Host Tissue Destruction Research Articles

Necrotizing periodontitis in the context of Takayasu s disease: Report of a case

Necrotizing periodontitis is characterized by gingival necrosis with bleeding and pain, and alveolar bone attachment loss. The disease is associated to spirochete and Prevotella intermedia infection, as well as to a series of bacterial infections, with an altered local host immune response and tissue destruction. We present the case of a 40-year-old woman with inflammatory vasculitis of the aorta and its main branches, known as Takayasu’s disease. In the course of treatment of the latter, she developed necrotizing periodontitis. In this case local factors such as abundant bacterial plaque accumulation secondary to a lack of tooth brushing, and the administration of methotrexate, may have played a role. The clinical condition improved notoriously after emergency treatment with local antiseptics and tartar removal

New Insights of an Old Defense System: Structure, Function, and Clinical Relevance of the Complement System

The complement system was discovered a century ago as a potent defense cascade of innate immunity. After its first description, continuous experimental and clinical research was performed, and three canonical pathways of activation were established. Upon activation by traumatic or surgical tissue damage, complement reveals beneficial functions of pathogen and danger defense by sensing and clearing injured cells. However, the latest research efforts have provided a more distinct insight into the complement system and its clinical subsequences. Complement has been shown to play a significant role in the pathogenesis of various inflammatory processes such as sepsis, multiorgan dysfunction, ischemia/reperfusion, cardiovascular diseases and many others. The three well-known activation pathways of the complement system have been challenged by newer findings that demonstrate direct production of central complement effectors (for example, C5a) by serine proteases of the coagulation cascade. In particular, thrombin is capable of producing C5a, which not only plays a decisive role on pathogens and infected/damaged tissues, but also acts systemically. In the case of uncontrolled complement activation, “friendly fire” is generated, resulting in the destruction of healthy host tissue. Therefore, the traditional research that focuses on a mainly positive-acting cascade has now shifted to the negative effects and how tissue damage originated by the activation of the complement can be contained. In a translational approach including structure-function relations of this ancient defense system, this review provides new insights of complement-mediated clinical relevant diseases and the development of complement modulation strategies and current research aspects.

Inactivation of staphylococcal virulence factors using a light-activated antimicrobial agent

BackgroundOne of the limitations of antibiotic therapy is that even after successful killing of the infecting microorganism, virulence factors may still be present and cause significant damage to the host. Light-activated antimicrobials show potential for the treatment of topical infections; therefore if these agents can also inactivate microbial virulence factors, this would represent an advantage over conventional antibiotic therapy. Staphylococcus aureus produces a wide range of virulence factors that contribute to its success as a pathogen by facilitating colonisation and destruction of host tissues.ResultsIn this study, the ability of the light-activated antimicrobial agent methylene blue in combination with laser light of 665 nm to inactivate staphylococcal virulence factors was assessed. A number of proteinaceous virulence factors were exposed to laser light in the presence of methylene blue and their biological activities re-determined. The activities of V8 protease, α-haemolysin and sphingomyelinase were shown to be inhibited in a dose-dependent manner by exposure to laser light in the presence of methylene blue.ConclusionThese results suggest that photodynamic therapy could reduce the harmful impact of preformed virulence factors on the host.

Saliva enables the antimicrobial activity of LL-37 in the presence of proteases of Porphyromonas gingivalis.

Proteolysis is a common microbial virulence mechanism that enables the destruction of host tissue and evasion from host defense mechanisms. Antimicrobial peptides, also known as host defense peptides, are effector molecules of the innate immunity that demonstrate a broad range of antimicrobial and immunoregulatory activities. Deficiency of the human LL-37 antimicrobial peptide was previously correlated with severe periodontal disease. Porphyromonas gingivalis, the major pathogen associated with periodontitis, is highly proteolytic. In this study, P. gingivalis was found capable of degrading LL-37 by utilizing its arginine-specific gingipains. Saliva collected from volunteers with a healthy periodontium protected LL-37 from proteolysis by P. gingivalis. Salivary protection of LL-37 was heat resistant and specific and enabled LL-37 to inhibit growth of Escherichia coli in the presence of the P. gingivalis proteases. Previously, saliva and other body fluids have been shown to inhibit the antimicrobial activity of LL-37. Here we demonstrate that at a cost of a small reduction in the bactericidal activity of LL-37, saliva enables the antibacterial activity of LL-37 despite the presence of proteases secreted by the main periodontopathogen.

