cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.

Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')>. We found that the presence of this 2'-5' linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2'-5'-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3'-5' phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules.

Species-specific detection of the antiviral small-molecule compound CMA by STING.

Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10-carboxymethyl-9-acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell-intrinsic type I IFN response, depending on the ER-resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C-terminal ligand-binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate (c-diGMP)-binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c-diGMP. Altogether, these results provide novel insight into ligand-sensing properties of STING and, furthermore, unravel unexpected species-specific differences of this innate sensor.

The epidemiology and burden of gastroparesis: Real-world data from a large healthcare provider in Israel.

BACKGROUND: Gastroparesis is a gastrointestinal motility dysfunction characterized by delayed gastric emptying in the absence of gastric mechanical obstruction. Data on the epidemiology of gastroparesis are sparse even though the condition substantially impairs patients' quality of life. The aim of this study was to describe the epidemiology and estimate the short-term healthcare resource use burden of gastroparesis in a large population. METHODS: This cross-sectional study utilized computerized data from Maccabi Healthcare Services, a 2.5-million member state-mandated health organization in Israel. Data were collected between 2003 and 2018 to assess the prevalence of gastroparesis. Definite gastroparesis was defined by gastroparesis diagnosis and gastric emptying test. Probable gastroparesis was defined by gastroparesis diagnosis only. To compare the healthcare resource utilization (HCRU), data were also collected on controls that were individually matched (1:2) for age, sex, and comorbidities. KEY RESULTS: A total of 522 patients with gastroparesis were identified (21.1 per 100,000 WHO age-standardized), including 204 with definite gastroparesis (8.6 per 100,000 WHO). Male to female ratio was 1:2 and mean ± SD age of 54.7 ± 17.1 years. Diabetes accounted for 25.9% of gastroparesis cases and the rest were idiopathic. Gastroparesis patients were more likely to have cardiovascular diseases (10% vs. 6.9% for controls, p = 0.034) and lower prevalence of obesity (17% vs. 24.4%, p < 0.001). HCRU within the 2 years after index date were higher with more hospitalizations than controls (26.4% vs. 15.4%, p < 0.001), and more emergency room visits (31.6% vs. 24.1%, p = 0.002). CONCLUSIONS & INFERENCES: Gastroparesis is uncommon or under-documented in community care settings. Gastroparesis in general is associated with cardiovascular morbidities, lower BMI, and elevated utilization of healthcare services.

Induction of type I IFNs by intracellular DNA-sensing pathways.

A successful antimicrobial immune response involves the coordinate action of cells and soluble factors, with the cytokine family of type I interferons (IFNs) having a central role. Type I IFNs are not only crucial in conferring immediate antimicrobial, most importantly antiviral effects, but they also have an essential role in bridging the innate with the adaptive immune response. Therefore, production of these key cytokines must be tightly controlled. To this effect the host has evolved a set of pattern recognition receptors (PRRs) that reliably and specifically detect the presence of microbial pathogens before mounting an IFN response. Most PRR pathways that are known to induce type I IFNs are triggered upon recognition of nucleic acids. This mode of sensing is not straightforward, as large amounts of RNA and DNA are also present within the host. Nevertheless, in some cases distinct molecular features that are present within foreign nucleic acids but absent in endogenous nucleic acids, allow the host to reliably discriminate between 'self' and 'non-self'. At the same time, compartmentalization of PRRs within subcellular organelles that are usually devoid of host nucleic acids, but are sites of pathogen localization, is another principle that enables the host to distinguish self from non-self. The latter mode of sensing applies to the detection of microbial DNA within the cytoplasm, a compartment in which host DNAs are usually not present. Despite the past years' tremendous progress in the field of innate immunity, our understanding of cytoplasmic DNA sensing mechanisms is only beginning to form/take form. In this review, we outline the recent advancements in the elucidation of intracellular DNA-sensing pathways and discuss the future directions of this emerging field.

Inflammasomes: current understanding and open questions.

The innate immune system relies on its capability to detect invading microbes, tissue damage, or stress via evolutionarily conserved receptors. The nucleotide-binding domain leucine-rich repeat (NLR)-containing family of pattern recognition receptors includes several proteins that drive inflammation in response to a wide variety of molecular patterns. In particular, the NLRs that participate in the formation of a molecular scaffold termed the "inflammasome" have been intensively studied in past years. Inflammasome activation by multiple types of tissue damage or by pathogen-associated signatures results in the autocatalytic cleavage of caspase-1 and ultimately leads to the processing and thus secretion of pro-inflammatory cytokines, most importantly interleukin (IL)-1ß and IL-18. Here, we review the current knowledge of mechanisms leading to the activation of inflammasomes. In particular, we focus on the controversial molecular mechanisms that regulate NLRP3 signaling and highlight recent advancements in DNA sensing by the inflammasome receptor AIM2.

