Development of Inflammation in the Gastrointestinal Tract of Rats after Prolonged Administration of Alcohol

The purpose of the work was to experimentally study the chronic alcohol intoxication on the indicators of inflammation and lipid peroxidation in the gastrointestinal system. Materials and methods. Ethyl alcohol was added to the water for 2-month-old male rats, ranging from 5% to 15% for 108 days. In homogenates of mucous membranes of the gastrointestinal tract and liver, the activity of elastase enzymes, acid phosphatase and the concentration of malonic dialdehyde were determined, in serum – elastase activity and malonic dialdehyde content. Results and discussion. Biochemical research of one of the markers of inflammation (elastase activity) in rats found a probable increase of elastase activity in different parts of the digestive tract after prolonged alcohol consumption, regardless of the sex of the animals. Thus, in the serum of rats after the introduction of ethanol, the activity of elastase increased by 71.7%, in the oral mucosa – by 29.2%, in the gastric mucosa – by 55.5%, in the liver – by 29.0%. In the small and large intestine, the level of this marker of inflammation has changed slightly. The level of elastase activity shows the degree of accumulation of leukocytes in the tissues as a result of the development of the inflammatory process. Acid phosphatase activity in the oral mucosa of rats treated with ethanol increased by 47.4%, in the gastric mucosa – by 30.3%, in the mucous membrane of the small intestine – by 37.4%, in the mucous membrane of the colon – by 40.4%, in the liver – by 112.6%. Activation of acid phosphatase, along with other lysosomal enzymes, is the primary inflammatory response that triggers the production of mediators, which in turn cause secondary tissue alteration in subsequent stages of the inflammatory process. Therefore, the results obtained on the activation of acid phosphatase along with elastase indicate the presence of inflammation in the mucous membranes of the digestive tract, and especially in the liver of rats chronically treated with ethanol. The introduction of alcohol also led to an increase in the concentration of malonic dialdehyde in the mucous membranes: the oral cavity – by 20.3%, the stomach – by 32.3%, the small intestine – by 96.6%, the colon – by 50.2%, in the liver – by 39.4%, in serum – by 33.3%. A significant increase in the level of malonic dialdehyde in the tissues of the digestive tract of rats after long-term intake of ethanol is a sign of activation of lipid peroxidation and intensification of oxidative stress reactions. Conclusion. The results of the study of elastase activity indicate the development of inflammation in the mucous membranes of the gastrointestinal tract, liver and serum of rats under the influence of chronic administration of ethanol. Increased acid phosphatase activity in the tissues of the gastrointestinal tract after prolonged use of ethanol indicates damage to cell membranes, which is a consequence of inflammation. A significant increase in the level of malonic dialdehyde in the mucous membranes of the gastrointestinal tract, liver and serum of rats after chronic ethanol intake is a sign of intensification of oxidative stress reactions

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.

α1-Antitrypsin attenuates acute rejection of orthotopic murine lung allografts

Background α1-Antitrypsin (AAT) is an acute phase glycoprotein, a multifunctional protein with proteinase inhibitory, anti-inflammatory and cytoprotective properties. Both preclinical and clinical experiences show that the therapy with plasma purified AAT is beneficial for a broad spectrum of inflammatory conditions. The potential effects of AAT therapy have recently been highlighted in lung transplantation (LuTx) as well. Methods We used a murine fully mismatched orthotopic single LuTx model (BALB/CJ as donors and C57BL/6 as recipients). Human AAT preparations (5 mg, n = 10) or vehicle (n = 5) were injected to the recipients subcutaneously prior to and intraperitoneally immediately after the LuTx. No immune suppressive drugs were administered. Three days after the transplantation, the mice were sacrificed, and biological samples were assessed. Results Histological analysis revealed significantly more severe acute rejection in the transplanted lungs of controls than in AAT treated mice (p < 0.05). The proportion of neutrophil granulocytes, B cells and the total T helper cell populations did not differ between two groups. There was no significant difference in serum CXCL1 (KC) levels. However, when compared to controls, human AAT was detectable in the serum of mice treated with AAT and these mice had a higher serum anti-elastase activity, and significantly lower proportion of Th1 and Th17 among all Th cells. Cleaved caspase-3-positive cells were scarce but significantly less abundant in allografts from recipients treated with AAT as compared to those treated with vehicle. Conclusion Therapy with AAT suppresses the acute rejection after LuTx in a mouse model. The beneficial effects seem to involve anti-protease and immunomodulatory activities of AAT.

