Publications by authors named "Scott McComb"

19 Publications

  • Page 1 of 1

Identification of transcriptional subtypes in lung adenocarcinoma and squamous cell carcinoma through integrative analysis of microarray and RNA sequencing data.

Sci Rep 2021 04 22;11(1):8709. Epub 2021 Apr 22.

Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada.

Classification of tumors into subtypes can inform personalized approaches to treatment including the choice of targeted therapies. The two most common lung cancer histological subtypes, lung adenocarcinoma and lung squamous cell carcinoma, have been previously divided into transcriptional subtypes using microarray data, and corresponding signatures were subsequently used to classify RNA-seq data. Cross-platform unsupervised classification facilitates the identification of robust transcriptional subtypes by combining vast amounts of publicly available microarray and RNA-seq data. However, cross-platform classification is challenging because of intrinsic differences in data generated using the two gene expression profiling technologies. In this report, we show that robust gene expression subtypes can be identified in integrated data representing over 3500 normal and tumor lung samples profiled using two widely used platforms, Affymetrix HG-U133 Plus 2.0 Array and Illumina HiSeq RNA sequencing. We tested and analyzed consensus clustering for 384 combinations of data processing methods. The agreement between subtypes identified in single-platform and cross-platform normalized data was then evaluated using a variety of statistics. Results show that unsupervised learning can be achieved with combined microarray and RNA-seq data using selected preprocessing, cross-platform normalization, and unsupervised feature selection methods. Our analysis confirmed three lung adenocarcinoma transcriptional subtypes, but only two consistent subtypes in squamous cell carcinoma, as opposed to four subtypes previously identified. Further analysis showed that tumor subtypes were associated with distinct patterns of genomic alterations in genes coding for therapeutic targets. Importantly, by integrating quantitative proteomics data, we were able to identify tumor subtype biomarkers that effectively classify samples on the basis of both gene and protein expression. This study provides the basis for further integrative data analysis across gene and protein expression profiling platforms.
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http://dx.doi.org/10.1038/s41598-021-88209-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062554PMC
April 2021

Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering.

CRISPR J 2021 02;4(1):104-119

Human Health Therapeutics Research Center, National Research Council Canada, Ottawa, Canada.

Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.
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http://dx.doi.org/10.1089/crispr.2020.0090DOI Listing
February 2021

Current Advances and Hurdles in Chimeric Antigen Receptor Technology.

Cancers (Basel) 2020 Nov 11;12(11). Epub 2020 Nov 11.

Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

Since tumor-specific T cells were first utilized to treat melanoma patients in 1986 [...].
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http://dx.doi.org/10.3390/cancers12113329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697884PMC
November 2020

TNFR2 is required for RIP1-dependent cell death in human leukemia.

Blood Adv 2020 10;4(19):4823-4833

Department of Oncology and Children's Research Centre, University Children's Hospital Zurich, Zürich, Switzerland.

Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)-dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.
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http://dx.doi.org/10.1182/bloodadvances.2019000796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556136PMC
October 2020

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.

Mol Ther Methods Clin Dev 2020 Mar 31;16:238-254. Epub 2020 Jan 31.

Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.
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http://dx.doi.org/10.1016/j.omtm.2020.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021643PMC
March 2020

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7.

Sci Adv 2019 07 31;5(7):eaau9433. Epub 2019 Jul 31.

Department of Oncology and Children's Research Centre, University Children's Hospital Zürich, 8032 Zürich, Switzerland.

Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.
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http://dx.doi.org/10.1126/sciadv.aau9433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669006PMC
July 2019

Introduction to the Immune System.

Methods Mol Biol 2019 ;2024:1-24

Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON, Canada.

The immune system in a broad sense is a mechanism that allows a living organism to discriminate between "self" and "nonself." Examples of immune systems occur in multicellular organisms as simple and ancient as sea sponges. In fact, complex multicellular life would be impossible without the ability to exclude external life from the internal environment. This introduction to the immune system will explore the cell types and soluble factors involved in immune reactions, as well as their location in the body during development and maintenance. Additionally, a description of the immunological events during an innate and adaptive immune reaction to an infection will be discussed, as well as a brief introduction to autoimmunity, cancer immunity, vaccines, and immunotherapies.
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http://dx.doi.org/10.1007/978-1-4939-9597-4_1DOI Listing
March 2020

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

J Biol Chem 2018 07 13;293(30):11913-11927. Epub 2018 Jun 13.

