Contribution of Kazal-Like Domains of the Serine Protease Inhibitor-1 from Toxoplasma gondii in Asthma Therapeutic Vaccination Effectiveness

<b><i>Background:</i></b> We have previously showed rTgPI-1 tolerogenic adjuvant properties in asthma treatment, turning it a promising candidate for allergen-specific immunotherapy. This therapy is an alternative treatment to control asthma that still presents several concerns related to its formulation. rTgPI-1 contains independent inhibitory domains able to inhibit trypsin and neutrophil elastase, both involved in asthma pathology. <b><i>Objectives:</i></b> In view of the need to design rational therapies, herein we investigated the contribution of the different inhibitory domains in rTgPI-1 therapeutic effectiveness. <b><i>Methods:</i></b> BALB/c mice were rendered allergic by intraperitoneal OVA-alum sensitization and airway challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with OVA combined with the full-length recombinant protein rTgPI-1 or its truncated versions, Nt (containing trypsin-inhibitory domains) or Ct (containing neutrophil elastase-inhibitory domains). Afterward, mice were aerosol re-challenged. <b><i>Results:</i></b> Asthmatic mice treated with the neutrophil elastase- or the trypsin-inhibitory domains separately failed to improve allergic lung inflammation. Only when all inhibitory domains were simultaneously administered, an improvement was achieved. Still, a better outcome was obtained when mice were treated with the full-length rTgPI-1. <b><i>Conclusions:</i></b> Adjuvant ability depends on the presence of all its inhibitory domains in a single entity, so it should be included in potential asthma treatment formulations as a full-length protein.

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.

The Role of Neutrophil Elastase in Aortic Valve Calcification

Background: Calcific aortic valve disease (CAVD) is the most commonly valvular disease in the western countries initiated by inflammation and abnormal calcium deposition. Currently, there is no clinical drugs for CAVD. Neutrophil elastase(NE) plays a causal role in inflammation and participates actively in cardiovascular diseases. However, the effects of NE on valve calcification remains unclear. So we next explore whether it is involved in valve calcification and the molecular mechanisms involved.Methods: NE expression and activity in calcific aortic valve stenosis (CAVS) patients (n=58) and healthy patients (n=30) were measured by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC). Porcine aortic valve interstitial cells (pVICs) were isolated and used in vitro expriments. The effects of NE on pVICs inflammation, apoptosis and calcification were detected by hochest 33258 staining, MTT assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot. The effects of NE knockdown and NE activity inhibitor Alvelestat on pVICs inflammation, apoptosis and calcification under osteogenic medium induction were also detected by RT-PCR, western blot, alkaline phosphatase staining and alizarin red staining. Changes of Intracellular signaling pathways after NE treatment were measured by western blot.Results: The level and activity of NE were evaluated in patients with CAVS and calcified valve tissues. NE promoted inflammation, apoptosis and phenotype transition in pVICs in the presence or absence of osteogenic medium. Under osteogenic medium induction, NE silencing or NE inhibitor Alvelestat both suppressed the osteogenic differentiation of pVICs. Mechanically, NE played its role in promoting osteogenic differentiation of pVICs by activating the NF-κB and AKT signaling pathway.Conclusions: Collectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

Influence of new antimicrobial peptides of the medicinal leech Hirudo medicinalis on the functional activity of neutrophil granule proteins

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms. AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins the main effector cells of the immune system. MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis. RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity. CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the bodys fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit if it necessary to protect against damage to the bodys own tissues.