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Last updated: 13th August 2010

New insights into the pathogenesis of Crohn's disease: are they relevant for therapeutic options?
Vavricka SR, Rogler G
Division of Gastroenterology and Hepatology, Department of Internal Medicine, University Hospital Zürich, Zürich, Switzerland.
Swiss Med Wkly 2009 Sep;139 (37-38):527-34
During the last few years significant advances have been achieved in the understanding of the pathogenesis of inflammatory bowel disease (IBD). A genetic susceptibility to Crohn's disease has been proven by identification of variations as risk factor NOD2/CARD15. Functional data on NOD2/CARD15 and NF-kappaB activation indicate that an inflammatory reaction of the intestinal mucosa, as an immediate response of the innate immune system, may be necessary for the maintenance of gut homeostasis. Crohn's disease is now also discussed as an impaired and inadequate immune reaction and no longer only as a hyper-responsiveness of the mucosal immune system. Data on NOD2/CARD15 expression suggest that macrophages and epithelial cells could be the locus of the primary pathophysiological defect and that T-cell activation might just be a secondary effect inducing chronification of the inflammation, perhaps as backup mechanism to insufficient innate immunity. In addition to NOD2/CARD15 there are more "innate" pathways by which commensal and pathogenic bacteria can directly be hindered to invade the human body (such as interaction with Toll like receptors, TLRs and defensins). The "germ-concept" and the "genetic concept" of IBD pathophysiology are converging. However, more time is needed until these important insights in IBD pathogenesis will make their way into routine diagnostic procedures and treatment of patients with IBD.
Novel venom proteins produced by differential domain-expression strategies in Beaded lizards and Gila monsters (genus Heloderma).
Fry BG, Roelants K, Winter K, Hodgson WC, Griesman L, Kwok HF, Scanlon D, Karas J, Shaw C, Wong L, Norman JA
Venomics Research Laboratory, Department of Biochemistry & Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 Australia.
Mol Biol Evol. 2010 Feb;27(2):395-407.
The origin and evolution of venom proteins in helodermatid lizards was investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: i) the first full-length sequences of Lethal Toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral mono-domain, mono-product gene (beta-defensin) which underwent three tandem domain duplications to encode a tetra-domain, mono-product with a possible novel protein fold; (ii) an ancestral mono-domain gene (encoding a natriuretic peptide) was medially extended to become a penta-domain, penta-product through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by post-translational proteolysis; and iii) an ancestral multi-domain, multi-product gene belonging to the VIP/glucagon family being mutated to encode for a mono-domain, mono-product (exendins) followed by duplication and diversification into two variant classes (exendins 1&2 and exendins 3&4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isofroms within each class. These results highlight the importance of utilising evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.
Inhibition of bactericidal activity is maintained in a mouse alpha-defensin precursor with proregion truncations.
Figueredo SM, Ouellette AJ
Department of Pathology & Laboratory Medicine, School of Medicine, College of Health Sciences, University of California, Irvine, CA 92697-4800, United States.
Peptides. 2010 Jan;31(1):9-15.
alpha-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-alpha-defensin proCrp4((20-92)), anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-alpha-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the alpha-defensin component of the precursor would alter proCrp4 inhibition. Accordingly, two proCrp4 deletion variants, (Delta44-53)-proCrp4 and (Delta44-58)-proCrp4, truncated in a manner corresponding to deletions between MMP-7 cleavage sites, were prepared and assayed for bactericidal peptide activity. Consistent with the properties of full-length proCrp4((20-92)), (Delta44-53)-proCrp4 and (Delta44-58)-proCrp4 were processed effectively by MMP-7, lacked bactericidal activity at high peptide levels over a 3h exposure period, and failed to induce permeabilization of live Escherichia coliin vitro. Thus, bringing the inhibitory proregion domain into greater proximity with the Crp4 component of the precursor did not alter the activity of this pro-alpha-defensin. Therefore, the conserved distance that separates inhibitory acidic proregion residues from the Crp4 peptide is not critical to maintaining proCrp4((20-92)) in an inactive state.
New defensins from hard and soft ticks: Similarities, differences, and phylogenetic analyses.
Chrudimská T, Chrudimskı T, Golovchenko M, Rudenko N, Grubhoffer L
Faculty of Science, University of South Bohemia, Branisovská 31, Ceské Budejovice 37005, Czech Republic; Institute of Parasitology, Biology Centre, ASCR, v.v.i., Branisovská 31, Ceské Budejovice 37005, Czech Republic.
Vet Parasitol. 2010 Feb 10;167(2-4):298-303.
Despite the importance of ticks as vectors of disease very little is known about their immune system. Antimicrobial peptides, including defensins (phylogenetically ancient antibacterial peptides) are major components of innate immunity in ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites. With the aim of studying the evolution of the genes involved in tick defense, we identified the preprodefensin genes from four Ornithodoros tick species (O. papillipes: isoforms A, B, and D; O. tartakovskyi and O. puertoricensis: isoforms A and B; O. rostratus: isoform A) and from two Dermacentor tick species (D. reticulatus and D. marginatus: one isoform) not previously described. Phylogenetic analyses revealed that Ornithodoros defensin isoforms (A, B, C, and D) form 4 separate clades, while hard tick defensins are divided into several branches based on particular tick species.
Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia.
Winder AA, Wohlford-Lenane C, Scheetz TE, Nardy BN, Manzel LJ, Look DC, McCray PB
Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Respir. Res. 2009 ;10 96
BACKGROUND: The recognition of microbial molecular patterns via Toll-like receptors (TLRs) is critical for mucosal defenses. METHODS: Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-alpha and IFN-gamma) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints. RESULTS: Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-alpha and IFN-gamma), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines +/- dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-kappaB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects. CONCLUSION: While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-alpha and IFN-gamma may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.