Use of R-spondin1, An Intestinotrophic Mitogen, in the Treatment of Murine Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for a variety of hematologic malignancies. However, overall success of HCT is limited due to the immunologic recognition and destruction of host tissues resulting in graft-versus-host-disease (GvHD). In GvHD, donor derived immune cells recognize and destroy recipient epithelial tissues; predominantly liver, gastrointestinal (GI) mucosa, and skin. It has been reported that GI toxicity and crypt loss is a strong predictor of GvHD morbidity and mortality. Therefore, GI protective strategies may diminish GvHD severity. R-spondin1 (Rspo1) is an epithelial mitogen that stimulates mucosal growth in both small and large intestines. Therapeutic administration of Rspo1 has been shown to reduce the loss of body weight, diarrhea and rectal bleeding in experimental mouse colitis models. Histological evaluation revealed that Rspo1 improved mucosal integrity by stimulating crypt cell growth and mucosal regeneration in colitis-induced mice. Because the symptoms and pathogenesis of GI-GvHD are similar to colitis models, we chose to examine the impact of Rspo1 on mice with GvHD. Conventional CD4+/CD8+ T cells (Tconv) isolated from FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice. Mice received either 100μg Rspo1 protein in 100μL PBS or 100μL PBS alone IV from day 1 to day 6 post-transplantation. Surprisingly, mice receiving Rspo1 had significantly increased rates of mortality. Mice receiving Rspo1 experienced 100% mortality by day 6 post-HCT. In contrast, mice receiving only PBS demonstrated 0% mortality at 20 days post-HCT. Mortality was not a result of Rspo1 toxicity, as no morbidity or mortality was observed in the absence of allogenic Tconv in mice transplanted with either syngenic HCT or allogeneic TCD-BM alone. In order to assess whether the higher mortality experienced by Rspo1 treated mice was due to an explosive proliferation of Tconv, the transplant experiment was repeated with donor Tconv isolated from luciferase transgenic (luc+) H-2q mice. Groups receiving Rspo1 versus PBS did not show significantly different bioluminescence imaging signal intensity, suggesting that rapid proliferation of donor Tconv was not the basis for the observed differences in mortality. The overall mouse mortality in this experiment was consistent with the previous experiment. In an attempt to uncover the mechanism, we found no differences in the Perforin-Granzyme B or Fas/Fas ligand expression between control and treatment groups. Cytokine milieu (IFNγ, TNFα, IL-17, and IL-2) assessment in transplanted mice did not show significant differences in cytokine profiles of control versus Rspo1 treatment. Mouse autopsy demonstrated that transplanted mice receiving Rspo1 had marked crypt hyperplasia, apoptotic cells, and increased mitoses when compared PBS recipients. All other organ systems were considered normal in both groups by a veterinary pathologist. Blood cultures of both groups were negative for bacteria. CBC, liver enzymes, and electrolyte panels were normal in both groups. In order to assess lethal irradiation as a contributing factor to the increased mortality of Rspo1 treated mice, we repeated the above transplant experiment including non-irradiated immunodeficient Rag2−/−, γ-chain−/− H-2d recipients in addition to the lethally irradiated H-2 d recipients. Our mortality findings in the lethally irradiated group were consistent with the above mentioned transplant experiments showing rapid mortality rates in recipient mice receiving Rspo1. However, non-irradiated Rag2−/− γ-chain−/− mice that received Rspo1 had almost identical mortality rates to those receiving PBS; all mice surviving over 20 days post HCT. This finding underlines the critical importance of lethal irradiation as a contributing factor in Rspo1 treatment mortality. These findings suggest that the beneficial effects of Rspo1 in the colitis model may not be reproducible in GvHD. Furthermore, our studies indicate that the lethal effects of Rspo1 may be due to a myriad of factors including allogenic mismatch and lethal irradiation.

Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum.

BackgroundBacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria.MethodsA novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out.ResultsA total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition.ConclusionOverall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.

Characterization of a Serine Proteinase Mediating Encystation of Acanthamoeba

Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, gamma-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (k cat/Km of 8.2 microm(-1) min(-1)) not l-Leu-p-NA (k cat/Km of 1.1 microm(-1) min(-1)). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, gamma-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.

A calculated response: control of inflammation by the innate immune system

Inflammation is a rapid yet coordinated response that can lead to the destruction of microbes and host tissue. Triggers capable of inducing an inflammatory response include tissue damage and infection by pathogenic and nonpathogenic microbes. Each of these triggers represents a qualitatively distinct stress to the host immune system, yet our understanding of whether they are interpreted as such remains poor. Accumulating evidence suggests that recognition of these distinct stimuli converges on many of the same receptors of the innate immune system. Here I provide an overview of these innate receptors and suggest that the innate immune system can interpret the context of an inflammatory trigger and direct inflammation accordingly.

A New Autocatalytic Activation Mechanism for Cysteine Proteases Revealed by Prevotella intermedia Interpain A

Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

Inflammatory cytokines and dendritic cells in acute graft-versus-host disease after allogeneic stem cell transplantation

The wider use of allogeneic stem cell transplantation (allo-SCT) is still limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). The role of inflammatory cytokines such as TNF-alpha and IL-1, and their impact on immune effectors (mainly CD4+ and CD8+ T) cells has been extensively studied in the context of GVHD occurring after standard myeloablative allo-SCT. However, recent data suggested that GVHD pathophysiology is likely to involve more complex interactions where antigen-presenting cells, especially dendritic cells (DCs), may play a major role at time of initiation of acute GVHD. In addition, the wider use of reduced intensity and less toxic conditioning (RIC) regimens prior to allo-SCT would allow better visualization of the fine functions of immune effectors, thereby offering a window of opportunities to better decipher the intimate pathophysiological mechanisms underlying GVHD. The aim of this work is to review the available research evidence on the role of DCs as in vivo regulators of alloimmune reactivity, and their interactions with other immune effectors.

