Dynamic polarization flip in nanoripples on photoexcited Ti surface near its surface plasmon resonance.

Both normal and abnormal sub-100-nanometer ripples (wavenumber ∼10 µm(-1)) were separately observed on Ti surfaces excited by linearly polarized IR femtosecond laser pulses at lower and higher fluences. Numerical modeling of dispersion curves for surface plasmon-polaritons on the photoexcited Ti surfaces demonstrates its surface plasmon resonance with the peak wavenumber ∼8 µm(-1) spectrally tuned by prompt surface optical response, prompt surface charging, and pre-oxidation, with normal/abnormal nanoripples appearing at its red/blue shoulders, respectively.

16 fs, 350 nJ pulses at 5 MHz repetition rate delivered by chirped pulse compression in fibers.

We demonstrate a simple approach for broadening and compression of intense pulses at megahertz repetition rates by self-phase modulation in nonlinear photonic crystal fibers. In order to avoid damage by self-focusing, we positively chirp the input pulses, which allows coupling of significantly more energy into the fiber, while maintaining the same spectral bandwidth and compression as compared to the Fourier-limited case at lower energy. Using a commercial long-cavity Ti:sapphire oscillator with 55 fs, 400 nJ pulses at 5 MHz, we generate 16 fs, 350 nJ pulses, which is a factor of 4 more energy than possible with unchirped input pulses. Self-phase-modulated spectra supporting 11 fs duration are also shown with 350 nJ pulse energy. Excellent stability is recorded over at least 1 h.

The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes.

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.

An antimicrobial activity of cytolytic T cells mediated by granulysin.

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

[Hepcidin and Plasmodium falciparum malaria in anemic school children in Mali]. / Hepcidine et infection due a Plasmodium falciparum chez un groupe d'ecoliers anémiés du Mali.

Hepcidin is a peptide produced by hepatocytes and detectable in blood and urine. Urinary hepcidin excretion appeared to be significantly increasing in humans with acute and chronic infections or inflammatory diseases. However, the effects of common tropical parasitic infections on hepcidin have not been sufficiently examined. We carried out a study in school children from Mali living in a neighborhood where Plasmodium falciparum malaria and Schistosoma haematobium infections are prevalent. Anemia (hemoglobin < 120 g/l) prevalence was very high among these children (68%); 24% had iron deficiency anemia. The prevalence of infections was also high (65% had at least one infection and 41% had C-reactive protein (CRP) levels > 10 mg/L). S. haematobium was diagnosed in 64%. We assessed first morning urine hepcidin excretion in a sub-sample of 15 children with either S. haematobium, P. falciparum malaria or none; 14 of these 15 children were included in the analyses. Children with P. falciparum malaria excreted significantly higher levels of hepcidin than those with S. haematobium (chi2 = 3.86; p = 0.05) or without any infection (chi2 = 5.95; p = 0.01). Urinary hepcidin correlated significantly with CRP (Spearman's r = 0.59; p = 0.001) and serum ferritin (Spearman's r = 0.73; p = 0.003). Our study confirms the still limited evidence of an association between human malaria and increased urinary hepcidin and points out the need for further studies to define the contribution of hepcidin to anemia associated with this disease.

Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations.

We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.

Monocyte-chemotactic activity of defensins from human neutrophils.

We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.

Intramolecular inhibition of human defensin HNP-1 by its propiece.

We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.

Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and "specific" granule deficiency.

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity.

Defensins are small, cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM. Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.

Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.

Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

Defensins. Natural peptide antibiotics of human neutrophils.

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.

Primary structures of three human neutrophil defensins.

The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.

Defensins.

Defensins are widely distributed and abundant 3-4 kDa antimicrobial peptides that are variable cationic and contain six disulfide-paired cysteines. Three structurally distinct peptide families have been identified: 'classical' defensins, beta-defensins and insect defensins. In many animal species, defensin genes are found in clusters with substantial sequence variability outside the core disulfide-linked cysteines. Defensin peptides have been found in the granules of phagocytes and intestinal Paneth cells, on epithelial surfaces of the intestine and the trachea, and in the hemolymph of insects. They are produced from larger precursors by stepwise, tissue-specific, proteolytic processing, a production resembling that of peptide hormones. Microbes in the phagocytic vacuoles of granulocytes and certain macrophages encounter high concentrations of defensins. Increased transcription of defensin genes and stimulus-dependent release of pre-synthesized defensin-containing cytoplasmic granules contribute to the local antimicrobial response.

