Increased warm water intrusions could cause mass loss in East Antarctica during the next 200 years.

The East Antarctic Ice Sheet (EAIS) is currently surrounded by relatively cool water, but climatic shifts have the potential to increase basal melting via intrusions of warm modified Circumpolar Deep Water (mCDW) onto the continental shelf. Here we use an ice sheet model to show that under the current ocean regime, with only limited intrusions of mCDW, the EAIS will likely gain mass over the next 200 years due to the increased precipitation from a warming atmosphere outweighing increased ice discharge due to ice-shelf melting. However, if the ocean regime were to become dominated by greater mCDW intrusions, the EAIS would have a negative mass balance, contributing up to 48 mm of SLE over this time period. Our modelling finds George V Land to be particularly at risk to increased ocean induced melting. With warmer oceans, we also find that a mid range RCP4.5 emissions scenario is likely to result in a more negative mass balance than a high RCP8.5 emissions scenario, as the relative difference between increased precipitation due to a warming atmosphere and increased ice discharge due to a warming ocean is more negative in the mid range RCP4.5 emission scenario.

Potent Inducers of Endogenous Antimicrobial Peptides for Host Directed Therapy of Infections.

A new concept for treatment of infections is induction of our own antimicrobial peptides and the presented novel class of inducer, aroylated phenylenediamines (APDs), gives up to 20 to 30-fold induction of the human antimicrobial peptide LL-37, in vitro. In addition, oral administration of an APD in a rabbit model of Shigellosis resulted in recovery from the infection in a few days implying that APD's are promising candidates for treatment of infections.

Host antimicrobial defence peptides in human disease.

Antimicrobial peptides or host defence peptides are endogenous peptide antibiotics, which have been confirmed as an essential part of the immune system. Apart from direct killing of bacteria, a role for the peptides in antiviral and immunomodulatory functions has recently been claimed. In this chapter we have focused on the host contact with microbes, where these host defence peptides are key players. The interplay with commensals and pathogens in relation to antimicrobial peptide expression is discussed, with specific emphasis on the respiratory and the alimentary systems. A possible novel difference in epithelial interactions between commensals and pathogens is considered in relation to disease.

Antimicrobial peptide LL-37 internalized by immature human dendritic cells alters their phenotype.

The human cathelicidin LL-37 has been shown to be involved in the barrier function of the innate immunity, being released from specific cells upon challenge and exerting immunomodulatory effects. We here demonstrate that LL-37 affects immature dendritic cells, derived from human peripheral blood monocytes (MDDC). LL-37 is internalized by MDDC with subsequent localization primarily in the cytoplasmic compartment. However, LL-37 could also be detected in the nuclei of MDDC, suggesting that LL-37 may be transported into the nucleus. The uptake of LL-37 is dose, time and energy dependent, indicating that the observed internalization process involves an endocytic pathway. Incubation of immature MDDC with LL-37 caused phenotypic changes, characterized by an increased expression of the antigen-presenting molecule HLA-DR, and the costimulatory molecule CD86. Taken together, these findings suggest that LL-37 released upon triggering of the innate immunity, may affect cellular adaptive immunity through an interaction with immature dendritic cells.

Malassezia sympodialis differently affects the expression of LL-37 in dendritic cells from atopic eczema patients and healthy individuals.

BACKGROUND: Atopic eczema (AE) is a multifactorial disease, which has increased in prevalence. The skin-colonizing yeast Malasezzia sympodialis can induce IgE- and T-cell reactivity in patients with AE. LL-37 is an endogenous peptide antibiotic belonging to the cathelicidin family. The aim of this study was to examine whether exposure to M. sympodialis would affect the expression of LL-37 in dendritic cells. METHODS: The presence of LL-37 was analyzed in monocyte-derived dendritic cells (MDDCs) generated from healthy individuals and patients with AE by Western blotting and the corresponding cDNA by real-time quantitative RT-PCR. Antibacterial activity was measured with an inhibition zone assay in fractions after reverse phase chromatography. RESULTS: For the first time we here present data, showing that LL-37 is produced by MDDCs. Notably, the secretion of LL-37 was substantially enhanced in M. sympodialis-exposed MDDCs generated from patients with a high degree of eczema, as measured by SCORAD, compared to healthy controls and patients with a low SCORAD. The relative expression of LL-37 transcript in MDDCs generated from patients was up-regulated after 1 h of exposure to M. sympodialis and declined gradually at the time points analyzed, whereas the transcription was unaffected in the MDDCs of healthy controls. CONCLUSIONS: Our results suggest that M. sympodialis can trigger the innate immune response differently in patients with AE and healthy individuals. The enhanced LL-37 secretion from the MDDCs in the patients with AE may reflect the severity of their inflammatory response to M. sympodialis.

Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions.

Vernix caseosa is a white cream-like substance that covers the skin of the foetus and the newborn baby. Recently, we discovered antimicrobial peptides/proteins such as LL-37 in vernix, suggesting host defence functions of vernix. In a proteomic approach, we have continued to characterize proteins in vernix and have identified 20 proteins, plus additional variant forms. The novel proteins identified, considered to be involved in host defence, are cystatin A, UGRP-1, and calgranulin A, B and C. These proteins add protective functions to vernix such as antifungal activity, opsonizing capacity, protease inhibition and parasite inactivation. The composition of the lipids in vernix has also been characterized and among these compounds the free fatty acids were found to exhibit antimicrobial activity. Interestingly, the vernix lipids enhance the antimicrobial activity of LL-37 in vitro, indicating interactions between lipids and antimicrobial peptides in vernix. In conclusion, vernix is a balanced cream of compounds involved in host defence, protecting the foetus and newborn against infection.

Phylogeny, processing and expression of the rat cathelicidin rCRAMP: a model for innate antimicrobial peptides.

A database search identified a rat cDNA clone which phylogenetic analysis revealed to encode a cathelicidin most similar to mouse cathelicidin CRAMP. The analysis also showed that the evolutionary pattern of the cathelicidin family is lineage specific. The rat cathelicidin is called rCRAMP. Its peptide was isolated from granulocytes, and determined to be 43 amino acids long by mass spectrometry and N-terminal sequencing. Synthetic rCRAMP had antimicrobial activity. The expression of rCRAMP was investigated by reverse-transcriptase polymerase chain reaction followed by Southern hybridization and by Western blot analysis. rCRAMP was identified in granulocytes, thymus, testis, lung, mouth mucosa, tongue, oesophagus, colon, caecum and small intestine, a distribution similar to cathelicidins of mouse and human. The rat is a small laboratory animal with additional disease models available compared to the mouse. Our results open up the possibility to use the rat as a model system to study responses connected to cathelicidin expression in health and disease.

Hsp90 binds CpG oligonucleotides directly: implications for hsp90 as a missing link in CpG signaling and recognition.

CpG motifs originating from bacterial DNA (CpG DNA) can act as danger signals for the mammalian immune system. These CpG DNA motifs like many other pathogen-associated molecular patterns are believed to be recognized by a member of the toll-like receptor family, TLR-9. Here we show results suggesting that heat shock protein 90 (hsp90) is also implicated in the recognition of CpG DNA. Hsp90 was characterized as a binder to oligodeoxynucleotides (ODNs) containing CpG motifs (CpG ODNs) after several purification steps from crude protein extracts of peripheral blood mononuclear cells. This finding was further supported by direct binding of CpG ODNs to commercially available human hsp90. Additionally, immunohistochemistry studies showed redistribution of hsp90 upon CpG ODN uptake. Thus, we propose that hsp90 can act as a ligand transfer molecule and/or play a central role in the signaling cascade induced by CpG DNA.

Expression of the cathelicidin LL-37 is modulated by short chain fatty acids in colonocytes: relevance of signalling pathways.

