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Last updated: 13th August 2010

MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.
Yamamoto-Furusho JK, Barnich N, Hisamatsu T, Podolsky DK
Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Inflamm Bowel Dis. 2010 May;16(5):736-42.
BACKGROUND:: Human neutrophil peptide 1 (HNP-1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP-1 expression in epithelial cell lines. METHODS:: Gentamicin protection assay using Salmonella typhimurium was performed in Caco-2 cells. The mRNA level was determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and defensin expression was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappaB activation was assessed using pIV luciferase and Renilla plasmids. A NOD2 mutant was generated by site-directed mutagenesis. RESULTS:: Among the defensins tested, only HNP-1 expression is induced in colonic epithelial model HCT116 cells after MDP-LD stimulation. HNP-1 secretion is significantly increased after MDP-LD stimulation in the cell supernatant of intestinal epithelial cells expressing endogenous NOD2, but not in cells that lack endogenous NOD2 expression. HNP-1 is required for NOD2-dependent NF-kappaB activation after MDP-LD stimulation since hnp-1 siRNA transfection abrogated the response to MDP-LD stimulation. The antibacterial function of NOD2 against S. typhimurium was impaired when expression of HNP-1 was blocked by siRNA. CONCLUSIONS:: HNP-1 secretion depends on NOD2 stimulation by MDP-LD and contributes to antibacterial activity in intestinal epithelial cells expressing endogenous NOD2, but not NOD2 3020insC mutant associated with increased susceptibility to Crohn's disease. (Inflamm Bowel Dis 2009).
A novel antifungal analog peptide derived from protaetiamycine.
Lee J, Hong HJ, Kim JK, Hwang JS, Kim Y, Lee DG
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu, 702-701, Korea.
Mol Cells. 2009 Nov;28(5):473-7.
Previously, the 9-mer analog peptides, 9Pbw2 and 9Pbw4, were designed based on a defensin-like peptide, protaetiamycine isolated from Protaetia brevitarsis. In this study, antifungal effects of the analog peptides were investigated. The antifungal susceptibility testing exhibited that 9Pbw4 contained more potent antifungal activities than 9Pbw2. A PI influx assay confirmed the effects of the analog peptides and demonstrated that the peptides exerted their activity by a membrane-active mechanism, in an energy-independent manner. As the noteworthy potency of 9Pbw4, the mechanism(s) of 9Pbw4 were further investigated. The membrane studies, using rhodamine-labeled giant unilamellar vesicle (GUV) and fluorescein isothiocyanate (FITC)-dextran loaded liposome, suggested that the membrane-active mechanism of 9Pbw4 could have originated from the poreforming action and the radii of pores was presumed to be anywhere from 1.8 nm to 3.3 nm. These results were confirmed by 3D-flow cytometric contour-plot analysis. The present study suggests a potential of 9Pbw4 as a novel antifungal peptide.
Enteric defensins are essential regulators of intestinal microbial ecology.
Salzman NH, Hung K, Haribhai D, Chu H, Karlsson-Sjoberg J, Amir E, Teggatz P, Barman M, Hayward M, Eastwood D, Stoel M, Zhou Y, Sodergren E, Weinstock GM, Bevins CL, Williams CB, Bos NA
[1] Division of Gastroenterology [2] Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Nat Immunol. 2010 Jan;11(1):76-82
Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell alpha-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if alpha-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human alpha-defensin gene (DEFA5) and in mice lacking an enzyme required for the processing of mouse alpha-defensins. In these complementary models, we detected significant alpha-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5-expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to alpha-defensins in regulating the makeup of the commensal microbiota.
Identification and characterization of a novel antibacterial peptide, avian beta-defensin 2 from ducks.
Ma D, Wang R, Liao W, Han Z, Liu S
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, PR China.
J. Microbiol. 2009 Oct;47 (5):610-8
In this study, a novel avian beta-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, -20 degrees C to 100 degrees C) and pH values (range, 3 to 12).
Innate antimicrobial host defense in small intestinal Crohn's disease.
Koslowski MJ, Beisner J, Stange EF, Wehkamp J
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany; University of Tübingen, Germany.
