Subcellular distribution of apoptosis associated speck-like protein mediates inflammasome assembly: A novel mechanism in the regulation of interleukin-1beta release

Inflammation is an essential component of the innate immune defense against pathogens. The process of inflammation is mediated by the rapid release of intercellular mediators, which function in the activation and recruitment of cellular components of both the innate and adaptive immune system. However, dysregulation of this process, resulting in sustained inflammation is the underlying cause of a number of common pathological conditions. These include hereditary autoinflammatory disorders, gout, arthritis, asthma, and cancer. Furthermore, IL-1beta is a key component of the tumor microenvironment, which serves to recruit tumor-associated macrophages, promote angiogenesis, enhance NF-kB activation, and suppress anti-tumor immunity. The studies presented here explore novel regulatory mechanisms of IL-1beta maturation through the modulation of the inflammasome adaptor protein ASC. ASC is an intriguing protein, which participates in both inflammatory and apoptotic pathways and is frequently silenced by aberrant methylation in a large number of cancers. It is a small protein, consisting of only two protein-interaction domains, an N-terminal PYD, and a C-terminal CARD. Numerous studies have found that it is required for inflammasome assembly following activation of a number of diverse cytoplasmic NLR receptors. Therefore, regulation of this protein has broad implications for IL-1beta maturation.;In the first study, we examined the endogenous localization of ASC. Several independent research groups had found that ectopic expression of ASC results in several distinct localization patterns including nuclear, cytoplasmic, and the formation of a characteristic perinuclear aggregate termed a \u27speck\u27. However, the subcellular distribution of endogenous ASC had never been evaluated. Therefore, we examined the subcellular localization pattern of ASC in several monocytic cell lines as well as in primary monocytes and macrophages. We discovered that in resting monocytes, ASC is localized diffusely throughout the nucleus. However, upon inflammatory stimulation of the cells, we determined that ASC was rapidly redistributed to the cytoplasm where it subsequently formed a perinuclear aggregate, reminiscent of the \u27specks\u27 observed upon overexpression of this protein. Furthermore, we determined that other core inflammasome proteins NLRP3 and caspase-1 co-localized with ASC in these aggregates, suggesting that they represent inflammasomes. Additionally, we found that nuclear export of ASC is necessary for proper inflammasome assembly and processing of IL-1beta.;In Study 2, we identified Mycoplasma sp. as a novel activator of the inflammasome. Infection with Mycoplasma sp. cause a number of organ-specific diseases in humans, and they have also been linked to a number of proinflammatory disorders. Therefore, we hypothesized that IL-beta played a role in the innate immune defense against these pathogens. We discovered that incubation of monocytes and macrophages with either live or heat-killed Mycoplasma sp. stimulated the secretion of mature IL-1beta at levels comparable to those observed with known inflammasome activators. Furthermore, we also determined that Mycoplasma infection induced the nuclear to cytoplasmic translocation of ASC, as well as the formation of perinuclear aggregates, just as we observed with known inflammasome activators in Study 1. Finally, we showed that Mycoplasma -induced release of IL-1beta was dependent upon ASC, further indicating the role of the inflammasome in this process. Based upon this data, we hypothesize that chronic infection with Mycoplasma sp. results in sustained production of IL-1beta, which leads to a prolonged inflammatory state.;In our third and final study, we identified and characterized three ASC isoforms with regards to their subcellular localization, and their ability to function as an inflammasome adaptor protein. ASCb lacks the linker domain, ASCc possesses an in-frame deletion in the PYD, which does not disrupt the linker region or the CARD, and ASCd expresses only the first 35 amino acids of the PYD, which is fused to a novel peptide. Upon ectopic expression, each isoform exhibited a different localization pattern, and none of them formed the classic perinuclear \u27speck\u27, which has become the hallmark of full-length ASCa. Upon co-expression with either NLRP3(R260W) or caspase-1, co-localization was only observed by those isoforms, which expressed a fully intact PYD or CARD, respectively. Functionally, only ASCb was able to mediate IL-1beta processing when co-expressed with NLRP3(R260W) and pro-caspase-1, although to a lesser extent, presumably because neither of its protein interaction domains were disrupted.;In summary, these studies show novel mechanisms by which IL-1beta release is regulated and induced. The discovery of novel ASC isoforms and the role its subcellular distribution plays in the regulation of IL-1beta processing provide us with new insights into how inflammasome assembly is regulated and fin-tuned. Furthermore, the nuclear to cytoplasmic redistribution of ASC provides us with a potential new therapeutic target that would prevent the release of IL-1beta without impacting the expression of ASC

SOST Inhibits Prostate Cancer Invasion.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings

Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) regulates inflammasomes

Background The apoptotic speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for caspase 1 mediated interleukin (IL)-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs), which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an inflammasome adaptor and its precise function and relation to ASC will need further investigation. Conclusions Alternative splicing and potentially other editing mechanisms generate ASC isoforms with distinct abilities to function as inflammasome adaptor, which is potentially utilized to regulate inflammasomes during the inflammatory host response

A thoracic surgery clinic dedicated to indeterminate pulmonary nodules: Too many scans and too little pathology?

ObjectiveWidespread application of computed tomographic scans has increased detection of asymptomatic pulmonary nodules. A dedicated clinic was established to encourage referral and manage large numbers of patients with such nodules.MethodsPatients were evaluated periodically by a nurse practitioner with surgeon oversight, and follow-up imaging was centralized. Patients were rescanned at intervals on the basis of radiologist recommendation.ResultsA total of 414 patients, 189 male and 225 female with a median age of 60.2 years (20.7–84.1 years), were evaluated since April 2000. Median follow-up was 1.51 years (0–6.65 years). Thirty-seven percent (153/414) were older than 60 years with at least 10 pack-years of tobacco use, whereas 30% (123/414) had never smoked. A total of 286 patients completed at least 2 years of follow-up computed tomographic evaluation. After 2 years, 24.2% (69/286) were deemed in stable condition and were discharged from further follow-up, whereas 22.4% (64/286) of patients were followed up longer than 2 years owing to the development of new nodules. Forty-five percent (127/286) of patients did not complete their recommended follow-up at our clinic. Overall, 3% (13/414) of our patients have been shown to have a malignant tumor. Only 5 patients underwent curative resection of a primary lung cancer.ConclusionIn a population of patients with indeterminate nodules in routine clinical practice, few patients required intervention and few cancers were detected. Although the benefits of a “nodule” clinic may include patient reassurance and convenience to referring physicians, a significant number of patients did not complete their follow-up in our clinic

Standardized Direct Observation Assessment Tool: Using a Training Video.

BACKGROUND: We developed a DVD training tool to educate physicians evaluating emergency residents on accurate Standardized Direct Observation Assessment Tool (SDOT) application. OBJECTIVE: Our goal was to assess whether this training video improved attendings\u27 and senior residents\u27 SDOT use. METHODS: Participants voluntarily completed SDOT evaluations based on a scripted test video. A DVD with positive and negative scenarios of proper SDOT use was viewed. It included education on appropriate recording of 26 behaviors. The test scenario was viewed again and follow-up SDOTs submitted. Performances by attendings and residents on the pre- and post-test SDOTs were compared. RESULTS: Twenty-six attendings and 26 senior residents participated. Prior SDOT experience was noted for 8 attendings and 11 residents. For 20 anchors, participants recorded observed behaviors with statistically significant difference on one each of the pretest (no. 20; p = 0.034) and post-test (no. 14; p = 0.041) SDOTs. On global competency assessments, pretest medical knowledge (p = 0.016) differed significantly between groups. The training intervention changed one anchor (no. 5; p = 0.035) and one global assessment (systems-based practice; p = 0.031) more negatively for residents. Recording SDOTs with exact agreement occurred 48.73% for attendings pretest and 54.41% post-test; resident scores were 45.86% and 49.55%, respectively. DVD exposure slightly raised attending scores (p = 0.289) and significantly lowered resident scores (p = 0.046). CONCLUSIONS: Exposure to an independently developed SDOT training video tended to raise attending scores, though without significance, while at the same time lowered senior resident scores statistically significantly. Emergency attendings\u27 and senior residents\u27 SDOT scoring rarely differed with significance; about half of anchor behaviors were recorded with exact agreement. This suggests senior residents, with appropriate education, may participate in SDOT assessment

Down regulation of E-Cadherin (ECAD) - a predictor for occult metastatic disease in sentinel node biopsy of early squamous cell carcinomas of the oral cavity and oropharynx

<p>Abstract</p> <p>Background</p> <p>Prognostic factors in predicting occult lymph node metastasis in patients with head and neck squamous-cell carcinoma (HNSCC) are necessary to improve the results of the sentinel lymph node procedure in this tumour type. The E-Cadherin glycoprotein is an intercellular adhesion molecule in epithelial cells, which plays an important role in establishing and maintaining intercellular connections.</p> <p>Objectives</p> <p>To determine the value of the molecular marker E-Cadherin in predicting regional metastatic disease.</p> <p>Methods</p> <p>E-Cadherin expression in tumour tissue of 120 patients with HNSCC of the oral cavity and oropharynx were evaluated using the tissue microarray technique. 110 tumours were located in the oral cavity (91.7%; mostly tongue), 10 tumours in the oropharynx (8.3%). Intensity of E-Cadherin expression was quantified by the Intensity Reactivity Score (IRS). These results were correlated with the lymph node status of biopsied sentinel lymph nodes. Univariate and multivariate analysis was used to determine statistical significance.</p> <p>Results</p> <p>pT-stage, gender, tumour side and location did not correlate with lymph node metastasis. Differentiation grade (<it>p </it>= 0.018) and down regulation of E-Cadherin expression significantly correlate with positive lymph node status (<it>p </it>= 0.005) in univariate and multivariate analysis.</p> <p>Conclusion</p> <p>These data suggest that loss of E-cadherin expression is associated with increased lymhogeneous metastasis of HNSCC. E-cadherin immunohistochemistry may be used as a predictor for lymph node metastasis in squamous cell carcinoma of the oral cavity and oropharynx.</p> <p><b>Level of evidence: 2b</b></p

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Global estimates of mortality associated with long-term exposure to outdoor fine particulate matter.

Exposure to ambient fine particulate matter (PM2.5) is a major global health concern. Quantitative estimates of attributable mortality are based on disease-specific hazard ratio models that incorporate risk information from multiple PM2.5 sources (outdoor and indoor air pollution from use of solid fuels and secondhand and active smoking), requiring assumptions about equivalent exposure and toxicity. We relax these contentious assumptions by constructing a PM2.5-mortality hazard ratio function based only on cohort studies of outdoor air pollution that covers the global exposure range. We modeled the shape of the association between PM2.5 and nonaccidental mortality using data from 41 cohorts from 16 countries-the Global Exposure Mortality Model (GEMM). We then constructed GEMMs for five specific causes of death examined by the global burden of disease (GBD). The GEMM predicts 8.9 million [95% confidence interval (CI): 7.5-10.3] deaths in 2015, a figure 30% larger than that predicted by the sum of deaths among the five specific causes (6.9; 95% CI: 4.9-8.5) and 120% larger than the risk function used in the GBD (4.0; 95% CI: 3.3-4.8). Differences between the GEMM and GBD risk functions are larger for a 20% reduction in concentrations, with the GEMM predicting 220% higher excess deaths. These results suggest that PM2.5 exposure may be related to additional causes of death than the five considered by the GBD and that incorporation of risk information from other, nonoutdoor, particle sources leads to underestimation of disease burden, especially at higher concentrations

The Monarch Initiative in 2024: an analytic platform integrating phenotypes, genes and diseases across species.

Bridging the gap between genetic variations, environmental determinants, and phenotypic outcomes is critical for supporting clinical diagnosis and understanding mechanisms of diseases. It requires integrating open data at a global scale. The Monarch Initiative advances these goals by developing open ontologies, semantic data models, and knowledge graphs for translational research. The Monarch App is an integrated platform combining data about genes, phenotypes, and diseases across species. Monarch\u27s APIs enable access to carefully curated datasets and advanced analysis tools that support the understanding and diagnosis of disease for diverse applications such as variant prioritization, deep phenotyping, and patient profile-matching. We have migrated our system into a scalable, cloud-based infrastructure; simplified Monarch\u27s data ingestion and knowledge graph integration systems; enhanced data mapping and integration standards; and developed a new user interface with novel search and graph navigation features. Furthermore, we advanced Monarch\u27s analytic tools by developing a customized plugin for OpenAI\u27s ChatGPT to increase the reliability of its responses about phenotypic data, allowing us to interrogate the knowledge in the Monarch graph using state-of-the-art Large Language Models. The resources of the Monarch Initiative can be found at monarchinitiative.org and its corresponding code repository at github.com/monarch-initiative/monarch-app