NK cells. In this work, known NK cell-specific stimulants were used in whole blood cultures (TruCulture) to investigate the specificity of the NK cell-activating stimuli in the high complexity of this culture system and to determine whether and to what extent co-activation of further immune cells of the peripheral blood occurs. For this purpose, it was necessary to generate appropriate test systems. Thus, two bead-based multiplex Luminex immunoassays, IMAP 1 and IMAP 2, were developed for the detection of nine (IL-4, -6, -8, 10, GM CSF, IFN-γ, MCP-1, MIP-1β, TNF-α) and six (IL-1β, -1Ra, -12p70, -13, VEGF, M-CSF) analytes respectively. Additionally, highly sensitive single-molecule arrays (Simoa) were established for IL 4 and IL 12p70 as single-plex assays and for IL-6 and TNF-α as 2-plex assays as these four analytes required higher sensitivities than those provided by the Luminex technology. During assay development, parameters such as the basic buffer system and detector antibody concentration were optimized for optimal performance and sensitivity, with cross-reactivity between multiplexed analytes evaluated and reduced. Developed assays were then validated to confirm their potential as an analytical method for the TruCulture system and to confirm their reproducibility and validity. Method suitability was confirmed for the majority of analytes. Only for VEGF the pre-defined acceptance criteria for precision were not met, while for IL 4, IL-12p70, GM-CSF, VEGF and M-CSF, as part of the IMAPs, parallelism could not be demonstrated. A method comparison (Luminex vs. Simoa) using Passing-Bablok regression and Blant-Altman plots was performed for IL-6 and TNF-α assays to be able to use data from both assays for analysis. This showed comparability for both technologies. The developed and validated assays were then used to assess NK cell activation in whole blood cultures, supplemented by flow cytometric analyses. Synergistic effects of the stimulant combinations IL-12 + IL-18, R848 + IL-2 and K562 cells + IL 2 were shown to induce the strongest activation states of NK cells. It was observed that R848 + IL-2 stimulated not only cytokine production but also the degranulation process as NK cell effector functions and led to a broad activation of all immune cell populations of the peripheral blood. In contrast, the combination IL-12 + IL-18 showed NK cell stimulation only in the direction of cytokine production and moderately activated other immune cells. Although it is not yet possible to store these cells at -20 °C, which is a prerequisite for their use as default stimulant in the TruCulture application, the most specific NK cell activation was observed by stimulation with K562 cells in combination with IL-2

Analytical performance of 17 commercially available point-of-care tests for CRP to support patient management at lower levels of the health system

Accurate and precise point-of-care (POC) testing for C-reactive protein (CRP) can help support healthcare providers in the clinical management of patients. Here, we compared the analytical performance of 17 commercially available POC CRP tests to enable more decentralized use of the tool. The following CRP tests were evaluated. Eight quantitative tests: QuikRead go (Aidian), INCLIX (Sugentech), Spinit (Biosurfit), LS4000 (Lansionbio), GS 1200 (Gensure Biotech), Standard F200 (SD Biosensor), Epithod 616 (DxGen), IFP-3000 (Xincheng Biological); and nine semi-quantitative tests: Actim CRP (ACTIM), NADAL Dipstick (nal von minden), NADAL cassette (nal von minden), ALLTEST Dipstick (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10-40-80 (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10–30 (Hangzhou Alltest Biotech), Biotest (Hangzhou Biotest Biotech), BTNX Quad Line (BTNX), BTNX Tri Line (BTNX). Stored samples (n = 660) had previously been tested for CRP using Cobas 8000 Modular analyzer (Roche Diagnostics International AG, Rotkreuz, Switzerland (reference standards). CRP values represented the clinically relevant range (10–100 mg/L) and were grouped into four categories ( 80mg/L) for majority of the semi-quantitative tests. Among the eight quantitative POC tests evaluated, QuikRead go and Spinit exhibited better agreement with the reference method, showing slopes of 0.963 and 0.921, respectively. Semi-quantitative tests with the four categories showed a poor percentage agreement for the intermediate categories and higher percentage agreement for the lower and upper limit categories. Analytical performance varied considerably for the semi-quantitative tests, especially among the different categories of CRP values. Our findings suggest that quantitative tests might represent the best choice for a variety of use cases, as they can be used across a broad range of CRP categories

The Peri-Implant and Periodontal Microbiota in Patients with and without Clinical Signs of Inflammation

Late implant failures, caused by the inflammation of surrounding tissues are a problem in implant dentistry. The path of bacterial transmission from teeth to implants is not completely understood. Therefore, the purpose of this study was to analyze intraindividual bacterial transmission characterizing subgingival microbiomes in teeth and implants, both in healthy subjects and in those with signs of periodontitis or peri-implantitis. Samples of peri-implant and dental sulcus fluid were collected. To identify the predominant microbiota, amplified fragments of bacterial 16S rRNA gene were separated by single strand conformation polymorphism analysis, sequenced and taxonomically classified. A total of 25 different predominant genera were found in the diseased group and 14 genera in the healthy group. Species richness did not differ significantly between implants, neighboring teeth and teeth with largest probing depth in the diseased group. Additionally, no differences between teeth and implants in the healthy group were detected. In contrast, microbial diversity varied between the different sampling points. Species richness is similar in healthy and diseased sites, but the composition of the bacterial community differed within the individual subjects. The underlying analyses strongly suggest that complete transmission from neighboring teeth to implants is unlikely

Systematic investigation of polyurethane biomaterial surface roughness on human immune responses in vitro

It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 μm to 18 μm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material

Anti-biofilm properties of laser-synthesized, ultrapure silver–gold-alloy nanoparticles against Staphylococcus aureus

Abstract Staphylococcus aureus biofilm-associated infections are a common complication in modern medicine. Due to inherent resilience of biofilms to antibiotics and the rising number of antibiotic-resistant bacterial strains, new treatment options are required. For this purpose, ultrapure, spherical silver–gold-alloy nanoparticles with homogenous elemental distribution were synthesized by laser ablation in liquids and analyzed for their antibacterial activity on different stages of S. aureus biofilm formation as well as for different viability parameters. First, the effect of nanoparticles against planktonic bacteria was tested with metabolic activity measurements. Next, nanoparticles were incubated with differently matured S. aureus biofilms, which were then analyzed by metabolic activity measurements and three dimensional live/dead fluorescent staining to determine biofilm volume and membrane integrity. It could be shown that AgAu NPs exhibit antibacterial properties against planktonic bacteria but also against early-stage and even mature biofilms, with a complete diffusion through the biofilm matrix. Furthermore, AgAu NPs primarily targeted metabolic activity, to a smaller extend membrane integrity, but not the biofilm volume. Additional molecular analyses using qRT-PCR confirmed the influence on different metabolic pathways, like glycolysis, stress response and biofilm formation. As this shows clear similarities to the mechanism of pure silver ions, the results strengthen silver ions to be the major antibacterial agent of the synthesized nanoparticles. In summary, the results of this study provide initial evidence of promising anti-biofilm characteristics of silver–gold-alloy nanoparticles and support the importance of further translation-oriented analyses in the future

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography–induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial–macrophage interaction