The balance between activating and inhibitory signals from the different FcγRs for IgG ensures homeostasis of many inflammatory responses. FCGR2C is the product of an unequal crossover of the FCGR2A and FCGR2B genes encoding the activating FcγRIIa (CD32a) and inhibitory FcγRIIb (CD32b), respectively. A single nucleotide polymorphism (SNP) in exon 3 of FCGR2C results in either expression of the activating FcγRIIc (CD32c) (FCGR2C-open reading frame [ORF]) or its absence because of a stop codon (FCGR2C-Stop). Two additional variations in FcγRIIb/c expression on leukocytes have now been identified. In case of “nonclassical” FCGR2C-ORF alleles, FcγRIIc expression was unexpectedly absent, because of novel splice site mutations near exon 7 leading to another stop codon. In some individuals with FCGR2C-Stop alleles FcγRIIb was detected on NK cells, which normally are devoid of this protein. Individuals with these nonclassical FCGR2C-Stop alleles carried a deletion of FCGR2C-FCGR3B that extends into the promoter region of the adjacent FCGR2B gene and probably deletes a negative regulatory element in the FCGR2B promoter in NK cells. FcγRIIb expression on NK cells effectively inhibited killing mediated by FcγRIIIa (CD16a) in Ab-dependent cytotoxicity tests. Our findings demonstrate a more extensive and previously unnoticed variation in FcγR expression with relevance to immunity and inflammation.
The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.
Ever since Thaler and Sunstein published their influential book Nudge, the book and the theory it presents have received great praise and opposition. Nudge theory, and more particularly, nudging may be considered an additional strategy providing some novel instruments to the already rich governance toolbox. But what is its value? The current debates on Nudge theory are often highly normative or ideologically driven and pay limited attention to more practical aspects of the theory. Is nudging evaluable as a theory and a practice, and if so how? Is there solid evidence available of nudge success over other governance interventions? What is to be considered a nudge success? What data and evaluative techniques may assist in evaluating nudging beyond individual cases? The current article seeks to explore these questions.
Many significant bacterial pathogens have evolved virulence mechanisms to evade degradation and exposure to reactive oxygen (ROS) and reactive nitrogen species (RNS), allowing them to survive and replicate inside their hosts. Due to the highly reactive and short-lived nature of ROS and RNS, combined with limitations of conventional detection agents, the mechanisms underlying these evasion strategies remain poorly understood. In this study, we describe a system that uses redox-sensitive GFP to nondisruptively measure real-time fluctuations in the intrabacterial redox environment. Using this system coupled with high-throughput microscopy, we report the intrabacterial redox dynamics of Salmonella enterica Typhimurium (S. Typhimurium) residing inside macrophages. We found that the bacterial SPI-2 type III secretion system is required for ROS evasion strategies and this evasion relies on an intact Salmonella-containing vacuole (SCV) within which the bacteria reside during infection. Additionally, we found that cytosolic bacteria that escape the SCV experience increased redox stress in human and murine macrophages. These results highlight the existence of specialized evasion strategies used by intracellular pathogens that either reside inside a vacuole or "escape" into the cytosol. Taken together, the use of redox-sensitive GFP inside Salmonella significantly advances our understanding of ROS and RNS evasion strategies during infection. This technology can also be applied to measuring bacterial oxidative and nitrosative stress dynamics under different conditions in a wide variety of bacteria.oxidative stress | reactive oxygen species | Salmonella | bacterial pathogenesis | redox-sensitive GFP
The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C -ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C -ORF haplotype showed strong LD with, among others, rs201218628 ( FCGR2A -Q27W, r 2 = 0.63). LD between these two variants was weaker ( r 2 = 0.17) in Africans, whereas the FCGR2C -ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C -ORF haplotype and rs1801274 ( FCGR2A -H131R) were in weak LD ( r 2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C -ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation.
Over the years layering has gained increasing attention in studies of institutional change. Notably, the concept has been subject to the exact mechanism it tries to explain: incremental change. This article reviews the use of the concept over a 60-year time span in order to elucidate its value for studying institutional change. The article especially looks at the use of the concept by one of the leading authors in the field: Kathleen Thelen. It concludes that layering provides a bridge between -seemingly conflicting -ideas on incremental change and punctuated equilibrium. Note1 The historical analysis presented in this section is based on a literature review of over 166 journal articles from the disciplines of economics, law, sociology, political science, public policy and public administration. I by no means claim that this search is exhaustive. My search was limited by the number of articles I could trace in JSTOR archives.
Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.Bacterial pathogens have evolved specialized secretion systems to transfer bacterial virulence proteins, also called effectors, from the bacterium to the host cell cytoplasm. The type three secretion system (T3SS) 9 is a large macromolecular machine consisting of three transmembrane complexes: an inner membrane ring, an outer membrane ring, and a translocon pore that forms within the host cell membrane. Effectors are transferred from bacteria to the host cell through a needleshaped structure that connects the outer membrane ring and the translocon (1). Effectors translocated by the T3SS are central to pathogenesis, as mutations that prevent proper assembly of the secretion machinery result in significant virulence defects and often completely hinder bacterial colonization of the host. Consequently, technology to identify and characterize effectors is the key to our understanding of bacterial disease.Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen that causes gastroenteritis in humans and a systemic infection resembling typhoid fever in mice (2). S. typhimurium virulence depends on two T3SSs that are encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2) (2, 3). The SPI-1 T3SS is key during the early stages of infection and effectors translocated by this secretion system mediate entry of S. typhimurium...
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