Last updated: 11th June 2008

Literature

Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections.

Kocan KM, de la Fuente J, Manzano-Roman R, Naranjo V, Hynes WL, Sonenshine DE

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA.

Exp. Appl. Acarol. 2008 Jun

Abstract Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.

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Regional Variation in Gene Expression in the Healthy Colon is Dysregulated in Ulcerative Colitis.

Noble CL, Abbas AR, Cornelius J, Lees CW, Ho GT, Toy K, Modrusan Z, Pal N, Zhong F, Chalasani S, Clark H, Arnott ID, Penman ID, Satsangi J, Diehl L

United Kingdom.

Gut 2008 Jun

Abstract OBJECTIVE: The aim of this study was to investigate differential intestinal gene expression in patients with ulcerative colitis (UC) and controls. DESIGN: Genome wide expression study (41058 expression sequence tags, 215 biopsies). SETTING: Western General Hospital, Edinburgh, UK, Genentech Inc, San Francisco, USA. PATIENTS: 67 UC and 31 control subjects- 23 normal and 8 inflamed non-inflammatory bowel disease patients. INTERVENTIONS: Paired endoscopic biopsies were taken from 5 specific anatomical locations for RNA extraction and histology. The Agilent microarray platform was used and confirmation of results was undertaken by real time PCR and immunohistochemistry. RESULTS: In healthy control biopsies, cluster analysis showed differences in gene expression between the right and left colon. (chi2=25.1, p<0.0001). Developmental genes HOXA13, (p=2.3x10-16), HOXB13 (p <1x10-45), GLI1 (p=4.0x10-24), and GLI3 (p=2.1x10-28) primarily drove this separation. When all UC biopsies and control biopsies were compared, 143 sequences had a fold change of >1.5 in the UC biopsies (0.01>p>10-45) and 54 sequences had a fold change of <-1.5 (0.01>p>10-20). Differentially upregulated genes in UC included SAA1 (p<10-45) the alpha defensins, DEFA5&6 (p=0.00003 and p=6.95x10-7 respectively), MMP3 (p=5.6x10-10) and MMP7 (p=2.3x10-7). Increased DEFA5&6 expression was further characterized to Paneth cell metaplasia by immunohistochemistry and in-situ hybridization. Sub-analysis of the IBD2 & IBD5 loci, and the ABC transporter genes revealed a number of differentially regulated genes in the UC biopsies. CONCLUSIONS: Key findings are the expression gradient in the healthy adult colon and the involvement of novel gene families, as well as established candidate genes in the pathogenesis of UC.

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The GraRS regulatory system controls Staphylococcus aureus susceptibility to antimicrobial host defenses.

Kraus D, Herbert S, Kristian SA, Khosravi A, Nizet V, Gotz F, Peschel A

BMC Microbiol. 2008 Jun;8 (1):85

Abstract ABSTRACT: BACKGROUND: Modification of teichoic acids with D-alanine by the products of the dlt operon protects Gram-positive bacteria against major antimicrobial host defense molecules such as defensins, cathelicidins, myeloperoxidase or phospholipase. The graRS regulatory genes have recently been implicated in the control of D-alanylation in Staphylococcus aureus. RESULTS: To determine the impact of the GraRS regulatory system on resistance to antimicrobial host defense mechanisms and virulence of S. aureus, we compared inactivation of S. aureus SA113 wild type and its isogenic graRS deletion mutant by the human cathelicidin LL-37 or human neutrophil granulocytes in vitro, and the ability to cause infection in vivo. We show here that graRS deletion considerably alters bacterial surface charge, increases susceptibility to killing by human neutrophils or the defense peptide LL-37, and attenuates virulence of S. aureus in a mouse infection model. CONCLUSION: Our results indicate that S. aureus can regulate its surface properties in order to overcome innate host defenses.

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Genetic Variants of the Copy Number Polymorphic beta-Defensin Locus Are Associated with Sporadic Prostate Cancer.

Huse K, Taudien S, Groth M, Rosenstiel P, Szafranski K, Hiller M, Hampe J, Junker K, Schubert J, Schreiber S, Birkenmeier G, Krawczak M, Platzer M

Genome Analysis, Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany.

