Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

BACKGROUND AND PURPOSE The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by ELISA. KEY RESULTS Pretreatment of the animals with BbCI (2.5 mg.kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1 beta or tumour necrosis factor-alpha, however, did not change. CONCLUSIONS AND IMPLICATIONS Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of similar to 7 kDa. It cross- reacted with polyclonal serum anti- commercial aprotinin. About 1 mu g porcine aprotinin inhibited 6 mu g trypsin whereas 1 mu g commercial soybean inhibitor inhibited only 1 mu g trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.

Caspase 8: Mediating the effects of a novel proteasome inhibitor, NPI-0052

Targeting the proteasome with the sole FDA approved proteasome inhibitor (PI), bortezomib, has been fruitful in specific cancers. Its success has generated an interest in next-generation PIs that might have a therapeutic advantage in cancers, such as leukemia, where bortezomib monotherapy was less effective. This study focuses on a novel, clinically relevant PI, NPI-0052. Experiments show that NPI-0052 targets chymotrypsin- and caspase-like activities more potently than the trypsin-like activity in leukemia cells. NPI-0052 induced apoptosis, as determined by caspase-3 activation and DNA fragmentation. Using caspase inhibitors and caspase-8 (I9.2) or FADD (I2.1) deficient cells revealed that caspase-8 was essential for NPI-0052-induced apoptosis. NPI-0052 killed cells via a caspase-8-tBid-mitochondrial pathway, relying on caspase-8, whereas bortezomib relies on several caspases. NPI-0052 increased reactive oxygen species (ROS) levels, which contributed towards cytotoxicity since an antioxidant conferred protection. To improve the clinical efficacy of PIs, NPI-0052 was combined with epigenetic anti-cancer agents, histone deacetylase inhibitors (HDACi). NPI-0052 with MS-275 or vorinostat (FDA approved HDACi), synergistically induced apoptosis more effectively than an HDACi/bortezomib regimen in Jurkat cells. Caspase-8 and ROS contributed towards NPI-0052/HDACi cytotoxicity and caspase-8 mediated superoxide production by NPI-0052 or NPI-0052/HDACi. The proximal targets of these agents: proteasome activity and histone acetylation were examined to determine if they contributed towards synergistic effects. HDACi targeted proteasomal β subunits and corresponding catalytic activities responsible for degrading proteins. Immunoblotting showed increases in histone-H3 expression and its acetylation with NPI-0052 or NPI-0052/HDACi in Jurkat and primary cells. Importantly, the hyper-acetylation by NPI-0052 was not detected with bortezomib, suggesting that this effect may be unique to NPI-0052. An antioxidant attenuated histone-H3 expression and acetylation induced by NPI-0052 alone or with HDACi. Furthermore, the hyper-acetylation by NPI-0052 relied on caspase-8. These novel results show that a PI is eliciting classical epigenetic alterations, demonstrated by hyper-acetylation of histone-H3. This alteration was oxidant and caspase-8 dependent. Overall, results reveal that caspase-8 mediates many effects induced by NPI-0052. Data show overlapping activities by NPI-0052 and HDACi which are contributing, along with caspase-8 activation and oxidative stress, to cytotoxic interactions in leukemia cells, reinforcing the potential clinical utility of combining these two compounds. ^

Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine.

Pancreatic proteases in the duodenum inhibit the release of cholecystokinin (CCK) and thus exert feedback control of pancreatic exocrine secretion. Exclusion of proteases from the duodenum either by the diversion of bile-pancreatic juice or by the addition of protease inhibitors stimulates exocrine pancreatic secretion. The mechanism by which pancreatic proteases in the duodenum regulate CCK secretion is unknown. In this study, we isolated a trypsin-sensitive peptide that is secreted intraduodenally, releases CCK, and stimulates pancreatic enzyme secretion in rats. This peptide was found to be identical to the porcine diazepam binding inhibitor by peptide sequencing and mass spectrometry analysis. Intraduodenal infusion of 200 ng of synthetic porcine diazepam binding inhibitor1-86 in rats significantly stimulated pancreatic amylase output. Infusion of the CCK antagonist MK-329 completely blocked the diazepam binding inhibitor-stimulated amylase secretion. Similarly, diazepam binding inhibitor33-52 [corrected] also stimulated CCK release and pancreatic secretion in a dose-dependent manner although it was 100 times less potent than the whole peptide. Using a perfusion system containing isolated mucosal cells from the proximal intestine of rats, porcine diazepam binding inhibitor 10(-12) M) dose dependently stimulated CCK secretion. In separate studies, it was demonstrated that luminal secretion of the diazepam binding inhibitor immunoreactivity (7.5 X 10(11) M) could be detected in rat's intestinal washing following the diversion of bile-pancreatic juice. The secretion of this peptide was inhibited by atropine. In conclusion, we have isolated and characterized a CCK-releasing peptide that has a sequence identical to the porcine diazepam binding inhibitor from pig intestinal mucosa and that stimulates CCK release when administered intraduodenally in rat. This peptide may mediate feedback regulation of pancreatic enzyme secretion.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar com- pound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aerugi- nosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

Icatibant, An Inhibitor Of Bradykinin Receptor 2, For Hereditary Angioedema Attacks: Prospective Experimental Single-cohort Study.

