text:
|Home | About | Journals | Submit | Contact Us | Français
Antimicrobial peptides (AMPs) are widely expressed and rapidly induced at epithelial surfaces to repel assault from diverse infectious agents including bacteria, viruses, fungi and parasites. Much information suggests that AMPs act by mechanisms that extend beyond their capacity to serve as gene-encoded antibiotics. For example, some AMPs alter the properties of the mammalian membrane or interact with its receptors to influence diverse cellular processes including cytokine release, chemotaxis, antigen presentation, angiogenesis and wound healing. These functions complement their antimicrobial action and favor resolution of infection and repair of damaged epithelia. Opposing this, some microbes have evolved mechanisms to inactivate or avoid AMPs and subsequently become pathogens. Thus, AMPs are multifunctional molecules that have a central role in infection and Inflammation.
Initially, the importance of antimicrobial peptides (AMPs) to mammalian immunity was underestimated compared to their role in less complex immune systems such as those found in plants and invertebrates. However, as the importance of innate immune defense systems of mammals was steadily uncovered, the essential role of AMPs in mammalian immunity became firmly established. Today, we are beginning to see that much of the importance of AMPs in mammals might lie in their multifunctional role. To understand this topic, we must first discuss the extreme diversity of molecules referred to as AMPs.
So far, more than 1200 antimicrobial peptides of different origins have been identified or predicted. For a list of some of these, see the Antimicrobial Peptide Database (APD: http://aps.unmc.edu/AP/main.php). Most of these peptides maintain certain common features, such as being small (12–50 amino acids), containing positive charge and an amphipathic structure. Based on their amino acid composition, size and conformational structures, AMPs can be divided into several categories, such as peptides with α-helix structures, peptides with β-sheet structures stabilized by disulfide bridges or peptides with extended or loop structures (Figure 1). The expression of AMPs differs depending on the cell and tissue type, but in most cases AMPs are co-expressed as groups that act together. For example, in skin, more than 20 antimicrobial peptides and proteins have been identified [1], including cathelicidins, β-defensins and others. Relatively little information is available on the immunological function for most of these peptides, and it is unlikely that the immunomodulatory actions of the mammalian AMPs are also a component of the biology of the hundreds of AMPs produced by most prokaryotes and invertebrates. Thus, the first important concept to confer in a discussion of the AMPs is that they are similar only by their capacity to directly kill or inhibit the growth of microbes. Because this function is often through their capacity to influence lipid membrane structures, AMPs have some distant structural similarities. However, the specific immunological effect of AMPs is likely to be highly specific and isolated in particular AMP families. For purposes of illustration, we briefly describe two of the most extensively studied mammalian gene families of AMPs, the cathelicidins and defensins.
The cathelicidins have been described in both invertebrate species such as hagfish and vertebrate species including fish, birds, snakes and mammals [2-6]. In humans, a single cathelicidin gene (CAMP) is known that encodes an inactive precursor protein with an approximate mass of 18 KDa, and is thus named hCAP18. Cathelicidins are so named based on the highly conserved N-terminal region known as the cathelin domain. This protein contains two disulfide bonds between cysteine residues C85-C96 and C107-C124 [7,8] and was given its name based on an ability to inhibit the protease cathepsin-L. The cathelicidins are only considered to be a gene family because of the conservation seen in this region of the gene, encoding a protein that acts as a cysteine protease inhibitor and also possesses some antimicrobial activity [9]. The peptides generated by cathelicidin genes found between species show little similarity to each other and are referred to as a group solely because of the similarity of the precursor protein that is predominated by the large cathelin domain. In human neutrophils and other cell types hCAP18 is processed to release from the carboxy (C-) terminus of the precursor protein an antimicrobial peptide of 37 amino acids beginning with two leucines, named LL37 hCAP18 is stored in neutrophil granules at a molar concentration as high as 40 μM or 630 μg 10−9 cells [10] and is also produced in other granulocytes such as NK-cells and mast cells. It is also expressed, released or secreted by epithelial cells in the skin, lung, gut, mammary gland and epididymis [11-14]. As a mature peptide, LL37 has rapid, potent and broad-spectrum antimicrobial activity when released from the C-terminus of hCAP18 [15]. Its processing is essential for activation of its antimicrobial activity in vivo [16] and is accomplished by serine proteases from keratinocytes such as kallikreins [17] and neutrophil proteases such as proteinase 3 [18].
The structures of the cathelicidin peptides vary both within and between species because cathelicidins tend to show very little homology beyond the N-terminal cathelin domain. In species such as the pig, multiple catbelicidin genes encode peptides that include those with linear structures and rich in amino acids such as tryptophan, proline or arginine, peptides that have disulfide bonds and tend to form a β-sheet structure or peptides that can adopt an α helical confirmation (Figure 1). In humans, LL37 is rich in lysine and arginine and adopts an α-helical structure in detergent micelles (Figure 1a) [19] or in solutions with ionic compositions similar to human plasma, interstitial fluid or intracellular fluid [20]. In human neutrophils, cathelicidin is usually processed to release LL37, whereas at the human skin surface, LL37 is further processed by serine proteases or other proteases produced by skin microflora into different derivatives (e.g. RK-31, KS-30 and K20) with varied activities [12]. Overall, cathelicidin peptides should be considered to be a highly heterogeneous group of molecules both within and between species [21].
In mammalian species, around 50 α-defensins and 90 β-defensins have been identified which are either stored in the granules of neutrophils and Paneth cells, or are generated by monocyte, macrophages [22]. keratinocytes or mucosal epithelial cells of the respiratory, digestive, urinary and reproductive systems. All defensins are cationic, microbicidal, lack glycosyl- or acyl- side-chain modifications and contain six highly conserved cysteine residues which form three pairs of intramolecular disulfide bonds (Figure 1b). Although mammalian defensin peptides exhibit a large variability in their sequences, the six cysteines are retained. Based on sequence homology and the connectivity of the six oonserved cysteine residues, mamma-lian defensins are classified into α-, β- and θ-defensins. The six cysteines in α-defensins are linked in a 1–6, 2–4 and 3–5 pattern, whereas the disulfide connectivity in β-defensins is in a 1–5, 2–4 and 3–6 pattern. The structure of θ-defensins is circular without a free N- or C-terminus (Figure 1d). The disulfide paring of θ-defensins is different from α- and β-defensins because θ-defensins are formed by two bemi-α-defensins, each of which contributes three cysteines. Similar to cathelicidins, defensins are activated by proteolytic processing from an inactive precursor [23]. However, unlike the cathelicidins, diversity in the mature peptides has not been described based on post-translational processing.
Most AMPs are encoded in groups in the genome. For example, the human α-defensins HNP1 and 4 and HD5 and 6, and the β-defensins HNP1 and 2 are all mapped to a similar chromosomal location, 8p23, whereas human cathelicidin is located in chromosome 3p21.3 [24]. Their expression can be regulated both at the transcriptional and post-transcriptional levels, and the coordinated transcriptional regulation of AMP genes can lead to expression of multiple AMPs at a single site. As discussed earlier, because AMPs are expressed as pro-peptides followed by proteolytic processing to release the biologically active peptides, regulation of function is as dependent on the expression of appropriate proteases for processing as it is on the expression of the AMP gene product itself. These are accomplished differentially depending on the specific peptide and the tissue or cell type.
Recent observations have shown that the constitutive expression of AMPs is under strict developmental control and influenced by age and sexual maturation. For example, high constitutive expression of the cathelin-related antimicrobial peptide (CRAMP) was observed in neonatal mouse small intestinal epithelium, a site at which cathelicidin is not normally expressed in the adult. The presence of the peptide was limited to the first two weeks after birth and gradually disappeared with the onset of increased stem cell proliferation and epithelial cell migration along the crypt-villus axis. In this context, CRAMP conferred in neonates' protection from the enteric pathogen Listeria monocytogenes indicating that CRAMP expression might regulate bacterial Colonization and the establishment of gut homeostasis [25]. Anothar example of developmental control of AMPs is Bin1b, a rat epididymis-specific β-defensin with antimicrobial activity. This is maximally expressed during sexual maturation and can be upregulated by inflammation [26,27]. Bin1b binds to the sperm head in different regions of the epididymis with varied binding patterns and then induces progressive sperm motility in immotile immature sperm.
