Search

ImportanceUrinary tract infection (UTI) is the second most common infection leading to hospitalization and is often associated with gram-negative multidrug-resistant organisms (MDROs). Clinicians overuse extended-spectrum antibiotics although most patients are at low risk for MDRO infection. Safe strategies to limit overuse of empiric antibiotics are needed.ObjectiveTo evaluate whether computerized provider order entry (CPOE) prompts providing patient- and pathogen-specific MDRO risk estimates could reduce use of empiric extended-spectrum antibiotics for treatment of UTI.Design, setting, and participantsCluster-randomized trial in 59 US community hospitals comparing the effect of a CPOE stewardship bundle (education, feedback, and real-time and risk-based CPOE prompts; 29 hospitals) vs routine stewardship (n = 30 hospitals) on antibiotic selection during the first 3 hospital days (empiric period) in noncritically ill adults (≥18 years) hospitalized with UTI with an 18-month baseline (April 1, 2017-September 30, 2018) and 15-month intervention period (April 1, 2019-June 30, 2020).InterventionsCPOE prompts recommending empiric standard-spectrum antibiotics in patients ordered to receive extended-spectrum antibiotics who have low estimated absolute risk (

ImportancePneumonia is the most common infection requiring hospitalization and is a major reason for overuse of extended-spectrum antibiotics. Despite low risk of multidrug-resistant organism (MDRO) infection, clinical uncertainty often drives initial antibiotic selection. Strategies to limit empiric antibiotic overuse for patients with pneumonia are needed.ObjectiveTo evaluate whether computerized provider order entry (CPOE) prompts providing patient- and pathogen-specific MDRO infection risk estimates could reduce empiric extended-spectrum antibiotics for non-critically ill patients admitted with pneumonia.Design, setting, and participantsCluster-randomized trial in 59 US community hospitals comparing the effect of a CPOE stewardship bundle (education, feedback, and real-time MDRO risk-based CPOE prompts; n = 29 hospitals) vs routine stewardship (n = 30 hospitals) on antibiotic selection during the first 3 hospital days (empiric period) in non-critically ill adults (≥18 years) hospitalized with pneumonia. There was an 18-month baseline period from April 1, 2017, to September 30, 2018, and a 15-month intervention period from April 1, 2019, to June 30, 2020.InterventionCPOE prompts recommending standard-spectrum antibiotics in patients ordered to receive extended-spectrum antibiotics during the empiric period who have low estimated absolute risk (

BackgroundDetection and containment of hospital outbreaks currently depend on variable and personnel-intensive surveillance methods. Whether automated statistical surveillance for outbreaks of health care-associated pathogens allows earlier containment efforts that would reduce the size of outbreaks is unknown.MethodsWe conducted a cluster-randomized trial in 82 community hospitals within a larger health care system. All hospitals followed an outbreak response protocol when outbreaks were detected by their infection prevention programs. Half of the hospitals additionally used statistical surveillance of microbiology data, which alerted infection prevention programs to outbreaks. Statistical surveillance was also applied to microbiology data from control hospitals without alerting their infection prevention programs. The primary outcome was the number of additional cases occurring after outbreak detection. Analyses assessed differences between the intervention period (July 2019 to January 2022) versus baseline period (February 2017 to January 2019) between randomized groups. A post hoc analysis separately assessed pre-coronavirus disease 2019 (Covid-19) and Covid-19 pandemic intervention periods.ResultsReal-time alerts did not significantly reduce the number of additional outbreak cases (intervention period versus baseline: statistical surveillance relative rate [RR]=1.41, control RR=1.81; difference-in-differences, 0.78; 95% confidence interval [CI], 0.40 to 1.52; P=0.46). Comparing only the prepandemic intervention with baseline periods, the statistical outbreak surveillance group was associated with a 64.1% reduction in additional cases (statistical surveillance RR=0.78, control RR=2.19; difference-in-differences, 0.36; 95% CI, 0.13 to 0.99). There was no similarly observed association between the pandemic versus baseline periods (statistical surveillance RR=1.56, control RR=1.66; difference-in-differences, 0.94; 95% CI, 0.46 to 1.92).ConclusionsAutomated detection of hospital outbreaks using statistical surveillance did not reduce overall outbreak size in the context of an ongoing pandemic. (Funded by the Centers for Disease Control and Prevention; ClinicalTrials.gov number, NCT04053075. Support for HCA Healthcare's participation in the study was provided in kind by HCA.).

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

ImportanceUniversal nasal mupirocin plus chlorhexidine gluconate (CHG) bathing in intensive care units (ICUs) prevents methicillin-resistant Staphylococcus aureus (MRSA) infections and all-cause bloodstream infections. Antibiotic resistance to mupirocin has raised questions about whether an antiseptic could be advantageous for ICU decolonization.ObjectiveTo compare the effectiveness of iodophor vs mupirocin for universal ICU nasal decolonization in combination with CHG bathing.Design, setting, and participantsTwo-group noninferiority, pragmatic, cluster-randomized trial conducted in US community hospitals, all of which used mupirocin-CHG for universal decolonization in ICUs at baseline. Adult ICU patients in 137 randomized hospitals during baseline (May 1, 2015-April 30, 2017) and intervention (November 1, 2017-April 30, 2019) were included.InterventionUniversal decolonization involving switching to iodophor-CHG (intervention) or continuing mupirocin-CHG (baseline).Main outcomes and measuresICU-attributable S aureus clinical cultures (primary outcome), MRSA clinical cultures, and all-cause bloodstream infections were evaluated using proportional hazard models to assess differences from baseline to intervention periods between the strategies. Results were also compared with a 2009-2011 trial of mupirocin-CHG vs no decolonization in the same hospital network. The prespecified noninferiority margin for the primary outcome was 10%.ResultsAmong the 801 668 admissions in 233 ICUs, the participants' mean (SD) age was 63.4 (17.2) years, 46.3% were female, and the mean (SD) ICU length of stay was 4.8 (4.7) days. Hazard ratios (HRs) for S aureus clinical isolates in the intervention vs baseline periods were 1.17 for iodophor-CHG (raw rate: 5.0 vs 4.3/1000 ICU-attributable days) and 0.99 for mupirocin-CHG (raw rate: 4.1 vs 4.0/1000 ICU-attributable days) (HR difference in differences significantly lower by 18.4% [95% CI, 10.7%-26.6%] for mupirocin-CHG, P

The Centers for Medicare and Medicaid Services require hospitals to report on quality metrics which are used to financially penalize those that perform in the lowest quartile. Surgical site infections (SSIs) are a critical component of the quality metrics that target healthcare-associated infections. However, the accuracy of such hospital profiling is highly affected by small surgical volumes which lead to a large amount of uncertainty in estimating standardized hospital-specific infection rates. Currently, hospitals with less than one expected SSI are excluded from rankings, but the effectiveness of this exclusion criterion is unknown. Tools that can quantify the classification accuracy and can determine the minimal surgical volume required for a desired level of accuracy are lacking. We investigate the effect of surgical volume on the accuracy of identifying poorly performing hospitals based on the standardized infection ratio and develop simulation-based algorithms for quantifying the classification accuracy. We apply our proposed method to data from HCA Healthcare (2014-2016) on SSIs in colon surgery patients. We estimate that for a procedure like colon surgery with an overall SSI rate of 3%, to rank hospitals in the HCA colon SSI dataset, hospitals that perform less than 200 procedures have a greater than 10% chance of being incorrectly assigned to the worst performing quartile. Minimum surgical volumes and predicted events criteria are required to make evaluating hospitals reliable, and these criteria vary by overall prevalence and between-hospital variability.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a major cause of cancer morbidity and mortality in Eastern Africa. The majority of patients with ESCC in Eastern Africa present with advanced disease at the time of diagnosis. Several palliative interventions for ESCC are currently in use within the region, including chemotherapy, radiation therapy with and without chemotherapy, and esophageal stenting with self-expandable metallic stents; however, the comparative effectiveness of these interventions in a low resource setting has yet to be examined.MethodsThis prospective, observational, multi-center, open cohort study aims to describe the therapeutic landscape of ESCC in Eastern Africa and investigate the outcomes of different treatment strategies within the region. The 4.5-year study will recruit at a total of six sites in Kenya, Malawi and Tanzania (Ocean Road Cancer Institute and Muhimbili National Hospital in Dar es Salaam, Tanzania; Kilimanjaro Christian Medical Center in Moshi, Tanzania; Tenwek Hospital in Bomet, Kenya; Moi Teaching and Referral Hospital in Eldoret, Kenya; and Kamuzu Central Hospital in Lilongwe, Malawi). Treatment outcomes that will be evaluated include overall survival, quality of life (QOL) and safety. All patients (≥18 years old) who present to participating sites with a histopathologically-confirmed or presumptive clinical diagnosis of ESCC based on endoscopy or barium swallow will be recruited to participate. Key clinical and treatment-related data including standardized QOL metrics will be collected at study enrollment, 1 month following treatment, 3 months following treatment, and thereafter at 3-month intervals until death. Vital status and QOL data will be collected through mobile phone outreach.DiscussionThis study will be the first study to prospectively compare ESCC treatment strategies in Eastern Africa, and the first to investigate QOL benefits associated with different treatments in sub-Saharan Africa. Findings from this study will help define optimal management strategies for ESCC in Eastern Africa and other resource-limited settings and will serve as a benchmark for future research.Trial registrationThis study was retrospectively registered with the ClinicalTrials.gov database on December 15, 2021,  NCT05177393 .

BackgroundThe profound changes wrought by coronavirus disease 2019 (COVID-19) on routine hospital operations may have influenced performance on hospital measures, including healthcare-associated infections (HAIs). We aimed to evaluate the association between COVID-19 surges and HAI and cluster rates.MethodsIn 148 HCA Healthcare-affiliated hospitals, from 1 March 2020 to 30 September 2020, and a subset of hospitals with microbiology and cluster data through 31 December 2020, we evaluated the association between COVID-19 surges and HAIs, hospital-onset pathogens, and cluster rates using negative binomial mixed models. To account for local variation in COVID-19 pandemic surge timing, we included the number of discharges with a laboratory-confirmed COVID-19 diagnosis per staffed bed per month.ResultsCentral line-associated blood stream infections (CLABSI), catheter-associated urinary tract infections (CAUTI), and methicillin-resistant Staphylococcus aureus (MRSA) bacteremia increased as COVID-19 burden increased. There were 60% (95% confidence interval [CI]: 23-108%) more CLABSI, 43% (95% CI: 8-90%) more CAUTI, and 44% (95% CI: 10-88%) more cases of MRSA bacteremia than expected over 7 months based on predicted HAIs had there not been COVID-19 cases. Clostridioides difficile infection was not significantly associated with COVID-19 burden. Microbiology data from 81 of the hospitals corroborated the findings. Notably, rates of hospital-onset bloodstream infections and multidrug resistant organisms, including MRSA, vancomycin-resistant enterococcus, and Gram-negative organisms, were each significantly associated with COVID-19 surges. Finally, clusters of hospital-onset pathogens increased as the COVID-19 burden increased.ConclusionsCOVID-19 surges adversely impact HAI rates and clusters of infections within hospitals, emphasizing the need for balancing COVID-related demands with routine hospital infection prevention.

BackgroundThe Practical, Robust Implementation and Sustainability Model (PRISM) was developed in 2008 as a contextually expanded version of the broadly used Reach, Adoption, Effectiveness, Implementation, and Maintenance (RE-AIM) framework. PRISM provides researchers a pragmatic and intuitive model to improve translation of research interventions into clinical and community practice. Since 2008, the use of PRISM increased across diverse topics, populations, and settings. This citation analysis and scoping systematic review aimed to assess the use of the PRISM framework and to make recommendations for future research.MethodsA literature search was conducted using three databases (PubMed, Web of Science, Scopus) for the period of 2008 and September 2020. After exclusion, reverse citation searches and invitations to experts in the field were used to identify and obtain recommendations for additional articles not identified in the original search. Studies that integrated PRISM into their study design were selected for full abstraction. Unique research studies were abstracted for information on study characteristics (e.g., setting/population, design), PRISM contextual domains, and RE-AIM outcomes.ResultsA total of 180 articles were identified to include PRISM to some degree. Thirty-two articles representing 23 unique studies integrated PRISM within their study design. Study characteristics varied widely and included studies conducted in diverse contexts, but predominately in high-income countries and in clinical out-patient settings. With regards to use, 19 used PRISM for evaluation, 10 for planning/development, 10 for implementation, four for sustainment, and one for dissemination. There was substantial variation across studies in how and to what degree PRISM contextual domains and RE-AIM outcomes were operationalized and connected. Only two studies directly connected individual PRISM context domains with RE-AIM outcomes, and another four included RE-AIM outcomes without direct connection to PRISM domains.ConclusionsThis is the first systematic review of the use of PRISM in various contexts. While there were low levels of 'integrated' use of PRISM and few reports on linkage to RE-AIM outcomes, most studies included important context domains of implementation and sustainability infrastructure and external environment. Recommendations are provided for more consistent and comprehensive use of and reporting on PRISM to inform both research and practice on contextual factors in implementation.

Pathogens use virulence factors to inhibit the immune system1. The guard hypothesis2,3 postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response1. Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC34 as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-15. Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response.

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.

In recent years, the global public health community has increasingly recognized the importance of antimicrobial stewardship (AMS) in the fight to improve outcomes, decrease costs, and curb increases in antimicrobial resistance around the world. However, the subject of antifungal stewardship (AFS) has received less attention. While the principles of AMS guidelines likely apply to stewarding of antifungal agents, there are additional considerations unique to AFS and the complex field of fungal infections that require specific recommendations. In this article, we review the literature on AMS best practices and discuss AFS through the lens of the global core elements of AMS. We offer recommendations for best practices in AFS based on a synthesis of this evidence by an interdisciplinary expert panel of members of the Mycoses Study Group Education and Research Consortium. We also discuss research directions in this rapidly evolving field. AFS is an emerging and important component of AMS, yet requires special considerations in certain areas such as expertise, education, interventions to optimize utilization, therapeutic drug monitoring, and data analysis and reporting.

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassarcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1-6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7-11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15-17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.

Mucormycosis is a difficult to diagnose rare disease with high morbidity and mortality. Diagnosis is often delayed, and disease tends to progress rapidly. Urgent surgical and medical intervention is lifesaving. Guidance on the complex multidisciplinary management has potential to improve prognosis, but approaches differ between health-care settings. From January, 2018, authors from 33 countries in all United Nations regions analysed the published evidence on mucormycosis management and provided consensus recommendations addressing differences between the regions of the world as part of the "One World One Guideline" initiative of the European Confederation of Medical Mycology (ECMM). Diagnostic management does not differ greatly between world regions. Upon suspicion of mucormycosis appropriate imaging is strongly recommended to document extent of disease and is followed by strongly recommended surgical intervention. First-line treatment with high-dose liposomal amphotericin B is strongly recommended, while intravenous isavuconazole and intravenous or delayed release tablet posaconazole are recommended with moderate strength. Both triazoles are strongly recommended salvage treatments. Amphotericin B deoxycholate is recommended against, because of substantial toxicity, but may be the only option in resource limited settings. Management of mucormycosis depends on recognising disease patterns and on early diagnosis. Limited availability of contemporary treatments burdens patients in low and middle income settings. Areas of uncertainty were identified and future research directions specified.

Nucleotide-binding domain, leucine-rich repeat (NLR) proteins constitute a diverse class of innate immune sensors that detect pathogens or stress-associated stimuli in plants and animals. Some NLRs are activated upon direct binding to pathogen-derived ligands. In contrast, we focus here on a vertebrate NLR called NLRP1 that responds to the enzymatic activities of pathogen effectors. We discuss a newly proposed 'functional degradation' mechanism that explains activation and assembly of NLRP1 into an oligomeric complex called an inflammasome. We also discuss how NLRP1 is activated by non-pathogen-associated triggers such as the anti-cancer drug Val-boroPro, or by human disease-associated mutations. Finally, we discuss how research on NLRP1 has led to additional biological insights, including the unexpected discovery of a new CARD8 inflammasome.

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

National evidence-based guidelines recommend offering hydroxyurea to patients with sickle cell anemia 9 months of age and older using shared decision making, but offer no strategies to aid implementation. We developed a hydroxyurea multicomponent decision aid via a needs assessment, clinic observations, and iterative feedback to address parent decision needs and promote a discussion between clinicians and parents. A total of 75 parents and 28 clinicians participated across all phases. The decision aid was rated as useful. Hydroxyurea knowledge improved and decisional conflict decreased supporting the potential for use to facilitate shared decision making in pediatric sickle cell anemia.

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity.

Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging-Alzheimer's Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Detection of cytosolic DNA by the enzyme cGAS triggers the production of cGAMP, a second messenger that binds and activates the adaptor protein STING, which leads to interferon (IFN) production. Here, we found that in vivo natural killer (NK) cell killing of tumor cells, but not of normal cells, depends on STING expression in non-tumor cells. Experiments using transplantable tumor models in STING- and cGAS-deficient mice revealed that cGAS expression by tumor cells was critical for tumor rejection by NK cells. In contrast, cGAS expression by host cells was dispensable, suggesting that tumor-derived cGAMP is transferred to non-tumor cells, where it activates STING. cGAMP administration triggered STING activation and IFN-β production in myeloid cells and B cells but not NK cells. Our results reveal that the anti-tumor response of NK cells critically depends on the cytosolic DNA sensing pathway, similar to its role in defense against pathogens, and identify tumor-derived cGAMP as a major determinant of tumor immunogenicity with implications for cancer immunotherapy.

Inflammasomes are cytosolic multiprotein complexes that initiate protective immunity in response to infection, and can also drive auto-inflammatory diseases, but the cell types and signalling pathways that cause these diseases remain poorly understood. Inflammasomes are broadly expressed in haematopoietic and non-haematopoietic cells and can trigger numerous downstream responses including production of IL-1β, IL-18, eicosanoids and pyroptotic cell death. Here we show a mouse model with endogenous NLRC4 inflammasome activation in Lysozyme2 + cells (monocytes, macrophages and neutrophils) in vivo exhibits a severe systemic inflammatory disease, reminiscent of human patients that carry mutant auto-active NLRC4 alleles. Interestingly, specific NLRC4 activation in Mrp8 + cells (primarily neutrophil lineage) is sufficient to cause severe inflammatory disease. Disease is ameliorated on an Asc -/- background, and can be suppressed by injections of anti-IL-1 receptor antibody. Our results provide insight into the mechanisms by which NLRC4 inflammasome activation mediates auto-inflammatory disease in vivo.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1AB6 and NLRP1BB6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.

Patient-centered health risk assessments (HRAs) that screen for unhealthy behaviors, prioritize concerns, and provide feedback may improve counseling, goal setting, and health. To evaluate the effectiveness of routinely administering a patient-centered HRA, My Own Health Report, for diet, exercise, smoking, alcohol, drug use, stress, depression, anxiety, and sleep, 18 primary care practices were randomized to ask patients to complete My Own Health Report (MOHR) before an office visit (intervention) or continue usual care (control). Intervention practice patients were more likely than control practice patients to be asked about each of eight risks (range of differences 5.3-15.8 %, p

PurposePrimary care needs new models to facilitate advance care planning conversations. These conversations focus on preferences regarding serious illness and may involve patients, decision makers, and health care providers. We describe the feasibility of the first primary care-based group visit model focused on advance care planning.MethodsWe conducted a pilot demonstration of an advance care planning group visit in a geriatrics clinic. Patients were aged at least 65 years. Groups of patients met in 2 sessions of 2 hours each facilitated by a geriatrician and a social worker. Activities included considering personal values, discussing advance care planning, choosing surrogate decision-makers, and completing advance directives. We used the RE-AIM framework to evaluate the project.ResultsTen of 11 clinicians referred patients for participation. Of 80 patients approached, 32 participated in 5 group visit cohorts (a 40% participation rate) and 27 participated in both sessions (an 84% retention rate). Mean age was 79 years; 59% of participants were female and 72% white. Most evaluated the group visit as better than usual clinic visits for discussing advance care planning. Patients reported increases in detailed advance care planning conversations after participating (19% to 41%, P = .02). Qualitative analysis found that older adults were willing to share personal values and challenges related to advance care planning and that they initiated discussions about a broad range of relevant topics.ConclusionA group visit to facilitate discussions about advance care planning and increase patient engagement is feasible. This model warrants further evaluation for effectiveness in improving advance care planning outcomes for patients, clinicians, and the system.

PurposeTo evaluate the association of subretinal hyperreflective material (SHRM) with visual acuity (VA), geographic atrophy (GA), and scar in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT).DesignProspective cohort study within a randomized clinical trial.ParticipantsThe 1185 CATT participants.MethodsMasked readers graded scar and GA on fundus photography and fluorescein angiography and graded SHRM on time-domain and spectral-domain (SD) optical coherence tomography (OCT) throughout 104 weeks. Measurements of SHRM height and width in the fovea, within the center 1 mm(2), or outside the center 1mm(2) were obtained on SD OCT images at 56 (n = 76) and 104 (n = 66) weeks.Main outcome measuresPresence of SHRM, as well as location and size, and associations with VA, scar, and GA.ResultsAmong CATT participants, the percentage with SHRM at enrollment was 77%, decreasing to 68% at 4 weeks after treatment and to 54% at 104 weeks. At 104 weeks, scar was present more often in eyes with persistent SHRM than in eyes with SHRM that resolved (64% vs. 31%; P < 0.0001). Among eyes with detailed evaluation of SHRM at weeks 56 (n = 76) and 104 (n = 66), mean VA letter score was 73.5 (standard error [SE], 2.8), 73.1 (SE, 3.4), 65.3 (SE, 3.5), and 63.9 (SE, 3.7) when SHRM was absent, present outside the central 1 mm(2), present within the central 1 mm(2) but not the foveal center, or present at the foveal center (P = 0.02), respectively. When SHRM was present, the median maximum height under the fovea, within the central 1 mm(2) including the fovea and anywhere within the scan, was 86 μm, 120 μm, and 122 μm, respectively. Visual acuity was decreased with greater SHRM height and width (P < 0.05).ConclusionsIn eyes with neovascular age-related macular degeneration (AMD), SHRM is common and often persists after anti-vascular endothelial growth factor treatment. At 2 years, eyes with scar were more likely to have SHRM than other eyes. Greater SHRM dimensions were associated with worse VA. In eyes with neovascular AMD, SHRM is an important morphologic biomarker.

