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INTRODUCTION: Topical corticosteroids (TCS), used to treat atopic dermatitis (AD), have been associated with type 2 diabetes and osteoporosis in epidemiological studies, possibly explained by systemic absorption.OBJECTIVES: We examined whether intensive daily whole-body TCS treatment over two weeks followed by twice weekly application for four weeks could elicit insulin resistance and increase bone resorption in adults with AD.METHODS: A randomized parallel-group double-blind double-dummy non-corticosteroid-based active-comparator study design was completed in Copenhagen, Denmark. Thirty-six non-obese, non-diabetic adults with moderate-to-severe AD were randomized to whole-body treatment with betamethasone 17-valerate 0.1% plus a vehicle once daily or tacrolimus 0.1% twice daily after washout. Insulin sensitivity assessed by the hyperinsulinemic-euglycemic clamp combined with tracer infusions and biomarkers of bone formation (P1NP) and resorption (CTX) were evaluated at baseline, after two weeks of daily treatment and after further four weeks of twice-weekly maintenance treatment.RESULTS: AD severity improved with both treatments and systemic inflammation was reduced. After two weeks, we observed similar increase in peripheral insulin sensitivity with use of betamethasone (n=18) and tacrolimus (n=18). Bone resorption biomarker, CTX, was unchanged, while bone formation marker, P1NP, decreased after betamethasone treatment after both two and six weeks but remained unchanged in the tacrolimus arm.CONCLUSIONS: Whole-body treatment with TCS leads to systemic exposure but appears not to compromise glucose metabolism during short-term use, which may be a result of reduced systemic inflammatory activity. The negative impact on bone formation could be regarded an adverse effect of TCS.

The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms-important in causing chronic and therapy resistant infections-is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.

BACKGROUND Tissue factor pathway inhibitor 2 (TFPI-2) is a matrix-associated serine protease inhibitor with an enigmatic function in vivo. Here, we describe that TFPI-2 is present in fibrin of wounds and also expressed in skin, where it is up-regulated upon wounding. METHODOLOGY AND PRINCIPAL FINDINGS Neutrophil elastase cleaved TFPI-2, and a C-terminal fragment was found to bind to bacteria. Similarly, a prototypic peptide representing this C-terminal part, EDC34, bound to bacteria and bacterial lipopolysaccharide, and induced bacterial permeabilization. The peptide also induced leakage in artificial liposomes, and displayed a random coil conformation upon interactions with liposomes as well as lipopolysaccharide. EDC34 was antibacterial against both Gram-negative and Gram-positive bacteria in physiological buffer conditions. CONCLUSIONS/SIGNIFICANCE The results demonstrate that the C-terminus of TFPI-2 encodes for antimicrobial activity, and may be released during wounding.

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages. Citation: Jena P, Mohanty S, Mohanty T, Kallert S, Morgelin M, et al. (2012) Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages. PLoS ONE 7(12): e50345. doi:10.1371/journal.pone.0050345

The Streptococcus pyogenes cysteine protease SpeB (streptococcal pyrogenic exotoxin B) is important for the invasive potential of the bacteria, but its production is down-regulated following systemic infection. This prompted us to investigate if SpeB potentiated the host immune response after systemic spreading. Addition of SpeB to human plasma increased plasma-mediated bacterial killing and prolonged coagulation time through the intrinsic pathway of coagulation. This effect was independent of the enzymatic activity of SpeB and was mediated by a non-covalent medium-affinity binding and modification of the serpin A1AT (a-1 antitrypsin). Consequently, addition of A1AT to plasma increased bacterial survival. Sequestration of A1AT by SpeB led to enhanced contact system activation, supported by increased bacterial growth in prekallikrein deficient plasma. In a mouse model of systemic infection, administration of SpeB reduced significantly bacterial dissemination. The findings reveal an additional layer of complexity to host-microbe interactions that may be of benefit in the treatment of severe bacterial infections.

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase ( PAP) have been reported to form amyloid fibrils called "SEVI'' and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

Udgivelsesdato: 2009-Oct-30, Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.

BACKGROUND: Cellular prion-related protein (PrP(c)) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c), and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c) could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.