Evaluation and validation of the detection of soluble triggering receptor expressed on myeloid cells 1 by enzyme-linked immunosorbent assay

Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application

Differential effects of glycoprotein B epitope-specific antibodies on human cytomegalovirus-induced cell–cell fusion

Attachment of, and cell–cell fusion induced by, human cytomegalovirus were studied in the presence of neutralizing monospecific antibodies against antigenic domains 1 (AD-1) or 2 (AD-2) of glycoprotein B (gB, gpUL55). Efficient inhibition of the virion-mediated fusion event was consistently observed for the human AD-2-specific antibody as determined by a reporter gene activation assay based on permissive astrocytoma cells. In contrast, antibodies directed against the major neutralizing gB epitope AD-1 reduced fusion only by 20–60 %. Virus attachment via heparan sulfate was unaffected by the antibodies under the conditions used. Virus receptor binding as examined by heparin treatment of adsorbed virus was significantly reduced only if the virus had been coated with the AD-2-specific antibody. Neutralization of virus infectivity by the AD-2-specific antibody thus seems most likely to result from interference with a receptor-binding event during initial virus–host cell interaction

Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediatedby the human endoprotease furin

AbstractInhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatmentof infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R × K/R R. Uncleaved gB precusor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone

Soluble Triggering Receptor Expressed on Myeloid Cells 1 Is Released in Patients with Stable Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is increasingly recognized as a systemic disease that is associated with increased serum levels of markers of systemic inflammation. The triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently identified activating receptor on neutrophils, monocytes, and macrophage subsets. TREM-1 expression is upregulated by microbial products such as the toll-like receptor ligand lipoteichoic acid of Gram-positive or lipopolysaccharides of Gram-negative bacteria. In the present study, sera from 12 COPD patients (GOLD stages I–IV, FEV1 51 ± 6%) and 10 healthy individuals were retrospectively analyzed for soluble TREM-1 (sTREM-1) using a newly developed ELISA. In healthy subjects, sTREM-1 levels were low (median 0.25 ng/mL, range 0–5.9 ng/mL). In contrast, levels of sTREM-1 in sera of COPD patients were significantly increased (median 11.68 ng/mL, range 6.2–41.9 ng/mL, P<.05). Furthermore, serum levels of sTREM-1 showed a significant negative correlation with lung function impairment. In summary, serum concentrations of sTREM-1 are increased in patients with COPD. Prospective studies are warranted to evaluate the relevance of sTREM-1 as a potential marker of the disease in patients with COPD

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

Physical activity impacts immune homeostasis and leads to rapid and marked increase in cell-free DNA (cfDNA). However, the origin of cfDNA during exercise remains elusive and it is unknown if physical activity could improve or interfere with methylation based liquid biopsy. We analyzed the methylation levels of four validated CpGs representing cfDNA from granulocytes, lymphocytes, monocytes, and non-hematopoietic cells, in healthy individuals in response to exercise, and in patients with hematological malignancies under resting conditions. The analysis revealed that physical activity almost exclusively triggered DNA release from granulocytes, highlighting the relevance as a pre-analytical variable which could compromise diagnostic accuracy

Regulatory T Cells and IL-10 Independently Counterregulate Cytotoxic T Lymphocyte Responses Induced by Transcutaneous Immunization

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T(reg)) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3(+) regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10(-/-)) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T(reg) in IL-10(-/-) mice and the use of B cell deficient JHT(-/-) mice, we can exclude T(reg) and B cells as source of IL-10 in the setting of TCI.We identify T(reg) and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T(reg) function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors

Optimized dithranol-imiquimod-based transcutaneous immunization enables tumor rejection

Introduction: Transcutaneous immunization (TCI) is a non-invasive vaccination method promoting strong cellular immune responses, crucial for the immunological rejection of cancer. Previously, we reported on the combined application of the TLR7 agonist imiquimod (IMQ) together with the anti-psoriatic drug dithranol as novel TCI platform DIVA (dithranol/IMQ based vaccination). In extension of this work, we further optimized DIVA in terms of drug dose, application pattern and established a new IMQ formulation. Methods: C57BL/6 mice were treated on the ear skin with dithranol and IMQ-containing ointments together with ovalbumin-derived peptides. T cell responses were determined by flow cytometry and IFN-ɤ ELISpot assay, local skin inflammation was characterized by ear swelling. Results: Applying the adjuvants on separate skin sites, a reduced number of specific CD8+ T cells with effector function was detectable, indicating that the local concurrence of adjuvants and peptide antigens is required for optimal vaccination. Likewise, changing the order of dithranol and IMQ resulted in an increased skin inflammatory reaction, but lower frequencies of antigen-specific CD8+ T cells indicating that dithranol is essential for superior T cell priming upon DIVA. Dispersing nanocrystalline IMQ in a spreadable formulation (IMI-Sol+) facilitated storage and application rendering comparable immune responses. DIVA applied one or two weeks after the first immunization resulted in a massive increase in antigen-specific T cells and up to a ten-fold increased memory response. Finally, in a prophylactic tumor setting, double but no single DIVA treatment enabled complete control of tumor growth, resulting in full tumor protection. Discussion: Taken together, the described optimized transcutaneous vaccination method leads to the generation of a strong cellular immune response enabling the effective control of tumor growth and has the potential for clinical development as a novel non-invasive vaccination method for peptide-based cancer vaccines in humans

Mast cell-derived mediators promote murine neutrophil effector functions

Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neutrophil phagocytosis and generation of reactive oxygen species. All these phenomena partly depend on mast cell-derived TNF and to a greater extend on GM-CSF. Furthermore, spontaneous apoptosis of neutrophils is greatly diminished due to the ability of mast cells to deliver antiapoptotic GM-CSF. Finally, we show in a murine model for acute lung inflammation that neutrophil phagocytosis is impaired in mast cell-deficient Kit W-sh /Kit W-sh mice but can be restored upon mast cell engraftment. Thus, a previously underrated feature of mast cells is their ability to boost neutrophil effector functions in immune response

Quality by design (QbD) approach for a nanoparticulate imiquimod formulation as an investigational medicinal product

The present article exemplifies the application of the concept of quality by design (QbD) for the systematic development of a nanoparticulate imiquimod (IMQ) emulsion gel formulation as an investigational medicinal product (IMP) for evaluation in an academic phase-I/II clinical trial for the treatment of actinic keratosis (AK) against the comparator Aldara (EudraCT: 2015-002203-28). The design of the QbD elements of a quality target product profile (QTPP) enables the identification of the critical quality attributes (CQAs) of the drug product as the content of IMQ, the particle-size distribution, the pH, the rheological properties, the permeation rate and the chemical, physical and microbiological stability. Critical material attributes (CMAs) and critical process parameters (CPPs) are identified by using a risk-based approach in an Ishikawa diagram and in a risk-estimation matrix. In this study, the identified CPPs of the wet media ball-milling process’s milling time and milling speed are evaluated in a central composite design of experiments (DoEs) approach, revealing criticality for both factors for the resulting mean particle size, while only the milling time is significantly affecting the polydispersity. To achieve a mean particle size in the range of 300–400 nm with a minimal PdI, the optimal process conditions are found to be 650 rpm for 135 min. Validating the model reveals a good correlation between the predicted and observed values. Adequate control strategies were implemented for intermediate products as in-process controls (IPCs) and quality control (QC) tests of the identified CQAs. The IPC and QC data from 13 “IMI-Gel” batches manufactured in adherence to good manufacturing practice (GMP) reveal consistent quality with minimal batch-to-batch variability