BackgroundEfforts to improve primary care diabetes management have assessed strategies across heterogeneous groups of patients and practices. However, there is substantial variability in how well practices implement interventions and achieve desired outcomes.ObjectiveTo examine practice contextual features that moderate intervention effectiveness.DesignSecondary analysis of data from a cluster randomized trial of three approaches for implementing the Chronic Care Model to improve diabetes care.ParticipantsForty small to mid-sized primary care practices participated, with 522 clinician and staff member surveys. Outcomes were assessed for 822 established patients with a diagnosis of type 2 diabetes who had at least one visit to the practice in the 18 months following enrollment.Main measuresThe primary outcome was a composite measure of diabetes process of care, ascertained by chart audit, regarding nine quality measures from the American Diabetes Association Physician Recognition Program: HgA1c, foot exam, blood pressure, dilated eye exam, cholesterol, nephropathy screen, flu shot, nutrition counseling, and self-management support. Data from practices included structural and demographic characteristics and Practice Culture Assessment survey subscales (Change Culture, Work Culture, Chaos).Key resultsAcross the three implementation approaches, demographic/structural characteristics (rural vs. urban + .70(p = .006), +2.44(p < .001), -.75(p = .004)); Medicaid: < 20 % vs. ≥ 20 % (-.20(p = .48), +.75 (p = .08), +.60(p = .02)); practice size: < 4 clinicians vs. ≥ 4 clinicians (+.56(p = .02), +1.96(p < .001), +.02(p = .91)); practice Change Culture (high vs. low: -.86(p = .048), +1.71(p = .005), +.34(p = .22)), Work Culture (high vs. low: -.67(p = .18), +2.41(p < .001), +.67(p = .005)) and variability in practice Change Culture (high vs. low: -.24(p = .006), -.20(p = .0771), -.44(p = .0019) and Work Culture (high vs. low: +.56(p = .3160), -1.0(p = .008), -.25 (p = .0216) were associated with trajectories of change in diabetes process of care, either directly or differentially by study arm.ConclusionsThis study supports the need for broader use of methodological approaches to better examine contextual effects on implementation and effectiveness of quality improvement interventions in primary care settings.

Inflammasomes comprise a family of cytosolic multi-protein complexes that sense infection, or other threats, and initiate inflammation via the recruitment and activation of the Caspase-1 protease. Although the precise molecular mechanism by which most inflammasomes are activated remains a subject of considerable debate, the NAIP/NLRC4 subfamily of inflammasomes is increasingly well understood. A crystal structure of NLRC4 was recently reported, and a domain in NAIPs that recognizes bacterial ligands was identified. In addition, gain-of-function mutations in NLRC4 have been shown to cause an auto-inflammatory syndrome in humans. Lastly, the NAIP/NLRC4 inflammasome has been shown to provide a novel form of cell intrinsic defense against Salmonella infection, involving expulsion of infected cells from the intestinal epithelium.

Despite documented laboratory and clinical benefits of hydroxyurea for children with sickle cell anemia (SCA), the drug's long-term safety and efficacy remains poorly defined. The HUSOFT trial and extension study examined feasibility, toxicity, and hematological efficacy of hydroxyurea in infants with SCA. This report describes HUSOFT participants who have continued hydroxyurea therapy for 15 years. With IRB approval, medical records were reviewed for clinical, laboratory, and growth parameters. Twenty-eight infants enrolled in the original 2-year HUSOFT study received open-label liquid hydroxyurea at 20 mg/kg/day; 17 completed the extension study with dose escalation to 30 mg/kg/day. Eight of these 17 (6 girls and 2 boys, all HbSS) have continued on daily hydroxyurea for at least 15 years (median age at last follow-up 17.6 years) without interruption. All hematologic indices (Hb concentration, mean corpuscular volume (MCV), fetal hemoglobin) showed sustained effect after 15 years. The median maximum tolerated dose of hydroxyurea has decreased from 30 to 26 mg/kg/day (range 19.5-31.2); neutropenia [absolute neutrophil count (ANC)

PurposeTo describe the frequency and patient-reported readiness to change, desire to discuss, and perceived importance of 13 health risk factors in a diverse range of primary care practices.MethodsPatients (n = 1,707) in 9 primary care practices in the My Own Health Report (MOHR) trial reported general, behavioral, and psychosocial risk factors (body mass index [BMI], health status, diet, physical activity, sleep, drug use, stress, anxiety or worry, and depression). We classified responses as "at risk" or "healthy" for each factor, and patients indicated their readiness to change and/or desire to discuss identified risk factors with providers. Patients also selected 1 of the factors they were ready to change as most important. We then calculated frequencies within and across these factors and examined variation by patient characteristics and across practices.ResultsOn average, patients had 5.8 (SD = 2.12; range, 0-13) unhealthy behaviors and mental health risk factors. About 55% of patients had more than 6 risk factors. On average, patients wanted to change 1.2 and discuss 0.7 risks. The most common risks were inadequate fruit/vegetable consumption (84.5%) and overweight/obesity (79.6%). Patients were most ready to change BMI (33.3%) and depression (30.7%), and most wanted to discuss depression (41.9%) and anxiety or worry (35.2%). Overall, patients rated health status as most important.ConclusionsImplementing routine comprehensive health risk assessments in primary care will likely identify a high number of behavioral and psychosocial health risks. By soliciting patient priorities, providers and patients can better manage counseling and behavior change.

Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP.

UnlabelledSTING (stimulator of interferon [IFN] genes) initiates type I IFN responses in mammalian cells through the detection of microbial nucleic acids. The membrane-bound obligate intracellular bacterium Chlamydia trachomatis induces a STING-dependent type I IFN response in infected cells, yet the IFN-inducing ligand remains unknown. In this report, we provide evidence that Chlamydia synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite not previously identified in Gram-negative bacteria, and that this metabolite is a prominent ligand for STING-mediated activation of IFN responses during infection. We used primary mouse lung fibroblasts and HEK293T cells to compare IFN-β responses to Chlamydia infection, c-di-AMP, and other type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment induced type I IFN responses in cells expressing STING but not in cells expressing STING variants that cannot sense cyclic dinucleotides but still respond to cytoplasmic DNA. The failure to induce a type I IFN response to Chlamydia and c-di-AMP correlated with the inability of STING to relocalize from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing such a response in Chlamydia-infected cells.ImportanceThis study shows that the Gram-negative obligate pathogen Chlamydia trachomatis, a major cause of pelvic inflammatory disease and infertility, synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite that thus far has been described only in Gram-positive bacteria. We further provide evidence that the host cell employs an endoplasmic reticulum (ER)-localized cytoplasmic sensor, STING (stimulator of interferon [IFN] genes), to detect c-di-AMP synthesized by Chlamydia and induce a protective IFN response. This detection occurs even though Chlamydia is confined to a membrane-bound vacuole. This raises the possibility that the ER, an organelle that innervates the entire cytoplasm, is equipped with pattern recognition receptors that can directly survey membrane-bound pathogen-containing vacuoles for leaking microbe-specific metabolites to mount type I IFN responses required to control microbial infections.

The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

ObjectivesThe study objectives were to determine whether massage therapy reduces symptoms of depression in subjects with human immunodeficiency virus (HIV) disease.DesignSubjects were randomized non-blinded into one of three parallel groups to receive Swedish massage or to one of two control groups, touch or no intervention for eight weeks.Settings/locationThe study was conducted at the Department of Psychiatry and Behavioral Neurosciences at Cedars-Sinai Medical Center in Los Angeles, California, which provided primary clinical care in an institutional setting.SubjectsStudy inclusion required being at least 16 years of age, HIV-seropositive, with a diagnosis of major depressive disorder. Subjects had to be on a stable neuropsychiatric, analgesic, and antiretroviral regimen for >30 days with no plans to modify therapy for the duration of the study. Approximately 40% of the subjects were currently taking antidepressants. All subjects were medically stable. Fifty-four (54) subjects were randomized, 50 completed at least 1 week (intent-to-treat; ITT), and 37 completed the study (completers).InterventionsSwedish massage and touch subjects visited the massage therapist for 1 hour twice per week. The touch group had a massage therapist place both hands on the subject with slight pressure, but no massage, in a uniform distribution in the same pattern used for the massage subjects.Outcome measuresThe primary outcome measure was the Hamilton Rating Scale for Depression score, with the secondary outcome measure being the Beck Depression Inventory.ResultsFor both the ITT and completers analyses, massage significantly reduced the severity of depression beginning at week 4 (p ≤ 0.04) and continuing at weeks 6 (p ≤ 0.03) and 8 (p ≤ 0.005) compared to no intervention and/or touch.ConclusionsThe results indicate that massage therapy can reduce symptoms of depression in subjects with HIV disease. The durability of the response, optimal "dose" of massage, and mechanisms by which massage exerts its antidepressant effects remain to be determined.

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin and PrgJ, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7-11), but is widely assumed to have only an auxiliary role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is a member of a small multigene family, raising the possibility of redundancy and functional specialization among Naip genes. Here we show in mice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2-NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5-NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP-NLRC4 inflammasomes.