In the adult, the expression of many AMPs is greatly increased following injury or infection, or the AMP is stored at high concentrations as an inactive precursor in intracellular granules. Induction of AMP expression often involves signaling mediated by pattern-recognition receptors such as Toll-like receptors (TLRs) or responses to the release of pro-inflammatory cytokines. In keratinocytes, hBD1 mRNA is constitutively expressed, but the expression of hBD2-hBD4 are only induced by stimulation with TLR ligands, tumor necrosis factor (TNF), interleukin-1β, interferon (IFN)-γ or phorbol myristate acetate [28]. By contrast, cathelicidin expression in humans is less abundantly induced directly by TLRs or cytokines, but rather relies on histone acetylation, the action of the Vitamin D receptor (VDRE) and the modulation of Vitamin D action in specific tissues [29-34]. Inhibitors of histone deacetylase can be an important inducer of cathelicidin [29,30,34]. For instance, increased histone acetylation by the histone deacetylase inhibitor butyrate or trichostatin A enhances the expression of cathelicidin in gastrointestinal cells and lung epithelial cells. This mode of regulation combined with the action of VDRE is complex, with several groups showing that vitamin D regulates cathelicidin expression in multiple ways. Hormonally active vitamin D(3)-1,25-dihydroxyvitamin D(3) (1,25 D3) directly induces cathelicidin expression via a consensus VDRE in the CAMP promoter [31]. This stimulation occurs in different cell types from humans, including isolated human keratinocytes, monocytes, neutrophils, in addition to normal human bone marrow (BM) -derived macrophages, but is less active in control of CAMP expression in colonic epithelial cells, which require inhibition of histone deacetylase [31-33]. Rapid upregulation of cathelicidin that is necessary for immunologically relevant responses in a local environment is possible through the enzymatic action of cytochrome P450-containing hydroxylase, CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3 [35]. Notably, histone acetylation further amplifies 1,25 D3-regulated cathelicidin in skin keratinocytes [34]. Therefore, 1,25 D3 acts as a signaling molecule in immunity by enhancing the expression of cathelicidin. Furthermore, 1,25 D3 increases pattern recognition by increasing CD14 and TLR2 [35,36], thus enabling the host to recognize and respond to microbes and to protect against infection. To summarize a complex pathway, local stored pools of 25 D3 are first rapidly CDoverted to 1,25 D3 following the activation of CYP27B1. Active 1,25 D3 induces AMPs directly and indirectly through the capacity to both increase trarncription of cathelicidin and pattern recognition molecules. Increased TLR and CD14 expression can further increase defensin expression and other innate responses triggered by the TLR pathways. This process is further ccntrolled by histone acetylation that provides fine control in some epithelia, such as that in the colon.
The flipside of AMP regulation occurs when expression is turned off and is often associated with increased susceptibility to infection by pathogens otherwise sensitive to the AMPs. For example, psychological stress has been shown to have adverse effects on cathelicidin expression through the induction of endogenous glucocorticoids (GCs). Indeed, psychological stress and systemic or topical GC administration has been observed to downregulate epidermal expression of murine cathelicidin and β-defensin 3, leading to increased severity of group A Streptococcus pyogenes skin infection [37]. The capacity to decrease AMP expression is also a microbial virulence factor and is discussed later.
In vitro, most AMPs act against many different types of microbes including gram-negative and gram-positive bacteria, protozoa, fungi and some viruses. This is particularly true for the mammalian AMPs that have maximal effectiveness against specific groups of organisms relevant to the tissue where the AMP is expressed. For instance, β-defensins exhibit activity against Staphylococcus aureus and Pseudomonas aerugirosa [38], which are relevant to skin infection, whereas α-defensins expressed in the gut and in granulocytes are able to inactivate certain enveloped viruses (such as HIV) in addition to killing important bacterial pathogens such as Salmonella [39]. Some mammalian AMPs have a role in many types of infection. For example in vivo, cathelicidin knockout mice (Camp-/-) have been found to be susceptible to Group A Streptococcus [40,41], herpes simplex virus [42]. Escherichia coli [43] and vaccinia virus [44]. Similarly, knocking-out mouse β-defensin 1 results in delayed clearance of Haemophilus influenzae from the lung [45] and increased colonization by Staphylococcus species in the bladder [46]. Usually, the expression of these AMPs is increased at the onset of infection in response to stimuli. Therefore, the antimicrobial activity of AMPs is one of most important countermeasures that the mammalian host has developed to contend with microbial invagion and/or infection.
How do antimicrobial peptides exert their antimicrobial activity? Depending on the specific AMP studied, most show the capacity to directly disrupt the microbial cell membrane and thereby result in killing. Disruption of lipid bilayers by AMPs occurs through a variety of mechanisms (Box 1) [47]. Although the models shown in Box 1 used for the illustration of AMPs mechanisms are based on specific cationic antimicrobial peptides and are not applicable to all antimicrobial peptides, the structure-function models of the Shai-Matsuzaki-Huang (SMH) model [48] provide a reasonable explanation fOr the antimicrobial activity of most of these ccmpounds [49]. In general, cationic AMPs are attracted by electrostatic forces to the negative phospholipid headgroups on the membrane surface provided by capsular polysaccharides, which include lipopolysaccarides (LPS) in Gram-negative bacteria and teichoic acids (TA), lipoteichoic acids (LTA) and lysylphosphatidylglycerol in Gram-positive bacteria. Once AMPs gain access to the cytoplasmic membrane they interact with the lipid bilayer [47], followed by displacement of lipids, alteration of membrane structure and the creation of a physical hole causing cellular contents to leak out. Collectively, all these are well established models representative of the mechanisms for AMPs, and each model provides a different view of peptide activity, but none of these models is capable of adequately explaining their effectiveness in vivo.
There are several mechanisms by which different classes of AMPs could act on the microbial target membrane [47]. In the 'carpet model'. peptides accumulate on and orient parallel to the membrane surface. At a certain high concentration of peptides, AMPs disrupt the bilayer membrane in a detergent-like manner, resulting in the formation of micelles and leakage of cellular contents. In the 'toroidal-hole model', the polar heads of the peptides face the polar head groups of the lipids, inducing the lipids to form a continuous bend from the top to the bottom in the fashion of a toroid which is lined by both the peptides and the lipid head groups. Some cationic AMPs adopt an α-helical configuration, attach to, aggregate and insert into oriented bilayers that are hydrated with water vapor, leading to the formation of 'barrel-stave' holes in the membrane. For this model, the hydrophobic peptide regions align with the lipid core region of the bilayer and the hydrophilic peptide regions form the interior region of the pore. Notably, the differences between the toroidal model from the barrel-stave model is that peptides in the former are always associated with the lipid head groups even when they are perpendicularly inserted in the lipid bilayer. Finally, although most AMPs have been shown to disrupt cell membranes and induce microbial killing, a few AMPs kill bacteria without any detectable lysis. These AMPs can penetrate the cell membrane and bind to different targets, such as DNA, to inhibit bacterial growth.
Because the mechanism of action for most of the cationic AMPs depends on the interaction between the peptide and the microbial membrane to promote membrane destabilization, a question arises as to how these cationic AMPs can function in vivo but are not toxic to host cells. To address this point, several hypotheses have been proposed. One commonly cited explanation is that AMPs produced by mammals will not act on eukaryotic cells because of the cholesterol content of eukaryotic membranes. Unlike eukaryotic cell membranes, bacterial cell membranes are free of cholesterol. For example, the AMP sarcotoxin IA Was shown to be less disruptive to liposomes containing cholesterol than cholesterol-free liposomes [50]. Because cholesterol is known to cause condensation of phospholipid bilayers, it might prevent AMPs from penetrating into the cytoplasmic membrane of eukaryotic cells. Besides cholesterol, the asymmetric distribution of phospholipids in the cytoplasmic membrane of eukaryotic cells might also contribute to the insensitivity of eukaryotic cells to AMPs. The outer monolayer of the host membrane consists of neutral phospholipids without net charge, whereas most of the lipids with negatively charged headgrouPs, which should have affinity to cationic AMPs, are segregated into the inner leaf1et facing the cytoplasm [49,51]. Moreover, the cytotoxic effects ofAMPs to eukaryotic host cells in vivo are also substantially attenuated by serum components such as apolipoprotein A-I and B, or other lipoproteins, which have been shown to bind to LL37 [52]. This binding mechanism indicates an important role for serum lipoproteins in protecting host cells from damage caused by LL37. However, the direct antimicrobial activity ofmost AMPs in vitro and in vivo is often blunted by the physiological concentration of salt (e.g. 150 mM NaCl), increasing concentrations of divalent cations and serum proteins [53]. In spite of this, under physiological conditions bacteria also change their membrane structure and gene expression in vivo and remain susceptible to AMPs even in high salt concentrations [54]. Thus, the membrane activity of AMPs might be enough to kill microbes, but it is not cytotoxic to host cells with distinct lipid composition and in sequestered environments in which ionic composition of the extracellular fluid, and serum or extracellular matrix proteins, act to protect the host.
Despite the selective activity of many AMPs for microbial membranes, several studies have also shown that these AMPs at high concentrations will also destroy eukaryotic cells in vitro, such as T lymphocytes [55], leading to the hypothesis that they might also have a role in tumor surveillance [56-58]. In spite of this, the cytotoxic concentrations of AMPs are, in general, higher than the concentrations required for elimination of microbes, revealing a cell-selective killing mechanism [59].
