Detection and Quantification of Citrullinated Chemokines

BACKGROUND: Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest. METHODOLOGY/PRINCIPAL FINDINGS: First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit(5)]CXCL8). Specific detection of [Cit(5)]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit(5)]CXCL8. CONCLUSION/SIGNIFICANCE: The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit(5)]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8

Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-γ and counteraction by interferon-γ

Introduction Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis ( CIA). Since interferon (IFN)-gamma inhibits Th17 cell development, IFN-gamma receptor knockout (IFN-gamma R KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-gamma on the effector mechanisms of IL-17 in an in vitro system was also investigated. Methods IFN-gamma R KO mice induced for CIA were treated with anti-IL-17 or control antibody. The collagen type II (CII)-specific humoral and cellular autoimmune responses, myelopoiesis, osteoclastogenesis, and systemic cytokine production were determined. Mouse embryo fibroblasts (MEF) were stimulated with IL-17, tumor necrosis factor (TNF)-alpha and the expression of cytokines and chemokines were determined. Results A preventive anti-IL-17 antibody treatment inhibited CIA in IFN gamma R KO mice. In the joints of anti-IL-17-treated mice, neutrophil influx and bone destruction were absent. Treatment reduced the cellular autoimmune response as well as the splenic expansion of CD11b(+) cells, and production of myelopoietic cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-6. IL-17 and TNF-alpha synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NF kappa B ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-gamma in a STAT-1 (signal transducer and activator of transcription-1)dependent way. Conclusions In the absence of IFN-gamma, IL-17 mediates its proinflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-gamma profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-gamma, this may be an additional mechanism through which IFN-gamma attenuates autoimmune diseases

Chemokine-Induced Macrophage Polarization in Inflammatory Conditions

Macrophages represent a heterogeneous cell population and are known to display a remarkable plasticity. In response to distinct micro-environmental stimuli, e.g., tumor stroma vs. infected tissue, they polarize into different cell subtypes. Originally, two subpopulations were defined: classically activated macrophages or M1, and alternatively activated macrophages or M2. Nowadays, the M1/M2 classification is considered as an oversimplified approach that does not adequately cover the total spectrum of macrophage phenotypes observed in vivo. Especially in pathological circumstances, macrophages behave as plastic cells modifying their expression and transcription profile along a continuous spectrum with M1 and M2 phenotypes as extremes. Here, we focus on the effect of chemokines on macrophage differentiation and polarization in physiological and pathological conditions. In particular, we discuss chemokine-induced macrophage polarization in inflammatory diseases, including obesity, cancer, and atherosclerosis

Noncoding after all : biases in proteomics data do not explain observed absence of lncRNA translation products

Over the past decade, long noncoding RNAs (lncRNAs) have emerged as novel functional entities of the eukaryotic genome. However, the scientific community remains divided over the amount of true noncoding transcripts among the large number of unannotated transcripts identified by recent large scale and deep RNA-sequencing efforts. Here, we systematically exclude possible technical reasons underlying the absence of lncRNA-encoded proteins in mass spectrometry data sets, strongly suggesting that the large majority of lncRNAs is indeed not translated

Role of C‐X‐C chemokines as regulators of angiogenesis in lung cancer

Lung cancer is the leading cause of malignancy‐related mortality in the U.S. and is predicted to increase over the remainder of this decade. Despite attempts to advance early diagnosis and use combination therapies, the clinical response of this cancer yields an overall 5‐year survival rate of less than 15%. Clearly, new strategies for therapy are indicated. Although carcinogenesis is complex, tumor growth beyond 1–2 mm3 is dependent on angiogenesis. One of the potential mechanisms that allows for tumorigenesis is dysregulation of the balance of angiogenic and angiostatic factors that favors net neovascularization within the primary tumor. Numerous studies have investigated the role of a variety of molecules in the regulation of angiogenesis. Recently, interleukin‐8 (IL‐8), a member of the C‐X‐C chemokine family, has been found to be an angiogenic factor. In contrast, platelet factor 4 (PF4), another C‐X‐C chemokine, has been shown to have angiostatic properties. It is interesting that the major structural difference between IL‐8 and PF4 is the presence of the NH2‐terminal ELR (Glu‐Leu‐Arg) motif that precedes the first cysteine amino acid residue of IL‐8 and is important in ligand/receptor interactions. We hypothesize that angiogenesis associated with tumorigenesis is dependent on members of the C‐X‐C chemokine family acting as either angiogenic or angiostatic factors. This paradigm predicts that the biological balance in the expression of these C‐X‐C chemokines dictates whether the neoplasm grows and develops metastatic potential or regresses. In this review we discuss our recent laboratory findings that support this contention and suggest that further elucidation of the biology of C‐X‐C chemokines in the context of neovascularization of nonsmall cell lung cancer will permit novel targeted therapy aimed specifically at attenuating tumor growth and metastasis. J. Leukoc. Biol. 57: 752–762; 1995.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141888/1/jlb0752.pd

Association of alcohol control policies with adolescent alcohol consumption and with social inequality in adolescent alcohol consumption : a multilevel study in 33 countries and regions

Funding: The Health Behaviour in School-aged Children (HBSC) is an international study carried out in collaboration with WHO Europe. This research is funded by public sources in each member country. Specifically, ELL, CMM, and ISQ are supported by the Ministry de Health, Spain. CMM is supported by the VI Plan Propiode Investigación de la Universidad de Sevilla 2018, under the action “II.5B Contrato de acceso al Sistema Españolde Ciencia, Tecnología e Innovación para el Desarrollo del Programa Propio de I + D + i de la Universidad de Sevilla”. JI is supported by the UK Medical Research Council (MC_UU_12017/12) and the Chief Scientist Office (SPHSU12).Background  Previous research found inconsistent associations between alcohol control policies and socioeconomic inequality with adolescent drinking outcomes. This study expands the focus beyond individual associations to examine whether a combination of policies is related to socioeconomic inequality in adolescent drinking outcomes and whether this relationship varies across survey years. Methods  Multilevel modelling of 4 waves of repeat cross-sectional survey data (2001/02, 2005/06, 2009/10, and 2013/14) from the Health Behaviour in School-aged Children (HBSC) study was carried out. The sample was composed of 671,084 adolescents (51% girls) aged 11, 13, and 15 (mean age=13.58; SD=1.65) from 33 European and North American countries/regions. The dependent variables were lifetime alcohol consumption, weekly alcohol consumption, and lifetime drunkenness. Independent variables were of three types: individual-level variables (age, sex, Family Affluence Scale, and the Perceived Family Wealth), time-level variable (survey year), and context-level variables (minimum legal drinking age, physical availability, advertising restrictions, a total alcohol policy index, and affordability of alcohol). Results  The total alcohol policy index showed a negative relationship with both lifetime and weekly consumption. Higher affordability of alcohol was related to higher lifetime and weekly consumption and higher lifetime drunkenness. Family Affluence Scale was positively related to all three alcohol measures and Perceived Family Wealth was negatively related to lifetime drunkenness, with these associations increasing across survey years. The total alcohol policy index buffered the associations of Family Affluence Scale and Perceived Family Wealth with adolescent drinking outcomes. Conclusion  A combination of alcohol control policies is more effective in reducing adolescent drinking outcomes than single policy measures. Reducing the affordability of alcohol stood out as the most successful single measure. Socioeconomic inequalities (i.e. higher alcohol consumption and drunkenness in adolescents with higher family affluence and higher drunkenness in adolescents perceiving their families to be poor) have persisted and even increased across survey years. A combined alcohol control policy can help in tackling them.PostprintPublisher PDFPeer reviewe