Int J Med Microbiol. 2010 Jan;300(1):34-40.
Paneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly the alpha-defensins HD-5 and -6 (DEFA5 und DEFA6). Both these PC products show a specific reduction in small intestinal Crohn's disease (CD) - a form of inflammatory bowel disease (IBD). Their decrease is independent of current inflammation and an association with a NOD2 frameshift mutation has been demonstrated. More recently, another independent and even more frequent mechanism has been found which is linked to diminished levels of the Wnt pathway transcription factor TCF7L2 (also known as TCF4). Besides regulating the expression of HD-5 and HD-6 as TCF4 target genes, the Wnt pathway also orchestrates Paneth cell differentiation and maturation and controls stem cell maintenance in the small intestine. Besides NOD2 (which is predominantly expressed in PC) and ATG16L1 (inter alia important in the exocytosis of PC products), TCF4 is the third gene which is associated with small intestinal CD and Paneth cell antimicrobial function. Thus, Paneth cells seem to be key player emphazising a paramount importance of antimicrobial host defense in small intestinal CD pathogenesis.
Expression of antiviral molecular genes in nasal polyp-derived cultured epithelial cells.
Watanabe S, Wang J, Matsukura S, Suzaki H
Department of Otorhinolaryngology, Showa University School of Medicine, Tokyo, Japan.
Acta oto-laryngologica. Supplementum 2009 Jun (562):101-4
CONCLUSION: The results of the present study indicate the existence of natural immunity mechanisms via which viruses are eliminated from nasal and paranasal sinus mucosa. OBJECTIVES: Acute sinusitis and acute aggravation of chronic sinusitis are often caused by bacteria, which are secondary to viral infection of the nose. Antiviral molecules are considered to be expressed and protect the host after viral infection. We investigated the expression of antiviral molecules after viral infection of the nose. MATERIALS AND METHODS: We assessed the expression of antiviral molecules, defensin and interferon mRNA, by the real-time polymerase chain reaction (PCR) technique after stimulating cultured nasal polyp cells with polyinosine-polycytidylic acid (poly(I:C)), which is an analog of double-stranded (ds) RNA. RESULTS: The expression of beta-defensin mRNA significantly increased after the stimulation. On the other hand, expression of both interferon-alpha mRNA and interferon-beta mRNA was recognized, but only the expression of interferon-beta mRNA increased after the stimulation.
Pivotal Advance: Glycyrrhizin restores the impaired production of {beta}-defensins in tissues surrounding the burn area and improves the resistance of burn mice to Pseudomonas aeruginosa wound infection.
Yoshida T, Yoshida S, Kobayashi M, Herndon DN, Suzuki F
*Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas, USA; andShriners Hospitals for Children, Galveston, Texas, USA.
J Leukoc Biol. 2010 Jan;87(1):35-41.
The decreased production of antimicrobial peptides in tissues surrounding the burn sites has been described in patients with severe burn injury. Small numbers of Pseudomonas aeruginosa spread easily to the whole body of burn mice when infected at burn site tissues. Gr-1(+)CD11b(+) cells, demonstrated in tissues surrounding the burn site, are inhibitory on the production of antimicrobial peptides by EK. In this paper, the decreased production of antimicrobial peptides by EK influenced by Gr-1(+)CD11b(+) cells was shown to be restored by glycyrrhizin. CCL2 and IL-10 were determined to be effector soluble factors for the suppressor activities of Gr-1(+)CD11b(+) cells on antimicrobial peptide production by EK. However, Gr-1(+)CD11b(+) cells, which were treated previously with glycyrrhizin, did not produce these soluble factors. Also, sepsis stemming from P. aeruginosa burn-site infection was not demonstrated in burn mice treated with glycyrrhizin. These results suggest that through the improved production of antimicrobial peptides in tissues surrounding the burn area, sepsis stemming from P. aeruginosa wound infection is controllable by glycyrrhizin in severely burned mice.
A protein phosphatase 2C, responsive to the bacterial effector AvrRpm1 but not to the AvrB effector, regulates defense responses in Arabidopsis.
Widjaja I, Lassowskat I, Bethke G, Eschen-Lippold L, Long HH, Naumann K, Dangl JL, Scheel D, Lee J
Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany.
