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Last updated: 13th August 2010

Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease.
Onishi RM, Gaffen SL
Department of Medicine, Division of Rheumatology & Clinical Immunology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Immunology 2010 Mar;129 (3):311-21
Interleukin-17 (IL-17) has emerged as a central player in the mammalian immune system. Although this cytokine exerts a host-defensive role in many infectious diseases, it promotes inflammatory pathology in autoimmunity and other settings. A myriad of studies have focused on how IL-17-producing cells are generated. However, the means by which IL-17 achieves its effects, either for the benefit or the detriment of the host, are due in large part to the induction of new gene expression. Whereas many IL-17 target genes are common to different disease states, in some cases the effects of IL-17 differ depending on the target cell, infectious site or pathogen. Gene products induced by IL-17 include cytokines (IL-6, granulocyte-colony-stimulating factor, tumour necrosis factor-alpha), chemokines (CXCL1, CXCL2, CCL20, among many others), inflammatory effectors (acute-phase protesins, complement) and antimicrobial proteins (defensins, mucins). Different cell types appear to respond differently to IL-17 in terms of target gene expression, with notable differences seen in mesenchymal and epithelial cells compared with cells of haematopoietic origin. Here, we summarize the major IL-17 target genes that mediate this cytokine´s activities in both autoimmune and chronic diseases as well as during various types of infections.
Severity of Staphylococcus aureus infection of the skin is associated with inducibility of human beta-defensin 3 but not human beta-defensin 2.
Zanger P, Holzer J, Schleucher R, Scherbaum H, Schittek B, Gabrysch S
Institut für Tropenmedizin, Eberhard Karls Universität, Tübingen, Germany.
Infect Immun. 2010 Jul;78(7):3112-7.
Gram-positive bacteria are the predominant cause of skin infections. Antimicrobial peptides (AMPs) are believed to be of major importance in skin's innate defense against these pathogens. This study aimed at providing clinical evidence for the contribution of AMP inducibility to determining the severity of Gram-positive skin infection. Using real-time PCR, we determined the induction of human beta-defensin 2 (HBD-2), HBD-3, and RNase 7 by comparing healthy and lesional mRNA levels in 32 patients with Gram-positive skin infection. We then examined whether AMP induction differed by disease severity, as measured by number of recurrences and need for surgical drainage in patients with Staphylococcus aureus-positive lesions. We found that HBD-2 and -3, but not RNase 7, mRNA expression was highly induced by Gram-positive bacterial infection in otherwise healthy skin. Less induction of HBD-3, but not HBD-2, was associated with more-severe S. aureus skin infection: HBD-3 mRNA levels were 11.4 times lower in patients with more than 6 recurrences (P = 0.01) and 8.8 times lower in patients reporting surgical drainage (P = 0.01) than in the respective baseline groups. This suggests that inducibility of HBD-3 influences the severity of Gram-positive skin infection in vivo. The physiological function of HBD-2 induction in this context remains unclear.
Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing.
Taudien S, Groth M, Huse K, Petzold A, Szafranski K, Hampe J, Rosenstiel P, Schreiber S, Platzer M
Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany.
BMC Genomics 2010 ;11 252
BACKGROUND: The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn´s disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel. RESULTS: Six PCR products spread over approximately 87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From approximately 142,000 reads, approximately 120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed. CONCLUSION: Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.
The DUB/USP17 deubiquitinating enzymes: a gene family within a tandemly repeated sequence, is also embedded within the copy number variable beta-defensin cluster.
Burrows JF, Scott CJ, Johnston JA
Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Faculty of Medicine, Health and Life Sciences, Queen´s University, Belfast, Northern Ireland.
BMC Genomics 2010 ;11 250
BACKGROUND: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the ´CAAX´ box protease RCE1. RESULTS: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable beta-defensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating. CONCLUSIONS: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.
Defensin-mRNA expression in the upper gastrointestinal tract is modulated in children with celiac disease and Helicobacter pylori-positive gastritis.
