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Li X, Lu C, Yang Y, Yu C, Rao Y. Site-specific targeted drug delivery systems for the treatment of inflammatory bowel disease. Biomed Pharmacother 2020; 129:110486. [PMID: 32768972 DOI: 10.1016/j.biopha.2020.110486] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Revised: 06/30/2020] [Accepted: 06/30/2020] [Indexed: 12/14/2022] Open
Abstract
Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis and manifests as a complex and dysregulated immune response. To date, there is no cure for IBD; thus, lifelong administration of maintenance drugs is often necessary. Since conventional IBD treatment strategies do not target the sites of inflammation, only limited efficacy is observed with their use. Moreover, the possibility of severe side effects resulting from systemic drug redistribution is high when conventional drug treatments are used. Therefore, a straightforward disease-targeted drug delivery system is desirable. Based on the pathophysiological changes associated with IBD, novel site-specific targeted drug delivery strategies that deliver drugs directly to the inflammation sites can enhance drug accumulation and decrease side effects. This review summarizes novel inflammation targeted delivery systems in the management of IBD. It also discusses the challenges and new perspectives in this field.
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Affiliation(s)
- Xin Li
- Department of Pharmacology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
| | - Chao Lu
- Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
| | - Yanyan Yang
- Department of Pharmacology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
| | - Chaohui Yu
- Department of Gastroenterology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
| | - Yuefeng Rao
- Department of Pharmacology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
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Markovic M, Ben-Shabat S, Keinan S, Aponick A, Zimmermann EM, Dahan A. Molecular Modeling-Guided Design of Phospholipid-Based Prodrugs. Int J Mol Sci 2019; 20:ijms20092210. [PMID: 31060339 PMCID: PMC6538990 DOI: 10.3390/ijms20092210] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Revised: 04/29/2019] [Accepted: 05/01/2019] [Indexed: 02/06/2023] Open
Abstract
The lipidic prodrug approach is an emerging field for improving a number of biopharmaceutical and drug delivery aspects. Owing to their structure and nature, phospholipid (PL)-based prodrugs may join endogenous lipid processing pathways, and hence significantly improve the pharmacokinetics and/or bioavailability of the drug. Additional advantages of this approach include drug targeting by enzyme-triggered drug release, blood–brain barrier permeability, lymphatic targeting, overcoming drug resistance, or enabling appropriate formulation. The PL-prodrug design includes various structural modalities-different conjugation strategies and/or the use of linkers between the PL and the drug moiety, which considerably influence the prodrug characteristics and the consequent effects. In this article, we describe how molecular modeling can guide the structural design of PL-based prodrugs. Computational simulations can predict the extent of phospholipase A2 (PLA2)-mediated activation, and facilitate prodrug development. Several computational methods have been used to facilitate the design of the pro-drugs, which will be reviewed here, including molecular docking, the free energy perturbation method, molecular dynamics simulations, and free density functional theory. Altogether, the studies described in this article indicate that computational simulation-guided PL-based prodrug molecular design correlates well with the experimental results, allowing for more mechanistic and less empirical development. In the future, the use of molecular modeling techniques to predict the activity of PL-prodrugs should be used earlier in the development process.
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Affiliation(s)
- Milica Markovic
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Shimon Ben-Shabat
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | | | - Aaron Aponick
- Department of Chemistry, University of Florida, Gainesville, FL 32603, USA.
| | - Ellen M Zimmermann
- Department of Medicine, Division of Gastroenterology, University of Florida, Gainesville, FL 32608, USA.