BACKGROUND AND AIMS: Short chain fatty acids (SCFA) exert profound effects on the colonic mucosa. In particular, SCFA modulate mucosal immune functions. The antimicrobial cathelicidin LL-37 is expressed by colon epithelial cells. In the present study the effect of SCFA on LL-37 expression was investigated. METHODS: LL-37 expression in vivo was assessed by immunohistochemistry. Real time quantitative reverse transcription-polymerase chain reaction was employed to determine LL-37 expression in colonocytes in vitro after treatment with various cytokines, SCFA, or flavone. LL-37 levels were correlated to cell differentiation which was determined by alkaline phosphatase (AP) activity. In addition, intracellular signalling pathways such as MEK-ERK (mitogen/extracellular signal protein kinase (MEK)/extracellular signal regulated protein kinase (ERK)) and p38/mitogen activated protein (MAP) kinase were explored. RESULTS: In vivo, LL-37 expression in healthy mucosa was restricted to differentiated epithelial cells in human colon and ileum. In colonocytes, increased LL-37 expression associated with cell differentiation was detected in vitro following treatment with butyrate, isobutyrate, propionate, and trichostatin A. Flavone induced LL-37 transcription but did not affect AP activity while cytokines had no effect. To dissect pathways mediating differentiation and LL-37 expression, specific inhibitors were applied. Inhibition of the protein kinase MEK enhanced butyrate induced AP activity while LL-37 expression in colon epithelial cells was blocked. In contrast, inhibition of p38/MAP kinase blocked cell differentiation without inhibiting LL-37 expression. CONCLUSIONS: Expression of the cathelicidin LL-37 in colonocytes and cellular differentiation are separately modulated by SCFA via distinct signalling pathways. These data may provide a rationale for dietary modulation of mucosal defence mechanisms.

The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa.

BACKGROUND: Peptide antibiotics are part of the surface defences against microbial intruders. However, the presence and significance of these innate immune effectors in the skin barrier of the newborn infant have not yet been appreciated. Erythema toxicum neonatorum is an inflammatory skin reaction of unknown aetiology and significance, commonly present in the healthy newborn infant. OBJECTIVES: As peptide antibiotics are upregulated in inflammatory skin disorders, we hypothesized that this also could be the case in erythema toxicum. We also investigated if the vernix caseosa, a cream-like white substance present on the skin of the infant at birth, might contribute to host defences. METHODS: The presence of the human antibacterial peptide LL-37 was investigated by immunohistochemistry and confocal imaging of skin biopsies from four 1-day-old infants with an erythema toxicum rash and four matched newborns without the rash. In addition, we analysed the expression of LL-37 and human beta defensin-1, an antibacterial peptide of epithelial origin, by reverse transcriptase-polymerase chain reaction. Finally, we screened for antibacterial components in vernix material obtained from six healthy newborns by inhibition zone assays. RESULTS: All biopsies from the lesions of erythema toxicum showed a dense, nodular infiltrate with numerous LL-37-expressing cells located in the dermal layer and a clear localization of the peptide within CD15-expressing neutrophils, EG2-expressing eosinophils and CD1a-expressing dendritic cells. LL-37 was also found to be located in CD1a-expressing Langerhans cells and a positive staining for the peptide was seen throughout the whole epidermal layer, both in infants with and without the rash. Skin samples from infants with the rash of erythema toxicum showed a constitutive expression of human beta defensin-1, while the expression of LL-37 seemed to be induced. Furthermore, LL-37 and lysozyme were detected in the protein fractions derived from the vernix caseosa, and these fractions exhibited a clear antibacterial activity. CONCLUSIONS: Peptide antibiotics are present in the vernix caseosa and in the skin of the healthy newborn infant, indicating effective innate immune protection already during fetal and neonatal life.

Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.

Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens. The human and mouse cathelicidins (LL-37 and CRAMP, respectively) are expressed at select epithelial interfaces where they have been proposed to kill a number of gram-negative and gram-positive bacteria. To determine if these peptides play a part in the protection of skin against wound infections, the anti-microbial activity of LL-37 and CRAMP was determined against the common wound pathogen group A Streptococcus, and their expression was examined after cutaneous injury. We observed a large increase in the expression of cathelicidins in human and murine skin after sterile incision, or in mouse following infection by group A Streptococcus. The appearance of cathelicidins in skin was due to both synthesis within epidermal keratinocytes and deposition from granulocyctes that migrate to the site of injury. Synthesis and deposition in the wound was accompanied by processing from the inactive prostorage form to the mature C-terminal peptide. Analysis of anti-microbial activity of this C-terminal peptide against group A Streptococcus revealed that both LL-37 and CRAMP potently inhibited bacterial growth. Action against group A Streptococcus occurred in conditions that typically abolish the activity of anti-microbial peptides against other organisms. Thus, cathelicidins are well suited to provide defense against infections due to group A Streptococcus, and represent an important element of cutaneous innate immunity.

The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations.

We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)-2, we isolated and characterized several antibacterial peptides/proteins from the supernatant-alpha-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B-although other active components were also present. We then used reverse transcriptase-polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, gammadelta T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several alphabeta T-cell lines. The alpha-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, gammadelta T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-gamma. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human alpha-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.