Effects of Alvelestat, an Oral Neutrophil Elastase Inhibitor, on Elevated Elastase and Collagen Turnover Biomarkers in Patients with Bronchiolitis Obliterans Syndrome after Hematopoietic Cell Transplantation

Introduction Bronchiolitis Obliterans Syndrome (BOS) is a rare but devastating complication of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) and is associated with a high morbidity and mortality. There is a dearth of treatment options for BOS and new strategies are needed. Airway neutrophilia is a hallmark of BOS, even in the absence of infection, and neutrophil elastase (NE) is an enzyme that has been implicated in the pathogenesis of BOS. We are conducting a phase 1b study of an oral NE inhibitor, alvelestat, in patients with BOS after HCT . Biomarkers, including the elastin breakdown peptides desmosine/isodesmosine (DES/IDES), and stimulated neutrophil elastase assess direct effect on NE activity. Neo-epitope by-products of collagen type 3 and 6 synthesis (PRO-C3 and PRO-C6) and degradation (C3M and C6M) are measured as biomarkers of fibrosis/tissue modelling Methods Patients age ≥18 years with BOS and chronic GVHD after HCT were recruited to the National Cancer Institute protocol (NCT02669251). This phase 1 study had 2 parts: 8-week intra-patient dose escalation period, followed by a continuation period that allowed for up to 6 months of treatment. Alvelestat was given orally starting at 60mg twice daily, increased every 2 weeks to 120mg twice daily, 180mg twice daily, and finally 240mg twice daily. Peripheral blood samples were collected at baseline and at the end of each dose-escalation stage. Plasma DES/IDES was measured by isotopic dilution liquid chromatography-tandem mass spectrometry (Huang et al Thorax 2012;67:502-508). Ex vivo zymosan stimulated neutrophil elastase activity was measured by ProteaseTag® immunoassay (ProAxsis Ltd, Northern Ireland). PRO-C3, PRO-C6, C3M and C6M were measured by competitive ELISA. (Nordic Biosciences, Denmark). Results are presented as Mean and Standard Error Mean (SEM). Results Between 2016 and 2018, 7 patients were enrolled (3 men and 4 women). Median FEV 1 after bronchodilator at time of enrollment was 44% predicted (range 38-74). All 7 patients were able to tolerate dose escalation of alvelestat up to the maximum dose 240mg twice daily. Preliminary clinical results were previously presented. DES/IDES was elevated at baseline (mean 0.464 (SEM 0.0508) ng/ml, with 6 of 7 subjects above the Upper Limit of Normal (ULN, 0.280 ng/ml)). Levels progressively declined during the dose escalation period to 0.380 (SEM 0.0419) ng/ml by week 8, representing a mean within subject % change from baseline (CFB) of -16.2% (SEM 6.794, Figure 1a) Ex vivo zymosan stimulated elastase activity also showed progressive decrease over the dose escalation period, with some subjects demonstrating 100% suppression (Figure 1b). Collagen synthesis as measured by PRO-C3 and PRO-C6 was increased above ULN at baseline and declined with alvelestat treatment (Figure 1c and 1d). There was no consistent change in collagen degradation biomarkers (C3M, C6M) There was consistency of a suppressive effect on biomarkers of elastase activity and collagen turnover in 6 of 7 treated patients, all of whom had improved or stable lung disease (ranging from change in FEV1 % predicted at end of treatment from +9% to -6%). Conclusion NE can damage lung tissue due to elastin breakdown, pro-inflammatory and pro-fibrotic effects (Sallenave J-M, J Leuk Biol 2015;98:137-139). This is the first evidence of elevated elastase activity as detected by elastin breakdown in patients with BOS and chronic GVHD. Treatment with the selective NE inhibitor, alvelestat was associated with progressive reduction of plasma desmosine levels over 8 weeks of within-subject dose escalation and reduction stimulated neutrophil elastase activity. The consistent suppression of elastase and of collagen synthesis/turnover biomarkers following alvelestat treatment is encouraging for its potential to impact progressive lung fibrosis in BOS and chronic GVHD. Disclosures Parkin: Mereo BioPharma Group: Current Employment. Moore: Mereo BioPharma Group: Current Employment. Pavletic: Center for Cancer Research: Research Funding; National Cancer Institute: Research Funding; National Institutes of Health: Research Funding; Celgene: Research Funding; Actelion: Research Funding; Eli Lilly: Research Funding; Pharmacyclics: Research Funding; Kadmon: Research Funding.