As discussed previously, AMPs exhibit multiple functions related to their capacity to disrupt membranes. They have the ability to confer protection against a variety of pathogens and the potential to act as cytotoxic agents against certain type of cancers. However, the direct antimicrobial activity implicit to the term 'AMP' has strongly biased interpretation of the function of these peptides as 'natural antibiotics', This bias perhaps has delayed discovery of their other roles in immunity.
The minimal inhibitory concentrations (MIC) of AMPs against microbes in vitro are typically much higher than the physiological concentrations of peptides found in vivo under resting conditions. For instance, the concentration of LL37 or β-defensins is less than 2 μg ml−1 at mucosal sites, whereas the MIC of LL37 in vitro for E.coli is more than 32 μg ml−1. Thus, the question arises; how do AMPs exert their antimicrobial function in vivo if the effective concentration is too low? One explanation for this is that they act synergistically with other classes of AMPs to exert their desired effect. For example, LL37 can act with BD2, lysozyme and lactoferrin that are co-expressed to exert optimal killing. Another explanation is that in situations of inflammation, AMPs often accumulate at a high local concentration sufficiently above the MIC, thus acting alone as a classic AMP. However, increasing evidence indicates that some AMPs can confer protection by an indirect mechanism and not simply because they can kill microbes. They can function as potent immune regulators, altering host gene expression, acting as chemokines and/or inducing chemokine production, inhibiting LPS- or hyaluronan-induced pro-inflammatory cytokine production, promoting wound healing and modulating the responses of dendritic cells or T cells of the adaptive immune response (Figure 2). In this way, AMPs can be seen as acting as a bridge between innate and adaptive immunity. All of these functions favor resolution of infection and reverse potentially harmful inflammation, and complement the direct antimicrobial action. Here, we review some of the important functions of AMPs that are not related to their direct antimicrobial action.
Upon stimulation by microbial pathogens, local cells release AMPs at the site of the infection or injury [60]. In addition to inhibiting microbial growth, an additional function of some of these AMPs is that they act to directly recruit leukocytes or induce the expression of chemokines or cytokines including CXCL8 (IL-8), CCL2 (monocyte chemoattractant protein, MCP-1) and IFN-α, thereby indirectly promoting recruitment of effector cells such as neutrophils, monocytes, macrophages, immature dendritic cells and T cells. For example, human α-defensins (HNP-1 and HNP-2, but not HNP-3) have chemotactic activity mediating the recruitment of monocytes to inflammatory sites [61]. Subsequently, the defensins hBD3 and 4 have been reported to be chemotactic for monocytes and macrophages [62], and hBD2 has chemoattractant activity for mast cells [63]. Notably, both human α and β-defensins are chemotactic for memory T cells and immature dendritic cells. Human α-defensins selectively induce the migration of human nalve CD4+CD45RA+ and CD8+ cells, but not CD4+CD45RA+ memory T cells [64]. whereas β-defensins are chemotactic for immature dendritic cells and CD4+CD45RO+ memory T cells. This chemotactic effect of human defensins on both T cells and dendritic cells is pertussis toxin-sensitive and inhibited by antibodies to CCR6 [65]. Therefore, the chemotactic activity of human defensins has been suggested to promote cellular immune responses by recruiting dendritic and T cells to the site of microbial invasion through interactions with the G protein-coupled receptor, CCR6 (Figure 3). However, structure-function studies have shown conflicting data in this process. On one hand, intramolecular disulfide bonding in hBD3 was reported to be required for binding and the activation of receptors for chemotaxis, but dispensable for its antimicrobial function [66]. On the other hand, recent data have also shown that chemoattractant activity of hBD3 is not dependent on disulfide bonds but is cysteine V-dependent [67] and CCR6 might not be a functional receptor for β-defensins in memory lymphocytes and dendritic cells [68]. These contrasting results might be because of mismatched intramolecular disulfide bonding of hBD3 or different cell lines and experimental conditions used, but at any rate serve to reinforce the function of defensins in chemotaxis and emphasize the complexity of these systems.
Like the βdefensins, cathelicidins have also been shown to be chemotactic. LL37, is chemotactic for neutrophils, monocytes and T cells, but not for dendritic cells. In some experimental sygtems, the chemotactic activity of this peptide is mediated by the G protein-coupled formyl peptide receptor-like 1 (FPRL-l) [69]. The mouse cathelicidin., CRAMP, like LL37, was found to be chemotactic for human monocytes, neutrophils, macrophages and mouse peripheral blood leukocytes [70]. CRAMP also induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRLI or mouse formyl peptide recaptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1 or mouse formyl peptide receptor-2 by CRAMP [70] (Figure 3).
In addition to direct chemotactic activity, AMPs can exert indirect chemotactic activity by stimulating chemokine and cytokine secretion from a variety of cell types through receptor-dependent mechanism [64,71]. LL37 synergistically enhances the IL-1β-induced production of cytokines (IL-6, IL-10) and chemokines such as MCP-1 and MCP-3 [72] in human peripheral blood monocytes (PBMC). hBD3 can induce expression of the costimulatory molecules CD80, CD86 and CD40 by interaction with TLRs 1 and 2 on monocytes and dendritic cells resulting in MyD88 signaling, leading to IL-1 receptor-associated kinase-1 phosphorylation [73]. In human kerationcytes, hBD-2, -3, -4 and cathelicidin, but not hBD-1, act through G-protein coupled receptors and increase gene expression and protein production of IL-6, IL-10, IP-10, MCP-1, MIP-3α and RANTES [74]. Interestingly, although structurally very different, β-defensins and cathelicidin show somewhat similar effects On the activation of the ERK mitogen activated protein kinase (MAPK) pathway to induce inflammatory cytokine production in epithelial cells [74,75]. Also in normal human keratinocytes, hBD-2, -3, -4 and LL37, but not hBD-1, induced. phosphorylation of the MAPKs ERK1/2 and p38, but not JNK. The activation of ERK1/2 led to the activation of the downstream transcription factor, Elk-1, resulting in the secretion of Elk-1-controlled IL-18, which activated T, B and NK cells to produce high levels ofIFN-γ, and increased IgE production in CD4+ T cells [76,77). Notably, the combination of peptides resulted in a synergistic effect on IL-18 secretion. Thus, the ability of hBDs and cathelicidin to induce cytokine and chemokine secretion demonstrates that these AMPs influence the outcome of infection in at least three ways; they act as direct antimicrobials, they are chemotactic and they induce the release of multiple cytokines and chemokines that further refine and amplify the innate immune response.
AMPs have diverse and complementary effects on cellular inflammatory responses that are beyond the stimulation of chemotaxis. In particular, mammalian AMPs have a crucial role in regulation of TLR-dependent inflammatory responses. Our studies in a mouse model of allergic contact dermatitis showed cathelicidins inhibited TLR4- and CD44-mediated induction of cytokine release in dendritic cells and macrophages [78,79]. In dendritic cells, cathelicidins inhibited TLR4- but not TLR2-mediated induction of dendritic cell maturation and cytokine release, and this inhibition was associated with an alteration of cell membrane function and structure by cathelicidin (Figure 3) [78]. A similar effect of cathelicidin was also observed in macrophages for another TLR4 ligand, small hyaluronan (HA), which is relesed following injury and is in endogenous ligand for TLR4 and CD44. Cathelicidin inhibited HA induced MTP-2 release from mouse bone-marrow-derived macrophages by interfering with membrane binding events mediated by HA in a CD44-dependent manner, but independent of G-protein coupled receptor or epidermal growth factor (EGF) receptor signaling [79]. Both observations provide evidence that cathelicidin can act to inhibit TLR4. In contrast to models in which AMPs have been proposed to act directly on the receptor, these results indicate that the membrane active peptide influences the local membrane environment of the receptor and thus alters its activation state (Figure 3). Similar phenomena have been observed in several other systems in which LL37 has been found to selectively suppress TNF-α release from human monocytes and macrophages stimulated by LPS or LTA, suggesting AMPs might have a role in preventing sepsis [80,81]. Genomic approaches showed that LL37 significantly inhibited the expression of specific proinflammatory genes upregu1ated by NF-κB in the presence of LPS, including NF-κBI (p105/p50) and TNF-α-induced protein 2 (TNFA1P2). In contrast, LL37 did not inhibit LPS-induced genes that antagonize inflammation, such as TNF-α-induced protein 3 (TNFAIP3) and the NF-κB inhibitor, NF-κBIA or certain chemokine genes that are classically considered proinflammatory [80]. However, an alternative mechanism for how LL37 influences LPS responses also lies in the capacity of this, and other cationic peptides, to bind and neutralize LPS [82-85]. In this scenario, the AMP acts as a scavenger for the activating ligand, removing it before it can trigger inflammation.
Collectively, all these observations indicate that AMPs have an important role in regulating and balancing inflammatory responses to microbes. Although central questions regarding the mechanisms of the immunomodulatory effects of AMPs are yet to be clarified, the overall capacity of AMPs to influence multiple steps in host cell activation leads to the conclusion that some AMPs are not only involved in suppressing uncontrolled microbial growth but also that they modify inflammation. Recent clinical observations in human disease states support this conclusion, and are discussed later.
Another important role suggested for AMPs is to promote wound healing. This can occur by many mechanisms. As mentioned earlier, cathelicidins in addition to the defensins hBD2 and hBD3 rapidly and dramatically increase at the wound bed and edge [86,87]. hBD2 stimulates migration, proliferation and tube formation of endothelial cells in wounds, leading to accelerated wound closure [87]. Cathelicidin peptides have also been shown in several studies to affect the wound repair process. Our initial observation that AMPs were present in mammalian skin was made when studying wound repair in a porcine model. Here, cathelicidin peptide in pigs (PR-39) induced the heparan sulfate proteoglycans syndecan-1 and -4 in wounds, which in turn regulate cell proliferation and migration induced by heparin-binding growth factors [88]. The human cathelicidin LL37 has been shown to initiate metalloproteinase-dependent cleavage of membrane bound heparin-binding-EGF (HB-EGF). This soluble form of HB-EGF then binds to and initiates phosphorylation of EGFR and subsequently activates STAT1 and STAT3 (Figure 3). This trans-activation of the EGFR has been suggested to lead to enhanced keratinocyte migration required for re-epithelialization of the wound [89]. LL37 has also been proposed to increase wound closure by inducing the transcription factors Snail and Slug and activating the MAPK, phosphoinositide 3-kinase (P13K) and Akt signaling pathways [90]. Furthermore, LL37 directly activates FPRL-1 expresaed on endothelial cells to increase proliferation and formation of vessel-like structures, and induces functional angiogenesis important for cutaneous wound neovascularization [91]. LL37 acts directly on dermal fibroblasts, inhibiting transforming growth factor (TGF)-induced collagen expression, resulting in anti-fibrotic effects that might benefit normal wound repair [92]. Together these observations indicate that antimicrobial peptides can serve both protective and regenerative roles after injury that are unrelated to direct defense against infection.
As discussed previously, AMPs have been shown to participate in alerting, mobilizing and amplifying innate and adaptive immune respooses of the host, and will confer protection against microbial infections. Decreased expression of AMPs can increase susceptibility to infectious diseases. For example, the expression of AMPs such as cathelicidin, hBD2 and hBD3 is not increased in individuals with atopic dermatitis, despite the presence of skin inflammation. By contrast, patients with psoriasis have high expression of these AMPs in their inflammatory lesions (Figure 4). The differences in AMP expression between psoriasis and atopic dermatitis gain immunOlogical relevance in light of the proven antimicrobial activity of these AMPs against known superinfecting pathogens of atopic dermatitis, including Group A Streptococcus (GAS) [60]. S.aureus [93] and vaccinia virus [44]. In addition, the variability in defensin gene copy numbers can contribute to differences in individual resistance to infections [94,95]. For instance, individuals with lower gene copy number of hBD2 (≤3) have a significantly higher risk of developing Crohn's disease (CD) than individuals with ≥4 copies [94].
The presence of AMPs can be a double-edged sword because they might also exaggerate inflammatory responses and lead to disease. In this context, two models for the action of AMPs in promoting undesirable inflammation have been reported. In one, associated with the human facial skin disease rosacea, an excess of cathelicidin in the form of LL37 drives inflammation and abnormal blood vessel growth by mechanisms of cell activation [96]. In a second and novel mechanism, the abundance LL37 in psoriasis has been proposed as a mechanism that breaks tolerance to self-DNA and drives autoimmunity [97]. Here, LL37 binds extracellular self-DNA fragments and enables them to enter plasmacytoid dendritic cells (pDCs). This in turn triggers a robust TLR9-mediated IFN-α response. As a consequence, local maturation of myeloid dendritic cells are triggered by the type I IFNs to activate autoreactive Th1 or Th17 cells, resulting in the production of IFN-γ, IL-22 and IL-17 [98-99) (Figure 4). It is not clear how patients without psoriasis are able to control this self-perpetuating cycle. Thus, although recent clinical studies have shown the expression of AMPs is associated with the pathogenesis of several diseases (Table 1, much remains to be learned about how they function. Clearly, regulation of AMP expression and processing has an important role.
Thus far, we have discussed AMPs as multifunctional peptides, acting directly to kill microbes and also through multiple indirect strategies that influence cellular function in the host. What is the benefit of a multidimensional strategy to respond to microbial invasion? The answer might lie in the lessons learned through use of pharmaceutically produced antibiotics, and how ineffective they can become with the evolution of microbial resistance. By acting through a single approach, antibiotic function can be completely evaded by a single resistance system. It is apparent today that many pathogenic microbes have evolved mechanisms to avoid the antimicrobial action of AMPs, yet the expression of AMPs still protects the host (Box 2).
Microbes have evolved several mechanisms to degrade AMPs, yet the expression of AMPs continues to provide protection against infection. Why? Several theories can be advanced to explain their effectiveness in mammalian immunity. First, even though most pathogens have developed some countermeasures against AMPs, strict regulation of the expression of AMPs at the infection site leads to extremely high concentrations of AMPs in vivo that cannot be completely overcome by the microbe [115,116]. This combined with simultaneous production of a variety of different AMPs, impedes microbial colonization and growth [25]. In addition, the high costs for microbes to develop and express resistance genes preclude modifications that allow total resistance. In particular, it is difficult to evade the membrane-disturbing activity of AMPs while preserving the functional and structural integrity of the microbial cell wall and membrane. Furthermore, a strategy of destruction of the AMPs also poses several problems for the microbe. Most peptides are created from sequences of amino acids lacking unique epitopes that could serve as the recognition site of a specifically evolved protease that would be required for selective destruction of the AMP. Thus, it is difficult to design a degradation mechanism that would not also degrade necessary host proteins for attachment or microbial proteins necessary for survival. Finally, we have seen that all multicellular organisms attack microbes with multiple peptides of different structural classes, hence destruction of one peptide might not suffice to avoid the lethal assault of a diverse group of AMPs [49].
Several mechanisms have been described for microbial resistance to AMPs. Some microbes use advanced strategies to downregulate the expression of AMPs or produce proteases to degrade AMPs. For instance, Shigella spp. (S.dysenteriae, S.flexmeri, S.boydii. and S.sonnei), highly contagious bacteria that cause bacillary dysentery of varying severity, release plasmid DNA to turn off the expression of antimicrobial peptides in epithelial cells or monocytes [100], and this helps the bacteria evade AMP function, Another strategy for resistance is for the microbe to enzymatically inactivate the AMP before it can act. S. aureus secretes proteases V8 and aureolysin to inactivate cationic antimicrobial peptide LL37 [101]. S. epidermidis produces metalloprotease sepA to degrade the anionic antimicrobial peptide dermcidin [102]. Another effective strategy used by microbes to evade the effects of AMPs is to change the composition of their cell membrane. Both S. aureus and S. epidermidis control the three-component antimicrobial peptide-sensing system (aps) to regulate L-lysine and D-alanine synthesis. This modifies membrane lipid phophatidylglycerol with lysine and adds D-alanine to teichoic acid, resulting in the reduction of the net negative charge of the bacterial membrane so as to repel cationic AMPs [97,103-105]. Similarly, under certain circumstances, Gram-negative bacteria also control their two-component signal transduction systems, PhoP-PhoQ and PmrA-PmrB, to modify the structure of their LPS [106]. Salmonella enterica Typhimurium can form hepta-acylated lipid A (via the addition of palmitate by the bacterial protein PagP), add phosphate and phosphoethanolamine to the core polysaccharide and modify lipid A phosphate groups with ethanolamine and aminoarabinose. These alterations, and severa1 others not discussed here [107-109], decrease the susceptibility of microbes to AMPs and contribute to their pathogenicity.
Great progress has been achieved in the last decade with respect to the mechanisms of AMP action and their complex role in our immune system. Along with the capacity of AMPs to directly kill microbes, AMPs also boost specific innate immune responses and exert selective immunomodulatory effects on the host. Upon exposure to danger, AMPs create an overall balance by inhibiting microbial growth, attenuating exacerbated inflammatory responses and stimulating certain beneficial aspects of inflammation. This combined functional profile highlights the unique tempora1 niche that AMPs occupy in microbial defense (i.e. they are ideally suited for rapid but transient responses). These characteristics also make AMPs promising agents as beneficial therapeutics. Therefore, understanding the function of antimicrobial peptides as 'natural antibiotics' and their mechanism of action as 'immune regulators' has great promise for yielding new strategies in the control of human disease.
Work in the laboratory of R.L.G. is supported by NIH grants R01AR052728, RO1AI052453, contract HHSN266200400029 and a VA Merit Award. We apologize to those authors whose work could not be cited because of space constraints.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: To get from Corato a San Giovanni in Fiore by train you can catch one of the 9 trains that everyday travel between Bisceglie and Crotone, the closest station to the city. The first train to Crotone leaves from Bisceglie at 04:56 and the last train departs at 13:45. If you choose a local train the trip lasts on average 06:24 hours. On Wanderio you can compare all schedules, fares and railway companies to Crotone and book the best itinerary starting from Bisceglie.

