The Antimicrobial Peptide, LL-37, Inhibits in vitro Osteoclastogenesis

Uncoupled bone resorption leads to net alveolar bone loss in periodontitis. The deficiency of LL-37, the only human antimicrobial peptide in the cathelicidin family, in patients with aggressive periodontitis suggests that LL-37 may play a pivotal role in the inhibition of alveolar bone destruction in periodontitis. We aimed to investigate a novel function of LL-37 in osteoimmunity by blocking osteoclastogenesis in vitro. Human osteoclast progenitor cells were isolated from a buffy coat of blood samples. The cells were cultured in the presence of various concentrations of LL-37 during an in vitro induction of osteoclastogenesis. Non-toxic doses of LL-37 could block multinuclear formation of the progenitor cells and significantly diminish the number of tartrate-resistant acid-phosphatase-positive cells and the formation of resorption pits (p < 0.05), whereas these concentrations induced cellular proliferation, as demonstrated by increased expression of proliferating cell nuclear antigen. Expression of several osteoclast genes was down-regulated by LL-37 treatment. It was demonstrated that nuclear translocation of nuclear- factor-activated T-cells 2 (NFAT2) was blocked by LL-37 treatment, consistent with a significant reduction in the calcineurin activity (p < 0.005). Collectively, our findings demonstrate that LL-37 inhibits the in vitro osteoclastogenesis by inhibiting the calcineurin activity, thus preventing nuclear translocation of NFAT2. Abbreviations: CALCR, calcitonin receptor; ClC-7, chloride-proton exchanger; CTSK, cathepsin K; DAPI, 4′,6-diamidino-2-phenylindole; EGTA, ethylene glycol tetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF/CSF1, macrophage-colony- stimulating factor; MMP-9, matrix metalloproteinase-9; MTT, [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; NFAT2, nuclear factor of activated T-cells 2; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; RT-PCR, reverse-transcription polymerase chain- reaction; TBS, Tris-buffered saline; TCIRG1, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3; TRAcP, tartrate-resistant acid phosphatase

Multifaceted effects of synthetic TLR2 ligand and Legionella pneumophilia on Treg-mediated suppression of T cell activation

Contains fulltext : 97133.pdf (publisher's version ) (Open Access)BACKGROUND: Regulatory T cells (Treg) play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2) agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff) cells and dendritic cells (DCs) individually and in co-cultures with Tregs. RESULTS: TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs) contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. CONCLUSIONS: These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion

NOX Enzymes and Pulmonary Disease

Abstract The primary function of the lung is to facilitate the transfer of molecular oxygen (O2; dioxygen) from the atmosphere to the systemic circulation. In addition to its essential role in aerobic metabolism, O2 serves as the physiologic terminal acceptor of electron transfer catalyzed by the NADPH oxidase (NOX) family of oxidoreductases. The evolution of the lungs and circulatory systems in vertebrates was accompanied by increasing diversification of NOX family enzymes, suggesting adaptive roles for NOX-derived reactive oxygen species in normal physiology. However, this adaptation may paradoxically carry detrimental consequences in the setting of overwhelming/persistent environmental stressors, both infectious and noninfectious, and during the process of aging. Here, we review current understanding of NOX enzymes in normal lung physiology and their pathophysiologic roles in a number of pulmonary diseases, including lung infections, acute lung injury, pulmonary arterial hypertension, obstructive lung disorders, fibrotic lung disease, and lung cancer. Antioxid. Redox Signal. 11, 2505-2516.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78108/1/ars.2009.2599.pd

The Influence of Oral Bacteria on Epithelial Cell Migration In Vitro

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a > 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission

Enzyme-Catalyzed Macrocyclization of Long Unprotected Peptides

A glutathione S-transferase (GST) catalyzed macrocyclization reaction for peptides up to 40 amino acids in length is reported. GST catalyzes the selective SNAr reaction between an N-terminal glutathione (GSH, γ-Glu-Cys-Gly) tag and a C-terminal perfluoroaryl-modified cysteine on the same polypeptide chain. Cyclic peptides ranging from 9 to 24 residues were quantitatively produced within 2 h in aqueous pH = 8 buffer at room temperature. The reaction was highly selective for cyclization at the GSH tag, enabling the combination of GST-catalyzed ligation with native chemical ligation to generate a large 40-residue peptide macrocycle.Massachusetts Institute of Technology (MIT startup funds)National Institutes of Health (U.S.) (grant GM101762)Damon Runyon Cancer Research Foundation (Award)Sontag Foundation (Distinguished Scientist Award)Amgen Inc. (Summer Graduate Research Fellowship

Functional antibodies against Plasmodium falciparum sporozoites are associated with a longer time to qPCR-detected infection among schoolchildren in Burkina Faso.

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo; epidemiological studies powered and designed to address this important question should become a research priority