The recombinant plant Bauhinia bauhinioides elastase inhibitor reduces rat thrombus without alterations in hemostatic parameters

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.

Potential of Topical Microemulsion Serum Formulations to Enhance In Vitro and Clinical Anti-Skin Wrinkle Benefits of Cordyceps Militaris Extracts

This research is the first to investigate the anti-skin wrinkle properties of Cordyceps mili-taris extracts both in vitro and in vivo. Anti-skin wrinkle activities of C. militaris were investigated by means of matrix metalloproteinase-1 (MMP-1), elastase, and hyaluronidase inhibitions. Microemulsions and topical serum formulation containing C. militaris extract were developed. The anti-skin wrinkle efficacy and irritation properties of the topical serum formulations were clinically investigated in human volunteers. Cordycepin was identified as a major component of C. miliaris extract that was responsible for MMP-1, elastase, and hyaluronidase inhibition. The C. miliaris water extract possessed the most potent inhibition on MMP-1 (77.9 ± 5.3%) and elastase (84.4 ± 4.0%). Interestingly, CW was as a potent MMP-1 and elastase inhibitor as oleanolic acid and EGCG. CW was incorporated into the microemulsion with the smallest internal droplet size (146.1 ± 1.5 nm) and further developed as a topical serum formulation. The resulting serum formulation effectively enhanced skin moisture (42.2 ± 14.2%), increased the skin elasticity (39.9 ± 7.3%), and induced no skin irritation in 30 human volunteers. The effectiveness on the skin was detected after 1 week of the applications. Therefore, it was suggested as an effective anti-skin wrinkle formulation.

Reduced neutrophil elastase inhibitor elafin and elevated TGFβ1 are linked to inflammatory response in sputum of CF patients with Pseudomonas aeruginosa

Research questionPulmonary disease progression in patients with cystic fibrosis (CF) is characterized by inflammation and fibrosis, and aggravated by Pseudomonas aeruginosa (Pa). We investigated the impact of Pa specifically 1) on protease/antiprotease balance and 2) inflammation, as well as 3) the link of both parameters to clinical parameters of CF-patients.MethodsTGFβ1, IL1β, IL8, neutrophil elastase (NE) and elastase inhibitor elafin were measured (ELISA assays), and gene expression of the NF-ĸB pathway was assessed (RT-PCR) in the sputum of 60 CF-patients with a minimum age of 5 years. Spirometry was assessed according to ATS guidelines.Results1) NE was markedly increased in Pa-positive sputum, whereas elafin was significantly decreased. 2) Increased IL1β/IL8 were associated with both Pa infection and reduced FEV1, as well as sputum TGFβ1 was elevated in Pa-infected CF-patients and linked to an impaired lung function. 3) Moreover, gene expression of NF-ĸB signaling components was increased in sputum of Pa-infected patients; these findings were positively correlated with IL8.ConclusionOur study links Pa infection to an imbalance of NE and NE-inhibitor elafin and increased inflammatory mediators. Moreover, our data demonstrate an association between high TGFβ1 sputum levels and a progress in chronic lung inflammation and pulmonary fibrosis in CF. Controlling the excessive airway inflammation by inhibition of NE and TGFβ1 might be promising therapeutic strategies in future CF therapy and a possible complement to CFTR-modulators.

Proteomics-Based Identification of Salivary Changes in Patients with Burning Mouth Syndrome

Burning mouth syndrome (BMS) is a chronic oral condition characterized by an intraoral burning sensation, taste alterations, and dry mouth sensations. Although a number of factors have been closely related to the appearance of the symptoms, including anxiety, depression, and sleep disturbances, the etiology of BMS remains unclear. Furthermore, currently no objective diagnostic tools exist, making its diagnosis challenging. Therefore, to contribute to the knowledge about BMS etiology and look for objective tools for its diagnosis, the present study was conducted. Thus, the aim of this study was to analyze the proteomic profile of the resting whole saliva of patients with BMS and age and sex-matched controls using two-dimensional electrophoresis. The results showed evidence of changes in saliva at the level of proteins related to important pathways such as stress (sAA), immune system (Ig), and inflammation (leukocyte elastase inhibitor). While some of our findings have been previously described others, such as the deregulation of the coiled-coin domain containing protein 25 in BMS, are presented here for the first time to our knowledge. Thus, saliva provides us with relevant information about BMS pathophysiology and could be considered a suitable biofluid for its study and/or diagnosis.

Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

Proteomic Analysis of the Hemolymph After Metschnikowia bicuspidata Infection in the Chinese Mitten Crab Eriocheir sinensis

The “milky disease” of the Chinese mitten crab, Eriocheir sinensis, is a highly lethal fungal disease caused by Metschnikowia bicuspidata infection. To elucidate the immune responses of the hemolymph of E. sinensis to M. bicuspidata infection, a comparative analysis of the hemolymph of E. sinensis infected with M. bicuspidata and that treated with phosphate buffered saline was performed using label-free quantitative proteomics. A total of 429 proteins were identified. Using a 1.5-fold change in expression as a physiologically significant benchmark, 62 differentially expressed proteins were identified, of which 38 were significantly upregulated and 24 were significantly downregulated. The upregulated proteins mainly included cytoskeleton-related proteins (myosin regulatory light chain 2, myosin light chain alkali, tubulin α-2 chain, and tubulin β-1 chain), serine protease and serine protease inhibitor (clip domain-containing serine protease, leukocyte elastase inhibitor, serine protein inhibitor 42Dd), catalase, transferrin, and heat shock protein 70. Upregulation of these proteins indicated that phenoloxidase system, phagocytosis and the ROS systems were induced by M. bicuspidata. The downregulated proteins were mainly organ and tissue regeneration proteins (PDGF/VEGF-related factor protein, integrin-linked protein kinase homing pat-4 gene) and hemagglutination-associated proteins (hemolymph clottable protein, hemocyte protein-glutamine gamma-glutamyltransferase). Downregulation of these proteins indicated that M. bicuspidata inhibited hemocyte regeneration and hemolymph agglutination. Fifteen differentially expressed proteins related to immunity were verified using a parallel reaction monitoring method. The expression trend of these proteins was similar to that of the proteome. To the best of our knowledge, this is the first report on the proteome of E. sinensis in response to M. bicuspidata infection. These results not only provide new and important information on the immune response of crustaceans to yeast infection but also provide a basis for further understanding the molecular mechanism of complex host pathogen interactions between crustaceans and fungi.