Inflammatory Bowel Disease Patients' Treatment Preferences Using a Discrete Choice Experiment Technique: The InPuT Study.

INTRODUCTION: The aim of this study was to investigate patients' preferences regarding the evolving treatment landscape in Crohn's disease (CD) and ulcerative colitis (UC) based on a discrete choice experiment. METHODS: Eligible patients (aged 18 years or older) had a confirmed diagnosis of CD or UC and were willing and able to participate in telephone interviews. The survey design is based on a prior literature review, a pilot study, and clinical expert discussions. Preferences related to clinical and practical features of advanced therapies, like tumor necrosis factor alpha inhibitors, anti-integrins, anti-interleukins, and Janus kinase inhibitors, were assessed. Patients were asked to choose between two different hypothetical treatment alternatives visualized in up to 11 choice scenarios. Based on these choices, the relative importance of treatment characteristics was derived from regression coefficients estimated by a conditional logit model. RESULTS: Of the 291 patients included, 219 (75%) were eligible for this analysis. Among the evaluated attributes in CD, 1-year remission rate was ranked highest, with 42.3% relevance for the overall decision. The second most important attribute was the frequency of serious adverse events (AE) (25.1%), followed by sustained remission over 2 years (17.8%). Lower importance was assigned to the administration mode (14.6%) and none to the frequency of non-serious AE (0.1%). In UC, preferences were driven by efficacy (25.3% for mucosal healing; 23.4% for corticosteroid-free remission) and the frequency of serious AE (18.3%), followed by the administration mode (18.1%). Also, non-serious AE were classified as relevant factors for decision-making (10.7%), while maintaining remission for at least 2 years showed no significant impact (4.4%). CONCLUSION: For both indications, efficacy outcomes were rated most important, followed by the frequency of serious AE. Variations were mainly found in the evaluation of non-serious AE and sustained remission. Considering patient preferences may improve the effectiveness of available therapies for moderate to severe CD and UC.

The thermal and enzymatic taxifolin-alphitonin rearrangement.

This report describes a detailed investigation of the thermal and enzymatic conversion of taxifolin to alphitonin. Chromatographic separation of the four dihydroquercetin stereoisomers 1-4 in combination with circular dichroism spectroscopy permitted elucidation of the kinetics of this rearrangement and characterization of the different reaction pathways involved. Our findings are corroborated by quantum chemistry calculations that reveal a unique cascade of tautomerization processes leading from taxifolin to alphitonin and also explain the racemization of alphitonin at room temperature. Furthermore, the substrate specificity toward (+)-taxifolin of an enzyme from Eubacterium ramulus catalyzing this intriguing rearrangement is demonstrated.

CCR2 mediates homeostatic and inflammatory release of Gr1(high) monocytes from the bone marrow, but is dispensable for bladder infiltration in bacterial urinary tract infection.

CCR2 is thought to recruit monocytes to sites of infection. Two subpopulations of murine blood monocytes differing in Gr1 and CCR2 expression have been described. The exact role of CCR2 in migration of CCR2(low)Gr1(low) and CCR2(high)Gr1(high) monocytes into nonlymphoid tissue is controversial. In this study, we have addressed this question in a murine model of bacterial urinary tract infection. Only Gr1(high) monocytes were recruited into the infected bladder. CCR2 deficiency reduced their frequency in this organ, indicating a requirement of this chemokine receptor. Importantly, CCR2-deficient mice also showed reduced Gr1(high) monocyte numbers in the blood, but not in the bone marrow (BM), indicating that CCR2 acted at the step of monocyte release into the circulation. The same was found also in noninfected mice, indicating a further involvement of CCR2 in steady-state BM egress. An additional requirement of CCR2 in monocyte recruitment from the blood into the bladder was excluded by tracking particle-labeled endogenous monocytes and by adoptive transfer of BM-derived monocyte subsets. These findings demonstrate that CCR2 governs homeostatic and infection-triggered release of Gr1(high) monocytes from the BM into the blood but is dispensable for recruitment into a nonlymphoid tissue.