Decreased pulmonary levels of the anti-inflammatory Clara cell 16 kDa protein after induction of airway inflammation in asthmatics.

The Clara cell 16 kDa protein (CC16) maps to an atopy-associated region of chromosome 11 and has been ascribed an anti-inflammatory function. Using reverse-phase HPLC and Western blot analysis, we have evaluated the polypeptide pattern in bronchoalveolar lavage (BAL) fluid retrieved from asthmatics, before and after induction of airway inflammation by low-dose allergen inhalation challenge. A prominent decrease of CC16 was seen after induction of inflammation, and a further CC16 decrease was observed in lavage fluid where surfactant had been removed. Reduced levels of pulmonary CC16 may cause loss of anti-inflammatory activity in the airways and contribute to the development of airway inflammation in asthma.

Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients.

Antibacterial peptides and proteins are an integral part of the epithelial defense barrier that provides immediate protection against bacterial invasion. In humans, alpha-defensins are mainly bactericidal effectors in circulating granulocytes, beta-defensin-1 is synthesized in epithelial cells, and LL-37 is produced in granulocytes but is also induced in skin epithelia during inflammation. To investigate the importance of these defense effectors in disease, we analyzed bronchoalveolar lavage fluid (BALF) for bactericidal activity. Antibacterial activity was found in BALF material from healthy individuals and sarcoidosis patients, with enhanced activity in BALF from the patients. The activity was present as several antibacterial components, of which we have so far characterized LL-37, lysozyme, alpha-defensins, and antileukoprotease. In addition, the antibacterial peptide LL-37 was located in alveolar macrophages, bronchial epithelial cells, and bronchial glands, suggesting that it has a defensive role in airway mucosa. In conclusion, the airway epithelium is protected by a complex antibacterial defense system. This is activated in sarcoidosis, and may explain why these patients seldom develop severe respiratory tract infections.

Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system.

The bactericidal machinery of mammalian neutrophils is built up of many components with different chemical properties, involving proteins, peptides and oxygen-dependent radicals. All these components work in synergy, leading to destruction and elimination of ingested microbes. During the eighties, it gradually became clear, that cationic peptides are a part of the oxygen-independent bactericidal effectors in phagocytic cells. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. These potent broad spectra peptides are included as immediate effector molecules in innate immunity. The detailed killing mechanism for these effectors is partly known, but nearly all of them have membrane affinity, and permeate bacterial membranes, resulting in lysis of the bacteria. This peptide-membrane interaction includes also eukaryotic membranes, that implicates cytotoxic effects on host cells. Studies in vitro have established that the microenvironment is critical for their activities. In connection to cystic fibrosis, the effects of microenvironment changes are apparent, causing inactivation of peptide defences and leading to repeated serious bacterial infections. Thus, the importance of the microenvironment is also supported in vivo. Additional functions of these peptides such as chemotactic, mitogenic and stimulatory in the wound healing process suggest further important roles for these peptides.

Cloning and characterization of ZNF189, a novel human Krüppel-like zinc finger gene localized to chromosome 9q22-q31.

A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37.

The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 microM, and at 13-25 microM the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.

The epithelia constitute a major barrier to the environment and provide the first line of defense against invading microbes. Antimicrobial peptides are emerging as participants in the defense system of epithelial barriers in general. Originally we isolated the human antimicrobial peptide LL-37 from granulocytes. The gene (CAMP or cathelicidin antimicrobial peptide) coding for this peptide belongs to the cathelicidin family, whose members contain a conserved pro-part of the cathelin type. The human genome seems to have only one gene of this family, whereas some mammalian species have several cathelicidin genes. In the present work we demonstrate up-regulation of this human cathelicidin gene in inflammatory skin disorders, whereas in normal skin no induction was found. By in situ hybridization and immunohistochemistry the transcript and the peptide were located in keratinocytes throughout the epidermis of the inflammatory regions. In addition, the peptide was detected in partially pure fractions derived from psoriatic scales by immunoblotting. These fractions also exhibited antibacterial activity. We propose a protective role for LL-37, when the integrity of the skin barrier is damaged, participating in the first line of defense, and preventing local infection and systemic invasion of microbes.

NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells.

NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.