| | - Arik Dahan
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
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Markovic M, Dahan A, Keinan S, Kurnikov I, Aponick A, Zimmermann EM, Ben-Shabat S. Phospholipid-Based Prodrugs for Colon-Targeted Drug Delivery: Experimental Study and In-Silico Simulations. Pharmaceutics 2019; 11:pharmaceutics11040186. [PMID: 30995772 PMCID: PMC6523355 DOI: 10.3390/pharmaceutics11040186] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2019] [Revised: 04/07/2019] [Accepted: 04/08/2019] [Indexed: 12/23/2022] Open
Abstract
In ulcerative colitis (UC), the inflammation is localized in the colon, and one of the successful strategies for colon-targeting drug delivery is the prodrug approach. In this work, we present a novel phospholipid (PL)-based prodrug approach, as a tool for colonic drug targeting in UC. We aim to use the phospholipase A2 (PLA2), an enzyme that is overexpressed in the inflamed colonic tissues of UC patients, as the PL-prodrug activating enzyme, to accomplish the liberation of the parent drug from the prodrug complex at the specific diseased tissue(s). Different linker lengths between the PL and the drug moiety can dictate the rate of activation by PLA2, and subsequently determine the amount of free drugs at the site of action. The feasibility of this approach was studied with newly synthesized PL-Fmoc (fluorenylmethyloxycarbonyl) conjugates, using Fmoc as a model compound for testing our hypothesis. In vitro incubation with bee venom PLA2 demonstrated that a 7-carbon linker between the PL and Fmoc has higher activation rate than a 5-carbon linker. 4-fold higher colonic expression of PLA2 was demonstrated in colonic mucosa of colitis-induced rats when compared to healthy animals, validating our hypothesis of a colitis-targeting prodrug approach. Next, a novel molecular dynamics (MD) simulation was developed for PL-based prodrugs containing clinically relevant drugs. PL-methotrexate conjugate with 6-carbon linker showed the highest extent of PLA2-mediated activation, whereas shorter linkers were activated to a lower extent. In conclusion, this work demonstrates that for carefully designed PL-drug conjugates, PLA2 overexpression in inflamed colonic tissues can be used as prodrug-activating enzyme and drug targeting strategy, including insights into the activation mechanisms in a PLA2 binding site.
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Affiliation(s)
- Milica Markovic
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Arik Dahan
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | | | | | - Aaron Aponick
- Department of Chemistry, University of Florida, Gainesville, FL 32603, USA.
| | - Ellen M Zimmermann
- Department of Medicine, Division of Gastroenterology, University of Florida, Gainesville, FL 32603, USA.
| | - Shimon Ben-Shabat
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
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Markovic M, Ben-Shabat S, Keinan S, Aponick A, Zimmermann EM, Dahan A. Prospects and Challenges of Phospholipid-Based Prodrugs. Pharmaceutics 2018; 10:pharmaceutics10040210. [PMID: 30388756 PMCID: PMC6321354 DOI: 10.3390/pharmaceutics10040210] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 10/24/2018] [Accepted: 10/26/2018] [Indexed: 12/19/2022] Open
Abstract
Nowadays, the prodrug approach is used already at the early stages of drug development. Lipidic prodrug approach is a growing field for improving a number of drug properties/delivery/therapy aspects, and can offer solutions for various unmet needs. This approach includes drug moiety bound to the lipid carrier, which can be triglyceride, fatty acids, steroid, or phospholipid (PL). The focus of this article is PL-based prodrugs, which includes a PL carrier covalently bound to the active drug moiety. An overview of relevant physiological lipid processing pathways and absorption barriers is provided, followed by drug delivery/therapeutic application of PL-drug conjugates, as well as computational modeling techniques, and a modern bioinformatics tool that can aid in the optimization of PL conjugates. PL-based prodrugs have increased lipophilicity comparing to the parent drug, and can therefore significantly improve the pharmacokinetic profile and overall bioavailability of the parent drug, join the endogenous lipid processing pathways and therefore accomplish drug targeting, e.g., by lymphatic transport, drug release at specific target site(s), or passing the blood-brain barrier. Moreover, an exciting gateway for treating inflammatory diseases and cancer is presented, by utilizing the PL sn-2 position in the prodrug design, aiming for PLA₂-mediated activation. Overall, a PL-based prodrug approach shows great potential in improving different drug delivery/therapy aspects, and is expected to grow.
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Affiliation(s)
- Milica Markovic
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | - Shimon Ben-Shabat
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
| | | | - Aaron Aponick
- Department of Chemistry, University of Florida, Gainesville, FL 32603, USA.
| | - Ellen M Zimmermann
- Department of Medicine, Division of Gastroenterology, University of Florida, Gainesville, FL 32610, USA.
