One year in Antarctica: mucosal immunity at three Australian stations.
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The effect of a year's isolation in Antarctica on the human mucosal immune system was assessed during the winter of 1992 at three Australian Antarctic stations: Casey, Davis and Mawson. Saliva samples were collected from each expeditioner prior to their departure from Australia and during each month in Antarctica. The concentrations of salivary immunoglobulins IgA and IgG were significantly different between the three stations, but there were no differences for salivary IgM and albumin. The mean concentrations of IgA were higher at Mawson (P < 0.008), and the mean concentrations of IgG were lower at Davis (P < 0.001) compared with the other stations. Ranges of values observed at the stations over the 12-13 months were similar. The variability of values within individuals showed station differences for salivary IgM and IgG only. The study revealed significant changes in salivary immunoglobulin values over the period in Antarctica, with similar patterns at the three Australian stations. The salivary IgA and IgM levels were lower in the first 4 months in Antarctica (January-April) and increased to maximum values in July-August, before returning to mean levels when isolation was broken in October-November. The patterns of salivary IgA and IgM suggest that stressors due to isolation may play a role in alterations of mucosal immunity in expeditioners in Antarctica. (+info)
Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated Drosophila ng-1 gene.
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The steroid hormone ecdysone controls multiple aspects of insect development, including larval moults and metamorphosis, and can induce specific genetic responses in different tissues. The definition of the molecular mechanisms able to mediate this tissue-specific responsiveness may greatly contribute to understanding how such an accurate genetic response is achieved. In this work we have identified, by transgenic analysis, the regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile. Our results show that an ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the FKH regulatory protein, which can potentially modulate the ecdysone genetic regulated response. (+info)
CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice.
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Antigen-specific IgE plays an important role in the pathogenesis of allergic disorders. Immunostimulatory CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential immunomodulators for switching on protectiveT(h)1-mediated immunity and preventing or potentially inhibiting T(h)2-dependent allergic responses. To date, allergic models used in CpG ODN studies have been established by immunization of mice with allergen in the presence of adjuvant. This, in addition to failure to assess specific IgE production in most of the studies, has limited understanding of the role of CpG ODN vaccination in allergic responses. Here, we examine the effects of synthesized CpG ODN on both developing and ongoing IgE responses in mice sensitized using a recombinant mosquito salivary antigen (rAed a 2) without adjuvant. Pretreatment of mice with CpG ODN mixed with rAed a 2 successfully inhibited subsequent induction of serum rAed a 2-specific IgE (but not IgG1) and antigen-induced IL-4 and IL-5 production in spleen cells. This was associated with an increase of serum IgG2a and IL-12, and increased IFN-gamma and IL-12 production by spleen cells. In this model, however, co-administration of CpG ODN with rAed a 2 to presensitized mice failed to down-regulate ongoing IgE responses despite significant up-regulation of serum IL-12 and specific IgG2a. Strikingly, a transient skin delayed-type hypersensitivity reaction occurred in CpG ODN-treated mice. These observations provide a new insight into the potential therapeutic application of CpG ODN to allergic disorders. (+info)
Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.
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One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity. (+info)
Zinc and copper bind to unique sites of histatin 5.
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Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif. (+info)
Effects of hard tissue-related hormones on the intracellular calcium ion of the rat odontoblasts.
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We examined the effects of PTH, calcitonin (CT) and parotin subunit on the intracellular Ca2+ of odontoblasts using a chelate reagent, FURA2-AM. Rat CT (rCT), at a final concentration of 0.01 microM, induced a gradual lowering of Ca2+, and addition of ATP, in the presence of CT, resulted in a partial and short-lasting recovery of Ca2+. At higher concentrations, it caused a rapid decrease of Ca2+. CTs of other animal species showed similar effects. Human PTH (hPTH) added at concentrations of 0.01, 0.1 and 1 microM, caused no significant changes in intracellular Ca2+. Parotin subunit caused a rapid lowering of Ca2+ which was seen already at 0.01 microM. rCT added after treatment with hPTH caused an immediate decrease of Ca2+ to zero level, showing that CT action was enhanced by pretreatment with hPTH. This enhancement was also confirmed by addition of hPTH after rCT, where at 1 microM, it caused further acute decrease in Ca2+. After intracellular Ca2+ was lowered by CT pretreatment, parotin, at 0.1 microM, induced a further but gradual decrease of Ca2+. The present results, together with our previous study indicating that hPTH increased cAMP production and that CT inhibited the PTH action, made it clear that all the hormones affect odontoblasts, and that CT and parotin act via Ca-related signal transduction system, while PTH acts via cAMP-PKA-related cascade. Possible crosstalk of both systems was also suggested. (+info)
Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.
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Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme. (+info)
Identification of an IL-2 binding protein in the saliva of the Lyme disease vector tick, Ixodes scapularis.
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A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2. (+info)