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Amsterdam, Netherlands
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Amsterdam, Netherlands

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News Article | May 1, 2017
Site: www.futurity.org

While brain inflammation is a major part of neurodegenerative diseases including multiple sclerosis, Alzheimer’s, Parkinson’s, ALS, and others, comparatively little is known about its causes and exact role in various diseases. Now, researchers may have made a key discovery about the molecular trigger for brain inflammation, a new study suggests. In a study published in the Journal of Experimental Medicine, researchers identified key molecules that drive brain inflammation in a mouse model of multiple sclerosis—molecules that are present at abnormally high levels in the brains of humans with the disease. The findings show that these inflammatory molecules are ripe targets for further study and potential targets for future multiple sclerosis treatments. The research may also lead to a better understanding of Alzheimer’s, traumatic brain injury, stroke, and other diseases that involve neuroinflammation. “We need to better understand brain inflammation at the molecular level in order to treat neurodegenerative conditions,” says Jenny Ting, a professor of genetics at University of North Carolina at Chapel Hill, who is also a member of the university’s Lineberger Comprehensive Cancer Center. “Our study shows how two proteins that control inflammation are crucial to a particular kind of brain inflammation.” The study began as an investigation of LPC (lysophosphatidylcholine), a fat-related signaling molecule that researchers have suspected stokes harmful brain inflammation in multiple sclerosis and other central nervous system diseases. In initial experiments, study co-lead authors—postdoctoral researcher Haitao Guo, graduate student Leslie Freeman, and former graduate student Sushmita Jha—found evidence that LPC triggers the inflammatory activation of mouse immune cells through two proteins called NLRP3 and NLRC4. NLRP3 and NLRC4 are components of the so-called innate immune system—a network of infection-fighting molecules and cells evolutionarily older than the better-known adaptive immune system’s T-cells, B-cells, and antibodies. Like other NLR-family proteins, NLRP3 and NLRC4 appear to have evolved to detect molecular patterns associated with certain microbes. The two proteins trigger inflammation in response to these microbes. There is evidence, too, that NLR-family proteins can trigger inflammation in response to non-microbial signals related to tissue damage. LPC is suspected to be one such kind of signal, and it is this sort of non-microbial tissue inflammation that researchers think is involved in neurodegenerative diseases. In previous studies, NLRP3 was shown to be a factor in brain inflammation in multiple sclerosis and Alzheimer’s disease. But no one had reported a brain inflammation role for NLRC4 in neurodegenerative diseases involving animal models. To investigate that possibility, Freeman, Guo, and Jha examined mouse astrocytes and microglia—resident brain cells that can perform immune functions in the nervous system. These cells are usually the main sources of inflammation in neurodegenerative diseases. Ting’s team found that LPC could induce an inflammatory response in these brain cells, as well, in a way dependent on NLRP3 and NLRC4. The researchers then worked with a mouse model of multiple sclerosis. They used a chemical called cuprizone to induce brain inflammation. This chemical also helped them strip the fatty layer surrounding nerve fibers. They found that the usual inflammatory activation of astrocytes and microglia, along with the stripping of nerve fibers, was greatly reduced when the mice lacked the genes for both NLRP3 and NLRC4. “Essentially, we saw a profound reduction of the inflammatory disease in these mice,” Guo says. “And where just one of those genes was absent, we didn’t see as pronounced a reduction of inflammation.” Underscoring the likely clinical relevance of these findings, the group found high levels of NLRC4 in astrocytes and microglia from the brain-inflamed mice, as well as in biopsied brain tissue from multiple sclerosis patients. Affected mouse and human brain tissue also showed abnormally high levels of an LPC cell receptor protein called G2A. “This is direct evidence of the importance of NLRC4 and NLRP3 in astrocytic and microglial inflammation, and we showed that this damage-associated molecule called LPC triggers the inflammation,” says Guo. The National Multiple Sclerosis Society and National Institutes of Health funded this work.


News Article | April 27, 2017
Site: www.biosciencetechnology.com

Brain inflammation is a key component of multiple sclerosis, Alzheimer’s, Parkinson’s, ALS, and most other major neurodegenerative diseases. How inflammation starts, how it’s sustained, and how it contributes to these diseases is not well understood, but scientists from the University of North Carolina School of Medicine have just found some important clues. In a study published in the Journal of Experimental Medicine, UNC researchers led by Jenny Ting, Ph.D., the William R. Kenan Distinguished Professor of Genetics, identified key molecules that drive brain inflammation in a mouse model of multiple sclerosis – molecules that are present at abnormally high levels in the brains of humans with the disease. The findings show that these inflammatory molecules are ripe targets for further study and potential targets for future multiple sclerosis treatments. The research may also lead to a better understanding of Alzheimer’s, traumatic brain injury, stroke and other diseases that involve neuroinflammation. “We need to better understand brain inflammation at the molecular level in order to treat neurodegenerative conditions,” said Ting, who is also a member of the UNC Lineberger Comprehensive Cancer Center. “Our study shows how two proteins that control inflammation are crucial to a particular kind of brain inflammation.” The study began as an investigation of LPC (lysophosphatidylcholine), a fat-related signaling molecule that researchers have suspected stokes harmful brain inflammation in multiple sclerosis and other central nervous system diseases. In initial experiments, study co-lead authors – UNC postdoctoral researcher Haitao Guo, PhD, graduate student Leslie Freeman, and former graduate student Sushmita Jha, PhD (now an assistant professor at the Indian Institute of Technology Jodhpur) – found evidence that LPC triggers the inflammatory activation of mouse immune cells through two proteins called NLRP3 and NLRC4. NLRP3 and NLRC4 are components of the so-called innate immune system – a network of infection-fighting molecules and cells evolutionarily older than the better-known adaptive immune system’s T-cells, B-cells, and antibodies. Like other NLR-family proteins, NLRP3 and NLRC4 appear to have evolved to detect molecular patterns associated with certain microbes. The two proteins trigger inflammation in response to these microbes. There is evidence, too, that NLR-family proteins can trigger inflammation in response to non-microbial signals related to tissue damage. LPC is suspected to be one such kind of signal, and it is this sort of non-microbial tissue inflammation that researchers think is involved in neurodegenerative diseases. In previous studies, NLRP3 was shown to be a factor in brain inflammation in multiple sclerosis and Alzheimer’s disease. But no one had reported a brain inflammation role for NLRC4 in neurodegenerative diseases involving animal models. To investigate that possibility, Freeman, Guo, and Jha examined mouse astrocytes and microglia – resident brain cells that can perform immune functions in the nervous system. These cells are usually the main sources of inflammation in neurodegenerative diseases. Ting’s team found that LPC could induce an inflammatory response in these brain cells, as well, in a way dependent on NLRP3 and NLRC4. The researchers then worked with a mouse model of multiple sclerosis. They used a chemical called cuprizone to induce brain inflammation. This chemical also helped them strip the fatty layer surrounding nerve fibers. They found that the usual inflammatory activation of astrocytes and microglia, along with the stripping of nerve fibers, was greatly reduced when the mice lacked the genes for both NLRP3 and NLRC4. “Essentially, we saw a profound reduction of the inflammatory disease in these mice,” Guo said. “And where just one of those genes was absent, we didn’t see as pronounced a reduction of inflammation.” Underscoring the likely clinical relevance of these findings, the group found high levels of NLRC4 in astrocytes and microglia from the brain-inflamed mice, as well as in biopsied brain tissue from multiple sclerosis patients. Affected mouse and human brain tissue also showed abnormally high levels of an LPC cell receptor protein called G2A. “This is direct evidence of the importance of NLRC4 and NLRP3 in astrocytic and microglial inflammation, and we showed that this damage-associated molecule called LPC triggers the inflammation,” said Guo.


A blood test that has shown promise in predicting how cancer will progress and what treatments will be most effective for a given patient may not be reliable for either, according to a new Penn Medicine study published this week in Cancer, a peer-reviewed journal of the American Cancer Society. Investigators have been looking for a biomarker in bladder cancer, and one emerging candidate is the neutrophil-to-lymphocyte ratio (NLR). Previous studies have linked an elevated NLR with worse overall survival after radical cystectomy, a surgery in which the entire bladder and nearby lymph nodes are removed. Other studies suggest NLR correlates with the amount of cancer found during surgery, meaning the blood test might predict which patients will benefit from pre-surgery chemotherapy to shrink their tumors. But new research led by Eric Ojerholm, MD, a resident physician in the department of Radiation Oncology in the Perelman School of Medicine at the University of Pennsylvania, raises doubts about NLR as a biomarker. In contrast to previous studies, Dr. Ojerholm's team found NLR is not effective at predicting the overall survival of patients with muscle-invasive bladder cancer. Further, they found NLR was not helpful in determining which patients would benefit from chemotherapy before surgery. Ojerholm said the discrepancy between the findings of this study and previous work comes in the methodology. "Dozens of earlier studies reported NLR as a biomarker for bladder cancer, and we hoped that this would be true," Ojerholm said. "Yet extraordinary claims require extraordinary evidence. And all prior studies relied on observational datasets. Many also used statistical methods that can lead to false positive results. So we decided to rigorously put NLR to the test." Ojerholm's team analyzed data that was collected in real-time during a prospective clinical trial, making this the first study of NLR in bladder cancer not to rely on observational data. The study analyzed SWOG 8710, which was a randomized Phase III trial of 317 patients with muscle-invasive bladder cancer. All patients were treated with radical cystectomies. Half had pre-surgery chemotherapy, while the other half did not. "The trial we used has a few big advantages to study NLR," Ojerholm said. "First, baseline blood samples were collected as part of the trial protocol. Second, the study's long-term follow-up gave us adequate 'statistical power,' meaning that if NLR really was a biomarker, then we should be able to detect it. Third, the trial randomly assigned some patients to receive pre-surgery chemotherapy. This allowed us to test NLR both as a prognostic and predictive biomarker." Of the 317 total patients, Ojerholm and his team identified 230 for a prognostic analysis to see if NLR could serve as a predictor of how long patients would live after curative treatment. They identified 263 others for a predictive analysis to see if NLR could tell which patients would respond to chemotherapy. There was a median follow-up of 18.6 years. For the prognostic analysis, NLR was not a significant factor in overall survival. The important factors were age and whether the patient received pre-surgery chemotherapy. For the predictive analysis, NLR did not predict which patients benefitted from chemotherapy. On the question of why most previous publications supported NLR as a biomarker, Ojerholm pointed to several factors beyond methodology and statistical design. "There's also the problem of publication bias," Ojerholm explained. "Sometimes authors won't submit negative results, and sometimes journals won't accept them. That could be a real issue as NLR research continues." Ojerholm stressed that no single study is definitive, and doctors must weigh results from the entire literature. "Yet this study does raise questions about NLR for bladder cancer," Ojerholm added, "and we need more evidence before using this biomarker in clinical practice."


News Article | October 27, 2016
Site: www.eurekalert.org

PHILADELPHIA--A blood test that has shown promise in predicting how cancer will progress and what treatments will be most effective for a given patient may not be reliable for either, according to a new Penn Medicine study published this week in Cancer, a peer-reviewed journal of the American Cancer Society. Investigators have been looking for a biomarker in bladder cancer, and one emerging candidate is the neutrophil-to-lymphocyte ratio (NLR). Previous studies have linked an elevated NLR with worse overall survival after radical cystectomy, a surgery in which the entire bladder and nearby lymph nodes are removed. Other studies suggest NLR correlates with the amount of cancer found during surgery, meaning the blood test might predict which patients will benefit from pre-surgery chemotherapy to shrink their tumors. But new research led by Eric Ojerholm, MD, a resident physician in the department of Radiation Oncology in the Perelman School of Medicine at the University of Pennsylvania, raises doubts about NLR as a biomarker. In contrast to previous studies, Dr. Ojerholm's team found NLR is not effective at predicting the overall survival of patients with muscle-invasive bladder cancer. Further, they found NLR was not helpful in determining which patients would benefit from chemotherapy before surgery. Ojerholm said the discrepancy between the findings of this study and previous work comes in the methodology. "Dozens of earlier studies reported NLR as a biomarker for bladder cancer, and we hoped that this would be true," Ojerholm said. "Yet extraordinary claims require extraordinary evidence. And all prior studies relied on observational datasets. Many also used statistical methods that can lead to false positive results. So we decided to rigorously put NLR to the test." Ojerholm's team analyzed data that was collected in real-time during a prospective clinical trial, making this the first study of NLR in bladder cancer not to rely on observational data. The study analyzed SWOG 8710, which was a randomized Phase III trial of 317 patients with muscle-invasive bladder cancer. All patients were treated with radical cystectomies. Half had pre-surgery chemotherapy, while the other half did not. "The trial we used has a few big advantages to study NLR," Ojerholm said. "First, baseline blood samples were collected as part of the trial protocol. Second, the study's long-term follow-up gave us adequate 'statistical power,' meaning that if NLR really was a biomarker, then we should be able to detect it. Third, the trial randomly assigned some patients to receive pre-surgery chemotherapy. This allowed us to test NLR both as a prognostic and predictive biomarker." Of the 317 total patients, Ojerholm and his team identified 230 for a prognostic analysis to see if NLR could serve as a predictor of how long patients would live after curative treatment. They identified 263 others for a predictive analysis to see if NLR could tell which patients would respond to chemotherapy. There was a median follow-up of 18.6 years. For the prognostic analysis, NLR was not a significant factor in overall survival. The important factors were age and whether the patient received pre-surgery chemotherapy. For the predictive analysis, NLR did not predict which patients benefitted from chemotherapy. On the question of why most previous publications supported NLR as a biomarker, Ojerholm pointed to several factors beyond methodology and statistical design. "There's also the problem of publication bias," Ojerholm explained. "Sometimes authors won't submit negative results, and sometimes journals won't accept them. That could be a real issue as NLR research continues." Ojerholm stressed that no single study is definitive, and doctors must weigh results from the entire literature. "Yet this study does raise questions about NLR for bladder cancer," Ojerholm added, "and we need more evidence before using this biomarker in clinical practice." Additional Penn authors of this study include Andrew Smith, Wei-Ting Hwang, Brian Baumann, Kai Tucker, Ronac Mamtani, Ben Boursi, and John Christodouleas. This information is embargoed until Thursday, October 27th. Penn Medicine is one of the world's leading academic medical centers, dedicated to the related missions of medical education, biomedical research, and excellence in patient care. Penn Medicine consists of the Raymond and Ruth Perelman School of Medicine at the University of Pennsylvania (founded in 1765 as the nation's first medical school) and the University of Pennsylvania Health System, which together form a $5.3 billion enterprise. The Perelman School of Medicine has been ranked among the top five medical schools in the United States for the past 18 years, according to U.S. News & World Report's survey of research-oriented medical schools. The School is consistently among the nation's top recipients of funding from the National Institutes of Health, with $373 million awarded in the 2015 fiscal year. The University of Pennsylvania Health System's patient care facilities include: The Hospital of the University of Pennsylvania and Penn Presbyterian Medical Center -- which are recognized as one of the nation's top "Honor Roll" hospitals by U.S. News & World Report -- Chester County Hospital; Lancaster General Health; Penn Wissahickon Hospice; and Pennsylvania Hospital -- the nation's first hospital, founded in 1751. Additional affiliated inpatient care facilities and services throughout the Philadelphia region include Chestnut Hill Hospital and Good Shepherd Penn Partners, a partnership between Good Shepherd Rehabilitation Network and Penn Medicine. Penn Medicine is committed to improving lives and health through a variety of community-based programs and activities. In fiscal year 2015, Penn Medicine provided $253.3 million to benefit our community.


News Article | December 12, 2016
Site: www.eurekalert.org

Immunologists at St. Jude Children's Research Hospital have discovered that a protein called NLRC3 plays a central role in inhibiting colon cells from becoming cancerous. The study, led by Thirumala-Devi Kanneganti, Ph.D., a member of the St. Jude Department of Immunology, appears online today in the journal Nature. Researchers found that deleting the protective NLRC3 protein exacerbates colon cancer development. They also identified key molecular components of the NLRC3 tumor suppressing pathway, offering targets for new drugs that restore the protective mechanism to treat colon cancer. NLRC3 is a member of a large family of NLR "sensor" proteins that regulate immune and other cell functions. However, until now, the role of NLRC3 in protecting against cancer development was unknown. Previous studies showed that tumors from colon cancer patients exhibited drastically reduced NLRC3 gene expression. Detailed analysis by Kanneganti and her colleagues revealed how NLRC3 regulates molecules in a key cellular pathway called the PI3K-mTOR pathway, which controls cell proliferation, immune response, inflammation and cancer. Using an induced colon cancer model, Kanneganti and her colleagues found mice that developed tumors showed significantly reduced NLRC3, just as human patients did. The researchers also found that NLRC3-deficient mice were much more prone to colon cancer. What's more, a mouse strain with a tendency to develop colon polyps showed much greater tumor development when they lacked NLRC3. In other studies with mice, the researchers established that NLRC3 acted mainly in epithelial cells in the colon, showing the protein's direct role in protecting the gut against inflammation caused by infection and colon tumor development. In studies with human colon cells, the scientists found that overexpressing the NLRC3 gene greatly reduced cell proliferation. "All of these complementary approaches to understanding NLRC3 allowed us to really nail it down that NLRC3 is important for protecting from abnormal colon cell growth, and when it is not present, tumors will develop," said Kanneganti. "This suggested that if we can somehow induce NLRC3 expression clinically, it will block the signaling pathways that lead to tumorigenesis." A key to understanding how to enhance NLRC3's activity was pinpointing which molecular pathway in the cell's biological machinery it regulated, Kanneganti said. The research revealed that NLRC3 maintains a brake on the PI3K-mTOR pathways. The scientists also found that the PI3K-mTOR pathway switched on early in the tumor-triggering process. "In developing drug therapies, it might be difficult to target the PI3K-mTOR pathway itself, because it is such a central node in cell signaling," said Kanneganti. "Thus, we could target NLRC3 itself and block tumorigenesis early on." Kanneganti said that NLRC3 likely plays a broader role than only preventing tumors. "We really do not know its role in infectious and inflammatory diseases,"she added. More broadly, she said, further studies will likely offer insights into the roles of other NLR family members. "NLRs have multiple functions in regulating immunity and inflammation and blocking tumorigenesis," she said. "None of us really thought NLRs could be involved in the PI3K-mTOR pathway. So, this study is really intriguing, because it opens up our ability to think more in depth about the function of NLRs and the diverse roles they play." Rajendra Karki, of St. Jude, and Si Ming Man, formerly of St. Jude, are the paper's first authors. The other authors are R. K. Subbarao Malireddi, Sannula Kesavardhana, Qifan Zhu, Amanda Burton, Bhesh Raj Sharma, Xiaopeng Qi, Stephane Pelletier and Peter Vogel, all of St. Jude; and Philip Rosenstiel of Christian-Albrechts-University Kiel, Germany. The research was supported in part by grants (AI101935, AI124346, AR056296, CA163507) from the National Institutes of Health and ALSAC.


News Article | December 21, 2016
Site: www.nature.com

Previous studies have shown that NLRC3 functions as a negative regulator of signalling pathways activated by Toll-like receptors (TLRs) and the DNA sensor STING in response to pathogen-associated molecular patterns or to virus infection13, 14. The physiological role of NLRC3 has, however, remained largely unknown. Using an established mouse model of colitis-associated colorectal tumorigenesis, we investigated the role of NLRC3 in colorectal cancer. To do this, we injected mice intraperitoneally with azoxymethane, followed by three rounds of dextran sulfate sodium (DSS) treatment (Extended Data Fig. 1a). All time points referred to hereafter indicate the number of days after injection of azoxymethane. The number of tumours was quantified at day 80. Qualitative reverse-transcriptase PCR analysis revealed a reduction in the expression of the gene encoding NLRC3 in tumour tissue compared with non-tumour-associated tissue in the colon of wild-type mice 80 days after injection of azoxymethane (day 80; Extended Data Fig. 1b). We injected cohorts of co-housed wild-type and Nlrc3−/− mice with azoxymethane, followed by three rounds of DSS treatment, and examined the prevalence of tumours in the colon of these mice at day 80 (Extended Data Fig. 1a, c). We found that Nlrc3−/− mice lost more body weight after the first two rounds of DSS treatment compared to wild-type mice and developed significantly more tumours (Fig. 1a–d). Histological hallmarks associated with thickening of the colon, inflammation, ulceration, hyperplasia and the extent or severity of damage were more frequently identified in the middle and distal colon and the rectum of Nlrc3−/− mice compared to the corresponding regions in wild-type mice (Fig. 1e, f and Extended Data Fig. 1d). All Nlrc3−/− mice suffered high-grade dysplasia, whereas wild-type mice suffered low-grade dysplasia (Extended Data Fig. 1e). We found that 63% of the Nlrc3−/− mice were positive for adenocarcinoma in the colon, compared to 0% of the wild-type controls at day 80 (Extended Data Fig. 1f). Although NLRC3 showed a gene-dose-dependent response to azoxymethane and DSS (Fig. 1g), it does not appear to have an effect on the normal mouse intestine or colon (Extended Data Fig. 1g). Nlrc3−/− mice lost significantly more body weight and suffered more severe shortening of, and damage to, the colon after only a single round of DSS treatment compared to their wild-type counterparts (Extended Data Fig. 2a–c). Certain members of the NLR protein family can form inflammasomes, driving maturation of IL-18, a cytokine important for mediating protection against colitis-associated tumorigenesis2, 3, 4, 15. We did not observe differential production of IL-18 in wild-type and Nlrc3−/− mice at day 14 or at day 80 (Extended Data Fig. 2d). Instead, production of the other inflammasome-associated cytokine IL-1β and inflammasome-independent cytokines IL-6, TNF and GCSF and the chemokines KC (also known as CXCL1), MCP-1 (also known as CCL2) and MIP-1α (also known as CCL3) was elevated in colon tissue of Nlrc3−/− mice compared to wild-type mice at day 14 (Extended Data Fig. 2d–g). We further confirmed these results and also found increased levels of circulating IL-6, GCSF, KC and MIP-1α in the sera of Nlrc3−/− mice compared to wild-type mice at day 14 (Extended Data Fig. 2h). The expression of IL-17 and IL-22 was also elevated in the colon tissue of Nlrc3−/− mice compared to wild-type mice at day 14, whereas the expression of IL-23, IFNβ and IFNγ remained unchanged (Extended Data Fig. 3a). Consistent with the observation that Nlrc3−/− mice had elevated levels of many pro-inflammatory mediators at day 14, we observed increased levels of IκBα and STAT3 phosphorylation in the colon tissue of Nlrc3−/− mice compared to wild-type mice (Extended Data Fig. 3b). However, differential phosphorylation of ERK was not observed (Extended Data Fig. 3b). Global increases in the production of inflammatory mediators and the activation of immune signalling pathways reflected the hyper-susceptibility of Nlrc3−/− mice to colitis. Using flow cytometry, we profiled the immune cell populations in the colons of untreated wild-type and Nlrc3−/− mice and wild-type and Nlrc3−/− mice at days 8 and 14. We observed an increased number of macrophages, neutrophils and natural killer cells in the colons of Nlrc3−/− mice compared to wild-type mice 14 days after azoxymethane and DSS treatment (Extended Data Fig. 3c, d), which is consistent with the increased levels of inflammation observed at this time point. However, we did not observe differences in the relative number of macrophages, CD11b+CD11c+ cells, neutrophils, B cells, CD4+ T cells, CD8+ T cells and natural killer cells between untreated wild-type and Nlrc3−/− mice or mice at day 8 (Extended Data Fig. 3d). NLRC3 has been implicated in the regulation of T-cell activation11; however, we did not observe a difference in the levels of IFNγ+ or TNF+ CD4+ T cells when wild-type and Nlrc3−/− splenocytes were stimulated with CD3 and CD28 in the presence of IL-2 (Extended Data Fig. 3e). We performed bone-marrow chimaera studies to identify the contribution of NLRC3 in haematopoietic cells versus radioresistant stromal cells during colitis-associated tumorigenesis. As we expected, Nlrc3−/− mice that received Nlrc3−/− bone marrow were more susceptible to tumorigenesis than wild-type mice that received wild-type bone marrow (Fig. 1h and Extended Data Fig. 4a). However, Nlrc3−/− mice that received wild-type bone marrow had a significantly increased tumour burden compared with wild-type mice that received wild-type bone marrow. In addition, wild-type mice that received Nlrc3−/− bone marrow had a significantly increased tumour burden compared to wild-type mice that received wild-type bone marrow (Fig. 1h and Extended Data Fig. 4a). We further confirmed our findings and generated mice that lacked NLRC3 in haematopoietic cells specifically (Vav1creNlrc3fl/fl), cells of the myeloid lineage (LysMcreNlrc3fl/fl) and intestinal epithelial cells (Vil1creNlrc3fl/fl). Mice lacking NLRC3 in intestinal epithelial cells developed the highest number of tumours, followed by mice lacking NLRC3 in haematopoietic cells (Fig. 1i and Extended Data Fig. 4b). Mice lacking NLRC3 in cells of the myeloid lineage had a similar number of tumours to wild-type mice (Fig. 1i and Extended Data Fig. 4b). These data support the observation that the oncogenic inhibitory effect of NLRC3 is more dominant in intestinal epithelial cells and more subtle in haematopoietic cells. A closer examination of the intestinal epithelial cells of Nlrc3−/− mice revealed a significant increase in numbers of both Ki67+ and PCNA+ (both proteins that are associated with cellular proliferation) cells per intestinal crypt compared to wild-type mice at day 14 (Fig. 2a). Additionally, colonic epithelial stem cells collected from Nlrc3−/− mice more readily developed into organoids in ex vivo culture compared to those collected from wild-type mice (Fig. 2b). The average diameters of the organoids derived from Nlrc3−/− mice were significantly increased compared to the average diameters of the organoids from wild-type mice (Fig. 2b). Expression of the stem-cell marker Lgr5 in the colon tissue was similar in wild-type and Nlrc3−/− mice (Extended Data Fig. 4c), suggesting that the differences in the number and size of intestinal organoids was due to differential colony-forming capacity rather than differences in the numbers of starting intestinal stem cells. To investigate the effect of NLRC3 on cell proliferation more directly, we overexpressed NLRC3 in the human colon cell line HCT116 and found that these cells exhibited reduced levels of proliferation compared to cells expressing a control (GFP) protein (Fig. 2c). Furthermore, primary Nlrc3−/− fibroblasts proliferated more rapidly than wild-type fibroblasts (Fig. 2d). Cellular proliferation can also be achieved when growth factors, nutrients and cellular energy activate metabolic pathways via the kinase mTOR16. We found increased phosphorylation levels of S6 kinase, 4E-BP1 and AKT at Ser473, the downstream targets of mTOR, in the colon tissue of Nlrc3−/− mice compared to wild-type mice at day 14 (Fig. 3a, b). Increased phosphorylation of these mTOR targets was also observed in Nlrc3−/− organoids compared to wild-type organoids (Fig. 3c, d). However, we found no difference in the expression of genes involved in the Wnt signalling pathway, including Wnt1, Ctnnb1, Lef1, Tcf4, Tcf7 and Axin2 (Extended Data Fig. 4c). The extent of nuclear localization of β-catenin was also similar between wild-type and Nlrc3−/− mice (Extended Data Fig. 4d). Dysregulation of the mTOR signalling pathways in Nlrc3−/− mice occurred very early, 8 days after injection of azoxymethane, whereas no difference in the production of inflammatory cytokines and mediators and phosphorylation of IκBα was observed at this time point (Extended Data Fig. 5). Moreover, the differential AKT–mTOR signalling observed at this time point was not caused by differences in the relative levels of immune cells recruited to the colon (Extended Data Fig. 3d). These data suggest that the dysregulated mTOR signalling observed at an earlier time point may lead to increased NF-κB signalling at a later time point. Phosphorylation and activation of mTOR is driven by a number of upstream signalling proteins in the PI3K–AKT–mTOR pathway. Phosphorylation of AKT at the Thr308 site by the kinase PDK1 allows AKT to activate mTOR17, 18, 19. To examine whether NLRC3 directly affects the apical molecules in the PI3K–AKT–mTOR pathway, we first investigated the phosphorylation status of AKT at the Thr308 site. We observed elevated phosphorylation of AKT at Thr308 in the colon tissue of Nlrc3−/− mice compared to wild-type mice at day 14 (Fig. 3e). Increased levels of AKT phosphorylation in colon tissue were observed predominately in epithelial cells and, to a lesser extent, in infiltrating cells (Fig. 3f). Similarly, increased phosphorylation of AKT at Thr308 was observed in Nlrc3−/− organoids treated with IGF-1 compared to wild-type controls (Fig. 3g, h). We further observed increased activation of PDK1 in the colon tissue of Nlrc3−/− mice treated with azoxymethane and DSS compared to wild-type mice (Fig. 3e). In addition, elevated degree of phosphorylation of AKT at Thr308 and of 4E-BP1 were observed in the colon of Nlrc3+/− heterozygous mice compared to littermate wild-type mice at day 14, although this increase was smaller than that observed in littermate Nlrc3−/− homozygous mice (Extended Data Fig. 6a). The gene-dose-dependent effect of NLRC3 on the suppression of the mTOR signalling pathways is reminiscent of the gene-dose-dependent effect of NLRC3 on the suppression of tumorigenesis (Fig. 1g). Activated mTOR is phosphorylated and migrates to lysosomal and late endosomal membranes20, 21. We observed increased phosphorylation of mTOR in the colon tissue of Nlrc3−/− mice (Fig. 3e). We also found an increased co-localization frequency between mTOR and LAMP1 puncta in IGF-1-treated primary Nlrc3−/− fibroblasts compared to IGF-1-treated primary wild-type fibroblasts (Fig. 4a). Increased mTOR signalling was observed in Nlrc3−/− fibroblasts or wild-type fibroblasts treated with short interfering RNAs (siRNAs) against Nlrc3 compared to their corresponding controls (Extended Data Figs 6b–e, 7a–d). We further investigated whether NLRC3 is able to restrict proliferation in a spontaneous mouse model of colon cancer. We crossed the mouse line containing a heterozygous mutation in the gene encoding adenomatous polyposis coli (ApcMin/+) with Nlrc3−/− mice and found that ApcMin/+Nlrc3−/− mice had a higher tumour burden than ApcMin/+ control mice (Extended Data Fig. 8a). Of the ApcMin/+Nlrc3−/− mice, 40% developed hyperplasia (compared to 0% in the littermate control group) and ApcMin/+Nlrc3−/− mice exhibited increased damage in the colon (Extended Data Fig. 8b, c). Notably, we observed increased number of Ki67+ proliferative cells and cells positive for phosphorylated S6 kinase in the colon of ApcMin/+Nlrc3−/− mice compared to ApcMin/+ mice (Extended Data Fig. 8c). Moreover, the capacity of ApcMin/+Nlrc3−/− intestinal stem cells to proliferate into organoids was greater than that of ApcMin/+ intestinal stem cells (Extended Data Fig. 8d). Treatment with the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin impaired the ability of intestinal stem cells to proliferate into organoids in both strains (Extended Data Fig. 8d). Treatment of the ApcMin/+ mice and ApcMin/+Nlrc3−/− mice with the PI3K–mTOR inhibitor NVP-BEZ235 reduced the tumour burden and phosphorylation of S6 kinase in the tumours and enterocytes of ApcMin/+Nlrc3−/− mice to a level observed in treated ApcMin/+ mice (Fig. 4b, c). Collectively, these data suggest that NLRC3 restricts cellular proliferation via the PI3K–mTOR axis during colon tumorigenesis. Following generation of inositol phospholipids by activated PI3Ks, AKT is recruited to the cell membrane where it undergoes a conformational change and is phosphorylated at Thr308 by PDK1 (refs 17, 18, 19). Co-immunoprecipitation assays showed that NLRC3 weakly interacted with PDK1 and did not interact with AKT (Fig. 4d). Instead, we found that NLRC3 co-immunoprecipitated with p85 subunits of PI3K (Fig. 4d). In addition, we observed increased levels of interaction between the p85 and p110α subunits of PI3K in Nlrc3−/− primary fibroblasts or mouse bone-marrow-derived macrophages (BMDMs) (Extended Data Fig. 9a, b). We also observed a higher level of phosphorylation and activation of p85 PI3K in the colon tissue of Nlrc3−/− mice treated with azoxymethane and DSS compared to wild-type mice (Fig. 4e). These data provide evidence to suggest that NLRC3 disrupts an association between the PI3K p85 and p110α subunits and reduces the activity of PI3K p85 itself. Deletion of the CARD, NACHT or LRR domain of NLRC3 impaired the ability of NLRC3 to interact with either the p85 or the p110α subunit of PI3K (Extended Data Fig. 9c–g). Reconstitution of NLRC3 in Nlrc3−/− fibroblasts reduced the degree of phosphorylation of AKT Thr308 and other downstream molecules to levels similar to those seen in wild-type fibroblasts upon stimulation with IGF-1 (Fig. 4f). In addition to growth factor receptors, activation of TLR4 can engage the PI3K–AKT–mTOR pathway22, 23, 24. We observed increased activation of the mTOR signalling pathways in lipopolysaccharide (LPS)-treated primary Nlrc3−/− BMDMs compared to LPS-treated wild-type BMDMs (Extended Data Fig. 10a,b). We further confirmed our findings in an independently generated line of NLRC3-deficient mice that we term Nlrc3ld/ld mice (NLRC3 large deletion, data not shown; see Methods). Collectively these findings identify NLRC3 as an inhibitory sensor of the PI3K–AKT–mTOR pathway, mediating protection against tumorigenesis in colorectal cancer (Extended Data Fig. 10c). NLRC3 does not act solely to protect against cancer. A previous study has shown that expression of NLRC3 is downregulated in patients with the autoimmune disease Wegener’s granulomatosis25. Moreover, a loss-of-function mutation in the gene encoding the NLRC3-like protein in zebrafish results in systemic inflammation26. These findings collectively provide evidence to support the cross-species functionality of NLRC3. Whether NLRC3 needs to be bound to a specific ligand or is engaged in the activation of its regulatory function in a ligand-independent manner remains to be explored. Understanding the precise functions of NLRC3 could open up new avenues in the treatment of infectious and autoinflammatory diseases and cancer.


Roelen A.L.C.,NLR | Lin P.H.,Technical University of Delft | Hale A.R.,Technical University of Delft
Safety Science | Year: 2011

Event analysis is needed to learn and improve safety. In air transport, 'occurrences' are routinely reported by pilots and air traffic controllers, and in-flight data analysis systems automatically monitor aircraft system behaviour and capture parameter threshold exceedances. The safety analyst of a large airline has to analyse dozens of occurrences each day. To understand why events happened the analyst has to go beyond the given information and make causal inferences. The analyst is able to do this for causal factors closely related in time and space to the event itself by applying individual knowledge and expertise. But typically the result of the analysis is ad hoc reaction to each individual event. Systematic analysis is needed to find areas of improvement for factors that are further removed from the event (latent factors). New tools are needed to help the analyst in this respect. There is a need for models that represent possible causal event sequence scenarios that include technical, human, and organisational factors. Building such models is a huge task, and requires the combination of detailed knowledge of all aspects of the system, processing huge amounts of data, a substantial mathematical background and the ability to capture this all in a user friendly software tool to be used by the safety analysts. Experience in Causal Modelling of Air Transportation System (CATS) in the Netherlands and similar projects in FAA and Eurocontrol in aviation shows that this is indeed a formidable task, but it has to be done to further improve safety. © 2010 Elsevier Ltd.


Arntzen M.,NLR | Simons D.G.,Technical University of Delft
Applied Acoustics | Year: 2014

Traditionally aircraft flyover noise is assessed by displaying contours of noise metrics. These models can be used to study noise mitigation measures but they lack the possibility to play-back the audible sound as predicted by their calculations. To that end, noise synthesis is an option that allows to experience differences due to noise abatement procedures or new aircraft designs. A noise synthesis technique for aircraft noise is demonstrated by predicting the noise at a noise monitoring location near an airport. By comparing the synthesized results to a recorded measurement, an indication on the capability of this technique has been acquired. Differences between the synthesized and measured sound remain. A large part of that difference is believed to be caused by the inherent uncertainty when using predictive empirical source noise models. It is shown that differences between departure routes can be captured, thereby illustrating the potential of this method to listen to different take-off procedures. Future improvements in source noise prediction and the inclusion of the effects of turbulence on propagation will further aid to the realism of synthesized aircraft noise. © 2013 Elsevier Ltd. All rights reserved.


Arntzen M.,NLR
INTER-NOISE 2015 - 44th International Congress and Exposition on Noise Control Engineering | Year: 2015

The general aviation airport of Lelystad (the Netherlands) will be transformed into a commercially operated regional airport. Regional aircraft are expected to fly passengers to their holiday destinations. This means that several communities will be affected by new aircraft noise. Besides noise contours, there was a strong desire to inform the public regarding the actual sound that aircraft would generate in their communities. To that end, auralizations of aircraft noise were created to inform communities on the expected sound in their environment. NLR's Virtual Community Noise Simulator allows combining auralization with visualization. Short videos of each individual scenario were made and presented to disseminate the information. Each video comprised locally recorded background noise and aircraft noise that was tailored to represent the projected future flight paths near each location. The resulting videos (32 in total) were presented at six consultation evenings to inform the general public. Besides a plenary presentation, the videos were also presented in smaller rooms in combination with a loudspeaker system and a recording device to replay the videos at a calibrated level. This setup proved to be very valuable and provided the additional information desired by the public in a comprehensive manner. © 2015 by ASME.


News Article | December 13, 2016
Site: www.medicalnewstoday.com

The molecule is called NLRC3 and is a member of the large NOD-like receptor (NLR) family of "sensor proteins" that are found inside cells, where they help to control immune and other functions. However, until now, scientists did not realize that NLRC3 might also protect cells from cancer. The new research - led by Dr. Thirumala-Devi Kanneganti, of the immunology department at St. Jude Children's Research Hospital in Memphis, TN - is published in the journal Nature. Cancer arises when cells in the body start to grow out of control. This can begin in almost any part of the body and may spread to other areas. Colorectal cancer starts in the epithelial cells that line the colon and rectum. Excluding skin cancers, colorectal cancer is the third most common cancer diagnosed in both men and women in the United States. Estimates from the American Cancer Society suggest that there will be 95,270 new cases of colon cancer and 39,220 new cases of rectal cancer in the U.S. during 2016. Colorectal cancer is the second leading cause of cancer-related deaths among those in the U.S. It is expected to cause about 49,190 deaths in 2016. In their report, the researchers note that previous studies have revealed tumor tissue from patients with colorectal cancer show a dramatically reduced expression of the gene that codes for NLRC3. This has been responsible for "highlighting an undefined potential function" for the sensor protein in the development of cancer. In their investigation, Dr. Kanneganti and colleagues found that NLRC3 regulates a key cellular process called the PI3K-mTOR pathway, which controls cell proliferation, immune response, inflammation, and cancer. For their study, the team used mice bred to develop colon cancer. They found their tumors had significantly lower levels of NLRC3, as previous studies had found in human patients. They also showed that mice lacking NLRC3 are much more prone to colitis and colorectal cancer. Furthermore, mice engineered to develop colon polyps also showed greater tumor development when they lacked NLRC3. In further studies with mice, the team found that NLRC3 plays an active role mainly in the colon's epithelial cells, helping to prevent inflammation and tumor development. The researchers also carried out experiments in human colon cells. There, they found that over-expression of the NLRC3 gene greatly reduced cell proliferation. Further investigation revealed that NLRC3 inhibits PI3K-mTOR pathways. The team also found these pathways are switched on early during tumor formation. Dr. Kanneganti says that altogether, the findings show that NLRC3 plays an important role in stopping abnormal cell growth. When it is not present, tumors develop. This raises the question of whether increasing NLRC3 expression might be a way to block the cell processes that lead to tumor formation (tumorigenesis). "In developing drug therapies, it might be difficult to target the PI3K-mTOR pathway itself, because it is such a central node in cell signaling," notes Dr. Kanneganti. "Thus, we could target NLRC3 itself and block tumorigenesis early on." Speculating further, Dr. Kanneganti suggests that NLRC3 is probably involved in many other cell processes aside from tumor prevention. "We really do not know its role in infectious and inflammatory diseases," she adds. Additional studies may uncover even more valuable clues about the role of other members of the NLR family. Learn how susceptibility to colon cancer may vary with gut microbes.

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