| | - Arik Dahan
- Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
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Markovic M, Ben‐Shabat S, Keinan S, Aponick A, Zimmermann EM, Dahan A. Lipidic prodrug approach for improved oral drug delivery and therapy. Med Res Rev 2018; 39:579-607. [DOI: 10.1002/med.21533] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 07/26/2018] [Accepted: 07/27/2018] [Indexed: 12/13/2022]
Affiliation(s)
- Milica Markovic
- Department of Clinical PharmacologySchool of Pharmacy, Faculty of Health Sciences, Ben‐Gurion University of the NegevBeer‐Sheva Israel
| | - Shimon Ben‐Shabat
- Department of Clinical PharmacologySchool of Pharmacy, Faculty of Health Sciences, Ben‐Gurion University of the NegevBeer‐Sheva Israel
| | | | - Aaron Aponick
- Department of ChemistryUniversity of FloridaGainesville Florida
| | - Ellen M. Zimmermann
- Department of MedicineDivision of Gastroenterology, University of FloridaGainesville Florida
| | - Arik Dahan
- Department of Clinical PharmacologySchool of Pharmacy, Faculty of Health Sciences, Ben‐Gurion University of the NegevBeer‐Sheva Israel
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Murgia A, Hinz C, Liggi S, Denes J, Hall Z, West J, Santoru ML, Piras C, Manis C, Usai P, Atzori L, Griffin JL, Caboni P. Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid, free fatty acids and amino acid levels. Metabolomics 2018; 14:140. [PMID: 30830399 DOI: 10.1007/s11306-018-1439-4] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Accepted: 10/05/2018] [Indexed: 01/08/2023]
Abstract
BACKGROUND Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression. OBJECTIVES The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples. METHODS A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis. RESULTS Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease. CONCLUSIONS Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.
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Affiliation(s)
- Antonio Murgia
- Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - Christine Hinz
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - Sonia Liggi
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - Jùlìa Denes
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - Zoe Hall
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - James West
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | | | - Cristina Piras
- Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy
| | - Cristina Manis
- Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
| | - Paolo Usai
- Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Cagliari, Italy
| | - Luigi Atzori
- Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy
| | - Julian L Griffin
- Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK
| | - Pierluigi Caboni
- Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
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Phospholipid-drug conjugates as a novel oral drug targeting approach for the treatment of inflammatory bowel disease. Eur J Pharm Sci 2017. [DOI: 10.1016/j.ejps.2017.06.022] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
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Miklavcic JJ, Hart TDL, Lees GM, Shoemaker GK, Schnabl KL, Larsen BMK, Bathe OF, Thomson ABR, Mazurak VC, Clandinin MT. Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease. World J Gastroenterol 2015; 21:10080-10090. [PMID: 26401073 PMCID: PMC4572789 DOI: 10.3748/wjg.v21.i35.10080] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2015] [Revised: 03/27/2015] [Accepted: 05/04/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease.
METHODS: Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn’s disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups.
RESULTS: Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC.
CONCLUSION: This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.
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Lipoprotein-associated phospholipase A2 and arterial stiffness evaluation in patients with inflammatory bowel diseases. J Crohns Colitis 2014; 8:936-44. [PMID: 24529818 DOI: 10.1016/j.crohns.2014.01.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Revised: 01/18/2014] [Accepted: 01/19/2014] [Indexed: 02/08/2023]
Abstract
BACKGROUND AND AIMS The association between inflammatory bowel diseases (IBD) and cardiovascular disease (CVD) remains equivocal. Arterial stiffness, as assessed by pulse wave velocity (PWV), and lipoprotein-associated phospholipase A2 (Lp-PLA2) are surrogates of CVD risk. AIM The aim of this study was to assess carotid-femoral PWV and Lp-PLA2 in patients with IBD without history of CVD. METHODS Established CVD risk factors, IBD characteristics, PWV and Lp-PLA2 activity were assessed in 44 patients with IBD, 29 with Crohn's disease (CD) and 15 with ulcerative colitis (UC), and 44 matched controls. RESULTS IBD patients had lower total and low density lipoprotein cholesterol (LDL-C) levels. There was no difference in PWV between patients and controls (6.8 vs. 6.4m/s), but patients with CD had higher PWV compared to those with UC (7 vs. 6.3m/s; p=0.044), and to controls. Smoking rates were significantly higher among CD patients. Factors associated with PWV were age, mean arterial pressure and smoking. Lp-PLA2 activity was significantly lower in patients with IBD (46.8 vs. 53.9 nmol/mL/min; p=0.011). There was no difference in Lp-PLA2 between CD and UC patients. LDL-C was the only significant predictor of Lp-PLA2. CONCLUSIONS Our study showed lower Lp-PLA2 activity in patients with IBD compared with controls, reflecting lower LDL-C in the former. There was no difference in PWV between the two groups. Arterial stiffness was higher in patients with CD, which is likely related to higher smoking rates. These findings challenge a possible association between IBD and CVD, but further studies are required.
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MC-12, an annexin A1-based peptide, is effective in the treatment of experimental colitis. PLoS One 2012; 7:e41585. [PMID: 22844504 PMCID: PMC3402399 DOI: 10.1371/journal.pone.0041585] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2012] [Accepted: 06/25/2012] [Indexed: 12/11/2022] Open
Abstract
Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E2 (PGE2), cytoplasmic phospholipase A2 (cPLA2) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A2; reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE2 in the colon and PGE2 metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.
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Huebner C, Petermann I, Lam WJ, Shelling AN, Ferguson LR. Characterization of single-nucleotide polymorphisms relevant to inflammatory bowel disease in commonly used gastrointestinal cell lines. Inflamm Bowel Dis 2010; 16:282-95. [PMID: 19653290 DOI: 10.1002/ibd.21043] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND The era of genome-wide association studies (GWAS) has led to the identification of many inflammatory bowel disease (IBD)-associated single-nucleotide polymorphisms (SNPs) with unknown function. The next step would be to identify the functional consequences of these polymorphisms in order to target them efficiently for therapeutic purposes. One way to study this type of genetic variation is the use of cell line models. However, to characterize the functional effect of a SNP, it is important to know if the selected cell line model itself carries the studied genetic variation. Here, we genotyped 50 IBD markers across 32 susceptibility genes in 9 commonly used gastrointestinal cell lines. METHODS We used Sequenom, TaqMan, and DNA sequencing for the genotyping. To determine the expression profile of the selected genes, we conducted real-time RT-PCR. RESULTS We found variant SNPs in all analyzed cell lines. Almost every minor allele was carried by at least one of the tested cell lines. We analyzed the effect of 4 SNPs in more detail using quantitative real-time RT-PCR (qRT-PCR) comprising genes ATG16L1, CD14, MDR1, and OCTN2. According to our data, only 2 of the commonly studied SNPs in MDR1 and CD14 have an impact on gene expression. CONCLUSIONS We have identified genotype variants in all analyzed cell lines. Some of them are functional and alter the response to drugs (MDR1) or affect bacterial recognition (TLR4, NOD2). Our results highlight that the genotype should not be neglected in experimental design when using model cell lines.
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Affiliation(s)
- Claudia Huebner
- Discipline of Nutrition, FM&HS, The University of Auckland, Auckland, New Zealand.
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Murakami M, Ohta T, Ito S. Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells. J Neurosci Res 2009; 87:2095-104. [PMID: 19235895 DOI: 10.1002/jnr.22036] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron-glia interaction in vitro. We examined the increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of interleukin-1beta (IL-1beta) or prostaglandin E(2) (PGE(2)) and COX-2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK-induced [Ca(2+)](i) increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca(2+)](i). The effects of LPS were blocked by pretreatment with an interleukin-1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL-1beta from enteric glial cells. The augmenting effects of IL-1beta on the BK-induced neural [Ca(2+)](i) increase and PGE(2) release from enteric glial cells were abolished by a phospholipase A(2) (PLA(2)) inhibitor and a COX inhibitor, and partly suppressed by a COX-2 inhibitor. IL-1beta up-regulated the COX-2 expression in enteric glial cells. LPS promotes IL-1beta secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE(2) release via glial PLA(2) and COX-2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease.