Vordenbäumen S, Pilic D, Otte JM, Schmitz F, Schmidt-Choudhury A
Department of Endocrinology, Diabetology, and Rheumatology, Heinrich Heine University Düsseldorf, Germany.
J. Pediatr. Gastroenterol. Nutr. 2010 Jun;50 (6):596-600
OBJECTIVES: Defensins are expressed in epithelial cells as cationic peptides with antimicrobial properties. Because of their role as immunologically important effector molecules, their contribution in maintaining a stable microenvironment in the gastrointestinal tract has recently received much attention. The present study was designed to further characterize expression patterns of defensins in diseases of the upper gastrointestinal tract in children, particularly in Helicobacter pylori (Hp)-associated gastritis or celiac disease (CD). PATIENTS AND METHODS: Semiquantitative real-time reverse transcriptase-polymerase chain reaction (PCR) was carried out with gene-specific primers for human beta-defensin 1 to 6 (hBD1 to 6) and human alpha-defensin 5 and 6 (hD5 and 6) in mucosal biopsies of children diagnosed as having CD (n = 11; 4.2-16.2 years) or Hp gastritis (n = 18; 3.2-16.7 years). Levels of expression were compared with those of healthy individuals (n = 21; 2.8-14.6 years). Expression levels in Hp-infected specimens were furthermore compared with those with histologic inflammation not associated with Hp infection (n = 30; 3.6-15.7 years). RESULTS: Expression of hBD2 was upregulated in the antrum and corpus of patients with Hp gastritis, whereas inflammation without detection of Hp was not associated with any change in defensin gene expression. In patients with CD, expression of hBD2 was upregulated in the antrum, whereas hBD1 and 4 were downregulated in duodenal biopsies. CONCLUSIONS: Different pathological conditions of the upper gastrointestinal tract lead to specific modulations of defensin gene expression in children. Especially the pathophysiological role of hBD2 in Hp infection and hBD1 and 4 in CD warrant further attention.
Expression of beta-Defensin-3 in Lungs of Immunocompetent Rats with Methicillin-Resistant Staphylococcus aureus Ventilator-Associated Pneumonia.
Wu Q, Gui P, Yao S, Zhu H, Li J, Li Y
Department of Anesthesiology, Union Hospital.
The Journal of surgical research 2010 Jan
BACKGROUND: Despite the rising incidence and high rate of treatment failure of ventilator-associated pneumonia (VAP) due to methicillin-resistant Staphylococcus aureus (MRSA), to date there has been no rat model specifically designed for antimicrobial evaluation. beta-Defensin-3 Acronym for beta-defensin-3 is correct as (BD-3) is an antimicrobial peptide and mainly expresses in the gastrointestinal and respiratory tract. It demonstrates a broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes, including multi-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Therefore, the authors hypothesized that the expression of BD-3 might change in lungs of rats with MRSA VAP, and this change might play an important role in the pathogenesis of VAP. MATERIALS AND METHODS: Eighty specific pathogen-free male Sprague-Dawley rats were randomly assigned to the following experimental groups: (1) N (nonventilated pneumonia) group (n=34): rats were infected intrabronchially with 0.2mL of 10(10) cfu/mL MRSA inoculum; (2)V (ventilator-associated pneumonia) group (n=34): rats were ventilated for 4h using the protective ventilation settings: 6mL/kg tidal volume, 5 cmH(2)O of positive end-expiratory pressure (PEEP), 88 breaths/min, and F(i)O(2)=0.21. After ventilation, rats were inoculated intrabronchially with the same amount of MRSA as in N group; (3) P (protective mechanical ventilation) group (n=6): 0.2mL of normal saline was instilled into lungs of rats after 4h of ventilation as in V group; (4) C (control) group (n=6): only 0.2mL of normal saline were instilled into lungs of rats. Rats from both P and C groups were killed 48h after instillation of normal saline. Rats from other two groups were killed 3, 6, 12, 24, 48h after inoculation. Pneumonia evaluation was performed by macroscopic, histopathologic, and microbiologic criteria. The expression of BD-3 in lungs was determined by immunohistochemistry staining and Western blot analysis. RESULTS: Rats inoculated after 4h of protective mechanical ventilation rapidly developed progressive pneumonia with heterogeneous distribution of lesions and different degrees of histologic evolution predominating in lower lobes. Lung bacterial concentrations in V group at each time point were significantly higher than those of N group. It was 12.2+/-0.9log(10) cfu/g of tissue at h 48 in V group, and 8.7+/-0.4 in N group. Bacteremia occurred in nine of 10 rats at h 48 in V group, while two of 10 rats in N group. Both C and P groups showed a very low level of BD-3. Compared with C group, the expression of BD-3 at h 48 in both N and V groups significantly increased. However, the latter was significantly lower than the former. CONCLUSIONS: Rat model of MRSA VAP was obtained by intrabronchially inoculating rats with 2x10(9) cfu MRSA after 4h of protective mechanical ventilation. VAP was more severe than nonventilated pneumonia in terms of histopathologic and microbiologic criteria, especially systemic spread. This may be associated with the inhibited up-regulation of BD-3 expression by mechanical ventilation.
