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  • Publication
    Lymphoid Susceptibility to the Aggregatibacter Actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is Dependent Upon Baseline Levels of the Signaling Lipid, Phosphatidylinositol-3,4,5-Triphosphate
    (2016-02-01) Shenker, Bruce J.; Walker, Lisa P.; Zekavat, Ali; Boesze-Battaglia, Kathleen
    The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1 (LFA-1), a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of 334LEEYSKR340, in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC336 motif of LtxA (CRAC336WT). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of RTX-producing organisms.
  • Publication
    Inhibition of Mast Cell Degranulation by a Chimeric Toxin Containing a Novel Phosphatidylinositol-3,4,5-Triphosphate Phosphatase
    (2011-11-01) Shenker, Bruce J.; Boesze-Battaglia, Kathleen; Zekavat, Ali; Walker, Lisa; Besack, Dave; Ali, Hydar
    It is well established that many cell functions are controlled by the PI-3K signaling pathway and the signaling lipid, phosphatidylinositol-3,4,5-triphosphate (PIP3). This is particularly true for mast cells which play a key regulatory role in allergy and inflammation through activation via high-affinity IgE receptors (FcɛRI) leading to activation of signaling cascades and subsequent release of histamine and other pro-inflammatory mediators. A pivotal component of this cascade is the activation of PI-3K and a rise in intracellular levels of PIP3. In this study, we developed a novel chimeric toxin that selectively binds to mast cells and which functions as a PIP3 phosphatase. Specifically, the chimeric toxin was composed of the FcɛRI binding region of IgE and the active subunit of the cytolethal distending toxin, CdtB, which we have recently demonstrated to function as a PIP3 phosphatase. We demonstrate that the chimeric toxin retains PIP3 phosphatase activity and selectively binds to mast cells. Moreover, the toxin is capable of altering intracellular levels of PIP3, block antigen-induced Akt phosphorylation and degranulation. These studies provide further evidence for the pivotal role of PIP3 in regulating mast cell activation and for this signaling lipid serving as a novel target for therapeutic intervention of mast cell-mediated disease. Moreover, these studies provide evidence for the utilization of CdtB as a novel therapeutic agent for targeting the PI-3K signaling pathway.
  • Publication
    In Vitro Replication of Cyanobacterial Plasmids from Synechocystis PCC 6803
    (1994-09-01) Yang, Xiaoyu; Daniell, Henry; McFadden, Bruce
    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.
  • Publication
    Subgingival Microbiota Dysbiosis in Systemic Lupus Erythematosus: Association with Periodontal Status
    (2017-03-20) Dias Corrêa, Jôice; Cerqueira Calderaro, Débora; Aparecida Ferreira, Gilda; Maria Souza Mendonça, Santuza; Fernandes, Gabriel R; Xiao, E; Lúcio Teixeira, Antônio; Leys, Eugene J; Graves, Dana T; Aparecida Silva, Tarcília
    Background Periodontitis results from the interaction between a subgingival biofilm and host immune response. Changes in biofilm composition are thought to disrupt homeostasis between the host and subgingival bacteria resulting in periodontal damage. Chronic systemic inflammatory disorders have been shown to affect the subgingival microbiota and clinical periodontal status. However, this relationship has not been examined in subjects with systemic lupus erythematosus (SLE). The objective of our study was to investigate the influence of SLE on the subgingival microbiota and its connection with periodontal disease and SLE activity. Methods We evaluated 52 patients with SLE compared to 52 subjects without SLE (control group). Subjects were classified as without periodontitis and with periodontitis. Oral microbiota composition was assessed by amplifying the V4 region of 16S rRNA gene from subgingival dental plaque DNA extracts. These amplicons were examined by Illumina MiSeq sequencing. Results SLE patients exhibited higher prevalence of periodontitis which occurred at a younger age compared to subjects of the control group. More severe forms of periodontitis were found in SLE subjects that had higher bacterial loads and decreased microbial diversity. Bacterial species frequently detected in periodontal disease were observed in higher proportions in SLE patients, even in periodontal healthy sites such as Fretibacterium, Prevotella nigrescens, and Selenomonas. Changes in the oral microbiota were linked to increased local inflammation, as demonstrated by higher concentrations of IL-6, IL-17, and IL-33 in SLE patients with periodontitis. Conclusions SLE is associated with differences in the composition of the microbiota, independently of periodontal status. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0252-z) contains supplementary material, which is available to authorized users.