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Affiliation(s)
- Matsuka Murakami
- Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
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Zhang F, Suarez G, Sha J, Sierra JC, Peterson JW, Chopra AK. Phospholipase A2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells. Cell Signal 2009; 21:1085-99. [DOI: 10.1016/j.cellsig.2009.02.018] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2009] [Revised: 02/24/2009] [Accepted: 02/24/2009] [Indexed: 11/28/2022]
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Di Mari JF, Saada JI, Mifflin RC, Valentich JD, Powell DW. HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts. Am J Physiol Gastrointest Liver Physiol 2007; 293:G719-28. [PMID: 17640979 DOI: 10.1152/ajpgi.00117.2007] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Proinflammatory cytokines and eicosanoids are central players in intestinal inflammation. IL-1, a key cytokine associated with intestinal mucosal inflammation, induces COX-2 expression in human colonic myofibroblasts (CMF) and increased prostaglandin E(2) secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1alpha-induced cyclooxygenase-2 (COX-2) expression is the result of NF-kappaB- and ERK-mediated transcription, as well as COX-2 message stabilization, which depends on p38, MAPKAPK-2 (MK-2) and human antigen R (HuR) RNA binding protein activation. Lipoxygenase (LOX)-derived hydroxyeicosatetraenoic acids (HETEs) are elevated in IBD and colonic adenomas and "cross talk" has been observed between the COX and LOX pathways. Since COX-2 expression is primarily in CMFs in colonic adenomas, we examined the impact of LOX metabolites, particularly HETEs, on IL-1alpha-induced COX-2 expression in human CMFs. Although 5(S)-, 12(R)-, and 15(S)-HETEs alone had little to no effect on COX-2 expression, they enhanced IL-1-mediated COX-2 expression 3.6 +/- 0.5-fold. Studies utilizing heterogeneous nuclear RNA amplification and 5,6-dichloro-beta-d-ribofuranosylbenzimidazole treatment were undertaken to measure COX-2 transcription and message stabilization, respectively. We found that HETEs enhanced IL-1-induced COX-2 mRNA levels in CMF as the result of increased p38, MK-2, and HuR activity, increasing message stability greater than that observed with IL-1 alone. Thus HETEs can act synergistically with IL-1alpha to induce COX-2 expression in human CMFs. HETEs may play a role in both colonic inflammation and in increasing the risk of CRC in IBD independently and via induction of COX-2-mediated prostaglandin secretion.
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Affiliation(s)
- J F Di Mari
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA.
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15
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Busserolles J, Payá M, D'Auria MV, Gomez-Paloma L, Alcaraz MJ. Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M. Biochem Pharmacol 2005; 69:1433-40. [PMID: 15857607 DOI: 10.1016/j.bcp.2005.01.020] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2004] [Accepted: 01/31/2005] [Indexed: 12/19/2022]
Abstract
Proinflammatory mediators, namely eicosanoids, reactive oxygen and nitrogen species and cytokines, are clearly involved in the pathogenesis of intestinal bowel disease. bolinaquinone (BQ) and petrosaspongiolide M (PT), two marine products with potent anti-inflammatory action, have been shown to control the production of mediators in acute and chronic inflammatory processes. Hence, we have tested here the hypothesis that BQ and PT could ameliorate inflammation and oxidative stress parameters in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in Balb/c mice. BQ and PT were given orally in doses of 10 or 20mg/kg/day. Treatment of the animals with BQ or PT at the highest dose significantly protected against TNBS-induced inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Neutrophilic infiltration, interleukin (IL)-1beta and prostaglandin E(2) (PGE(2)) levels, as well as cyclooxygenase-2 (COX-2) protein expression were inhibited by both compounds. Colonic nitrite and nitrate levels and protein expression of inducible nitric oxide synthase (iNOS) were also lower in the treated groups in comparison to the TNBS control. BQ and PT reduced nitrotyrosine immunodetection and colonic superoxide anion production. Neither compound inhibited the expression of the protective protein heme oxygenase-1 (HO-1), although they reduced the extension of apoptosis. Our study also indicated that PT could interfere with the translocation of p65 into the nucleus, a key step in nuclear factor-kappaB (NF-kappaB) activation. Altogether, the results suggest that BQ and PT can have potential protective actions in intestinal inflammatory diseases.