ObjectiveTo clarify previous findings that diabetes distress is related to glycemic control and self-management whereas measures of depression are not, using both binary and continuous measures of depression.Research design and methodsFour hundred and sixty-three type 2 patients completed measures of diabetes distress (Diabetes Distress Scale [DDS]) and clinical depression (Patient Health Questionnaire 8 [PHQ8]). PHQ8 was employed as either a binary (>or=10) or continuous variable. Dependent variables were A1C, diet, physical activity (PA), and medication adherence (MA).ResultsThe inclusion of a binary or continuous PHQ8 score yielded no differences in any equation. DDS was significantly associated with A1C and PA, whereas PHQ8 was not; both DDS and PHQ8 were significantly and independently associated with diet and MA.ConclusionsThe lack of association between depression and glycemic control is not due to the use of a binary measure of depression. Findings further clarify the significant association between distress and A1C.

ObjectiveTo determine the concurrent, prospective, and time-concordant relationships among major depressive disorder (MDD), depressive symptoms, and diabetes distress with glycemic control.Research design and methodsIn a noninterventional study, we assessed 506 type 2 diabetic patients for MDD (Composite International Diagnostic Interview), for depressive symptoms (Center for Epidemiological Studies-Depression), and for diabetes distress (Diabetes Distress Scale), along with self-management, stress, demographics, and diabetes status, at baseline and 9 and 18 months later. Using multilevel modeling (MLM), we explored the cross-sectional relationships of the three affective variables with A1C, the prospective relationships of baseline variables with change in A1C over time, and the time-concordant relationships with A1C.ResultsAll three affective variables were moderately intercorrelated, although the relationship between depressive symptoms and diabetes distress was greater than the relationship of either with MDD. In the cross-sectional MLM, only diabetes distress but not MDD or depressive symptoms was significantly associated with A1C. None of the three affective variables were linked with A1C in prospective analyses. Only diabetes distress displayed significant time-concordant relationships with A1C.ConclusionsWe found no concurrent or longitudinal association between MDD or depressive symptoms with A1C, whereas both concurrent and time-concordant relationships were found between diabetes distress and A1C. What has been called "depression" among type 2 diabetic patients may really be two conditions, MDD and diabetes distress, with only the latter displaying significant associations with A1C. Ongoing evaluation of both diabetes distress and MDD may be helpful in clinical settings.

Although tobacco smoking, which has been linked to depression, is a major public health problem, little is known about the neurobiological factors that confer vulnerability to smoking in youngsters and the effects of adolescent smoking on the course of depression. This study examined whether hypothalamic-pituitary-adrenal (HPA) activity and stressful life experiences are related to smoking behavior in depressed and non-depressed adolescents, and whether smoking predicts a worsening course of depression. Smoking history and stressful experiences were assessed in 151 adolescents (48 with no personal or family history of psychiatric disorder, 48 with no psychiatric history, but at high risk for depression by virtue of parental depression, and 55 with current major depressive disorder). Evening salivary cortisol and nocturnal urinary-free cortisol were measured for three consecutive evenings. The participants were then followed at regular intervals for up to 5 years to assess smoking history, clinical course of depression and stressful experiences during the follow-up period. Increased evening/night-time cortisol levels were associated with both initiation and persistence of smoking during follow-up. Stressful life experiences further increased the risk for smoking in depressed as well as non-depressed youth. Smoking was also associated with a higher frequency of depressive episodes during follow-up. A model that included stressful experiences and cortisol levels reduced the contribution of smoking per se to depression. High evening/night-time cortisol level appears to be a vulnerability marker for smoking in adolescents, with stressful experiences further increasing the risk for smoking in vulnerable youth. High evening/night-time cortisol levels and stressful experiences accounted, at least partially, for the association between depressive illness and smoking behavior.

Previous work has demonstrated reliable electroencephalographic (EEG) sleep and hypothalamic-pituitary-adrenal (HPA) changes associated with adult major depressive disorder. These changes might be evident before clinical manifestation of the illness in at-risk persons. The aim of the study was to identify depression-related EEG sleep and HPA changes in healthy adolescents at high risk for depression, and to examine the relationship between EEG sleep (or HPA) changes and the onset of depression. Forty-eight adolescent volunteers with no personal history of a psychiatric illness, including depression, but who were at high risk for developing depression by virtue of parental depression (high-risk group), and 48 adolescent volunteers with no personal or family history of a psychiatric disorder (normal controls) were recruited. EEG sleep and HPA measures were collected on three consecutive evenings and nights at baseline. Clinical follow-up evaluations were conducted at regular intervals over a 5-year period. Compared with normal controls, adolescents at high risk for depression had shorter latency to rapid eye movement (REM) sleep, increased phasic REM sleep, more REM sleep and elevated nocturnal urinary-free cortisol (NUFC) excretion at baseline. Shorter REM latency, higher REM density and elevated NUFC (measured at baseline) were associated with the development of depression during follow-up. The findings that REM sleep abnormalities and elevated HPA activity occur before the onset of depression in at-risk adolescents suggest that these variables serve as vulnerability markers for the illness.