Chapter 16: Innate Immunity
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RIPA Buffer Lysis Protocol
PROVOST & WALLERT RESEARCH Investigating the Biochemistry and Cellular Physiology of NHE1 EST. 1998 Whole Cell Lysate Preparation using RIPA Buffer – for membrane and soluble proteins RIPA Buffer (Radio-Immune Precipitation Assay) is used to lyse cultured cells to prepare protein extraction from cytoplasmic, membrane and nuclear proteins. Samples prepared with RIPA Buffer can easily be used with a BCA protein assay, western blot, immuno assays or other biochemical determintion. To maintain proteins in the state at time of lysis it is critical to keep cells on ice and only use ice cold buffer throughout to reduce protease, kinase, phosphatase or other enzymatic activity of lysates. T-25 flasks: 1) Remove old media and rinse cells with 2-3 ml of ice cold PBS. 2) Tilt flask 1-2 min on ice to drain residual PBS and remove by aspiration. 3) Add 0.5 ml of ice cold PBS and scrape cells. Transfer to labeled chilled microfuge tube. 4) Rinse cells with additional 1 ml of PBS and add to scraped cells. 5) Centrifuge at 2,500 x g for 5 min at 4oC. Decant supernate, keep pelleted cells. 6) Resuspend pellet in 0.25 ml of RIPA Buffer – use a pipet tip to suspend cells. 7) Lyse cells by sonication for 2, 30 second pulses (50% power) while on ice. 8) Shake mixture gently on ice for 15 min. 9) Centrifuge samples at ~14,000 x g for 15 min to pellet cell debris. Keep supernatant soln. 10) Perform BCA (Pierce) NOT Bradford/biorad protein assay. RIPA Buffer 50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40 1% Sodium Deoxycholic acid, 0.1% sodium dodecylsulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, (add fresh) Protease Inhibitors (add fresh - see manufacturer’s instructions) . -
Influence of Infection and Inflammation on Biomarkers of Nutritional Status
A2.4 INFLUENCE OF INFECTION AND INFLAMMATION ON BIOMARKERS OF NUTRITIONAL STATUS A2.4 Influence of infection and inflammation on biomarkers of nutritional status with an emphasis on vitamin A and iron David I. Thurnham1 and George P. McCabe2 1 Northern Ireland Centre for Food and Health, University of Ulster, Coleraine, United Kingdom of Great Britain and Northern Ireland 2 Statistics Department, Purdue University, West Lafayette, Indiana, United States of America Corresponding author: David I. Thurnham; [email protected] Suggested citation: Thurnham DI, McCabe GP. Influence of infection and inflammation on biomarkers of nutritional status with an emphasis on vitamin A and iron. In: World Health Organization. Report: Priorities in the assessment of vitamin A and iron status in populations, Panama City, Panama, 15–17 September 2010. Geneva, World Health Organization, 2012. Abstract n Many plasma nutrients are influenced by infection or tissue damage. These effects may be passive and the result of changes in blood volume and capillary permeability. They may also be the direct effect of metabolic alterations that depress or increase the concentration of a nutrient or metabolite in the plasma. Where the nutrient or metabolite is a nutritional biomarker as in the case of plasma retinol, a depression in retinol concentrations will result in an overestimate of vitamin A deficiency. In contrast, where the biomarker is increased due to infection as in the case of plasma ferritin concentrations, inflammation will result in an underestimate of iron deficiency. Infection and tissue damage can be recognized by their clinical effects on the body but, unfortunately, subclinical infection or inflammation can only be recognized by measur- ing inflammation biomarkers in the blood. -
The Gut Microbiota and Inflammation
International Journal of Environmental Research and Public Health Review The Gut Microbiota and Inflammation: An Overview 1, 2 1, 1, , Zahraa Al Bander *, Marloes Dekker Nitert , Aya Mousa y and Negar Naderpoor * y 1 Monash Centre for Health Research and Implementation, School of Public Health and Preventive Medicine, Monash University, Melbourne 3168, Australia; [email protected] 2 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia; [email protected] * Correspondence: [email protected] (Z.A.B.); [email protected] (N.N.); Tel.: +61-38-572-2896 (N.N.) These authors contributed equally to this work. y Received: 10 September 2020; Accepted: 15 October 2020; Published: 19 October 2020 Abstract: The gut microbiota encompasses a diverse community of bacteria that carry out various functions influencing the overall health of the host. These comprise nutrient metabolism, immune system regulation and natural defence against infection. The presence of certain bacteria is associated with inflammatory molecules that may bring about inflammation in various body tissues. Inflammation underlies many chronic multisystem conditions including obesity, atherosclerosis, type 2 diabetes mellitus and inflammatory bowel disease. Inflammation may be triggered by structural components of the bacteria which can result in a cascade of inflammatory pathways involving interleukins and other cytokines. Similarly, by-products of metabolic processes in bacteria, including some short-chain fatty acids, can play a role in inhibiting inflammatory processes. In this review, we aimed to provide an overview of the relationship between the gut microbiota and inflammatory molecules and to highlight relevant knowledge gaps in this field. -