Plant J. 2010 Jan 1;61(2):249-258.
Summary Using a proteomics approach, a PP2C-type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB-LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1- and AvrB-dependent activation of RPM1. AvrRpm1-induced expression of the pathogenesis-related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense-related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1-induced responses, and confers dual (both positive and negative) regulation of defense gene expression.
HIV type 1 fails to trigger innate immune factor synthesis in differentiated oral epithelium.
Nittayananta W, Hladik F, Klausner M, Harb S, Dale BA, Coombs RW
Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
AIDS Res. Hum. Retroviruses 2009 Oct;25 (10):1013-21
The oral mucosa is relatively resistant to human immunodeficiency virus type 1 (HIV-1) transmission. The mechanisms contributing to this resistance remain incompletely understood, but may include HIV-induced synthesis of innate immune factors. We used fully differentiated oral epithelium as a surrogate for the oral mucosa in vivo, exposed it to X4- and R5-tropic HIV-1 in culture, and quantified mRNA expression of six innate immune factors. Neither virus increased expression of human beta defensin 2 (hBD-2) mRNA over supernatants from uninfected lymphoblast controls. HIV-1 also failed to induce mRNA of four additional innate immunity-related genes. Similar results were obtained with oral monolayer epithelial cells. Interestingly, the X4-tropic virus inhibited mRNA expression of hBD-2, and of three of the other factors, at higher dosages in the differentiated oral epithelium but not the monolayers. The failure of HIV-1 to induce innate immune factors in the differentiated epithelium was not due to a lack of tissue penetration, as we detected fluorescence-tagged virions up to 30 mum deep from the apical surface. HIV-1 does not trigger de novo innate immune factor synthesis in oral epithelium, pointing to the role of a constitutive innate immunity for protection against HIV-1 in the oral cavity.
Development of a novel immunoassay for the iron regulatory peptide hepcidin.
Busbridge M, Griffiths C, Ashby D, Gale D, Jayantha A, Sanwaiya A, Chapman RS
Department of Clinical Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust, London.
Br. J. Biomed. Sci. 2009 ;66 (3):150-7
To date there have been few published immunoassays for the important iron regulator hepcidin. This study describes a novel competitive radioimmunoassay (RIA) for the bioactive hepcidin peptide. A rabbit anti-hepcidin polyclonal antibody was produced using synthetic hepcidin radiolabelled with 125I to produce a competitive RIA. Normal patient (n=47) samples were collected and assayed for hepcidin to determine a reference range. Other patient groups collected were ulcerative colitis (UC; n=40), iron deficiency anaemia (IDA; n=15), chronic kidney disease not requiring dialysis (CKD; n=45) and chronic kidney disease requiring dialysis (HCKD; n=94). Detection limit of the assay was determined as 0.6 ng/mL. Intra-assay precision was 5 ng/mL (7.2%) and 50 ng/mL (5.8%), interassay precision was 5 ng/mL (7.6%) and 50 ng/mL (6.7%). Analytical recovery was 98% (5 ng/mL), 94% (10 ng/mL) and 97% (50 ng/mL). The assay was linear up to 200 ng/mL. No demonstrable cross-reactivity with human insulin, glucagon I, angiotensinogen I, beta-defensin 1-4, alpha-defensin-1 and plectasin was observed. There was significant correlation (r=0.96, P < or = 0.0001) between the hepcidin RIA and an established hepcidin SELDI-TOF-MS method. Analysis of the normal human samples gave a reference range of 1.1-55 ng/mL for hepcidin. Further statistical evaluation revealed a significant difference between male and female hepcidin levels. There was significant correlation between hepcidin and ferritin in the control group (r=0.6, P < or = 0.0001). There was also a significant difference between the normal and disease groups (P < or = 0.0001). Healthy volunteers (n=10) showed a diurnal increase in plasma hepcidin at 4.00 pm compared to 8.00 am. A robust and optimised immunoassay for bioactive hepcidin has been produced and the patient sample results obtained further validates the important role of hepcidin in iron regulation, and will allow further investigation of this important peptide and its role in iron homeostasis.