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

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

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

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

Single low dose primaquine to reduce gametocyte carriage and Plasmodium falciparum transmission after artemether-lumefantrine in children with asymptomatic infection: a randomised, double-blind, placebo-controlled trial

Background: A single low dose (0.25 mg/kg) of primaquine is recommended as a gametocytocide in combination with artemisinin-based combination therapies for Plasmodium falciparum but its effect on post-treatment gametocyte circulation and infectiousness to mosquitoes has not been quantified. Methods: In this randomised, double-blind, placebo-controlled trial, 360 asymptomatic parasitaemic children aged 2-15 years were enrolled and assigned to receive: artemether-lumefantrine (AL) and a dose of placebo; AL and a 0.25 mg/kg primaquine dose; or AL and a 0.40 mg/kg primaquine dose. On days 0, 2, 3, 7, 10 and 14, gametocytes were detected and quantified by microscopy, Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA), and quantitative reverse-transcriptase PCR (qRT-PCR). For a subset of participants, pre- and post-treatment infectiousness was assessed by mosquito feeding assays on days -1, 3, 7, 10 and 14. Results: Both primaquine arms had lower gametocyte prevalences after day 3 compared to the placebo arm, regardless of gametocyte detection method. The mean (95 % confidence interval) number of days to gametocyte clearance in children with patent gametocytes on day 0 (N = 150) was 19.7 (14.6 – 24.8), 7.7 (6.3 – 9.1) and 8.2 (6.7 – 9.6) for the AL-placebo, the 0.25 mg/kg primaquine dose and the 0.40 mg/kg primaquine dose arms, respectively. While 38.0 % (30/79) of selected gametocytaemic individuals were infectious before treatment, only 1/251 participant, from the AL-placebo group, infected mosquitoes after treatment. Conclusions: We observed similar gametocyte clearance rates after 0.25 and 0.40 mg/kg primaquine doses. Infectivity to mosquitoes after AL was very low and absent in primaquine arms

Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes.

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was approximately 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was approximately 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only approximately 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing