Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease.

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

Monitoring of Cerebrovascular Reactivity in Intracerebral Hemorrhage and Its Relation with Survival.

INTRODUCTION: Neuromonitoring analysis for intracerebral hemorrhage (ICH) is still rare, especially regarding vascular reactivity patterns. Our goal was to analyze neuromonitoring data and 28-day mortality for ICH patients. METHODS: Neuromonitoring records were retrospectively reviewed from a cohort of ICH patients admitted to a neurocritical care unit between 2013 and 2016. Variables considered were intracranial pressure (ICP), cerebral perfusion pressure (CPP), optimal CPP, and pressure reactivity index (PRx), as well as ICP dose, PRx dose, and time percentage above critical value (T%abv). Information regarding demographics, surgical drainage, external ventricular drain placement, and 28-day mortality was recorded. Statistical analysis was performed using the t-test and Kaplan-Meier curves. RESULTS: Forty-six patients were analyzed, with a mean of 263 ± 173 h of signal records and a median length of stay in the intensive care unit of 22 (interquartile range of 13) days. The mean age was 62.6 ± 11.8 years old, and 24 (52%) of the patients were male. Patients who died within 28 day (37.0%) had significantly higher mean ICP, PRx, ICP dose, PRx dose, and T%abv. Although their mean ICP was under 20 mmHg, they presented PRx > 0.25, indicating impaired cerebrovascular reactivity (0.30 ± 0.26). Also, patients with PRx > 0.25 had a lower survival rate, with a proportion of 14% at 28 days, as opposed to 85% of those with PRx < 0.25 (p < 0.001). CONCLUSION: The data suggest that autoregulation indexes are associated with 28-day mortality for ICH patients.

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation.

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patient's life quality and reduction of costs on health care system.

Design of a chromogenic substrate for elastase based on split GFP system-Proof of concept for colour switch sensors.

Recent studies have demonstrated that human neutrophil elastase (HNE) can be used as marker for inflammation/infection of chronic wounds since it was found to be present in high concentration in exudate collected from chronic wounds. Biosensors used in wound care benefit from a chromogenic signalling due to the readiness of signal interpretation, but the most common use faint yellow chromogenic molecules such as p-nitroaniline (pNa). In addition, if to be converted into smart dressings, the colour of the detection system should not be masked by the exudate's colour. In this work, we designed a chromogenic substrate for HNE aiming to be incorporated in a smart dressing as a colour switch sensor. The substrate was developed using the GFP-like chromoprotein ultramarine (UM), following the split GFP technology. The cleavage sequence for HNE (Ala-Ala-Pro-Val) was embedded into the sensing moiety of the substrate corresponding to the 11th ß-sheet. In the presence of HNE, the 11th ß-sheet is able to interact to the signalling moiety composed of the ß1-ß10 incomplete barrel, allowing the re-establishment of the chromophore environment and, hence, the colour production. Structural homology and molecular dynamics simulations were conducted to aid on the disclosure of the structural changes that are the base of the mechanism of action of this HNE switch substrate. Our findings explore the possible application of GFP-like chromogenic sensors in point-of-care devices for the evaluation of the wounds status, representing a major step in the medical field.