MICROSPORIDIAN PARASITES OF MOSQUITOES

The Microsporidia are a large diverse group of obligate, intracellular parasites. They are singlecelled eukaryotic microorganisms that have small genomes in the size range of prokaryotic cells and are now thought to be highly evolved fungi (Keeling and Fast 2002). They are exclusive parasites of other eukaryotes and possess a unique and highly specialized mechanism for invading host cells via infectious spores. Spores are the only stage that can exist outside a living host cell and they are the primary vehicles for horizontal transmission between hosts (peroral) and vertical transmission (transovarial) within the host. Spores are diagnostic, especially at the ultrastructural level (Fig. 1), and are distinguished by their small size (2–20 mm), thick walls (consisting of an endospore and exospore), and presence of a unique set of organelles that function as an extrusion apparatus. These include a tightly coiled polar filament (tube) that is attached to an anchoring disc at the anterior pole of the spore, a membranous polaroplast, and a posterior vacuole which collectively function to explosively inoculate the spore content or ‘‘sporoplasm’’ through the polar filament into a host cell to initiate infection. Microsporidia are ubiquitous in nature and exhibit a very broad host range within the animal kingdom. They have been described as parasites in all classes of vertebrates, including humans, and most invertebrates, but are particularly common to arthropods and fish. Microsporidia represent one of the largest and most diverse groups of parasitic organisms associated with mosquito populations in nature. They have been described from 14 different genera worldwide, and it is quite likely that all mosquitoes serve as hosts for one or more of these parasites. The group currently includes a heterogeneous assemblage of over 150 described species from 23 recognized genera, 15 of which are monotypic (only one species is known) (Table 1). Members of these genera exhibit extensive variation in their development and life cycles but generally fall into 2 broad categories. The first includes the monomorphic forms such as Anncaliia (formerly Nosema and then Brachiola) (Franzen et al. 2006) and Vavraia. These microsporidia have comparatively simple life cycles involving only one sporogonic sequence (Fig. 2). They develop asexually (merogony or schizogony) and produce a single spore type that is orally infectious to mosquito larvae. Vertical transmission may additionally occur via oral ingestion of spores on contaminated eggs (transovum), but there is no separate developmental sequence leading to ovarian infection in female hosts. These microsporidia have a very broad host range and are mildly pathogenic to mosquitoes, generally producing low larval mortality. Other genera from which only one developmental sequence has been described include Aedispora, Crepidulospora, Polydispyrenia, Senoma, Trichotosporea, and Tricornia. However, the complete life cycles and modes of transmission of members of these genera have yet to be resolved and they may be polymorphic. The second group includes the true polymorphic forms. These parasites are more common in nature and exhibit some of the most complex life cycles yet described for any microsporidia (Fig. 2). These include elements of asexual (schizogony, merogony, and sporogony) and sexual (karyogamy, gametogenesis, and plasmogamy) reproduction; the formation of multiple spore types in various stages of the host; host sexand tissue-dependent development; and separate developmental sequences leading to vertical (transovarial) and horizontal transmission. Many species, such as Edhazardia aedis, require two successive host generations to complete their life cycle, and at least 4 genera, Amblyospora, Duboscqia, Hyalinocysta and Parathelohania require obligatory development in an intermediate copepod host. These microsporidia generally exhibit higher levels of host specificity and although they do not cause any acute mortality or detectable morbidity in adult female hosts that go on to transmit infections transovarially, they have at least one phase of development that typically kills larval hosts during the last stadium. Mortality in larvae results from destruction of various host tissues and subsequent depletion of essential energy reserves necessary for pupation. The production of entomopathogenic toxins has never been documented. Copyright E 2007 by The American Mosquito Control Association, Inc.

Synergistic Up-Regulation of Epithelial Cell Matrix Metalloproteinase-9 Secretion in Tuberculosis

Mycobacterium tuberculosis (MTb) kills approximately 2 million people each year. MTb must drive host tissue destruction to disseminate and also to cause pulmonary cavitation. Matrix metalloproteinase-9 (MMP-9, gelatinase B) is implicated in this Tb-related immunopathology. We demonstrate that conditioned media from MTb-infected monocytes (CoMTb), but not direct infection with MTb, up-regulates MMP-9 gene expression and secretion from primary human bronchial epithelial cells (NHBE). MMP-9 secretion was increased 8.7-fold by CoMTb (P < 0.05) as assayed by gelatin zymography. A549 and 16HBE14o epithelial cell MMP secretion was significantly less than primary NHBE secretion. MMP-9 secretion was decreased 53.2% by inhibition of the p38 mitogen-activated protein kinase (MAPK) by SB203580 (P < 0.01) and 48.3% by inhibition of extracellular signal-regulated kinase with PD98059 (P < 0.05). MMP-9 secretion was prostaglandin independent. TNF-alpha was necessary but not sufficient for MMP-9 up-regulation by the monocyte-epithelial cell network. Soluble factors derived from Tb culture synergized with TNF-alpha to increase MMP-9 secretion by NHBE 6-fold (P < 0.01 compared with either stimulus alone). Together, these data reveal a new mechanism by which host- and pathogen-derived factors act together in MTb infection to drive MAPK-dependent MMP-9 secretion from respiratory epithelial cells.

