A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation

Introduction

Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.

Methods

Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.

Results

RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.

Conclusions

RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.     

High-performance liquid chromatography with spectrophotometric and electrochemical detection of a series of manganese(III) cationic porphyrins

Recent studies have revealed potent pharmacological activities of manganese-containing cationic porphyrins. An analytical method employing high-performance liquid chromatography with spectrophotometric and electrochemical detection (HPLC-UV/EC) suitable for in vivo applications is described for a series of manganese(III) cationic porphyrins with good separation and resolution. In particular, this method resolved the four atropisomers of manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+ or AEOL-10113), verified by mass spectrometry. Electrochemical and spectrophotometric methods of detection were compared using manganese(III) meso-tetrakis(1,3-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP5+ or AEOL-10150), the lead catalytic antioxidant of this series. Both methods of detection were quantitative, but electrochemical detection, although less specific for in vivo applications, appears to be considerably more sensitive than spectrophotometric detection.     

Efficient Imputation of Missing Markers in Low-Coverage Genotyping-by-Sequencing Data from Multiparental Crosses

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.     

A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.     

Conserved and species-specific alternative splicing in mammalian genomes

Background  

Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.