Tumour Biol. 2008 Jun;29 (2):83-92

Abstract Background/Aims: Prostate cancer represents the cancer with the highest worldwide prevalence in men. Chromosome 8p23 has shown suggestive genetic linkage to early-onset familial prostate cancer and is frequently deleted in cancer cells of the urogenital tract. Within this locus some beta-defensin genes (among them DEFB4, DEFB103, DEFB104) are localized, which are arranged in a gene cluster shown to exhibit an extensive copy number variation in the population. This structural variation considerably hampers genetic studies. In a new approach considering both sequence as well as copy number variations we aimed to compare the defensin locus at 8p23 in prostate cancer patients and controls. Methods: We apply PCR/cloning-based haplotyping and high-throughput copy number determination methods which allow assessment of both individual haplotypes and gene copy numbers not accessible to conventional SNP-based genotyping. Results: We demonstrate association of four common DEFB104 haplotypes with the risk of prostate cancer in two independent patient cohorts. Moreover, we show that high copy numbers (>9) of the defensin gene cluster are significantly underrepresented in both patient samples. Conclusions: Our findings imply a role of the antibacterial defensins in prostate cancerogenesis qualifying distinct gene variants and copy numbers as potential tumor markers.

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Antimicrobial Peptides in Lung Transplant Recipients with Bronchiolitis Obliterans Syndrome.

Anderson RL, Hiemstra PS, Ward C, Forrest IA, Murphy D, Proud D, Lordan J, Corris PA, Fisher AJ

Freeman Hospital, Newcastle upon Tyne Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine Newcastle University, Newcastle, UK; and Dept of Pulmonology, Leiden University Medical Centre, Leiden, The Netherlands.

Eur. Respir. J. 2008 May

Abstract Mechanisms other than classical alloimmunity are implicated in the pathogenesis of Bronchiolitis Obliterans Syndrome (BOS). We hypothesised that antimicrobial peptides (AMPs), elements of lung innate immune response, have a role in BOS pathogenesis.We measured pulmonary expression of the neutrophil-derived AMPs hCAP-18/LL-37 and alpha-defensins (HNP1-3), and the epithelial-cell derived AMPs human beta-defensin-2 (hBD-2), elafin and secretory leukoprotease inhibitor (SLPI) in stable lung transplant recipients and those with BOS, and examined the relationship between airway pathogens and AMP levels.Bronchoalveolar lavage (BAL) was performed on 46 lung transplant recipients (30 stable, 14 BOS). BAL was cultured for pathogens and ELISAs for AMPs were performed.Presence of airway pathogens was associated with significantly increased levels of neutrophil-derived and epithelial-derived AMPs. When patients without pathogens in BAL fluid were analysed, recipients with BOS (n=8) had elevated hCAP-18/LL-37 (p=<0.001) and HNP1-3 (p=0.004) compared to stable recipients (n=25). hBD-2 and elafin levels were comparable in BOS and stable recipients, but SLPI levels were reduced (p=<0.001) in BOS.BOS is associated with elevated airway hCAP18/LL-37 and HNP 1-3 from activated neutrophils, even in the absence of pathogens. Together with reduced airway SLPI this may favour non-alloimmune airway injury with reduced anti-protease defence and increased neutrophil degranulation.

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The role of the multifunctional peptide LL-37 in host defense.

Kai-Larsen Y, Agerberth B

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

Front. Biosci. 2008 ;13 3760-7

Abstract Neutrophil granules contain several antimicrobial peptides (AMPs) that are important effector molecules of innate immunity. In mammals, the main families of these peptides are the cathelicidins and defensins. Several defensins have been characterized in humans, while there is only one human cathelicidin, designated LL-37. This peptide is stored in specific granules of neutrophils in an inactive proform, which is processed extracellularly to the mature active peptide LL-37 and the propart cathelin after neutrophil degranulation. Apart from exhibiting a broad antimicrobial spectra, it is now evident that LL-37 possesses several additional functions that are related to host defense. Examples of such functions are chemotactic, endotoxin neutralizing, angiogenic and wound healing activities. These effects of LL-37 reveal a role as a mediator between innate and adaptive immunity. This review is giving an overview of the different immunological effects exerted by LL-37 and the physiological significance of these functions in immunity.

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Using RNA interference to determine the role of varisin in the innate immune system of the hard tick Dermacentor variabilis (Acari: Ixodidae).