Hereditary angioedema (HAE) with C1 inhibitor deficiency manifests as recurrent episodes of edema involving the skin, upper respiratory tract and gastrointestinal tract. It can be lethal due to asphyxia. The aim here was to evaluate the response to therapy for these attacks using icatibant, an inhibitor of the bradykinin receptor, which was recently introduced into Brazil. Prospective experimental single-cohort study on the efficacy and safety of icatibant for HAE patients. Patients with a confirmed HAE diagnosis were enrolled according to symptoms and regardless of the time since onset of the attack. Icatibant was administered in accordance with the protocol that has been approved in Brazil. Symptom severity was assessed continuously and adverse events were monitored. 24 attacks in 20 HAE patients were treated (female/male 19:1; 19-55 years; median 29 years of age). The symptoms were: subcutaneous edema (22/24); abdominal pain (15/24) and upper airway obstruction (10/24). The time taken until onset of relief was: 5-10 minutes (5/24; 20.8%); 10-20 (5/24; 20.8%); 20-30 (8/24; 33.4%); 30-60 (5/24; 20.8%); and 2 hours (1/24; 4.3%). The time taken for complete resolution of symptoms ranged from 4.3 to 33.4 hours. Adverse effects were only reported at injection sites. Mild to moderate erythema and/or feelings of burning were reported by 15/24 patients, itching by 3 and no adverse effects in 6. HAE type I patients who received icatibant responded promptly; most achieved improved symptom severity within 30 minutes. Local adverse events occurred in 75% of the patients.

Key Issues In Inhibitor Management In Patients With Haemophilia.

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Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

A Longitudinal Evaluation Of Anti-fviii Antibodies Demonstrated Igg4 Subclass Is Mainly Correlated With High-titre Inhibitor In Haemophilia A Patients.

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII-binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti-FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti-FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross-sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen-Bethesda assay. Anti-FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma-derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII-binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high-response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non-pathologic antibody subclass with anti-inflammatory properties in other situations, IgG4 is correlated with the presence of high-titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.

Short-Term effect of COX-2 selective inhibitor as an adjunct for the treatment of periodontal disease: a clinical double-blind study in humans

Adjunctive therapeutic strategies that modulate the inflammatory mediators can play a significant role in periodontal therapy. In this double-blind, placebo-controlled study, 60 subjects diagnosed as periodontitis patients were evaluated for 28 days after periodontal treatment combined with selective cyclooxygenase-2 (COX-2) inhibitor. The experimental group received scaling and root planning (SRP) combined with the Loxoprofen antiinflammatory drug (SRP+Loxoprofen). The control group received SRP combined with placebo (SRP+placebo). Plaque index (PI), probing pocket depth (PD) and bleeding on probing (BOP) were monitored with an electronic probe at baseline and after 14 and 28 days. Both groups displayed clinical improvement in PD, PI and BOP. They also showed statistically similar values (p>0.05) of PD reduction on day 14 (0.4 mm) and on day 28 (0.6 mm). At the baseline, few deeper sites (>7 mm) from SRP+Loxoprofen group were responsible and most PD reduction was observed after 14 days (p<0.05). The percentage of remaining deep pockets (>7 mm) after 14 days in the SRP+Loxoprofen group was significantly lower (p<0.05) than in the SRP+placebo group. Loxoprofen presents potential effect as an adjunct of periodontal disease treatment, but long-term clinical trials are necessary to confirm its efficacy.

Effect of nitric oxide inhibitor and donor substances on the infammatory process caused by endodontic irrigants

Nitric oxide (NO) has been considered a key molecule in infammation. OBJECTIVE: The aim of this study was to evaluate the effect of treatment with L-NAME and sodium nitroprussiate, substances that inhibit and release NO, respectively, on tissue tolerance to endodontic irrigants. MATERIAL AND METHODS: The vital dye exudation method was used in a rat subcutaneous tissue model. Injections of 2% Evans blue were administered intravenously into the dorsal penial vein of 14 male rats (200-300 g). The NO inhibitor and donor substances were injected into the subcutaneous tissue in the dorsal region, forming two groups of animals: G1 was inoculated with L-NAME and G2 with sodium nitroprussiate. Both groups received injections of the test endodontic irrigants: acetic acid, 15% citric acid, 17% EDTA-T and saline (control). After 30 min, analysis of the extravasated dye was performed by light absorption spectrophotometry (620 nm). RESULTS: There was statistically signifcant difference (p<0.05) between groups 1 and 2 for all irrigants. L-NAME produced a less intense infammatory reaction and nitroprussiate intensifed this process. CONCLUSIONS: Independently of the administration of NO inhibitors and donors, EDTA-T produced the highest irritating potential in vital tissue among the tested irrigating solutions.

A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 ± 2.47 µmol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.