Publications

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

JTD Keywords: cells, code, elisa, il-6, inflammation, kits, pathogenesis, procalcitonin, release, Cytokines, Humans, Immunoassay, Immunologic tests, Interleukin-6, Tumor necrosis factor-alpha

© 2020 Elsevier Ltd Central nervous system (CNS) injuries do not heal properly in contrast to normal tissue repair, in which functional recovery typically occurs. The reason for this dichotomy in wound repair is explained in part by macrophage and microglial malfunction, affecting both the extrinsic and intrinsic barriers to appropriate axonal regeneration. In normal healing tissue, macrophages promote the repair of injured tissue by regulating transitions through different phases of the healing response. In contrast, inflammation dominates the outcome of CNS injury, often leading to secondary damage. Therefore, an understanding of the molecular mechanisms underlying this dichotomy is critical to advance in neuronal repair therapies. Recent studies highlight the plasticity and complexity of macrophages and microglia beyond the classical view of the M1/M2 polarization paradigm. This plasticity represents an in vivo continuous spectrum of phenotypes with overlapping functions and markers. Moreover, macrophage and microglial plasticity affect many events essential for neuronal regeneration after injury, such as myelin and cell debris clearance, inflammation, release of cytokines, and trophic factors, affecting both intrinsic neuronal properties and extracellular matrix deposition. Until recently, this complexity was overlooked in the translation of therapies modulating these responses for the treatment of neuronal injuries. However, recent studies have shed important light on the underlying molecular mechanisms of this complexity and its transitions and effects on regenerative events. Here we review the complexity of macrophages and microglia after neuronal injury and their roles in regeneration, as well as the underlying molecular mechanisms, and we discuss current challenges and future opportunities for treatment.

JTD Keywords: chemokines and cytokines, macrophages, microglia, neuroinflammation, neuronal injury, regeneration, Chemokines and cytokines, Macrophages, Microglia, Neuroinflammation, Neuronal injury, Regeneration

Background: Acquired dysfunctional immunity in cirrhosis predisposes patients to frequent bacterial infections, especially spontaneous bacterial peritonitis (SBP), leading to systemic inflammation that is associated with poor outcome. But systemic inflammation can also be found in the absence of a confirmed infection. Detection of bacterial DNA has been investigated as a marker of SBP and as a predictor of prognosis. Data is, however, contradictory. Here we investigated whether levels of IL-6 and IL-8 putatively produced by myeloid cells in ascites are associated with systemic inflammation and whether inflammation depends on the presence of specific bacterial DNA. Methods and Materials: We enrolled 33 patients with decompensated liver cirrhosis from whom we collected paired samples of blood and ascites. IL-6 and IL-8 were measured in serum samples of all patients using ELISA. In a subset of 10 representative patients, bacterial DNA was extracted from ascites and whole blood, followed by 16S rRNA gene amplicon sequencing. Results: There were significantly higher levels of IL-6 in ascites fluid compared to blood samples in all patients. Interestingly, IL-6 levels in blood correlated tightly with disease severity and surrogates of systemic inflammation, while IL-6 levels in ascites did not. Moreover, patients with higher blood CRP levels showed greater SBP prevalence compared to patients with lower levels, despite similar positive culture results. Bacterial richness was also significantly higher in ascites compared to the corresponding patient blood. We identified differences in microbial composition and diversity between ascites and blood, but no tight relationship with surrogates of systemic inflammation could be observed. Discussion: In decompensated cirrhosis, markers of systemic inflammation and microbiota composition seem to be dysregulated in ascites and blood. While a relationship between systemic inflammation and microbiota composition seems to exist in blood, this is not the case for ascites in our hands. These data may suggest compartmentalization of the immune response and interaction of the latter with the microbiota especially in the blood compartment.

JTD Keywords: Acute-on-chronic liver failure, Ascites, Cirrhosis, Cytokines, Microbiome, Myeloid cells, Systemic inflammation

Animal cells undergo a dramatic series of shape changes as they divide, which depend on re-modeling of cell-substrate adhesions. Here, we show that while focal adhesion complexes are disassembled during mitotic rounding, integrins remain in place. These integrin-rich contacts connect mitotic cells to the underlying substrate throughout mitosis, guide polarized cell migration following mitotic exit, and are functionally important, since adherent cells undergo division failure when removed from the substrate. Further, the ability of cells to re-spread along pre-existing adhesive contacts is essential for division in cells compromised in their ability to construct a RhoGEF-dependent (Ect2) actomyosin ring. As a result, following Ect2 depletion, cells fail to divide on small adhesive islands but successfully divide on larger patterns, as the connection between daughter cells narrows and severs as they migrate away from one another. In this way, regulated re-modeling of cell-substrate adhesions during mitotic rounding aids division in animal cells.

JTD Keywords: Division, Mitotic-rounding, Integrin-based adhesion, Cytokinesis

Background Bacillus Calmette-Guérin (BCG) prevents tumour recurrence and progression in non–muscle-invasive bladder cancer (BC). However, common adverse events occur, including BCG infections. Objective To find a mycobacterium with similar or superior antitumour activity to BCG but with greater safety. Design In vitro, ex vivo, and in vivo comparisons of the antitumour efficacy of nonpathogenic mycobacteria and BCG. Intervention The in vitro antitumour activity of a broad set of mycobacteria was studied in seven different BC cell lines. The most efficacious was selected and its ex vivo capacity to activate immune cells and its in vivo antitumour activity in an orthotopic murine model of BC were investigated. Outcome measurements and statistical analysis Growth inhibition of BC cells was the primary outcome measurement. Parametric and nonparametric tests were use to analyse the in vitro results, and a Kaplan-Meier test was applied to measure survival in mycobacteria-treated tumour-bearing mice. Results and limitations Mycobacterium brumae is superior to BCG in inhibiting low-grade BC cell growth, and has similar effects to BCG against high-grade cells. M. brumae triggers an indirect antitumour response by activating macrophages and the cytotoxic activity of peripheral blood cells against BC cells. Although no significant differences were observed between BCG and M. brumae treatments in mice, M. brumae treatment prolonged survival in comparison to BCG treatment in tumour-bearing mice. In contrast to BCG, M. brumae does not persist intracellularly or in tumour-bearing mice, so the risk of infection is lower. Conclusions Our preclinical data suggest that M. brumae represents a safe and efficacious candidate as a therapeutic agent for non–muscle-invasive BC. Patient summary We investigated the antitumour activity of nonpathogenic mycobacteria in in vitro and in vivo models of non–muscle-invasive bladder cancer. We found that Mycobacterium brumae effectively inhibits bladder cancer growth and helps the host immune system to eradicate cancer cells, and is a promising agent for antitumour immunotherapy.

JTD Keywords: Animal models, Bacillus Calmette-Guérin, Cytokines, Immunomodulation, Immunotherapy, Mycobacteria, Urothelial cell line