[Construction of HBD-3 gene mammary-specific expression vector and eukaryotic expression]
Peng W, Lan Z, Ma J, Wang B, Zhang Y
Institute of Biotechnology, College of Animal Veterinary Medicine, Northwest Agiculture and Forestry University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao 2009 Jul;25 (7):968-74
To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human beta-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5' and 3' regulating region of beta-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human beta-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human beta-defensin-3 in the supernant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.
The antimicrobial activity of CCL28 is dependent on C-terminal positively charged amino acids.
Liu B, Wilson E
Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
Eur J Immunol. 2009 Oct 14;40(1):186-196.
Several chemokines have been shown to act as antimicrobial proteins, suggesting a direct contribution to innate immune protection. Based on the study of defensins and other antimicrobial peptides, it has been proposed that cationic amino acids in these proteins play a key role in their antimicrobial activity. The primary structure requirements necessary for the antimicrobial activity of chemokines however, has not yet been elucidated. Using mouse CCL28, we have identified a C terminal region of highly charged amino acids (RKDRK) that is essential to the antimicrobial activity of the murine chemokine. Additionally, other positively charged amino acids in the C-terminus of the protein contribute to the observed antimicrobial effect. Charge reversal and deletion mutations support our hypothesis that C-terminal positively charged amino acids are essential for the antimicrobial activity of CCL28. Results also demonstrate that although the C-terminal region of the chemokine is essential, it is not sufficient for full antimicrobial activity of CCL28.
A 3' UTR transition within DEFB1 is associated with chronic and aggressive periodontitis.
Schaefer AS, Richter GM, Nothnagel M, Laine ML, Rühling A, Schäfer C, Cordes N, Noack B, Folwaczny M, Glas J, Dörfer C, Dommisch H, Groessner-Schreiber B, Jepsen S, Loos BG, Schreiber S
Institute for Clinical Molecular Biology, University Medical Center Schleswig-Holstein, Kiel, Germany.
Genes Immun. 2010 Jan;11(1):45-54.
Periodontal diseases are complex inflammatory diseases and affect up to 20% of the worldwide population. An unbalanced reaction of the immune system toward microbial pathogens is considered as the key factor in the development of periodontitis. Defensins have a strong antimicrobial function and are important contributors of the immune system toward maintaining health. Here, we present the first systematic association study of DEFB1. Using a haplotype-tagging single nucleotide polymorphism (SNP) approach, including described promoter SNPs of DEFB1, we investigated the associations of the selected variants in a large population (N=1337 cases and 2887 ethnically matched controls). The 3' untranslated region SNP, rs1047031, showed the most significant association signal for homozygous carriers of the rare A allele (P=0.002) with an increased genetic risk of 1.3 (95% confidence interval: 1.11-1.57). The association was consistent with the specific periodontitis forms: chronic periodontitis (odds ratio=2.2 (95% confidence interval: 1.16-4.35), P=0.02), and aggressive periodontitis (odds ratio=1.3 (95% confidence interval 1.04-1.68), P=0.02). Sequencing of regulatory and exonic regions of DEFB1 identified no other associated variant, pointing toward rs1047031 as likely being the causative variant. Prediction of microRNA targets identified a potential microRNA-binding site at the position of rs1047031.Genes and Immunity advance online publication, 15 October 2009; doi:10.1038/gene.2009.75.
Modulation of human beta-defensin-2 expression by 17beta-estradiol and progesterone in vaginal epithelial cells.
Han JH, Kim MS, Lee MY, Kim TH, Lee MK, Kim HR, Myung SC
Department of Urology, KEPCO Medical Foundation Hanil General Hospital, Seoul, Republic of Korea.
Cytokine. 2010 Feb;49(2):209-14.
We investigated the expression of HBD-1 and -2 in vaginal epithelial cells treated with lipopolysaccharide (LPS) and the effects on HBD-2 expressions by 17beta-estradiol and progesterone. Primary vaginal epithelial cells were isolated from a segment of normal anterior vaginal wall obtained during vaginoplasty and were cultured in keratinocyte growth medium and were allowed to undergo their 3rd passage. Expression of HBD-1 and -2 by different stimuli using LPS 0.5mug/ml, 17beta-estradiol 2nM and progesterone 1muM was measured by RT-PCR, ELISA and real-time RT-PCR, respectively. HBD-1 was produced constitutively in vaginal epithelial cells and the production of HBD-1 was not influenced by LPS, 17beta-estradiol and progesterone, but the production of HBD-2 was increased inducibly by LPS. 17beta-Estradiol and progesterone did not change the production of HBD-2 in normal state, but 17beta-estradiol increased the production of HBD-2 and progesterone suppressed the production of HBD-2 under the circumstances with infection. The HBD-2 plays an important role at innate host defense on genitourinary tract. The lacks of estrogen during menopause or uses of a progesterone-based oral contraceptive in sexually active women may influence production of HBD-2 in vaginal epithelium and may increase susceptibility to bacterial vaginitis or recurrent UTI.
Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis.
Kamsteeg M, Jansen PA, van Vlijmen-Willems IM, van Erp PE, Rodijk-Olthuis D, van der Valk PG, Feuth T, Zeeuwen PL, Schalkwijk J
Department of Dermatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Br J Dermatol. 2010 Mar;162(3):568-78.
ABSTRACT Background: Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis have revealed genes with disease-specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease-specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general. Objectives: To analyze if gene expression signatures as found in purified epidermal cells from atopic dermatitis are also present in other eczematous conditions such as allergic and irritant contact dermatitis. Methods: We used real-time quantitative PCR, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analyzed for comparison. Results: Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in atopic dermatitis epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, atopic dermatitis, and allergic contact dermatitis but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response. Conclusions: These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.