From the Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M8, Canada,

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIP), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIP may allow inflammatory macrophages to participate in pathogen control for a longer duration.
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http://dx.doi.org/10.1074/jbc.RA118.003614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066316PMC
July 2018

MAP4K4 controlled integrin β1 activation and c-Met endocytosis are associated with invasive behavior of medulloblastoma cells.

Oncotarget 2018 May 1;9(33):23220-23236. Epub 2018 May 1.

University Children's Hospital Zürich, Department of Oncology, Children's Research Center, Zürich, Switzerland.

Local tissue infiltration of Medulloblastoma (MB) tumor cells precedes metastatic disease but little is still known about intrinsic regulation of migration and invasion in these cells. We found that MAP4K4, a pro-migratory Ser/Thr kinase, is overexpressed in 30% of primary MB tumors and that increased expression is particularly associated with the frequently metastatic SHH β subtype. MAP4K4 is a driver of migration and invasion downstream of c-Met, which is transcriptionally up-regulated in SHH MB. Consistently, depletion of MAP4K4 in MB tumor cells restricts HGF-driven matrix invasion and brain tissue infiltration . We show that these pro-migratory functions of MAP4K4 involve the activation of the integrin β-1 adhesion receptor and are associated with increased endocytic uptake. The consequent enhanced recycling of c-Met caused by MAP4K4 results in the accumulation of activated c-Met in cytosolic vesicles, which is required for sustained signaling and downstream pathway activation. The parallel increase of c-Met and MAP4K4 expression in SHH MB could predict an increased potential of these tumors to infiltrate brain tissue and cause metastatic disease. Molecular targeting of the underlying accelerated endocytosis and receptor recycling could represent a novel approach to block pro-migratory effector functions of MAP4K4 in metastatic cancers.
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http://dx.doi.org/10.18632/oncotarget.25294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955425PMC
May 2018

Triad3a induces the degradation of early necrosome to limit RipK1-dependent cytokine production and necroptosis.

Cell Death Dis 2018 05 22;9(6):592. Epub 2018 May 22.

Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses.
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http://dx.doi.org/10.1038/s41419-018-0672-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964080PMC
May 2018

IRAK4 is essential for TLR9-induced suppression of Epstein-Barr virus BZLF1 transcription in Akata Burkitt's lymphoma cells.

PLoS One 2017 31;12(10):e0186614. Epub 2017 Oct 31.

Laboratory for Experimental Infectious Diseases and Cancer Research of the Division of Infectious Diseases and Hospital Epidemiology and the Division of Oncology, University Children's Hospital of Zurich, University of Zurich, Zurich, Switzerland.

Burkitt's lymphoma (BL) is the most common childhood cancer in equatorial Africa, and is endemic to areas where people are chronically co-infected with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The contribution of these pathogens in the oncogenic process remains poorly understood. We showed earlier that the activation of Toll-like receptor (TLR) 9 by hemozoin, a disposal product formed from the digestion of blood by P. falciparum, suppresses the lytic reactivation of EBV in BL cells. EBV lytic reactivation is regulated by the expression of transcription factor Zta (ZEBRA), encoded by the EBV gene BZLF1. Here, we explore in the BL cell line Akata, the mechanism involved in repression by TLR9 of expression of BZLF1. We show that BZLF1 repression is mediated upon TLR9 engagement by a mechanism that is largely independent of de novo protein synthesis. By CRISPR/Cas9-induced inactivation of TLR9, MyD88, IRAK4 and IRAK1 we confirm that BZLF1 repression is dependent on functional TLR9 and MyD88 signaling, and identify IRAK4 as an essential element for TLR9-induced repression of BZLF1 expression upon BCR cross-linking. Our results unprecedentedly show that TLR9-mediated inhibition of lytic EBV is largely independent of new protein synthesis and demonstrate the central roles of MyD88 and IRAK4 in this process contributing to EBV's persistence in the host's B-cell pool.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186614PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663394PMC
November 2017

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9.