subset: commoncrawl


text:
A 3 bedroom, 2 bathroom detached villa situated on a large level plot with extensive land suitable for a variety of uses and close to Jalon. The property is accessed via a long gravel driveway leading to a large parking area and car port. The villa overlooks a large tiled terrace area with 10 x 5 swimming pool and has a few steps to the main entrance. The accommodation comprises Naya, lounge/ dining room, fitted kitchen and an entrance hall leading to three bedrooms and a family bathroom. The main bedroom has an en suite bathroom and most of the bedrooms have built in wardrobes and air conditioning. A door leads from the kitchen to a rear courtyard with useful storage sheds and workshops. There is also a separate large work shop close to the access drive. The swimming pool terrace is very private and there are attractive gardens. The villa is in excellent condition and has a beautiful private position and is suitable for those looking for a large plot with much potential.
We can help you to get a mortgage on favourable terms. A residence permit can be obtained with the purchase of this property. Please ask our team for more information.


text:
We're women. We're famous for it. We hide our power, give it away, put ourselves down, hold ourselves back, or we don't acknowledge our own real power … to shine, to make the most of our lives, to accept and to feel that we truly deserve the best for ourselves in our own lives. I'm not talking about the outdated macho definition of power, as in to "power over" someone or "power through" something difficult….not that power. I'm also not talking about the confidence and bold courage we earn as we feel our fears and take steps anyway. I mean the power that comes from living a life in line with our values, knowing our goals, and the REAL reasons we want to accomplish them. It's the electric current fueled by our soul- that inner knowing of what we want, what we are here to do, and why – that gives you the feeling that you can do anything. But all too often on the path to owning our power we give it away or hide it. We've become REALLY good at finding creative ways to do it. Like:
1) We say"I think" rather than "I know".


text:
Yet Culver and Clark argue the public's supposed mistrust is often misplaced or overblown.
How hacking is changing the news →
Roy Peter Clark, senior scholar at the Poynter Institute, a Florida-based journalism think tank, said that just because a name becomes public doesn't mean news organizations have to race to publish it.
Release of names in prostitution case stirs debate →


text:
The Big Red are planning a number of trips this season.
Posts Tagged: Denison (M)
Denison's goal for the season is to finish 25th in the nation.
In this special edition, we recognize a player from each division of the tournament.
Denison is taking control of their destiny by expanding their schedule.
Is a return to the glory days in store for Denison?
Denison finished last season with a .500 record.
The Denison University squash club has named Stephen Neal as its head coach.
Kenyon recently hosted the Midwest Round Robin.
Denison is still in a rebuilding cycle, but the team is up for the challenge.
Notre Dame coach Geoff McCuen prepared a video on the College Squash Association Midwest Singles Tournament.


text:
Quick Links
RSS 2.0 Feeds
Lottery News
Event Calendar
Latest Forum Topics
Web Site Change Log
RSS info, more feeds
Oct 5, 2005, 11:57 am
4 comments
A former Texas lottery official, who claimed that then-Gov. George W. Bush's desire to cover up his National Guard record helped steer decisions about a key lottery contract, said he wants to talk to senators about Supreme Court nominee Harriet Miers' possible role in that effort.
"If I were to be subpoenaed to come to the thing, I would come," said Lawrence Littwin, who filed a lawsuit after he was fired as the lottery's executive director in 1997. "I would say the committee, I think, would be interested."
Littwin claimed in a federal lawsuit that lottery operator GTECH held sway over the Texas Lottery Commission because former GTECH lobbyist Ben Barnes was involved in helping get Bush into the Texas Air National Guard during the Vietnam War.
GTECH, which settled the suit in 1999 and paid Littwin $300,000 without admitting wrongdoing, said in court filings that Littwin's Guard-related claims were "preposterous."
A Bush appointee, Miers served as chairwoman of the Texas Lottery Commission when it was mired in controversy. President Bush cited that record Monday in announcing his nomination of his longtime friend and adviser to replace retiring Justice Sandra Day O'Connor.
Littwin was hired in 1997 to replace Nora Linares, who had been fired after it was revealed that her boyfriend was working as a consultant for GTECH, the Rhode Island-based firm that has run the Texas Lottery since it began in 1992.
Littwin was fired after five months on the job. He said he was let go because of the aggressive approach that he advocated in scrutinizing GTECH's performance, including investigating whether the company made illegal contributions to public officials.
Littwin sued the company, seeking $2.6 million and claiming that it had arranged his firing. The lawsuit cited GTECH lobbyist Ben Barnes' claims that as Texas House speaker he had helped get Bush into the Guard. Littwin's suit said GTECH had been given preferential treatment by the commission, which controlled the contract.
Under pressure from the lottery commission, GTECH had severed ties with Barnes before Littwin was hired as executive director. GTECH paid Barnes and partner Ricky Knox $23 million to end their consulting contract.
Barnes, who for years had remained silent about his role in getting Bush into the Guard, was forced to discuss it during a September 1999 deposition in the Littwin lawsuit. Barnes' lawyers issued a statement saying that when he was House speaker, Barnes called the head of the Texas Air National Guard to put in a good word for Bush at the request of Bush family friends.
In the statement, Barnes' lawyer said no one from the Bush family had contacted him about the Guard slot.
Littwin, citing confidentiality provisions in the settlement with GTECH, has declined to discuss Miers' role. A federal judge, ruling against GTECH, said Miers did not have to give a deposition in the case.
Before the settlement, Littwin had questioned Miers' performance at the commission, charging that she ignored state law requiring annual audits of GTECH.
Under terms of the settlement, Littwin would have to forfeit $50,000 if he violates the confidentiality agreement. He said Monday that his lawyer told him he could testify if subpoenaed by the Senate.
In the agreement, Littwin said that he had "no personal knowledge of any of the criminal activity alleged in support of his claims against GTECH."
When the case was settled, GTECH said a "business decision" caused it to opt for the settlement instead of taking the case to trial.
Related Links
Cox News
We'd love to see your comments here! Register for a FREE membership — it takes just a few moments — and you'll be able to post comments here and on any of our forums. If you're already a member, you can Log In to post a comment.
Of course there is no personal bitterness or agenda involved. Just a patriot!
|||::> *'`*:-.,_,.-:*''*:--->>> Chewie <<<---.*''*:-.,_,.-:*''* <:::|||
I only trust myself - and that's a questionable choice
Sounds like a to me!
Support our Troops
Maybe this will sink the Mier nomination so we can get a real conservative on the Supreme Court!
Corruption, GTECH and TLC go hand in hand and are synonyms of each other, also sinister is their synonym.
Devilish people and corporations, unholy alliances.
BibleOnline ParishesOnline ChristianRadioOnline MassOnline Mass
"Ten measures of beauty descended to the world, nine were taken by Jerusalem."
If you haven't already, check out the About Us page that explains the main features of our site.
Also, please take a few moments and review the rules for posting at Lottery Post.
Any time you see a gray-underlined link, you can click the link to see a popup menu of options.
Popular features:
We try to include instructions on each page. Check for help content on the page that's giving you trouble.
The next place to seek help is our dedicated Help forum that contains detailed assistance for frequently requested topics.
If something isn't working properly, our automated Browser Test page can quickly identify common problems.
Finally, the Contact Us page has an incredible wealth of self-help material that answers 95% of the questions we are asked.
We always welcome feedback and suggestions.
About Us • Site Map • Membership Features • Advertising Opportunities • Change Log • Rules • Linking to Lottery Post
Index of Feeds • Lottery News • Event Calendar • Latest Forum Posts • Web Site Change Log
Search the news, forums, blogs, and even your private messages at our Search page.
This page was generated in 0.1037 seconds.Copyright © 1999-2017 Speednet Group. All rights reserved.