Electrical Lysis: Dynamics Revisited and Advances in On-Chip Operation
Critical Reviews™ in Biomedical Engineering, 41(1):37–50 (2013) Electrical Lysis: Dynamics Revisited and Advances in On-chip Operation Bashir I. Morshed,1,* Maitham Shams,2 & Tofy Mussivand3 1Department of Electrical & Computer Engineering, University of Memphis, Memphis, TN 38152; 2Department of Elec- tronics, Carleton University, Ottawa, ON, Canada; 3Medical Devices Innovation Institute, University of Ottawa, Ottawa, ON, Canada * Address all correspondence to: Bashir I. Morshed, Ph.D., Assistant Professor, 204C Engineering Science Building, Department of Electrical & Computer Engineering, University of Memphis, Memphis, TN 38152; Tel.: 901-678-3650; Fax: 901-678-5469: [email protected]. ABSTRACT: Electrical lysis (EL) is the process of breaking the cell membrane to expose the internal contents under an applied high electric field. Lysis is an important phenomenon for cellular analysis, medical treatment, and biofoul- ing control. This paper aims to review, summarize, and analyze recent advancements on EL. Major databases includ- ing PubMed, Ei Engineering Village, IEEE Xplore, and Scholars Portal were searched using relevant keywords. More than 50 articles published in English since 1997 are cited in this article. EL has several key advantages compared to other lysis techniques such as chemical, mechanical, sonication, or laser, including rapid speed of operation, ability to control, miniaturization, low cost, and low power requirement. A variety of cell types have been investigated for including protoplasts, E. coli, yeasts, blood cells, and cancer cells. EL has been developed and applied for decon- tamination, cytology, genetics, single-cell analysis, cancer treatment, and other applications. On-chip EL is a promis- ing technology for multiplexed automated implementation of cell-sample preparation and processing with micro- or nanoliter reagents. -
C3b Catalog Number: A114 Sizes Available
Name: C3b Catalog Number: A114 Sizes Available: 250 µg/vial Concentration: 1.0 mg/mL (see Certificate of Analysis for actual concentration) Form: Liquid Purity: >90% by SDS-PAGE Buffer: 10 mM sodium phosphate, 145 mM NaCl, pH 7.3 Extinction Coeff. A280 nm = 1.03 at 1.0 mg/mL Molecular Weight: 176,000 Da (2 chains) Preservative: None, 0.22 µm filtered Storage: -70oC or below. Avoid freeze/thaw. Source: Normal human serum (shown by certified tests to be negative for HBsAg and for antibodies to HCV, HIV-1 and HIV-II). Precautions: Use normal precautions for handling human blood products. Origin: Manufactured in the USA. General Description C3b is derived from native C3 upon cleavage and release of C3a. It is prepared by cleavage with the alternative pathway C3 convertase. This is important because only a single bond in C3 is cleaved by the native convertase, while cleavage by other proteases, such as trypsin, results in multiple cleavages at many other sites in the protein. Native human C3b is a glycosylated (~2.8%) polypeptide containing two disulfide-linked chains. C3b is central to the function of all three pathways of complement (Law, S.K.A. and Reid, K.B.M. (1995)). Initiation of each pathway generates proteolytic enzyme complexes (C3 convertases) which are bound to the target surface. These enzymes cleave a peptide bond in C3 releasing the anaphylatoxin C3a and activating C3b. For a brief time (~60 µs) this nascent C3b is capable of reacting with and covalently coupling to hydroxyl groups on the target surface. -
Adaptive Tdetect Fact Sheet for Recipient
FACT SHEET FOR RECIPIENTS Coronavirus Adaptive Biotechnologies Corporation September 2, 2021 Disease 2019 T-Detect COVID Test (COVID-19) You are being given this Fact Sheet because your coughing, difficulty breathing, etc.). A full list of sample is being tested or was tested for an adaptive T- symptoms of COVID-19 can be found at the following cell immune response to the virus that causes link: https://www.cdc.gov/coronavirus/2019- Coronavirus Disease 2019 (COVID-19) using the T- ncov/symptoms-testing/symptoms.html. Detect COVID Test. How are people tested for COVID-19? Two main kinds of tests are currently available for You should not interpret the results of this COVID-19: diagnostic tests and adaptive immune response tests. test to indicate the presence or absence of immunity or protection from COVID-19 • A diagnostic test tells you if you have a current infection. infection. • An adaptive immune response test tells you if you may have had a previous infection This Fact Sheet contains information to help you understand the risks and benefits of using this test to What is the T-Detect COVID Test? evaluate your adaptive immune response to SARS-CoV- This test is similar to an antibody test in that it measures 2, the virus that causes COVID-19. After reading this your adaptive immune response to SARS-CoV-2, the Fact Sheet, if you have questions or would like to virus that causes COVID-19. However in this case it discuss the information provided, please talk to your specifically measures your adaptive T-cell immune healthcare provider. -