Identification of genes differentially expressed in cauliflower associated with resistance to Xanthomonas campestris pv. campestris.
Jiang H, Song W, Li A, Yang X, Sun D
College of Life Sciences, Nankai University, Tianjin, 300071, People´s Republic of China.
Molecular biology reports 2010 Apr
Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.
The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region.
Zhou L, Liu SP, Chen LY, Li J, Ong LB, Guo L, Wohland T, Tang CC, Lakshminarayanan R, Mavinahalli J, Verma C, Beuerman RW
Singapore Eye Research Institute, Singapore, Singapore.
Amino acids 2010 Apr
Understanding the molecular mechanisms of antimicrobial peptide-membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3-5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)(2)KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3-9.7 muM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 mug/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5-10 muM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 muM. The concentration of 5-10 muM for Y2 from FCS correlated with the concentration of 5 muM (6.25 mug/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5-5 muM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.
Identification and characterization of an avian beta-defensin orthologue, avian beta-defensin 9, from quails.
Wang R, Ma D, Lin L, Zhou C, Han Z, Shao Y, Liao W, Liu S
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, 150030, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Jul;87(4):1395-405.
In this study, a newly identified avian beta-defensin (AvBD) orthologue was isolated from Chinese painted quail (Coturnix chinensis) lung and bone marrow tissues. The complete nucleotide sequence of the gene contained a 204-bp open-reading frame encoding 67 amino acids. Homology, characterization, and comparison of the gene with AvBD from other avian species confirmed that it was quail AvBD9. To analyze and compare the expression pattern of AvBD9 in tissues from young and adult quails, layer hens, and broilers, reverse transcription polymerase chain reaction was performed using mRNA isolated from 21 different tissues. The AvBD9 expression pattern distribution was differed among quails of different ages, layer hens, and broilers. It was widely expressed in all the tissues except the trachea, liver, and kidney and was highly expressed in the lung and heart of young quails. Similarly, it was widely expressed in all the tissues of adult quails except for the liver, kidney, spleen, thymus, and Harderian gland. In layer hens, AvBD9 was widely expressed in all the tissues except the trachea, glandular stomach, and cecum. Similarly, it was widely expressed in all the tissues of broilers except for the trachea, glandular stomach, rectum, cecum, bone marrow, and cecal tonsil. Recombinant AvBD9 (rAvBD9) was produced and purified by expressing the gene in Escherichia coli. Additionally, peptide according to quail AvBD9 sequence was synthesized, named sAvBD9. As expected, both rAvBD9 and sAvBD9 exhibited strong bactericidal properties against 11 strains of bacteria, including Gram-positive and Gram-negative forms.
Blocking of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes.
Kokoza V, Ahmed A, Woon Shin S, Okafor N, Zou Z, Raikhel AS
Department of Entomology and Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA.
Proc. Natl. Acad. Sci. U.S.A. 2010 May;107 (18):8111-6
To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and antiPlasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum, the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with antiPlasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite.