Synthesis and characterization of TEMPO-oxidized peptide-cellulose conjugate biosensors for detecting human neutrophil elastase

Here we describe the synthesis and characterization of a peptide-cellulose conjugate biosensor based on TEMPO-oxidized nanofibrillated cellulose (tNFC) for detecting elevated levels of human neutrophil elastase (HNE) in chronic wounds. The fluorescent peptide HNE substrate constructed from n-succinyl-Ala-Pro-Ala-7-amino-4-methyl-coumarin was attached to the TEMPO-oxidized cellulose surface via polyethylene glycol linker. The characterization of the biosensor conjugate shows a high degree of peptide incorporation onto the surface with the degree of substitution of 0.057. The relatively small crystallite size of 26.0 Å compared to other cellulose- and nanocellulose-based materials leads to a large specific surface area which can promote access of HNE to the enzyme substrates due to decreased steric interactions. Likewise, the porosity for tNFC was found to be higher than all other samples, including the nanocellulosic aerogel, lending to its hydrogel-like nature. The properties of tNFC were compared to other cellulose-based materials. The volume of each crystallite and volume ratio to the largest sample was calculated. tNFC was found to occupy the smallest space resulting in high amounts of sensors per crystallite unit volume. With a small crystallite volume and large number of sensors, the tNFC peptide-cellulose conjugate biosensor could provide a more sensitive system and is a good candidate for point of care diagnostic devices for detecting elevated protease levels in humans.

Aptamer Selection against Alpha-defensin Human Neutrophil Peptide 1 on an Integrated Microfluidic System for Diagnosis of Periprosthetic Joint Infections

Periprosthetic joint infections (PJI) arising from joint arthroplasty are dreadful, yet difficult to diagnose in subtle cases. Definite diagnosis requires microbiological culture to confirm the causative pathogens. However, up to...

Indoleamine 2,3-Dioxygenase Cannot Inhibit Chlamydia trachomatis Growth in HL-60 Human Neutrophil Granulocytes

AimsNeutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized.MethodsGlobal gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS).ResultsOur results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition.ConclusionsIDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

Iridal-Type Triterpenoids Displaying Human Neutrophil Elastase Inhibition and Anti-Inflammatory Effects from Belamcanda chinensis

The aim of this study is to explore anti-inflammatory phytochemicals from B. chinensis based on the inhibition of pro-inflammatory enzyme, human neutrophil elastase (HNE) and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. Three stereoisomers of iridal-type triterpenoids (1–3) were isolated from the roots of B. chinensis and their stereochemistries were completely identified by NOESY spectra. These compounds were confirmed as reversible noncompetitive inhibitors against HNE with IC50 values of 6.8–27.0 µM. The binding affinity experiment proved that iridal-type triterpenoids had only a single binding site to the HNE enzyme. Among them, isoiridogermanal (1) and iridobelamal A (2) displayed significant anti-inflammatory effects by suppressing the expressions of pro-inflammatory cytokines, such as iNOS, IL-1β, and TNF-α through the NF-κB pathway in LPS-stimulated RAW264.7 cells. This is the first report that iridal-type triterpenoids are considered responsible phytochemicals for anti-inflammatory effects of B. chinensis.

Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human Neutrophil Peptides (HNPs)

Antimicrobial peptides (AMPs) are known best for their role in innate immunity against bacteria, viruses, parasites and fungi. However, not only are they showing increasing promise as potential antimicrobial drug candidates, recently, it has been reported that certain AMPs also show a cytotoxic effect against cancer cells. Their possible antitumor effect could make AMPs interesting candidate cancer biomarkers and a possible lead for new anticancer therapy. Due to their cyclic structure, detection and identification of AMPs is challenging, however, mass spectrometry (imaging; MSI) has been shown as a powerful tool for visualization and identification of (unknown) cyclic AMPs. In this chapter, we will discuss how mass spectrometry (imaging), combined with the use of electron-transfer dissociation (ETD) as fragmentation technique, can be used as a reliable method to identify AMPs in their native cyclic state. Using this approach, we have previously detected and identified human neutrophil peptides (HNPs) as important AMPs in cancer, of which a detailed bacterial, viral and cancer-related overview will be presented.