Antimicrobial peptides in mammalian and insect host defence.

During the past year, additional insights into systems that regulate antimicrobial peptide production in Drosophila were reported. Granulysin, a peptide stored in the cytoplasmic granules of human natural killer cells and cytolytic T cells, was shown to kill Mycobacterium tuberculosis. More data implicating antimicrobial peptides in the pathogenesis of bronchopulmonary infections in cystic fibrosis appeared. Studies that examined the potential contributions of antimicrobial peptides to regional innate immunity gained in prominence. Efforts to design peptide analogues to prevent or treat infections continued.

Antimicrobial peptides of vertebrates.

The past year brought several discoveries that focused attention on antimicrobial peptides on epithelial surfaces. The malfunction of these substances was implicated as a cause of airway infections in cystic fibrosis. Other highlights included new insights into the relative selectivity of antimicrobial peptides for microbial membranes, their primary site of action.

Hepcidin--a peptide hormone at the interface of innate immunity and iron metabolism.

Hepcidin is a cationic amphipathic peptide made in the liver, released into plasma and excreted in urine. Hepcidin is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores, but it is also markedly induced during infections and inflammation. Under the influence of hepcidin, macrophages, hepatocytes, and enterocytes retain iron that would otherwise be released into plasma. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter that is expressed in the small intestine, and in hepatocytes and macrophages. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading, and decreased by anemia and hypoxia. Hepcidin is also rapidly induced by cytokines, including IL-6. The resulting decrease in plasma iron levels eventually limits iron availability to erythropoiesis and contributes to the anemia associated with infection and inflammation. The decrease in extracellular iron concentrations due to hepcidin probably limits iron availability to invading microorganisms, thus contributing to host defense.

Defensins.

Defensins are a family of small cationic, antibiotic peptides that contain six cysteines in disulfide linkage. The peptides are abundant in phagocytes and small intestinal mucosa of humans and other mammals and in the hemolymph of insects. They contribute to host defense against microbes and may participate in tissue inflammation and endocrine regulation during infection. Bioengineered defensins are potentially useful as prophylactic and therapeutic agents in infections.

Human defensin 5 expression in intestinal metaplasia of the upper gastrointestinal tract.

BACKGROUND: Upper gastrointestinal tract intestinal metaplasia (IM) is termed Barrett's oesophagus (BO) or gastric intestinal metaplasia (GIM), depending on its location. BO and GIM are associated with chemical exposure resulting from gastro-oesophageal reflux and chronic Helicobacter pylori infection, respectively. Paneth cells (PCs), characterised by cytoplasmic eosinophilic granules, are found in a subset of IM at these sites, but histology may not accurately detect them. AIM: To determine human defensin 5 (HD5; an antimicrobial peptide produced by PCs) expression in BO and GIM, and to investigate its association with H pylori infection. METHODS: Endoscopic biopsies from 33 patients with BO and 51 with GIM, and control tissues, were examined by routine histology and for H pylori infection and HD5 mRNA and protein expression. RESULTS: In normal tissues, HD5 expression was specific for PCs in the small intestine. Five patients with BE and 42 with GIM expressed HD5, but few HD5 expressing cells in IM had the characteristic histological features of PCs. Most HD5 positive specimens were H pylori infected and most HD5 negative specimens were not infected. CONCLUSIONS: HD5 immunohistochemistry was often positive in IM when PCs were absent by conventional histology. Thus, HD5 immunohistochemistry may be superior to histology for identifying metaplastic PCs and distinguishing GIM from BO. The higher frequency of HD5 expression in GIM than in BO is associated with a higher frequency of H pylori infection, suggesting that in IM PCs may form part of the mucosal antibacterial response.