Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut

Abstract Background The importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian β-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds. Results Time-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05). Conclusion Amino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota

The Peripheral Blood Transcriptome Identifies the Presence and Extent of Disease in Idiopathic Pulmonary Fibrosis

<div><h3>Rationale</h3><p>Peripheral blood biomarkers are needed to identify and determine the extent of idiopathic pulmonary fibrosis (IPF). Current physiologic and radiographic prognostic indicators diagnose IPF too late in the course of disease. We hypothesize that peripheral blood biomarkers will identify disease in its early stages, and facilitate monitoring for disease progression.</p> <h3>Methods</h3><p>Gene expression profiles of peripheral blood RNA from 130 IPF patients were collected on Agilent microarrays. Significance analysis of microarrays (SAM) with a false discovery rate (FDR) of 1% was utilized to identify genes that were differentially-expressed in samples categorized based on percent predicted D_LCO and FVC.</p> <h3>Main Measurements and Results</h3><p>At 1% FDR, 1428 genes were differentially-expressed in mild IPF (D_LCO >65%) compared to controls and 2790 transcripts were differentially- expressed in severe IPF (D_LCO >35%) compared to controls. When categorized by percent predicted D_LCO, SAM demonstrated 13 differentially-expressed transcripts between mild and severe IPF (< 5% FDR). These include CAMP, CEACAM6, CTSG, DEFA3 and A4, OLFM4, HLTF, PACSIN1, GABBR1, IGHM, and 3 unknown genes. Principal component analysis (PCA) was performed to determine outliers based on severity of disease, and demonstrated 1 mild case to be clinically misclassified as a severe case of IPF. No differentially-expressed transcripts were identified between mild and severe IPF when categorized by percent predicted FVC.</p> <h3>Conclusions</h3><p>These results demonstrate that the peripheral blood transcriptome has the potential to distinguish normal individuals from patients with IPF, as well as extent of disease when samples were classified by percent predicted D_LCO, but not FVC.</p> </div

Mouse Background Strain Profoundly Influences Paneth Cell Function and Intestinal Microbial Composition

Increasing evidence supports the central role of Paneth cells in maintaining intestinal host-microbial homeostasis. However, the direct impact of host genotype on Paneth cell function remains unclear. Here, we characterize key differences in Paneth cell function and intestinal microbial composition in two widely utilized, genetically distinct mouse strains (C57BL/6 and 129/SvEv). In doing so, we demonstrate critical influences of host genotype on Paneth cell activity and the enteric microbiota.Paneth cell numbers were determined by flow cytometry. Antimicrobial peptide (AMP) expression was evaluated using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), acid urea-polyacrylamide gel electrophoresis, and mass spectrometry. Effects of mouse background on microbial composition were assessed by reciprocal colonization of germ-free mice from both background strains, followed by compositional analysis of resultant gut bacterial communities using terminal restriction fragment length polymorphism analysis and 16 S qPCR. Our results revealed that 129/SvEv mice possessed fewer Paneth cells and a divergent AMP profile relative to C57BL/6 counterparts. Novel 129/SvEv á-defensin peptides were identified, including Defa2/18v, Defa11, Defa16, and Defa18. Host genotype profoundly affected the global profile of the intestinal microbiota, while both source and host factors were found to influence specific bacterial groups. Interestingly, ileal α-defensins from 129/SvEv mice displayed attenuated antimicrobial activity against pro-inflammatory E. coli strains, a bacterial species found to be expanded in these animals.This work establishes the important impact of host genotype on Paneth cell function and the composition of the intestinal microbiota. It further identifies specific AMP and microbial alterations in two commonly used inbred mouse strains that have varying susceptibilities to a variety of disorders, ranging from obesity to intestinal inflammation. This will be critical for future studies utilizing these murine backgrounds to study the effects of Paneth cells and the intestinal microbiota on host health and disease

Odorranalectin Is a Small Peptide Lectin with Potential for Drug Delivery and Targeting

BACKGROUND: Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein-sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting. PRINCIPAL FINDINGS: Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. (125)I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a beta-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form. CONCLUSION: These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection

Protein C Inhibitor—A Novel Antimicrobial Agent

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which subsequently leads to their permeabilization. As shown by negative staining electron microscopy, treatment of Escherichia coli or Streptococcus pyogenes bacteria with PCI triggers membrane disruption followed by the efflux of bacterial cytosolic contents and bacterial killing. The antimicrobial activity of PCI is located to the heparin-binding site of the protein and a peptide spanning this region was found to mimic the antimicrobial activity of PCI, without causing lysis or membrane destruction of eukaryotic cells. Finally, we show that platelets can assemble PCI on their surface upon activation. As platelets are recruited to the site of a bacterial infection, these results may explain our finding that PCI levels are increased in tissue biopsies from patients suffering from necrotizing fasciitis caused by S. pyogenes. Taken together, our data describe a new function for PCI in innate immunity

A Novel Non-Lens βγ−Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals

Novel Sulfated Polysaccharides Disrupt Cathelicidins, Inhibit RAGE and Reduce Cutaneous Inflammation in a Mouse Model of Rosacea

Rosacea is a common disfiguring skin disease of primarily Caucasians characterized by central erythema of the face, with telangiectatic blood vessels, papules and pustules, and can produce skin thickening, especially on the nose of men, creating rhinophyma. Rosacea can also produce dry, itchy eyes with irritation of the lids, keratitis and corneal scarring. The cause of rosacea has been proposed as over-production of the cationic cathelicidin peptide LL-37.We tested a new class of non-anticoagulant sulfated anionic polysaccharides, semi-synthetic glycosaminoglycan ethers (SAGEs) on key elements of the pathogenic pathway leading to rosacea. SAGEs were anti-inflammatory at ng/ml, including inhibition of polymorphonuclear leukocyte (PMN) proteases, P-selectin, and interaction of the receptor for advanced glycation end-products (RAGE) with four representative ligands. SAGEs bound LL-37 and inhibited interleukin-8 production induced by LL-37 in cultured human keratinocytes. When mixed with LL-37 before injection, SAGEs prevented the erythema and PMN infiltration produced by direct intradermal injection of LL-37 into mouse skin. Topical application of a 1% (w/w) SAGE emollient to overlying injected skin also reduced erythema and PMN infiltration from intradermal LL-37.Anionic polysaccharides, exemplified by SAGEs, offer potential as novel mechanism-based therapies for rosacea and by extension other LL-37-mediated and RAGE-ligand driven skin diseases

Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

Synbiotic approach restores intestinal homeostasis and prolongs survival in leukaemia mice with cachexia

Cancer cachexia is a multifactorial syndrome that includes muscle wasting and inflammation. As gut microbes influence host immunity and metabolism, we investigated the role of the gut microbiota in the therapeutic management of cancer and associated cachexia. A community-wide analysis of the caecal microbiome in two mouse models of cancer cachexia (acute leukaemia or subcutaneous transplantation of colon cancer cells) identified common microbial signatures, including decreased Lactobacillus spp. and increased Enterobacteriaceae and Parabacteroides goldsteinii/ASF 519. Building on this information, we administered a synbiotic containing inulin-type fructans and live Lactobacillus reuteri 100-23 to leukaemic mice. This treatment restored the Lactobacillus population and reduced the Enterobacteriaceae levels. It also reduced hepatic cancer cell proliferation, muscle wasting and morbidity, and prolonged survival. Administration of the synbiotic was associated with restoration of the expression of antimicrobial proteins controlling intestinal barrier function and gut immunity markers, but did not impact the portal metabolomics imprinting of energy demand. In summary, this study provided evidence that the development of cancer outside the gut can impact intestinal homeostasis and the gut microbial ecosystem and that a synbiotic intervention, by targeting some alterations of the gut microbiota, confers benefits to the host, prolonging survival and reducing cancer proliferation and cachexia