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Affiliation(s)
- Jérôme Busserolles
- Departamento de Farmacología, Universidad de Valencia, Facultad de Farmacia, Av. Vicent Andrés Estellés s/n, Valencia 46100, Spain
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16
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Abstract
Induction of immune responses following oral immunization is frequently dependent upon the co-administration of appropriate adjuvants that can initiate and support the transition from innate to adaptive immunity. The three bacterial products with the greatest potential to function as mucosal adjuvants are the ADP-ribosylating enterotoxins (cholera toxin and the heat-labile enterotoxin of Escherichia coli), synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN), and monophosphoryl lipid A (MPL). The mechanism of adjuvanticity of the ADP-ribosylating enterotoxins is the subject of considerable debate. Our own view is that adjuvanticity is an outcome and not an event. It is likely that these molecules exert their adjuvant function by interacting with a variety of cell types, including epithelial cells, dendritic cells, macrophages, and possibly B- and T-lymphocytes. The adjuvant activities of CpG and MPL are due to several different effects they have on innate and adaptive immune responses and both MPL and CpG act through MyD88-dependent and -independent pathways. This presentation will summarize the probable mechanisms of action of these diverse mucosal adjuvants and discuss potential synergy between these molecules for use in conjunction with plant-derived vaccines.
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Affiliation(s)
- L C Freytag
- Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA
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17
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Abstract
Inflammatory bowel disease includes Crohn's disease and ulcerative colitis, and is characterized by chronic inflammation of the intestines. The advances in understanding of the inflammatory process have resulted in improved treatment of inflammatory bowel disease. The systemic complications of inflammatory bowel disease involve many organs, eyes included. The ophthalmic complications are usually of inflammatory origin. Some of these complications, like scleritis, may reflect overall disease activity. Treatment of intestinal inflammation-either medical or surgical-usually helps resolution of ophthalmic complications. This review describes recent developments in the diagnosis and management of the inflammatory bowel disease and its ophthalmic complications.
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Affiliation(s)
- Faruque D Ghanchi
- Bradford Teaching Hospitals, Royal Infirmary, Bradford, West Yorkshire, UK
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18
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Krimsky M, Yedgar S, Aptekar L, Schwob O, Goshen G, Gruzman A, Sasson S, Ligumsky M. Amelioration of TNBS-induced colon inflammation in rats by phospholipase A2 inhibitor. Am J Physiol Gastrointest Liver Physiol 2003; 285:G586-92. [PMID: 12724134 DOI: 10.1152/ajpgi.00463.2002] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PLA2 inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PLA2 activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PLA2 inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation.
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Affiliation(s)
- M Krimsky
- Department of Biochemistry, Hadassah Medical School, Hebrew University Faculty of Medicine, Jerusalem, Israel 91120
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19
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Jun JY, Yeum CH, Park YW, Jang IY, Kong ID, Sim JH, So I, Kim KW, You HJ. Effects of arachidonic acid on ATP-sensitive K+ current in murine colonic smooth muscle cells. JAPANESE JOURNAL OF PHARMACOLOGY 2002; 90:81-7. [PMID: 12396031 DOI: 10.1254/jjp.90.81] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The effects of arachidonic acid (AA) and the mechanism through which it modulates ATP-sensitive K+ (K(ATP)) currents were examined in single smooth muscle cells of murine proximal colon. In the current-clamping mode, AA and glibenclamide induced depolarization of membrane potential. Using 0.1 mM ATP and 140 mM K+ solution in the pipette and 90 mM K+ in the bath solution at a -80 mV of holding potential, pinacidil activated the glibenclamide-sensitive inward current. The potential of these currents was reversed to near the equilibrium potential of K+ by 60 mM K+ in the bath solution. AA inhibited K(ATP) currents in a dose-dependent manner. This inhibition was not changed when 1 mM GDPbetaS was present in the pipette. Chelerythrine, protein kinase C inhibitor, did not block the AA effects. Superoxide dismutase and metabolic inhibitors (indomethacin and nordihydroguaiacretic acid) of AA did not affect the AA-induced inhibition. Eicosatetraynoic acid, a nonmetabolizable analogue of AA, inhibited the K(ATP) currents. These results suggest that AA-induced inhibition of K(ATP) currents is not mediated by G-protein or protein kinase C activation. The inhibitory action is likely to be a possible mechanism of AA-induced membrane depolarization.
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Affiliation(s)
- Jae Yeoul Jun
- Department of Physiology, College of Medicine, Chosun University, Kwangju, Korea
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20
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Ribardo DA, Kuhl KR, Peterson JW, Chopra AK. Role of melittin-like region within phospholipase A(2)-activating protein in biological function. Toxicon 2002; 40:519-26. [PMID: 11821123 DOI: 10.1016/s0041-0101(01)00247-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Phospholipase A(2)-activating protein (PLAA) has been implicated in the production of prostaglandins (e.g. PGE(2)) via activation of phospholipases in various stimulated cell types. Human PLAA, with 738 amino acid (aa) residues, contains a region of 38% homology (aa 503-538) with the 26-aa long melittin peptide, a major component of bee venom and a reported regulator of phospholipase A(2) and phospholipase D activity. To learn more about the role of PLAA in the production of eicosanoids and other inflammatory mediators, we synthesized a murine PLAA peptide (36-aa long) having homology to melittin, as well as to human and rat PLAA. The PLAA peptide and melittin increased the expression of genes encoding the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and cyclooxygenase-2 (COX-2), which is involved in PGE(2) production. We determined that the C-terminal region of the PLAA peptide (aa 515-538) was essential, since truncation of the C-terminal end of the PLAA peptide significantly reduced expression of genes encoding TNFalpha and COX-2 in macrophages. We concluded that PLAA could be important in the regulation of the inflammatory response because of its stimulatory effects on eicosanoid and cytokine synthesis. Consequently, control of plaa gene expression could be a target for the development of new drugs to control the inflammatory response.
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Affiliation(s)
- Deborah A Ribardo
- Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070, USA
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21
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Nardone G, Holicky EL, Uhl JR, Sabatino L, Staibano S, Rocco A, Colantuoni V, Manzo BA, Romano M, Budillon G, Cockerill FR, Miller LJ. In vivo and in vitro studies of cytosolic phospholipase A2 expression in Helicobacter pylori infection. Infect Immun 2001; 69:5857-63. [PMID: 11500464 PMCID: PMC98704 DOI: 10.1128/iai.69.9.5857-5863.2001] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2000] [Accepted: 06/11/2001] [Indexed: 02/07/2023] Open
Abstract
Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.
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Affiliation(s)
- G Nardone
- Department of Clinical and Experimental Medicine, Federico II University of Naples, Naples, Italy.
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22
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Ribardo DA, Crowe SE, Kuhl KR, Peterson JW, Chopra AK. Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D. J Biol Chem 2001; 276:5467-75. [PMID: 11094054 DOI: 10.1074/jbc.m006690200] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.
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Affiliation(s)
- D A Ribardo
- Department of Microbiology and Immunology and Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1070, USA
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23
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Abstract
Phospholipase A2 (PLA2) is an enzyme that catalyzes the hydrolysis of membrane phospholipids. This article reviews the source and structure of PLA2, the involvement of the enzyme in various biological and pathological phenomena, and the usefulness of PLA2 assays in laboratory diagnostics. Of particular importance is the role of PLA2 in the cellular production of mediators of inflammatory response to various stimuli. Assays for PLA2 activity and mass concentration are discussed, and the results of enzyme determinations in plasma from patients with different pathological conditions are presented. The determination of activity and mass concentration in plasma is particularly useful in the diagnosis and prognosis of pancreatitis, multiple organ failure, septic shock, and rheumatoid arthritis. A very important result is the demonstration that PLA2 is an acute phase protein, like CRP. Indeed, there is a close correlation between PLA2 mass concentration and CRP levels in several pathological conditions. Although the determination of C-reactive protein is much easier to perform and is routinely carried out in most clinical laboratories, the assessment of PLA2 activity or mass concentration has to be considered as a reliable approach to obtain a deeper understanding of some pathological conditions and may offer additional information concerning the prognosis of several disorders.
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Affiliation(s)
- E Kaiser
- Department of Medical Chemistry, University of Vienna, Austria
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24
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Chopra AK, Ribardo DA, Wood TG, Prusak DJ, Xu XJ, Peterson JW. Molecular characterization of cDNA for phospholipase A2-activating protein. BIOCHIMICA ET BIOPHYSICA ACTA 1999; 1444:125-30. [PMID: 9931468 DOI: 10.1016/s0167-4781(98)00249-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
A phospholipase A2-activating protein (PLAP) cDNA was cloned and sequenced from a human monocyte cDNA library, and expressed as a histidine-tagged fusion protein. The DNA-deduced aa sequence of human PLAP was 80,826 Da; however, SDS-PAGE analysis revealed a 72-74 kDa protein which matched the size of native PLAP from human monocytes. Anti-sense plap oligonucleotide blocked cholera toxin-induced release of 3H-labeled arachidonic acid from cells, indicating a potential role for PLAP in regulating phospholipase A2 activity.
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Affiliation(s)
- A K Chopra
- Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA.
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25
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Xu XJ, Ferguson MR, Popov VL, Houston CW, Peterson JW, Chopra AK. Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants. Infect Immun 1998; 66:3501-9. [PMID: 9673226 PMCID: PMC108379 DOI: 10.1128/iai.66.8.3501-3509.1998] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i. p., compared to 3.0 x 10(5) for the wild-type Aeromonas. Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act's biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections.
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Affiliation(s)
- X J Xu
- Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1070, USA
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26
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Groot J. Correlation between electrophysiological phenomena and transport of macromolecules in intestinal epithelium. Vet Q 1998. [DOI: 10.1080/01652176.1998.9694968] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/15/2022] Open
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27
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Bresci G, Gambardella L, Parisi G, Federici G, Bertini M, Rindi G, Metrangolo S, Tumino E, Bertoni M, Cagno MC, Capria A. Colonic disease in cirrhotic patients with portal hypertension: an endoscopic and clinical evaluation. J Clin Gastroenterol 1998; 26:222-7. [PMID: 9600375 DOI: 10.1097/00004836-199804000-00016] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Fifty cirrhotic patients with portal hypertension but without colonic or systemic disease underwent lower gastrointestinal endoscopy in order to investigate the effects, if any, of portal hypertension on the colon. Fifty patients without liver or systemic disease, examined by colonoscopy because of irritable bowel syndrome in the same period served as controls. Rectosigmoid varices were observed in 34% of the cirrhotic patients and 2% of the controls. Hemorrhoids were observed in 70% of the cirrhotic patients and 48% of the controls. Multiple vascular-appearing lesions were found in 16% of the cirrhotic patients and 6% of the controls. Nonspecific inflammatory changes were noted in 10% of the cirrhotic patients and 4% of the controls. Simultaneous presence, in the same patient, of rectosigmoid varices, hemorrhoids, multiple vascular-appearing lesions, and nonspecific inflammatory changes, was observed in only five (10%) of the cirrhotic patients. We found polyps in 12% of the cirrhotic patients and 14% of the controls, and a malignant tumor in 4% of the cirrhotic patients. The patients with normal colonoscopic findings were 8% of the cirrhotic patients and 36% of the controls. All patients and controls were followed up for 1 year; there was no gastrointestinal hemorrhage among controls, whereas 34% of the cirrhotic patients had an upper gastrointestinal hemorrhage (88% from esophageal varices, 12% from the stomach) and 4% had a lower gastrointestinal hemorrhage (one from rectosigmoid varices and one from nonspecific inflammatory lesions). Colonic lesions were significantly more frequent in the cirrhotic patients (92%) than in the control group (64%); however, such lesions did not seem specific to the disease and were not statistically correlated with the degree of esophageal varices by Child's grading, the etiology of cirrhosis, or the bleeding risk from the lower gastrointestinal tract.
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Affiliation(s)
- G Bresci
- Unità Operativa Gastroenterologia, Azienda Ospedaliera Pisana, Ospedale Cisanello, Pisa, Italy
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28
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Ferguson MR, Xu XJ, Houston CW, Peterson JW, Coppenhaver DH, Popov VL, Chopra AK. Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila. Infect Immun 1997; 65:4299-308. [PMID: 9317040 PMCID: PMC175616 DOI: 10.1128/iai.65.10.4299-4308.1997] [Citation(s) in RCA: 53] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems. Maximum toxin yield was obtained with the pTRX vector. Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector systems. The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography-based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies. Purified mature toxin migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots. The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously. Binding of the toxin to erythrocytes was temperature dependent, with no binding occurring at 4 degrees C. However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min. It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy. Calcium chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin. A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol. With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated. None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act. The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis. As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited.
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Affiliation(s)
- M R Ferguson
- Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston 77555-1070, USA
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29
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Saini SS, Peterson JW, Chopra AK. Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity. Biochem Biophys Res Commun 1997; 238:436-42. [PMID: 9299527 DOI: 10.1006/bbrc.1997.7295] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.
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Affiliation(s)
- S S Saini
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019, USA
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