Hynes WL, Stokes MM, Hensley SM, Todd SM, Sonenshine DE

Department of Biological Sciences, Old Dominion University, Norfolk, VA, 23529-0266, USA, whynes@odu.edu.

Exp. Appl. Acarol. 2008 May

Abstract Defensins are an important component of the innate immune system of ticks. These small peptides are produced by various genera of ticks, and expressed in various tissues. In this study we used RNA interference to silence the expression of the defensin varisin produced by the hemocytes of the American dog tick, Dermacentor variabilis. Ticks were injected with double stranded varisin RNA prior to being placed on a rabbit. After feeding, the ticks were removed, bled, and the hemolymph plasma and hemocytes separated. Hemocytes were screened for the presence (or absence) of both varisin transcript and peptide. Varisin peptide was below detectable levels and the transcript showed a greater than 99% knockdown. The antimicrobial activity of the hemolymph plasma was reduced 2-4 fold compared to that of control injected ticks indicating varisin accounts for a large portion of the antimicrobial activity of the hemolymph.

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Innate immunity and disorders of the female reproductive tract.

Horne AW, Stock SJ, King AE

The Queen's Medical Research Institute, Reproductive and Developmental Sciences, Centre for Reproductive Biology, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.

Reproduction 2008 Jun;135 (6):739-49

Abstract Sexually transmitted infections, and their associated sequelae, such as tubal infertility, ectopic pregnancy and preterm labour, are a major worldwide health problem. Chlamydia trachomatis infection is thought to be the leading global cause of tubal infertility and tubal ectopic pregnancy. Preterm birth occurs in around 10% of all deliveries, and nearly 30% of preterm deliveries are associated with intrauterine infection. The mucosal innate immune system of the female reproductive tract has evolved to eliminate such sexually transmitted pathogens whilst maintaining its ability to accommodate specialized physiological functions that include menstruation, fertilization, implantation, pregnancy and parturition. The aim of this review was to describe the role and distribution of key mediators of the innate immune system, the natural antimicrobial peptides (secretory leukocyte protease inhibitor, elafin and the defensins) and the pattern recognition toll-like receptors in the normal female reproductive tract and in the context of these pathological processes.

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Binding and activation of human and mouse complement by Cryptosporidium parvum (Apicomplexa) and susceptibility of C1q- and MBL-deficient mice to infection.

Petry F, Jakobi V, Wagner S, Tessema TS, Thiel S, Loos M

Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University Mainz, Augustusplatz/Hochhaus, D-55101 Mainz, Germany.

Mol. Immunol. 2008 May

Abstract Cryptosporidium parvum is a protozoan parasite (Apicomplexa) that causes gastrointestinal disease in animals and humans. Whereas immunocompetent hosts can limit the infection within 1 or 2 weeks, immunocompromised individuals develop a chronic, life-threatening disease. The importance of the adaptive cellular immune response, with CD4+ T-lymphocytes being the major players, has been clearly demonstrated. Several non-adaptive immune mechanisms have been suggested to contribute to the host defence, such as interferon-gamma (IFN-gamma) from NK cells, certain chemokines, beta-defensins and pro-inflammatory cytokines, but the influence of the complement systems has been less well studied. We analysed the in vitro binding and activation of the human and mouse complement systems and tested the susceptibility to infection in complement-deficient mouse strains. We found that C. parvum can activate both the classical and lectin pathways, leading to the deposition of C3b on the parasite. Using real-time PCR, parasite development could be demonstrated in adult mice lacking mannan-binding lectin (MBL-A/C(-/-)) but not in mice lacking complement factor C1q (C1qA(-/-)) or in wild type C57BL/6 mice. The contribution of the complement system and the lectin pathway in particular to the host defence against cryptosporidiosis may become apparent in situations of immunodeficiency such as HIV infections or in early childhood.

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The conserved salt bridge in human alpha -defensin 5 is required for its precursor processing and proteolytic stability.

Rajabi M, de Leeuw E, Pazgier M, Li J, Lubkowski J, Lu W

Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201.

J. Biol. Chem. 2008 May

Abstract Mammalian a-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical ss-bulge are totally conserved in the otherwise diverse sequences of all known mammalian a-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg6-Glu14 salt bridge in human a-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, while significantly reducing oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro, but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg13-Gln14 peptide bond in the loop region - a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against S. aureus, but substantially enhanced the killing of E. coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature a-defensin in vivo.

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