Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist

Background

Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers.

Methods

Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later.

Results

Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B₄ (39%) and CXCL8 (52%).

Conclusions

AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00860821","term_id":"NCT00860821"}}NCT00860821.     

Dual character of Toll-like receptor signaling: Pro-tumorigenic effects and anti-tumor functions

As a major class of pattern-recognition receptors, Toll-like receptors (TLRs) play a critical role in defense against invading pathogens. Increasing evidence demonstrates that, in addition to infection, TLRs are involved in other important pathological processes, such as tumorigenesis. Activation of TLRs results in opposing outcomes, pro-tumorigenic effects and anti-tumor functions. TLR signaling can inhibit apoptosis and promote chronic inflammation-induced tumorigenesis. TLR activation in tumor cells and immune cells can induce production of cytokines, increase tumor cell proliferation and apoptosis resistance, promote invasion and metastasis, and inhibit immune cell activity resulting in tumor immune escape. In contrast, the engagement of other TLRs directly induces growth inhibition and apoptosis of tumor cells and triggers activation of immune cells enhancing anti-tumor immune responses. Thus, the interpretation of the precise function of each TLR in tumors is very important for targeting TLRs and using TLR agonists in tumor therapy. We review the role of TLR signaling in tumors and discuss the factors that affect outcomes of TLR activation.     

Relationship between chemokine (C–X–C motif) ligand 12 gene variant (rs1746048) and coronary heart disease: Case–control study and meta-analysis

The goal of our study is to evaluate the contribution of CXCL12 rs1746048 (hg19, chr10:44775574) to the risk of CHD in Han Chinese, and to summarize its role in CHD through meta-analysis of existing studies among various ethnic groups. Significant association is observed between rs1746048-C and an increased risk of CHD in Han Chinese (χ² = 5.41, df = 1, P = 0.02). Post hoc analysis reveals an even stronger association of rs1746048 with the risk of CHD for subjects aged 65 years or older (genotype: χ² = 8.39, df = 2, P = 0.015; allele: χ2 = 9.13, df = 1, P = 0.003, odd ratio (OR) = 1.91, 95% confidential interval (CI) = 1.25–2.91). A break down analysis by gender shows that rs1746048 is likely a CHD risk factor under the recessive model in males (CC + CT versus TT: P = 0.05, χ² = 3.59, df = 1, OR = 1.72, 95% CI = 1.00–3.04). In addition, a meta-analysis of ten studies among over 107,000 individuals confirms that rs1746048 is a risk factor of CHD (P < 0.0001, OR = 1.12, 95% CI = 1.09–1.15) and this agrees with the findings of our case–control study in Han Chinese.     

HIV-1 envelope determinants for cell tropism and chemokine receptor use

Isolates of human immunodeficiency virus type-1 (HIV-1) display marked differences in their ability to replicate in macrophages and transformed T-cell lines in vitro, a property that has important implications for disease pathogenesis. The restriction in replication between these two CD4-positive cell types is largely at the level of viral entry and is regulated by the viral envelope (env) gene. The envelope protein (Env) is responsible for fusion of the viral and host membranes, and a particular region of Env called the V3-loop has been implicated in regulating viral tropism. However, other regions of Env, such as the V1- and V2-loops, have been shown to modulate the effects of the V3-loop. The discovery that Env initially binds the CD4 molecule on the target cell surface and then makes subsequent interactions with one of several members of the chemokine receptor family has greatly enhanced the molecular understanding of HIV-1 entry. The differential use of chemokine receptors by different viral isolates and their expression in different cell types largely explains viral tropism. The same regions in Env responsible for virus tropism have also been shown to play an important role in mediating chemokine receptor use. The recent crystallization of HIV-1 Env in complex with CD4 illuminates the architecture of the components involved in mediating fusion between the viral and host membranes. The spatial relationship between variable structures of Env previously implicated in tropism and chemokine receptor use and conserved Env structures potentially involved in chemokine receptor binding are discussed.     