Differential susceptibility of leukocyte subsets to cytotoxic T cell killing: Implications for HIV immunopathogenesis

Cytotoxic T lymphocytes (CTL) are crucial for the host defense against viral infection. In many cases, this anti-viral immune response contributes to host pathogenesis, through inflammation and tissue destruction. Few studies have explored the relative susceptibility of infected cells to CTL killing, and the range of cell types that may be effectively killed by CTLs in vivo, both of which are key to understanding both immune control of infection and immune-related pathogenesis. We developed and optimized a highly sensitive method to quantify the relative susceptibility of leukocyte subsets to CTL-mediated killing. Maximal sensitivity was achieved by uniquely measuring cell death occurring during the assay culture. We found that leukocyte subsets have a wide range of susceptibility to antigen-specific CTL-mediated lysis. Generally, T cells were more susceptible than B or NK cells, with CD4 T cells being more susceptible than CD8 T cells. In all lymphocyte lineages, susceptibility was greater for more differentiated subsets compared with their naïve counterparts; however, for dendritic cells, immature cells are more susceptible than mature cells. We focused on the susceptibility of T cell subsets, and found that naïve cells are far more resistant than memory cells, and in particular, CCR5+ or HLA-DR+ memory cells are highly susceptible to CTL-mediated killing. These results provide an explanation for the observation that certain subsets of CD4 T cells are ablated during chronic HIV infection, and indicate which subsets are most likely to contain the persistent viral reservoir.

The dual role of p55 tumour necrosis factor-alpha receptor in Actinobacillus actinomycetemcomitans-induced experimental periodontitis: host protection and tissue destruction.

Inflammatory immune reactions in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. Thus, we examined the mechanisms by which the proinflammatory cytokine tumour necrosis factor (TNF)-alpha modulates the outcome of Actinobacillus actinomycetemcomitans-induced periodontal disease in mice. Our results showed that TNF-alpha receptor p55-deficient mice [p55TNF-knock-out (KO)] developed a less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significantly less alveolar bone loss and inflammatory reaction. Real-time polymerase chain reaction (PCR) demonstrated that levels of chemokines (CXCL1, 3 and 10; CCL3 and 5) and their receptors (CXCR2 and 3, CCR5) were lower in p55TNF-KO mice, as were matrix metalloproteinase (MMP)-1, 2 and 9 and receptor activator of nuclear factor kB ligand (RANKL) mRNA levels. However, the absence of the TNF-alpha p55 results in an impairment of protective immunity to A. actinomycetemcomitans infection, characterized by increased bacterial load and higher levels of C-reactive protein during the course of disease. Such impaired host response may be the result of the reduced chemoattraction of lymphocytes, neutrophils and macrophages, and reduced inducible nitric oxide synthase expression (iNOS) and myeloperoxidase (MPO) production in periodontal tissues of p55 TNF-KO mice. Our results demonstrate the mechanisms involved determining periodontal disease severity by TNF-alpha receptor p55, and its role in providing immune protection to A. actinomycetemcomitans periodontal infection.

Inhibition of Nuclear Factor-κB Enhances the Capacity of Immature Dendritic Cells to Induce Antigen-Specific Tolerance in Experimental Autoimmune Encephalomyelitis

Autoimmune disorders develop as a result of deregulated immune responses that target self-antigens and cause destruction of healthy host tissues. Because dendritic cells (DCs) play an important role in the maintenance of peripheral immune tolerance, we are interested in identifying means of enhancing their therapeutic potential in autoimmune diseases. It is thought that during steady state, DCs are able to anergize potentially harmful T cells bearing T cell receptors that recognize self-peptide-major histocompatibility complexes. The tolerogenic capacity of DCs requires an immature phenotype, which is characterized by a reduced expression of costimulatory molecules. On the contrary, activation of antigen-specific naive T cells is enhanced by DC maturation, a process that involves expression of genes controlled by the transcription factor nuclear factor (NF)-kappaB. We evaluated the capacity of drugs that inhibit NF-kappaB to enhance the tolerogenic properties of immature DCs in the experimental autoimmune encephalomyelitis (EAE) model. We show that andrographolide, a bicyclic diterpenoid lactone, and rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, were able to interfere with NF-kappaB activation in murine DCs. As a result, treated DCs showed impaired maturation and a reduced capacity to activate antigen-specific T cells. Furthermore, NF-kappaB-blocked DCs had an enhanced tolerogenic capacity and were able to prevent EAE development in mice. The tolerogenic feature was specific for myelin antigens and involved the expansion of regulatory T cells. These data suggest that NF-kappaB blockade is a potential pharmacological approach that can be used to enhance the tolerogenic ability of immature DCs to prevent detrimental autoimmune responses.

A unique Rab GTPase, EhRabA, is involved in motility and polarization of Entamoeba histolytica cells

Entamoeba histolytica, an enteric protozoan parasite, infects 10% of the world's population leading to 50 million cases of invasive amoebiasis annually. Motility, which requires cell polarization, is important to the virulence of this pathogen, as it may result in destruction of host tissues and invasion. To gain insight into these processes in Entamoeba, a unique Rab GTPase, EhRabA, which localizes to the leading edge of cells, was characterized. Cell lines expressing a dominant negative version of EhRabA (EhRabA-DN) were generated. These mutant cells exhibited alterations in cell shape, polarity, and motility, supporting a role for this Rab in the regulation of these processes. Consistent with the notion that a dynamic actin cytoskeleton is crucial to cell polarity and motility, these mutants also exhibited alterations in the actin cytoskeleton. Cells expressing EhRabA-DN also displayed defects in several virulence functions including the ability to adhere to host cells, destroy host cells, and release cysteine proteases. Mislocalization of a prominent adhesion molecule, the galactose/N-acetylgalactosamine (Gal/GalNAc) adherence lectin and reorganization of ordered lipid domains, known as lipid rafts, also accompanied expression of EhRabA-DN. Interestingly, several endocytic processes were unaffected by expression of EhRabA-DN. Together, these data suggest that EhRabA may be involved in the regulation of polarization, motility and actin cytoskeletal dynamics: functions that participate in the pathogenicity of Entamoeba.

The pathogenicity of Entamoeba histolytica is related to the capacity of evading innate immunity

The host and parasite factors that influence susceptibility to Entamoeba histolytica infection and disease are not well understood. Entamoeba histolytica pathogenicity has been considered by focusing principally on parasite rather than host factors. Thus, research has concentrated on explaining the molecular differences between pathogenic E. histolytica and non-pathogenic E. dispar. However, the amoeba molecules considered most important for host tissue destruction (amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteinases) are present in both pathogenic E. histolytica and non-pathogenic E. dispar. In addition, the genetic differences in pathogenicity among E. histolytica isolates are unlikely to completely explain the different outcomes of infection. Considering that the principal difference between pathogenic and non-pathogenic amoebas lies in their surface coats, we propose that pathogenicity of the amoebas is related to the composition and properties of the surface coat components (or pathogen-associated molecular patterns, PAMPs), and the ability of innate immune response to recognize these components and eliminate the parasite. According to this hypothesis, a key feature that may distinguish pathogenic (E. histolytica) from non-pathogenic (E. dispar) strains is whether or not they can overcome innate immune defences. A corollary of this hypothesis is that in susceptible individuals the PAMPs are either not recognized or they are recognized by a set of Toll-like receptors (TLRs) that leads to an inflammatory response. In both cases, the result is tissue damage. On the contrary, in resistant individuals the innate/inflammatory response, induced through the activation of a different set of TLRs, eliminates the parasite.

Solution Structure of the Pore-forming Protein of Entamoeba histolytica

Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica. Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic diseases. Amoebapore A belongs to the superfamily of saposin-like proteins that are characterized by a conserved disulfide bond pattern and a fold consisting of five helices. Membrane-permeabilizing effector molecules of mammalian lymphocytes such as porcine NK-lysin and the human granulysin share these structural attributes. Several mechanisms have been proposed to explain how saposin-like proteins form membrane pores. All mechanisms indicate that the surface charge distribution of these proteins is the basis of their membrane binding capacity and pore formation. Here, we have solved the structure of amoebapore A by NMR spectroscopy. We demonstrate that the specific activation step of amoebapore A depends on a pH-dependent dimerization event and is modulated by a surface-exposed histidine residue. Thus, histidine-mediated dimerization is the molecular switch for pore formation and reveals a novel activation mechanism of pore-forming toxins.