Bio Protoc 2017 Apr 5;7(7):e2222. Epub 2017 Apr 5.

Department of Oncology and Children's Research Center, University Children's Hospital Zürich, Zürich, Switzerland.

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.
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http://dx.doi.org/10.21769/BioProtoc.2222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410427PMC
April 2017

Activation of concurrent apoptosis and necroptosis by SMAC mimetics for the treatment of refractory and relapsed ALL.

Sci Transl Med 2016 05;8(339):339ra70

Department of Oncology and Children's Research Centre, University Children's Hospital Zürich, 8032 Zürich, Switzerland.

More precise treatment strategies are urgently needed to decrease toxicity and improve outcomes for treatment-refractory leukemia. We used ex vivo drug response profiling of high-risk, relapsed, or refractory acute lymphoblastic leukemia (ALL) cases and identified a subset with exquisite sensitivity to small-molecule mimetics of the second mitochondria-derived activator of caspases (SMAC) protein. Potent ex vivo activity of the SMAC mimetic (SM) birinapant correlated with marked in vivo antileukemic effects, as indicated by delayed engraftment, decreased leukemia burden, and prolonged survival of xenografted mice. Antileukemic activity was dependent on simultaneous execution of apoptosis and necroptosis, as demonstrated by functional genomic dissection with a multicolored lentiCRISPR approach to simultaneously disrupt multiple genes in patient-derived ALL. SM specifically targeted receptor-interacting protein kinase 1 (RIP1)-dependent death, and CRISPR-mediated disruption of RIP1 completely blocked SM-induced death yet had no impact on the response to standard antileukemic agents. Thus, SM compounds such as birinapant circumvent escape from apoptosis in leukemia by activating a potent dual RIP1-dependent apoptotic and necroptotic cell death, which is not exploited by current therapy. Ex vivo drug activity profiling could provide important functional diagnostic information to identify patients who may benefit from targeted treatment with birinapant in early clinical trials.
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http://dx.doi.org/10.1126/scitranslmed.aad2986DOI Listing
May 2016

Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages.

Proc Natl Acad Sci U S A 2014 Aug 21;111(31):E3206-13. Epub 2014 Jul 21.

Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1N 6N5;

Myeloid cells play a critical role in perpetuating inflammation during various chronic diseases. Recently the death of macrophages through programmed necrosis (necroptosis) has emerged as an important mechanism in inflammation and pathology. We evaluated the mechanisms that lead to the induction of necrotic cell death in macrophages. Our results indicate that type I IFN (IFN-I) signaling is a predominant mechanism of necroptosis, because macrophages deficient in IFN-α receptor type I (IFNAR1) are highly resistant to necroptosis after stimulation with LPS, polyinosinic-polycytidylic acid, TNF-α, or IFN-β in the presence of caspase inhibitors. IFN-I-induced necroptosis occurred through both mechanisms dependent on and independent of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) and led to persistent phosphorylation of receptor-interacting protein 3 (Rip3) kinase, which resulted in potent necroptosis. Although various IFN-regulatory factors (IRFs) facilitated the induction of necroptosis in response to IFN-β, IRF-9-STAT1- or -STAT2-deficient macrophages were highly resistant to necroptosis. Our results indicate that IFN-β-induced necroptosis of macrophages proceeds through tonic IFN-stimulated gene factor 3 (ISGF3) signaling, which leads to persistent expression of STAT1, STAT2, and IRF9. Induction of IFNAR1/Rip3-dependent necroptosis also resulted in potent inflammatory pathology in vivo. These results reveal how IFN-I mediates acute inflammation through macrophage necroptosis.
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http://dx.doi.org/10.1073/pnas.1407068111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128105PMC
August 2014

Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase.

J Immunol 2014 Jun 5;192(12):5671-8. Epub 2014 May 5.

Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.
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http://dx.doi.org/10.4049/jimmunol.1303380DOI Listing
June 2014

Introduction to the immune system.

Methods Mol Biol 2013 ;1061:1-20

Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada.

The immune system in a broad sense is a mechanism that allows a living organism to discriminate between "self" and "non-self." Examples of immune systems occur in multicellular organisms as simple and ancient as sea sponges. In fact, complex multicellular life would be impossible without the ability to exclude external life from the internal environment. This introduction to the immune system explores the cell types and soluble factors involved in immune reactions, as well as their location in the body during development and maintenance. Additionally, a description of the immunological events during an innate and adaptive immune reaction to an infection is discussed, as well as a brief introduction to autoimmunity and cancer immunity.
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http://dx.doi.org/10.1007/978-1-62703-589-7_1DOI Listing
March 2014

From mice to women: the conundrum of immunity to infection during pregnancy.

J Reprod Immunol 2013 Mar;97(1):62-73

Human Health Therapeutics, Division of Life Sciences, National Research Council, Ottawa, ON, Canada.

Resistance to infection is the ability of the host to evoke a strong immune response sufficient to eliminate the infectious agent. In contrast, maternal tolerance to the fetus necessitates careful regulation of immune responses. Successful pregnancy requires the maternal host to effectively balance the opposing processes of maternal immune reactivity and tolerance to the fetus. However, this balance can be perturbed by infections which are recognized as the major cause of adverse pregnancy outcome including pre-term labor. Select pathogens also pose a serious threat of severe maternal illness. These include intracellular and chronic pathogens that have evolved immune evasive strategies. Murine models of intracellular bacteria and parasites that mimic pathogenesis of infection in humans have been developed. While human epidemiological studies provide insight into maternal immunity to infection, experimental infection in pregnant mice is a vital tool to unravel the complex molecular mechanisms of placental infection, congenital transmission and maternal illness. We will provide a comprehensive review of the pathogenesis of several infection models in pregnant mice and their clinical relevance. These models have revealed the immunological function of the placenta in responding to, and resisting infection. Murine feto-placental infection provides an effective way to evaluate new intervention strategies for managing infections during pregnancy, adverse fetal outcome and long-term effects on the offspring and mother.
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http://dx.doi.org/10.1016/j.jri.2012.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748615PMC
March 2013

Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium.

Nat Immunol 2012 Oct 26;13(10):954-62. Epub 2012 Aug 26.

National Research Council of Canada-Institute for Biological Sciences, Ottawa, Canada.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a virulent pathogen that induces rapid host death. Here we observed that host survival after infection with S. Typhimurium was enhanced in the absence of type I interferon signaling, with improved survival of mice deficient in the receptor for type I interferons (Ifnar1(-/-) mice) that was attributed to macrophages. Although there was no impairment in cytokine expression or inflammasome activation in Ifnar1(-/-) macrophages, they were highly resistant to S. Typhimurium-induced cell death. Specific inhibition of the kinase RIP1 or knockdown of the gene encoding the kinase RIP3 prevented the death of wild-type macrophages, which indicated that necroptosis was a mechanism of cell death. Finally, RIP3-deficient macrophages, which cannot undergo necroptosis, had similarly less death and enhanced control of S. Typhimurium in vivo. Thus, we propose that S. Typhimurium induces the production of type I interferon, which drives necroptosis of macrophages and allows them to evade the immune response.
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http://dx.doi.org/10.1038/ni.2397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005791PMC
October 2012

Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo.

PLoS One 2010 Dec 22;5(12):e15328. Epub 2010 Dec 22.

Department of Biochemistry, NRC-Institute for Biological Sciences, University of Ottawa, Ottawa, Canada.

Background: CD8(+) T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8(+) T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8(+) T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8(+) T cell responses has yet to be examined.

Methods And Findings: We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8(+) T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8(+) T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8(+) cells remained active caspase-3(low) throughout the contraction phase.

Conclusions: Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells. Furthermore, the contraction of CD8(+) T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015328PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008739PMC
December 2010
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