CXCR3 and its ligands in a murine model of obliterative bronchiolitis: regulation and function

Lung transplantation remains the only effective therapy for patients with end-stage lung disease, but survival is limited by the development of obliterative bronchiolitis (OB). The chemokine receptor CXCR3 and two of its ligands, CXCL9 and CXCL10, have been identified as important mediators of OB. However, the relative contribution of CXCL9 and CXCL10 to the development of OB and the mechanism of regulation of these chemokines has not been well defined. In this study, we demonstrate that CXCL9 and CXCL10 are up-regulated in unique patterns following tracheal transplantation in mice. In these experiments, CXCL9 expression peaked 7 days posttransplant, while CXCL10 expression peaked at 1 day and then again 7 days posttransplant. Expression of CXCL10 was also up-regulated in a novel murine model of lung ischemia, and in bronchoalveolar lavage fluid taken from human lungs 24 h after lung transplantation. In further analysis, we found that 3 h after transplantation CXCL10 is donor tissue derived and not dependent on IFN-gamma or STAT1, while 24 h after transplantation CXCL10 is from recipient tissue and regulated by IFN-gamma and STAT1. Expression of both CXCL9 and CXCL10 7 days posttransplant is regulated by IFN-gamma and STAT1. Finally, we demonstrate that deletion of CXCR3 in recipients reduces airway obliteration. However, deletion of either CXCL9 or CXCL10 did not affect airway obliteration. These data show that in this murine model of obliterative bronchiolitis, these chemokines are differentially regulated following transplantation, and that deletion of either chemokine alone does not affect the development of airway obliteration.     

Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity

The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.     

Sequence variants in the melatonin-related receptor gene (GPR50) associate with circulating triglyceride and HDL levels

The gene encoding the melatonin-related receptor (GPR50) is highly expressed within hypothalamic nuclei concerned with the control of body weight and metabolism. We screened GPR50 for mutations in an obese cohort and identified an insertion of four amino acid residues (TTGH) at position 501, two common coding polymorphisms (T528A and V602I), and one noncoding polymorphism (C-16X2GPR50T). Single-nucleotide polymorphisms were then typed in 500 English Caucasian subjects, and associations were sought to intermediate obesity phenotypes. Although no association was seen with body mass index, carriers of two copies of the mutant allele at C-16X2GPR50T, Ins501Del, and A1582G had significantly higher fasting circulating triglyceride levels (P < 0.05). In a separate set of 585 subjects, the associations were replicated, with statistically significant effects of similar magnitude and direction. The association of C-16X2GPR50T with fasting triglycerides was highly significant (P < 0.001). In addition, a significant association between C-16X2GPR50T and circulating HDL levels was observed in the combined population, with C-16X2GPR50T carriers having significantly lower circulating HDL-cholesterol levels (1.39 mM) than wild-type subjects (1.47 mM) (P < 0.01). These findings suggest a previously unexpected role for this orphan receptor in the regulation of lipid metabolism that warrants further investigation.     

A novel role for lbx1 in Xenopus hypaxial myogenesis

We have examined lbx1 expression in early X. laevis tadpoles. In contrast to amniotes, lbx1 is expressed in all of the myoblasts that contribute to the body wall musculature, as well as in a group of cells that migrate into the head. Despite this different expression, the function of lbx1 appears to be conserved. Morpholino (MO) knockdown of lbx1 causes a specific reduction of body wall muscles and hypoglossal muscles originating from the somites. Although myoblast migratory defects are observed in antisense MO injected tadpoles targeting lbx1, this results at least in part from a lack of myoblast proliferation in the hypaxial muscle domain. Conversely, overexpression of lbx1 mRNA results in enlarged somites, an increase in cell proliferation, but a lack of differentiated muscle. The control of cell proliferation is linked to a strong downregulation of myoD expression in gain-of-function experiments. Co-injection of myoD mRNA with lbx1 mRNA eliminates the overproliferation phenotype observed when lbx1 is injected alone. The results indicate that a primary function of lbx1 in hypaxial muscle development is to repress myoD, allowing myoblasts to proliferate before the eventual onset of terminal differentiation.     

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.     

Eosinophils contribute to killing of adult Onchocerca ochengi within onchocercomata following elimination of Wolbachia

Many filarial nematodes, including Onchocerca volvulus (the cause of human 'River Blindness'), have a mutually dependent relationship with Wolbachia bacteria. There has been much interest in Wolbachia as a chemotherapeutic target, since there are no macrofilaricidal drugs (i.e., lethal to adult worms) of low toxicity. Using the bovine parasite O. ochengi, we previously demonstrated that combined intensive and intermittent (COM) oxytetracycline treatment induces a sustained depletion of Wolbachia and is macrofilaricidal, whereas a short intensive regimen (SIR) is non-macrofilaricidal. To understand how targeting Wolbachia with oxytetracycline can lead to worm death, O. ochengi nodules (onchocercomata) were sequentially excised from cattle administered COM or SIR therapy, and cell infiltrates were microscopically quantified. Pre-treatment, worms were surrounded by neutrophils, with eosinophils rare or absent. At 8-12weeks after either regimen, eosinophils increased around worms and were observed degranulating on the cuticle. However, with the SIR treatment, neutrophils returned to predominance by 48weeks, while in the COM group, eosinophilia persisted. These observations suggest that accumulation of degranulating eosinophils over a prolonged period is a cause rather than an effect of parasite death, and the macrofilaricidal mechanism of antibiotics may relate to facilitation of eosinophil infiltration around worms by ablation of Wolbachia-mediated neutrophilia.     

SBMLToolbox: an SBML toolbox for MATLAB users

SUMMARY: We present SBMLToolbox, a toolbox that facilitates importing and exporting models represented in the Systems Biology Markup Language (SBML) in and out of the MATLAB environment and provides functionality that enables an experienced user of either SBML or MATLAB to combine the computing power of MATLAB with the portability and exchangeability of an SBML model. SBMLToolbox supports all levels and versions of SBML. AVAILABILITY: SBMLToolbox is freely available from http://sbml.org/software/sbmltoolbox     

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.