Regulation of alternative macrophage activation by galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.     

Liver X receptor agonists increase airway reactivity in a model of asthma via increasing airway smooth muscle growth

The liver X receptors (LXRalpha/beta) are orphan nuclear receptors that are expressed in a large number of cell types and have been shown to have anti-inflammatory properties. Nuclear receptors have previously proved to be amenable targets for small molecular mass pharmacological agents in asthma, and so the effect of an LXR ligand was assessed in models of allergic airway inflammation. LXR agonist, GW 3965, was profiled in rat and mouse models of allergic asthma. In the Brown Norway rats, GW 3965 (3-30 mg/kg) was unable to reduce the bronchoalveolar lavage eosinophilia associated with this model and had no impact on inflammatory biomarkers (eotaxin and IL-1beta). The compound did significantly stimulate ABCA-1 (ATP-binding cassette A1) mRNA expression, indicating that there was adequate exposure/LXR activation. In the mouse model, the LXR ligand surprisingly increased airway reactivity, an effect that was apparent in both the Ag and nonchallenged groups. This increase was not associated with a change in lung tissue inflammation or number of mucus-containing cells. There was, however, a marked increase in airway smooth muscle thickness in both treated groups. We demonstrated an increase in contractile response to exogenous methacholine in isolated airways taken from LXR agonist-treated animals compared with the relevant control tissue. We corroborated these findings in a human system by demonstrating increased proliferation of cultured airway smooth muscle. This phenomenon, if evidenced in man, would indicate that LXR ligands may directly increase airway reactivity, which could be detrimental, especially in patients with existing respiratory disease and with already compromised lung function.     

IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-beta. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1(+) cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA(+) cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1(+) cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.     

The combination of raloxifene and epigallocatechin gallate suppresses growth and induces apoptosis in MDA-MB-231 cells

Previous studies have demonstrated that raloxifene induces apoptosis in a variety of cancer cell lines. We aimed to determine if this effect was enhanced by combining raloxifene with epigallocatechin gallate (EGCG). Results demonstrated that EGCG (25 microM) and raloxifene (1-5 microM) produced enhanced cytotoxicity toward MDA-MB-231 breast cancer cells compared to either drug alone following 7 days of treatment. The combination of 5 microM raloxifene and EGCG was the most effective as it decreased cell number by 96% of control, and time-course studies demonstrated that significant cytotoxicity began 36 h after treatment. Potential mechanisms for this effect were then investigated. Flow cytometry experiments demonstrated that apoptosis was significantly increased following 12 h of combination treatment compared to all other treatment groups. A maximal increase in the proportion of cells in the G(1)-phase of the cell cycle (116% of control) occurred following 24 h of combination treatment, 12 h after the significant increase in apoptosis, and thus was not considered to be a viable mechanism for the enhancement of apoptosis. While raloxifene was a competitive inhibitor of microsomal UDP-glucuronosyltransferase activity (K(i) of 24 microM), it did not decrease the metabolism of EGCG as the rate of disappearance of EGCG from the media was the same for cells treated with either EGCG or EGCG+raloxifene. Finally, the combination treatment reduced the phosphorylation of EGFR and AKT proteins by 21.2+/-3.3% and 31.5+/-1.7% from control, respectively. In conclusion, the synergistic cytotoxicity elicited by the combination of EGCG and raloxifene results from an earlier and greater induction of apoptosis. This is likely to be a result of reduced phosphorylation of EGFR and AKT signaling proteins.     

A computational model of the human thyroid

The thyroid, the largest gland in the endocrine system, secretes hormones that help promote bodily growth and development. This gland regulates hormonal secretion rate in spite of changes in dietary iodine which is a key ingredient in the hormone's biosynthesis. The thyroid relies on several feedback mechanisms for this regulation, and in this paper we use recent molecular-level and clinical observations to engineer a computational thyroid model. We use simulation and analysis to show that this models captures known aspects of thyroid physiology. We identify features in the model that are responsible for hormonal regulation, and use the model to identify and evaluate competing hypotheses associated with Wolff-Chaikoff escape.     

Adenovirus serotype 5 hexon mediates liver gene transfer

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.     

Increased fitness of Pseudomonas fluorescens Pf0-1 leucine auxotrophs in soil

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.     

Enterohemorrhagic Escherichia coli exploits EspA filaments for attachment to salad leaves

Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.