text:
Luz Sanchez brings more than 25 years of experience in healthcare to his faculty role at Saint Mary's. He currently works as a consultant to the Health and Education Assessment Alliance in Arizona, where he assists independent health care providers with program evaluation, health care compliance, quality improvement, health improvement and chronic disease prevention strategies. He previously worked for Arizona's Maricopa County Department of Public Health, where he led teams in developing community-based research and evidence-based public health programs in oral health, nutrition, family health, HIV/AIDS planning and outreach, and other areas.
Luz also has extensive prior teaching experience in public health, including six years an assistant professor Midwestern University's College of Pharmacy. His early career experience includes a decade as a professor teaching medical and graduate students on public health topics at various universities in Bogota, Columbia.
Luz earned his Doctor of Nursing Practice from the University of Minnesota. He also holds an MSN from the University of Pennsylvania, a BSN from the University of Minnesota, and a B.A. in Biology from the College of St. Benedict.


text:
- University of Wisconsin-Eau Claire
- Music + Theatre Arts
- Academic Offerings
- Explore Opportunities
- Careers + Alumni
- News + Events
- About
- Contact Us
The project is expected to be completed in fall 2018, and the footbridge will be closed from late May to late August in both 2017 and 2018.More information
Go ahead — go after that dream! Pursuing excellence in music and theatre arts can translate into some pretty cool career paths. Picking a major or minor in any one of these areas opens up a world of great facilities, studios, lessons, classroom learning, performances and camaraderie with others in your specialty. Plus, you'll be surrounded by a variety of the other arts as well, which means creative inspiration is never too far away.
Whether you prefer the theory and history of the arts, or your greatest desire is to teach others how to hone their artistic craft, these categories of study contain majors and minors that will guide you into your profession. Whatever path you take, you'll get a broad and comprehensive education that not only dives into technical competence, but literature, sensitivity in style, and an insight into the role and effect of the arts in society.
View specific majors and minors under these categories.
You'll need to audition to be a part of the music program. Take a look at the program auditions page to learn the requirements and recommended preparation and repertoire.
Hear from a few of our theatre majors about why the education at UWEC is something special.
University of Wisconsin-Eau Claire
105 Garfield Avenue P.O. Box 4004 Eau Claire, WI 54702-4004 715-836-4636
Copyright © 2017 UW-Eau Claire and the Board of Regents of the University of Wisconsin SystemPrivacy Statement
Thanks for your interest in UW-Eau Claire. Let's get started!
Select a student category below for admission guidelines, important deadlines and instructions.
NOTE: To complete your application to UW-Eau Claire you will be taken off of this website to access the UW System online admission application.
If you want to apply for employment, please click here.


text:
|Reviews for Into Darkness
| Scorpio Serpent chapter 5 . 7/30/2004
Quite interesting! *Adds to favs*
| Mistress-of-chapters chapter 5 . 7/3/2004
| silverfire2005 chapter 1 . 6/20/2004
| jumpman72001 chapter 1 . 6/13/2004
| silly chapter 1 . 5/29/2004
its pretty good...strange i'll contineue to read it
| Gimpy chapter 5 . 5/20/2004
Hope this isn't the last one i want it too continue
| Gimpy chapter 1 . 5/20/2004
Can't wait for the lemon jk. i could care less as long as it is romantic in some ways but not too romantic but just enough u now.
anywas yes the chap was short but still good. didn't see any spelling mistakes at all.
From desk of G-Master
| Lalita chapter 5 . 4/23/2004
*screams* Suspense! Suspense! I can't take it! Update soon!
| Just me and myself chapter 5 . 4/23/2004
| PartnersInDenial chapter 5 . 4/18/2004
Great fic! Please Please UPDATE! It's awesome! Can't wait what's going to happen next!
| Ana chapter 5 . 4/17/2004
Another R/R fic. THis is my lucky day *giggles* Sorry I'm a little hyper. Um lets see I really likve this fic so far. I hope that u update soon. .~
| Raven A. Star chapter 5 . 4/15/2004
*blinks* Holy crap, that is a really good story...I'm off to read the InuYasha story right now.
| Killer Moth chapter 1 . 4/15/2004
I already gave my review on but still I really like this. I hope there is a happy ending here or you could make a sequel and continue the angst. Whichever you prefer. As you update I don't care. Later.
| Heterolifemate chapter 5 . 4/14/2004
Dang! This story is the hotness! What is Raven gonna do? Will Slade have his way with her before she can stop him? CANNOT WAIT FOR NEXT CHAPPIE!
| Quidditch-Gal chapter 4 . 3/29/2004


text:
Back to the hotel startpage
Please note!
Due to technical issues, you can't book rooms at Scandic Sluseholmen on our website. Please book by calling us on +46 8 517 517 20. We apologise for the inconvenience and we are working hard to fix the problem. We are looking forward welcoming you on Scandic Sluseholmen.


text:
Estate Betteridge Collection Ruby & Diamond Heart Brooch
Ruby and diamond heart brooch, set with 88 circular-cut rubies weighing approximately 7.42 total carats and 27 baguette-cut diamonds weighing approximately 2.29 total carats, mounted in platinum and 18k yellow gold. Dimensions: 1.5" x 1.2".
- Price: $16,500
- Ref #: EJPIN09369
- In Stock
- Available in: Greenwich
- Add to Shopping Bag
Please Sign In
Please sign in or create an account to use this feature.Sign In Cancel


text:
Your search did not match any record.
Search term Dymkowski Krzysztof inside index author did not match any record. Nearest terms in any collection are:
2 Dylan J Irvine 1 Dylan Mathys 1 Dylan Roth 1 Dylan Roth Mathys 1 Dylan Tatti 1 Dymkowski, K 1 Dymkowski, Krzysztof 1 Dyn, N 1 Dyn, Nira 1 Dyonis Rutz


text:
Six percent of world's total inventory of aluminum found hidden under tarps in remote Mexican desert
// September 8th, 2016 // No Comments » // News
A California businessman hired a private pilot to fly over a factory in a remote Mexican desert. There he found nearly one million metric tons of aluminum covered with tarp and bales of hay. The cache represents about 6% of the world's total aluminum inventory and is estimated to be worth over $2 billion dollars.