Extracorporeal Photopheresis Induces Netosis in Neutrophils Derived from Patients with Graft-Versus-Host Disease

Introduction Extracorporeal photopheresis (ECP) serves as a second line treatment for patients with acute or chronic graft versus host disease (GVHD) and demonstrates efficacy in ameliorating GVHD in this setting. The mechanism by which ECP acts against GVHD has not been fully elucidated yet. Preliminary data point to an association between GVHD and increased neutrophil extracellular trap (NET) formation (NETosis) although much is unknown regarding the pathologic implication of this association. In this study we performed a preliminary assessment of the influence of ECP on NETosis among patients with GVHD. Methods Six patients after allogeneic transplantation treated with ECP for severe GVHD post allogeneic transplant at the Rabin Medical Center in Israel were enrolled to the study. Blood samples were obtained at 3 different time points: before an ECP cycle, immediately after the end of the ECP and 24h after the initiation of the ECP cycle. Neutrophils were obtained from whole blood samples using a percoll gradient. NETosis was assessed by measurement of neutrophil elastase activity using a commercial NETosis assay kit by Cayman Chemical and by immunofluorescence staining for DNA (DAPI (4',6-diamidino-2-phenylindole)) and for citrullinated H3 (H3Cit), a marker of NET formation. All assessments were executed in unstimulated neutrophils and in neutrophils that were stimulated with phorbol myristate acetate (PMA)(100nmol) for 4 hours. Results Six patients (4 males) with chronic GVHD were included in the study. The underlying hematologic disease was acute myeloid leukemia (AML) in four patients, B-cell acute lymphocytic leukemia (B-ALL) and myelodysplastic syndrome (MDS) each in one patient. ECP was executed for moderate to severe GVHD. We observed a sharp increase in the formation of NETs following treatment with ECP among all study participants. The level of neutrophil elastase activity was significantly elevated from a mean value of 2.21mU/mL (±0.6mU/mL) at baseline to a mean value of 13.82mU/mL (±5.54mU/mL) immediately after the treatment (p-value=0.0022). The mean level of neutrophil elastase activity was significantly elevated to a mean peak value of 17.35mU/mL (±10.74mU/mL) 24h following the initiation of the ECP cycle (p-value 0.0063 for the difference between the first and the last time points). Pre-incubation of the neutrophils with PMA yielded similar results, as the mean neutrophil elastase activity was 6.05mU/mL (±3.63mU/mL), 20.16mU/mL (±6.72mU/mL), and 24.57mU/mL (±13.59mU/mL) before the ECP cycle initiation, immediately after treatment, and 24h following ECP onset, respectively (Figure 1). In agreement, the expression of H3cit was also increased in neutrophils derived from patients with GVHD after ECP treatment (Figure 2). Conclusion Our preliminary data indicate that ECP induces NET formation among patients with GVHD. NETosis might play a role in the therapeutic effects of ECP in this setting. These initial results might set the stage for future studies in this field. Figure 1 Figure 1. Disclosures Goldberg: MSD Israel: Consultancy. Yeshurun: Astellas: Consultancy; Janssen: Consultancy. Wolach: Janssen: Consultancy; Abbvie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy; Amgen: Research Funding; Novartis: Consultancy; Neopharm: Consultancy.

Development and Evaluation of Polyherbal Based Novel Antiaging Synergistic Formulation

Aim: The present work was aimed to develop and evaluate polyherbal-based novel antiaging formulation. Place and Duration of Study: Faculty of pharmacy, B N University Udaipur, Department of Pharmacognosy, Divine College of pharmacy Satana between Feb 2019 to Apr 2021. Methods: The selected plant extract Moringa oleifera hydroalcoholic extract (2%), Juglans regia aqueous extract (1%), Vitis vinifera ethanolic extract (1.5%), Camellia sinensis cold water extract (1.8%), Punica granatum aqueous extract (2%) were optimized in o/w type herbal cream by incorporation of different concentration of stearic acid and Tween 60. The total 9 formulations were evaluated on the basis of preliminary, phytochemical screening, and accelerated stability study and confirm the C7 polyherbal formulation (PHF) were more stable, safe, and showed pseudo plastic flow. Results: The In-vitro free radical scavenging assay (DPPH Assay - IC50 56 ± 0.04 µg/ml and H2O2 scavenging assay - IC50 = 67±0.68 µg/ml) compared with standard Ascorbic acid and In-vitro anti-collagenase and anti-elastase activity validate PHF as antioxidant and antiaging activity. The in vitro Anti-collagenase activity showed 89.5% inhibition at 100 μg/mL concentration of C7 formulation (IC50 = 54.62 μg/mL) and the standard Gallic acid showed 74.6% inhibition at 100 μg/mL (IC50 = 67.83 μg/mL). The in vitro Anti-elastase activity showed 67.5% inhibition at 250 μg/mL of C7 formulation (IC50 = 193.65 μg/mL) and copper sulphate solution used as standard showed 70.6% inhibition (IC50 = 772.42 μg/mL). The percent inhibition activity was observed that the C7 formulation is potential antiaging activity as compared to positive control. Conclusion: These studies conclude that the composition of PHF with a cream base increases the production of collagen and elastin which is responsible for the noteworthy synergistic antiaging activity.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti–SARS-CoV-2 systemic and mucosal immunity

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti–SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

Protease–Antiprotease Imbalance in Bronchiectasis

Airway inflammation plays a central role in bronchiectasis. Protease–antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases’ over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease–antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.

Influence of fat-free, fat and sucrose diets on rat periodont condition

Aim. To investigate the effect of consumption of diets with different content of fat and sugar on the state of the periodontium of rats.Methods. The rats received a semi-synthetic fat-free diet (FFD), a fat diet (5 % sunflower oil), and a sucrose diet (50 % sucrose) for 30 days. Determined the degree of periodontal atrophy, tooth decay and in the gum homogenate - the activity of elastase, catalase, urease, lysozyme and the content of malondialdehyde (MDA).Results. Sugar diet increases the degree of periodontal atrophy and tooth decay, but decreases elastase activity in the gums and increases catalase activity. Fat diet does not affect periodontal atrophy and tooth decay, but it reduces catalase activity.Conclusion. The increase in the intensity of caries and periodontal atrophy under the influence of a sucrose diet is probably due to the stimulation of the formation of lactic acid. Fat nutrition does not affect the intensity of dental caries and the degree of periodontal atrophy.

Effects of (–)-Loliolide against Fine Dust Preconditioned Keratinocyte Media-Induced Dermal Fibroblast Inflammation

At present air pollution in parts of East Asia is at an alarming level due to elevated levels of fine dust (FD). Other than pulmonary complications, FD was found to affect the pathogenesis of ROS-dependent inflammatory responses via penetrating barrier-disrupted skin, leading to degradation of extracellular matrix components through the keratinocyte-fibroblast axis. The present study discloses the evaluation of human dermal fibroblast (HDF) responses to FD preconditioned human keratinocyte media (HPM) primed without and with (-)-loliolide (HTT). HPM-FD treatment increased the ROS level in HDFs and activated mitogen-activated protein kinase-derived nuclear factor (NF)-κB inflammatory signaling pathways with a minor reduction of viability. The above events led to cell differentiation and production of matrix metalloproteinases (MMP), increasing collagenase and elastase activity despite the increase of tissue inhibitors of metalloproteinases (TIMP). Media from HTT primed keratinocytes stimulated with FD indicated ameliorated levels of MMPs, inflammatory cytokines, and chemokines in HDFs with suppressed collagenase and elastase activity. Present observations help to understand the factors that affect HDFs in the microenvironment of FD exposed keratinocytes and the therapeutic role of HTT as a suppressor of skin aging. Further studies using organotypic skin culture models could broaden the understanding of the effects of FD and the therapeutic role of HTT.