Blocking Properdin Prevents Complement-Mediated Hemolytic Uremic Syndrome and Systemic Thrombophilia
BASIC RESEARCH www.jasn.org Blocking Properdin Prevents Complement-Mediated Hemolytic Uremic Syndrome and Systemic Thrombophilia Yoshiyasu Ueda,1 Takashi Miwa,1 Damodar Gullipalli,1 Sayaka Sato,1 Daisuke Ito,1 Hangsoo Kim,1 Matthew Palmer,2 and Wen-Chao Song1 Departments of 1Systems Pharmacology and Translational Therapeutics and 2Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania ABSTRACT Background Properdin (P) is a positive regulator of the alternative pathway of complement activation. Although P inhibition is expected and has been shown to ameliorate the alternative pathway of comple- ment-mediated tissue injury in several disease models, it unexpectedly exacerbated renal injury in a murine model of C3 glomerulopathy. The role of P in atypical hemolytic uremic syndrome (aHUS) is uncertain. Methods We blocked P function by genetic deletion or mAb-mediated inhibition in mice carrying a factor H (FH) point mutation, W1206R (FHR/R), that causes aHUS and systemic thrombophilia with high mortality. Results Pdeficiency completely rescued FHR/R mice from premature death and prevented thrombocyto- penia, hemolytic anemia, and renal disease. It also eliminated macrovessel thrombi that were prevalent in FHR/R mice. All mice that received a function-blocking anti-P mAb for 8 weeks survived the experimental period and appeared grossly healthy. Platelet counts and hemoglobin levels were significantly improved in FHR/R mice after 4 weeks of anti-P mAb treatment. One half of the FHR/R mice treated with an isotype control mAb but none of the anti-P mAb-treated mice developed stroke-related neurologic disease. Anti-P mAb-treated FHR/R mice showed largely normal renal histology, and residual liver thrombi were detected in only three of 15 treated mice. -
WO 2016/147053 Al 22 September 2016 (22.09.2016) P O P C T
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/147053 Al 22 September 2016 (22.09.2016) P O P C T (51) International Patent Classification: (71) Applicant: RESVERLOGIX CORP. [CA/CA]; 300, A61K 31/551 (2006.01) A61P 37/02 (2006.01) 4820 Richard Road Sw, Calgary, AB, T3E 6L1 (CA). A61K 31/517 (2006.01) C07D 239/91 (2006.01) (72) Inventors: WASIAK, Sylwia; 431 Whispering Water (21) International Application Number: Trail, Calgary, AB, T3Z 3V1 (CA). KULIKOWSKI, PCT/IB20 16/000443 Ewelina, B.; 31100 Swift Creek Terrace, Calgary, AB, T3Z 0B7 (CA). HALLIDAY, Christopher, R.A.; 403 (22) International Filing Date: 138-18th Avenue SE, Calgary, AB, T2G 5P9 (CA). GIL- 10 March 2016 (10.03.2016) HAM, Dean; 249 Scenic View Close NW, Calgary, AB, (25) Filing Language: English T3L 1Y5 (CA). (26) Publication Language: English (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (30) Priority Data: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, 62/132,572 13 March 2015 (13.03.2015) US BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 62/264,768 8 December 2015 (08. 12.2015) US DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, [Continued on nextpage] (54) Title: COMPOSITIONS AND THERAPEUTIC METHODS FOR THE TREATMENT OF COMPLEMENT-ASSOCIATED DISEASES (57) Abstract: The invention comprises methods of modulating the complement cascade in a mammal and for treating and/or preventing diseases and disorders as sociated with the complement pathway by administering a compound of Formula I or Formula II, such as, for example, 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)- 5,7-dimethoxyquinazolin-4(3H)-one or a pharmaceutically acceptable salt thereof. -
Activation Regulates Alternative Pathway Complement Necrotic
Properdin Binds to Late Apoptotic and Necrotic Cells Independently of C3b and Regulates Alternative Pathway Complement Activation This information is current as of September 27, 2021. Wei Xu, Stefan P. Berger, Leendert A. Trouw, Hetty C. de Boer, Nicole Schlagwein, Chantal Mutsaers, Mohamed R. Daha and Cees van Kooten J Immunol 2008; 180:7613-7621; ; doi: 10.4049/jimmunol.180.11.7613 Downloaded from http://www.jimmunol.org/content/180/11/7613 References This article cites 56 articles, 27 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/180/11/7613.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Properdin Binds to Late Apoptotic and Necrotic Cells Independently of C3b and Regulates Alternative Pathway Complement Activation1,2 Wei Xu,* Stefan P. Berger,* Leendert A. -
The 'C3ar Antagonist' SB290157 Is a Partial C5ar2 Agonist
bioRxiv preprint doi: https://doi.org/10.1101/2020.08.01.232090; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The ‘C3aR antagonist’ SB290157 is a partial C5aR2 agonist Xaria X. Li1, Vinod Kumar1, John D. Lee1, Trent M. Woodruff1* 1School of Biomedical Sciences, The University of Queensland, St Lucia, 4072 Australia. * Correspondence: Prof. Trent M. Woodruff School of Biomedical Sciences, The University of Queensland, St Lucia, 4072 Australia. Ph: +61 7 3365 2924; Fax: +61 7 3365 1766; E-mail: [email protected] Keywords: Complement C3a, C3aR, SB290157, C5aR1, C5aR2 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.01.232090; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abbreviations used in this article: BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; C3aR, C3a receptor C5aR1, C5a receptor 1; CHO-C3aR, Chinese hamster ovary cells stably expressing C3aR; CHO-C5aR1, Chinese hamster ovary cells stably expressing C5aR1; DMEM, Dulbecco's Modified Eagle's Medium; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, foetal bovine serum; HEK293, human embryonic kidney 293 cells; HMDM, human monocyte-derived macrophage; i.p., intraperitoneal; i.v., intravenous; rhC5a, recombinant human C5a; RT, room temperature; S.E.M. -
Eosinophil Extracellular Traps and Inflammatory Pathologies—Untangling the Web!
REVIEW published: 26 November 2018 doi: 10.3389/fimmu.2018.02763 Eosinophil Extracellular Traps and Inflammatory Pathologies—Untangling the Web! Manali Mukherjee 1*, Paige Lacy 2 and Shigeharu Ueki 3 1 Department of Medicine, McMaster University and St Joseph’s Healthcare, Hamilton, ON, Canada, 2 Department of Medicine, Alberta Respiratory Centre, University of Alberta, Edmonton, AB, Canada, 3 Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan Eosinophils are an enigmatic white blood cell, whose immune functions are still under intense investigation. Classically, the eosinophil was considered to fulfill a protective role against parasitic infections, primarily large multicellular helminths. Although eosinophils are predominantly associated with parasite infections, evidence of a role for eosinophils in mediating immunity against bacterial, viral, and fungal infections has been recently reported. Among the mechanisms by which eosinophils are proposed to exert their protective effects is the production of DNA-based extracellular traps (ETs). Remarkably, Edited by: DNA serves a role that extends beyond its biochemical function in encoding RNA and Moncef Zouali, protein sequences; it is also a highly effective substance for entrapment of bacteria Institut National de la Santé et de la and other extracellular pathogens, and serves as valuable scaffolding for antimicrobial Recherche Médicale (INSERM), France mediators such as granule proteins from immune cells. Extracellular -
Adaptive Immune Systems
Immunology 101 (for the Non-Immunologist) Abhinav Deol, MD Assistant Professor of Oncology Wayne State University/ Karmanos Cancer Institute, Detroit MI Presentation originally prepared and presented by Stephen Shiao MD, PhD Department of Radiation Oncology Cedars-Sinai Medical Center Disclosures Bristol-Myers Squibb – Contracted Research What is the immune system? A network of proteins, cells, tissues and organs all coordinated for one purpose: to defend one organism from another It is an infinitely adaptable system to combat the complex and endless variety of pathogens it must address Outline Structure of the immune system Anatomy of an immune response Role of the immune system in disease: infection, cancer and autoimmunity Organs of the Immune System Major organs of the immune system 1. Bone marrow – production of immune cells 2. Thymus – education of immune cells 3. Lymph Nodes – where an immune response is produced 4. Spleen – dual role for immune responses (especially antibody production) and cell recycling Origins of the Immune System B-Cell B-Cell Self-Renewing Common Progenitor Natural Killer Lymphoid Cell Progenitor Thymic T-Cell Selection Hematopoetic T-Cell Stem Cell Progenitor Dendritic Cell Myeloid Progenitor Granulocyte/M Macrophage onocyte Progenitor The Immune Response: The Art of War “Know your enemy and know yourself and you can fight a hundred battles without disaster.” -Sun Tzu, The Art of War Immunity: Two Systems and Their Key Players Adaptive Immunity Innate Immunity Dendritic cells (DC) B cells Phagocytes (Macrophages, Neutrophils) Natural Killer (NK) Cells T cells Dendritic Cells: “Commanders-in-Chief” • Function: Serve as the gateway between the innate and adaptive immune systems.