text:
With Instructables you can share what you make with the world, and tap into an ever-growing community of creative experts.
Tell us about yourself!
Weather Forecast Cloud
Power Supply From Old PSU
Hard Drive Sander
Turn Nearly Anything Into a Speaker
Circuit Board Design Class
Bookcase/Wine Rack/Guitar Holder
6 Button RGB Controller.
Cigar Box Stomp Box
'3D Printed' Reinforced Concrete Business Cards
Complete Circuit Board Lab & POV Business Card
DIY Arduino Watch Sport 2.0
Wrapping Up
Laser Cutting Class
3D Print a Programmable Musical Instrument
I2C Between Arduinos
Create Faceted Papercraft-objects
ITunes Arduino Controller (Mac)
Modular Puzzle
Lampduino - an 8x8 RGB Floor Lamp
Persistence of Vision Wand
Laser Cut Cryptex - Puzzle Box
3D Printed Electronic Puzzle Box
Table Saw Class
Artcam Class
Welding Class
Mold Making & Casting Class
Pasta Making Class
Wearable Electronics Class
Glue Class
Robots Class
Getting Started with Electronics
Bread Class
Raspberry Pi Class
Arduino Class
LEDs and Lighting Class
How to Build Circuits
Arduino Based Stranger Things Lights
Make your own 10x10 LED Matrix
3D Printed Geneva Drive
PCB making guide
How to make a printed circuit board (PCB) using the UV light LED method.
Laser Engraved Playing Cards & Wooden Box
Program Arduino Pro Mini Using Arduino Uno
Arduino Mini-Shields
$12 Secret Code Box
Wireless Electricity Transmission (Simple things required to be collected from scrap)
Simple 10w Bluetooth Portable Speaker
Make a Custom Bic Brander
Glowing LED Mushroom Log Lamp
Easy 3D topographical maps
Capacitive touch vibrating box
LinkIt One Capacitive Tutorial
Resistive Touch Screen on the DP32
Arduino Touch Capacitive MIDI Keyboard
Making a great LED triangle
Cowboy Duel Game
Conductive Rubber: Make Touch Sensitive Robot Skin
Touch-Sensitive Musical Painting
RFID Reader Detector and Tilt-Sensitive RFID Tag
Simple Vault Mechanism
How to build an Arduino Uno on a BreadBoard
10 Breadboard Projects For Beginners
DIY Arduino based PHOTO BOOTH
DIY Wedding Photobooth with Easy Disassembly (pics only)
Portable Photobooth (iPad+DSLR)
Wedding Photobooth (iPad+DSLR)
LEGO Nikon IR Remote
Hand Sewn Pixel Shirts
Using Maya to create a professional product render quickly for Shapeways
How To Make A Raspberry Pi SuperComputer!
Silc Circuits: High Performance Conductive Silicone
World's easiest silicone mold.
3D painting: Layered Resin and Acrylic Paint
Stickytape Sensors
Scent-imental Notification System
Interactive LED Beer Pong Table 2.0 (BPT X5)
Conversational Gloves
How to start selling on Etsy
action figure lamp
Wooden Swords and How I Learned to Love Leather-Work
Beginner Arduino
3 Things You Can Make From Pringles Cans
Woven Wire Rings
Make yourself anonymous on the web!
DIY AA Batteries!
How to Cut Perfect Circles with A Table Saw
Let your inbox help you discover our best projects, classes, and contests. Instructables will help you learn how to make anything!
© 2016 Autodesk, Inc.


text:
Lateral Movement – the moving of an attacker from one compromised host throughout your domain until they find what they are looking for – is something we see many just about all attackers doing during compromise. I've talked a lot about the attacker behavior and how to stop it – strong protective controls can serve as powerful detective controls.
The problem for most of our customers is that they lack the visibility/documentation to comfortably put Lateral Movement preventions in place without breaking everything. Even before we put the protective controls in place we can put some of the detective controls in place – which will give us greater insight into what is currently happening in the environment so we can get the protective controls in faster.
One very powerful detective control I mentioned in my Ignite Australia session on monitoring : Special Groups. Special Groups were introduced in Windows 2008/Vista as a way to track when particular accounts log onto a system. Which is pretty awesome from a security/Lateral Account Movement perspective. Especially since you get to define just what group is a special group! For Lateral Account Movement and credential hygiene, we want to know if administrative accounts or highly privileged (Tier 0 in MSFT speak) accounts logon to systems, especially systems in the workstation or member server tiers. When you enable Special Groups, you get a unique Event ID (4964) in the Security log that you can send to Windows Event Forwarding (which naturally you have in your environment now because of my blog post or the in depth Microsoft Virtual Academy session . 🙂 )
Getting Event ID 4964 to show up does require some configuration, which is best done via Group Policy Preferences.
First, we need to make sure the audit policies applied to our machines are in fact auditing for Special Logon. Configure your GPO so Computer Configuration\Windows Settings\Security Settings\Advanced Audit Configuration\Account Management\Audit Special Logon is set to log Success and Failure (we do want failure here, it's very useful once we get those protective controls in place.)
Next we need to determine who is a Special Group in the organization. For the purposes of tracking Bad Guy Behaviors as well as preparing for Lateral Account Movement lockdowns I'd really like at least the following things considered "Special Groups."
- Local Account : S-1-5-113 – this will help track the local accounts people may be using on your network so you can put the Lateral Movement blocks for Local Account in later without breaking things, as well as track Bad Guy Behavior.
- Domain Admins : S-1-5-21domain-512 – we need to know if there are any workflows that are currently in place in an organization that would lead to credential exposure before putting protective controls in place, and also alerts on Bad Guy Behavior.
- Enterprise Admins : S-1-5-21root domain-519 – same as Domain Admins, just Enterprise wide. Even if you only have one domain, monitor this.
- Special Group: I recommend creating a group specifically for things you want to track with Special Logon later, and adding it to the registry policy now. You could nest the other groups into it as well, but you might want those specifically defined. You can use this group to add Service Accounts to for instance, then if one is used across workstations, you know you have a problem.
I'd also like to see Built In Administrators : S-1-5-32-544 – we want to know when someone is logging on with admin privileges in general from a forensic stand point often. But this may be just far too noisy in some environments for the first pass. Give it a try if you can.
We're using Special Group not just to track Bad Guy Behavior, but potentially dangerous administrator behaviors that will be blocked by Lateral Movement mitigations. One of the workflows to worry about is Domain Admins using "Run as a Different User" on their normal workstations. Many people now have secondary admin accounts (which is great!) but use RunAs when they are doing admin activities – such as running Active Directory Users and Computers from the same computer they are logged on and surfing the web/checking email. This behavior may seem safe, but a RunAs is a full interactive logon that leaves credentials behind in memory.
(This is why we stress the need for a hardware separation between regular and administrative credentials so much in the Pass the Hash Whitepapers.)
We need to now assemble our SIDs. Even for Domain/Enterprise Admins you can get the SID of from attribute editor, under the objectSID attribute.
You can also use the power of https://support.microsoft.com/en-us/kb/243330 to know the Domain Admins SID is always S-1-5-21domain-512 and fill in the "domain" part based on another group in your domain, or you could logon on to a Domain Controller as a Domain Admin and run whoami /groups. If you pipe it out to a text file as shown, you can get the SID from there (useful trick for other stuff too.)
So now that we have our SIDs, either make a new GPO or edit one that applies where you need it (which, is pretty much everywhere. Except maybe Tier 0/Domain Controllers to reduce initial noise.) Navigate to Computer Configuration\Preferences\Windows Settings\Registry\ and create a New Registry Item. You'll want to fill it in like the screenshot below, with the key HKEY_LOCAL_MACHINE\System\CurrentControlSet\Control\Lsa\Audit\SpecialGroups set to a REG_SZ (string) value that contains your SIDs of interest separated by ; (semi colons.)
Public Service Announcement : whenever you're making a Group Policy Preference like a registry key, make sure to check the box on the Common tab for "Remove this item when it is no longer applied." This will make sure when you unlink the GPO the setting is cleaned up, otherwise you can sometimes get weird tattoos on your registry of settings you unlinked ages ago.
Once you're done, the GPO should look something like this.
So now, when Bob the Domain Admin does the dangerous behavior of Shift+ Right Click +Run as Different User on his Bob the Regular User workstation we get a very descriptive alert telling us what Bob did and what group it was that triggered the alert.
Now that we have an Event ID, we can make a Subscription for this in Windows Event Forwarding. I already covered the basics of WEF in another post, so if you haven't read that go back to get the basics.
Our 4964 subscription can be quite simple :
Now you have way more insight into what is happening on your network – both in preparation for Lateral Movement lockdowns and to keep track of Bad Guy Behaviors.
Another barrier to getting Lateral Account Movement lockdowns in place or removing Service Accounts from high privileged groups like Domain Admins is often the accounts are in those groups for legacy reasons, and have been for years. When nobody knows why the account is there, or what might break it can make even the very real and very easy to exploit threat of a Service Account in the Domain Admins difficult to mitigate for some customers. This is another case where Windows Event Forwarding can come to the rescue!
Creating a WEF subscription for "Where all did Service Account X log in" is very easy to do. I recommend making these subscriptions one or two accounts at a time, to make it easier to narrow down what it's doing, and I recommend making sure the subscription is targeted to both Domain Computers and Domain Controllers for visibility into any local/NTLM logons that might not hit the Domain Controllers.
I've covered the basics of Windows Event Forwarding in both an Ignite Session on Channel 9 and a previous blog post so we'll skip to just the Xpath filter you'll need to track down a specific account :
<Query Id="0″ Path="Security">
<Select Path="Security">
(*[EventData[Data[@Name="TargetUserName"] = "Interesting Account Name 1″]]) or
(*[EventData[Data[@Name="TargetUserName"] = "Interesting Account Name 2″]]) or
(*[EventData[Data[@Name="TargetUserName"] = "Interesting Account Name …."]])
(*[System[Provider[@Name='Microsoft-Windows-Security-Auditing'] and (EventID=4624 or EventID=4625 or EventID=4648)]])
Replace Interesting Account name with the user names you're interested in. EventID 4624 will get you all of the logon types, service/batch/interactive so should go a long way to tracking down what is going on, but 4625 will track failed logons for you – which help not just with potential security issues but with troubleshooting after you reduce privileges/credential exposure. Event ID 4648 is for explicit credential logon, which is used by bad guys doing lateral movement and sysadmins doing things like runas/mapping drives, so it's definitely an Event ID you'll want. 🙂
Getting these monitors enabled in your domain will not only go a long way to helping you lock down and monitor Lateral Account Movement, they'll also be a huge help if you ever need an Incident Response.
Good luck and happy logging!
-Jessica @jepayneMSFT


text:
Dr. Jill Clark-Hamilton MD
- Adolescent Medicine Summit, NJ
Patient Reviews
Reviews reflect patients' level of satisfaction with the care they received from this provider. Dr. Jill Clark-Hamilton xhas an overall rating of 3.7.
Dr. Jill Clark-Hamilton, MD is a Doctor primarily located in Summit, NJ, with another office in Morristown, NJ. She has 34 years of experience. Her specialties include Adolescent Medicine and Pediatrics. Dr. Clark-Hamilton is affiliated with Overlook Medical Center and Morristown Medical Center. She speaks English.
Find out the languages this doctor/ or staff may speak.
View all
Learn where this doctor attended medical school.
View all
Specialties & Expertise
Dr. Jill Clark-Hamilton has the following 11 areas of expertise, 2 specialties and 1 board certification.
For the best health care, select a doctor who specializes in your medical condition.
View all
See areas of expertise for treating specific conditions, procedures and surgeries for this doctor.
View all
See the board certifications this doctor has received. Board certifications provide confidence that this doctor meets the nationally recognized standards for education, knowledge and experience.
View all
Health Guides
Attention Deficit Hyperactivity Disorder (ADHD), including the different types and who gets them.
Locations & Directions
Atlantic Health Adolescent Medicine & Eating Disorders Program
Nearby Doctors
Dr. Jill Clark-Hamilton, MD is similar to these 3 Doctors near Summit, NJ.


text:
Makita 18 Volt Cordless Bare Tools
Makita 18 volt bare tools are great for adding a needed cordless power tool to your existing collection of 18v tools all the while saving money by not purchasing extra batteries or chargers.People who purchase a Makita 18v combo kit often don't want to go out and purchase another kit for only one battery or single too, they want to purchase that single tool by itself. Those single tools are called Bare Tools. Makita cordless 18-volt power tool kits usually come with two batteries and a charger so purchasing a tool on it's
Makita XDT01Z 18-Volt LXT Brushless Cordless 3-Speed Impact Driver, Bare Tool
List Price: $199.00Our Price: $94.95You save: $104.05Free Shipping
Makita XPT03Z 18V LXT Hybrid 4-Function Impact, Hammer, Driver-Drill, Bare Tool
List Price: $279.00Our Price: $208.99You save: $70.01
Makita XSH03Z 18-Volt LXT Brushless 6-1/2-inch Cordless Circular Saw, Bare Tool
List Price: $249.00Our Price: $199.00You save: $50.00
Makita XRM04B 18-Volt LXT Lithium-Ion Cordless Bluetooth Radio, Bare Tool
List Price: $199.00
Makita XPH03Z 18-Volt LXT Cordless 1/2-inch Hammer Driver-Drill, Bare Tool
List Price: $179.00Our Price: $119.00You save: $60.00
Makita XLT02Z 18-Volt LXT Cordless 3/8-inch Angle Impact Wrench, Bare Tool
List Price: $299.00Our Price: $239.00You save: $60.00
Makita XST01Z 18V LXT Cordless Oil-Impulse 3-Speed Impact Driver, Bare Tool
List Price: $299.00Our Price: $179.99You save: $119.01Free Shipping
Makita XPH01Z 18V LXT Cordless 1/2" Hammer Drill Bare Tool Replaces LXPH01
List Price: $129.00Our Price: $42.89You save: $86.11
Makita XSF03Z 18V Lithium-Ion Brushless Cordless Drywall Screwdriver, Bare Tool
List Price: $179.99Our Price: $149.00You save: $30.99
Makita RJ01ZW 12-Volt MAX Lithium-Ion Cordless VS Recipro Saw, Bare Tool Only
List Price: $119.00Our Price: $99.00You save: $20.00Free Shipping
Makita LC01Z 12-Volt Lithium-Ion Cordless Vacuum Cleaner, Bare Tool Only
List Price: $79.00Our Price: $45.99You save: $33.01Free Shipping
Makita DT01ZW 12-Volt Lithium-Ion 1/4-inch Cordless Impact Driver, Bare Tool
List Price: $119.00Our Price: $79.00You save: $40.00Free Shipping
Makita XRH03Z 7/8-inch Lithium-Ion Cordless Combination Rotary Hammer, Bare Tool
List Price: $310.99Our Price: $228.99You save: $82.00Free Shipping
Makita XWT04Z 18-Volt LXT Cordless 1/2-inch High Torque Impact Wrench, Bare Tool
List Price: $229.00Our Price: $179.00You save: $50.00
Makita XHU02Z 18-Volt LXT Lithium-Ion Cordless Hedge Trimmer, Bare Tool Only
List Price: $199.00Our Price: $138.89You save: $60.11
Makita XCU02Z 18-Volt X2 LXT Lithium-Ion 36V Cordless Chain Saw Bare Tool Only
List Price: $499.00Our Price: $199.00You save: $300.00


text:
Medium S05E06 Apocalypse Now PROPER HDTV XviD FQM
Anonymous and Free Direct Download 300GB+
Torrent info
Name:Medium S05E06 Apocalypse Now PROPER HDTV XviD FQM
Total Size: 349.89 MB
Magnet: Magnet Link
Seeds: 0
Leechers: 0
Stream: Watch Online @ Movie4u
Last Updated: 2015-08-22 17:44:37 (Update Now)
Torrent added: 2009-08-23 14:45:45
Torrent Files List
medium.s05e06.hdtv.xvid-fqm.avi (Size: 349.89 MB) (Files: 1)
medium.s05e06.hdtv.xvid-fqm.avi349.89 MB
trackerleech seeds
Torrent description
Proudly Presents
Release Type.....: HDTV
Series...........: Medium
Genre............: Drama
Episode..........: S05E06
Title............: Apocalypse...Now?
Production Code..:
Orig. Air Date...: 09 Mar 2009
Release Date.....: 10 Mar 2009
Source...........: 1080i HDTV
Video Format.....: XviD
Video Resolution.: 624x352
Video Bitrate....: 999 kbps
Video Framerate..: 23.976
Audio Format.....: MP3 VBR
Audio Bitrate....: 153 kbps average
Audio Sample Rate: 48 KHz
Length...........: 41:57.306
Size.............: 366,886,024
Packs............: 19*20MB = 349MB
Allison awakes to an earthquake after having a dark dream
about an apocalypse and is certain this means the end
is near. The dreams continue to consume Allison so much
that she becomes preoccupied with preparing for the end
of civilization by stocking up on emergency supplies and
seeking guidance from a man who seems to be an expert
in survival. Meanwhile, Ariel finds herself strangely
attracted to an upperclassman that seeks her help in
Geometry class and her company on a date.
Release Notes
!nuke Medium.S05E06.HDTV.XviD-HTV
Group Greets
CTU, ORENJi, 2HD, DHD
and all other groups that choose quality over speed
Contact Info
We are currently looking for:
* US/CA cappers with HDTV and/or DSR feed - min 60 kB/s
GROUP NEWS: No, we will not affil your brand new/DR/colo site,
so please don't ask. Thanks!
contact: Ask your friends. Email is dead.
related torrents
Torrent namehealth leech seeds Size
Medium s05e06 apocalypse now proper hdtv xvid fqm1 0 349.00 MB 0
Apocalypse Now Now - Charlie Human epub0 0 1.33 MB 0