Insights into the dynamic interactions at chemokine-receptor interfaces and mechanistic models of chemokine binding

Chemokine receptors are the central signaling hubs of several processes such as cell migration, chemotaxis and cell positioning. In this graphical review, we provide an overview of the structural and mechanistic principles governing chemokine recognition that are currently emerging. Structural models of chemokine-receptor co-complexes with endogenous chemokines, viral chemokines and therapeutics have been resolved that highlight multiple interaction sites, termed as CRS1, CRS1.5 etc. The first site of interaction has been shown to be the N-terminal domain of the receptors (CRS1 site). A large structural flexibility of the N-terminal domain has been reported that was supported by both experimental and simulation studies. Upon chemokine binding, the N-terminal domain appears to show constricted dynamics and opens up to interact with the chemokine via a large interface. The subsequent sites such as CRS1.5 and CRS2 sites have been structurally well resolved although differences arise such as the localization of the N-terminus of the ligand to a major or minor pocket of the orthosteric binding site. Several computational studies have highlighted the dynamic protein-protein interface at the CRS1 site that seemingly appears to resolve the differences in NMR and mutagenesis studies. Interestingly, the differential dynamics at the CRS1 site suggests a mixed model of binding with complex signatures of both conformational selection and induced fit models. Integrative experimental and computational approaches could help unravel the structural basis of promiscuity and specificity in chemokine-receptor binding and open up new avenues of therapeutic design.     

DNA疫苗的分子佐剂应用研究进展

DNA疫苗因在动物尤其是大型动物与人类中诱发较低的免疫反应而严重影响其推广应用。介绍提高与调节DNA疫苗诱导反应的策略：（1）以细胞因子表达质控为佐剂;（2）以质粒编码的趋化因子与共刺激分子为佐剂;（3）以CPG ODN为佐剂。     

Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein,M-T7, through site directed mutagenesis

Viral chemokine modulating proteins provide new and extensive sources for therapeutics. Purified M-T7, a poxvirus-derived secreted immunomodulatory protein, reduces mononuclear cell invasion and atheroma in rodent models of angioplasty injury as well as aortic and renal transplant, improving renal allograft survival. M-T7 is a rabbit species-specific interferon gamma receptor (IFNγR) homolog, but also inhibits chemokine/glycosaminoglycan (GAG) interactions for C, CC and CXC chemokines, with cross-species specific inhibitory activity. M-T7 anti-atheroma activity is blunted in GAG deficient mouse aortic transplants, but not in CC chemokine receptor deficient transplants, supporting M-T7 interference in chemokine/GAG interactions as the basis of the atheroma-inhibitory activity. We have assessed point mutants of M-T7 both in vivo in a mouse angioplasty model and in vitro in tissue culture and binding assays, in order to better define the primary mechanism of anti-atheroma activity. Of these M-T7 mutants, the R¹⁷¹E and E²⁰⁹I M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE^−/− mice after angioplasty injury and R¹⁷¹E, moreover, greatly exacerbated plaque growth and inflammation. F¹³⁷D retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His_6X, F¹³⁷D, R¹⁷¹E, and E²⁰⁹I all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, R¹⁷¹E and E²⁰⁹I had significantly reduced binding to RANTES and IFNγ, whereas F¹³⁷D retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, R¹⁷¹E and E²⁰⁹I, exhibited reduced anti-inflammatory properties in vivo after mouse angioplasty with a loss of in vitro binding to RANTES and IFNγ, indicating these point mutations partially disrupt M-T7 ligand-binding activities. Unexpectedly, the M-T7 mutants all retained inhibitory activity for human monocyte THP-1 cell migration ex vivo, suggesting additional inhibitory properties against human monocyte THP-1 cells that are independent of chemokine inhibition.     

Design and synthesis of novel small molecule CCR2 antagonists: Evaluation of 4-aminopiperidine derivatives

A novel N-(2-oxo-2-(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide series of human CCR2 chemokine receptor antagonists was identified. With a pharmacophore model based on known CCR2 antagonists a new core scaffold was designed, analogues of it synthesized and structure–affinity relationship studies derived yielding a new high affinity CCR2 antagonist N-(2-((1-(4-(3-methoxyphenyl)cyclohexyl)piperidin-4-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide.     

Plasma membrane signaling in HIV-1 infection

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4 + T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.