  • Publication
    Aggregatibacter Actinomycetemcomitans Leukotoxin Cytotoxicity Occurs Through Bilayer Destabilization
    (2012-06-01) Brown, Angela C.; Boesze-Battaglia, Kathleen; Du, Yurong; Stefano, Frank P.; Kieba, Irene R.; Epand, Richard M.; Lally, Edward T.
    The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.
  • Publication
    Expression and Functional Evaluation of Biopharmaceuticals Made in Plant Chloroplasts
    (2017-06-01) Zhang, Bei; Shanmugaraj, Balamurugan; Daniell, Henry
    After approval of the first plant-made biopharmaceutical by FDA for human use, many protein drugs are now in clinical development. Within the last decade, significant advances have been made in expression of heterologous complex/large proteins in chloroplasts of edible plants using codon optimized human or viral genes. Furthermore, advances in quantification enable determination of in-planta drug dosage. Oral delivery of plastid-made biopharmaceuticals (PMB) is affordable because it eliminates prohibitively expensive fermentation, purification processes addressing major challenges of short shelf-life after cold storage. In this review, we discuss recent advances in PMBs against metabolic, inherited or infectious diseases, and also mechanisms of post-translational modifications (PTM) in order to increase our understanding of functional PMBs.
  • Publication
    Technetium-99 Conjugated with Methylene Diphosphonate Ameliorates Ovariectomy–induced Osteoporotic Phenotype without Causing Osteonecrosis in the Jaw
    (2012-12-01) Zhao, Yinghua; Wang, Lei; Liu, Yi; Akiyama, Kentaro; Chen, Chider; Atsuta, Ikiru; Zhou, Tao; Duan, Xiaohong; Jin, Yan; Shi, Songtao
    Technetium-99 conjugated with methylene diphosphonate (99Tc-MDP) is a novel bisphosphonate derivative without radioactivity and has been successfully used to treat arthritis in China for years. Since bisphosphonate therapy has the potential to induce bisphosphonate-associated osteonecrosis of the jaw (BRONJ), we examine whether 99Tc-MDP represents a new class of bisphosphonate for anti-resorptive therapy to ameliorate estrogen deficiency–induced bone resorption with less risk of causing BRONJ. We showed that 99Tc-MDP-treated ovariectomized (OVX) mice had significantly improved bone mineral density (BMD) and trabecular bone volume in comparison to the untreated OVX group by inhibiting osteoclasts and enhancing osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). To determine the potential of inducing BRONJ, 99Tc-MDP/dexamethasone (Dex) or zoledronate/Dex were administered into C57BL/6J mice via the tail vein, followed by extraction of maxillary first molars. Interestingly, 99Tc-MDP treatment showed less risk to induce osteonecrosis in the maxillary bones compared to zoledronate treatment group, partially because 99Tc-MDP neither suppressed adaptive regulatory T cells (Tregs) nor activated the inflammatory T-helper-producing interleukin 17 cells (Th17). Taken together, our findings demonstrate that 99Tc-MDP therapy may be a promising approach in the treatment of osteoporosis with less risk of causing BRONJ.
  • Publication
    Differentiation and Regenerative Capacities of Human Odontoma-Derived Mesenchymal Cells
    (2010-06-28) Song, Jin-Seon; Stefanik, Derek; Damek-Poprawa, Monika; Alawi, Faizan; Akintoye, Sunday O
    Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and βIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin-pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.
  • Publication
    Oral Delivery of Human Biopharmaceuticals, Autoantigens and Vaccine Antigens Bioencapsulated in Plant Cells
    (2013-06-15) Kwon, Kwang-Chul; Verma, Dheeraj; Singh, Nameirakpam D.; Herzog, Roland; Daniell, Henry
    Among 12 billion injections administered annually, unsafe delivery leads to >20 million infections and >100 million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections.