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

Natural ligand motifs of H-2E molecules are allele specific and illustrate homology to HLA-DR molecules

Motifs of peptldes naturally associated with H-2Ek and Ed molecules were determined by (i) pool sequencing of natural ligand mixtures and (ii) sequencing of individual natural ligands followed by their alignment to the basic motif suggested by pool sequencing. The data reveal nine amino acid motifs with interaction sites at relative positions P1, P4, P6 and P9, with specificities that are identical at some but different at other anchor positions between Ed and Ek motifs, illustrating the different requirements for peptides to be presented by these two MHC molecules. The anchors with the most restricted specificity are P1 and P9. P1 is aliphatic for Ek and predominantly aromatic for Ed. P9 is positively charged for both molecules. P4 and P6 show a totally different amino acid preference between Ek and Ed ligand motifs. An alignment of Ed and Ek protein sequences to the recently reported HLA-DR1 pocket residues is in agreement with observed anchor residues in Ek and Ed motifs, thus confirming the predicted similarity of mouse class II E molecules with human DR molecules. Furthermore, this alignment was extended to the putative pockets of class II Eb and E* molecules, and allowed, together with sequence information of previously Identified natural ligands of Eb and E5 molecules, a prediction of their respective motifs. The information obtained by this study should be useful to identify putative class II E epltopes in proteins and to design peptides for blocking class II E molecule

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

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

Biological and Pharmacokinetic Studies with β-Peptides

Interactions and cleavage reactions of β-amino acids and β-oligopeptides (up to nine residues, carrying the side chains of Ala, Val, Leu, Ile, Phe, Ser, Lys, and Hop) with biological systems, such as the most potent peptidases (pronase, proteinase K, 20S proteasome), microorganisms (Pseudomonas aeruginosa and Pseudomonas putida), and mammalian blood (intravenous application to rats) have been investigated and compared with ?-peptides. The results are: i) the three peptidases do not cleave β-peptides at all (within 24 h), and they are not inhibited by a β-peptide; ii) except for certain 3-aminobutanoic-acid (β-HAla) derivatives, neither free, nor N-acetyl-β-amino acids, nor β-peptides (offered as sole N and C source) lead to growth of the two bacteria tested; iii) two water-soluble β-heptapeptides (with Lys side chains) were shown to have elimination half-lives t1/2(β) of 3 and 10 h at 100- and 30-ng/ml levels, respectively, in the rodent blood – much larger than those of α-peptides. Thus, the preliminary results described here confirm the much greater stability of β-peptides, as compared to α-peptides, towards metabolization processes, but they also suggest that there may be interactions (by hitherto unknown mechanisms) between the worlds of α- and β-peptides

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