Chapter 5 Soluble Mediators of the Immune System - PowerPoint Presentation

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Chapter 5

Soluble Mediators of the Immune System

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Main Topics for today

Complement System

Cytokines, Chemokines, Growth Factors

Expanding on the Immune Response

Review innate and natural immune response

Antigen capture, presentation

Primary and Secondary immune response

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The Complement System

Classic Pathway:

immune complex triggered.

Apoptotic cells

Certain viruses and Gram-negative bacteria

C-Reactive protein bound to ligand

Alternate Pathway:

Various bacteria, fungi, viruses, or tumor cells

Mannose-Binding Lectin Pathway:

bacteria containing terminal mannose groups

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Physiologic Activities of the Complement system

Host defense against infection

Interface between innate and adaptive immunity

Disposal of waste

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The Complement System

Activation of Complement

Normally, complement components are present in the circulation in an inactive form.

Complement can become activated by antigen-antibody complexes in solution or circulation, bacteria, fungi, mannose containing bacterial components.

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Three Activation Pathways of Complement

(Redrawn from Walport MJ: Complement,

N Engl J Med

344[14]:1058, 2001.)

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Enzyme activation occurs after complement is initially activated; each enzyme precursor is activated by the previous complement component or complex in a cascade-like fashion

The pathways leading to the cleavage of C3 are triggered enzyme cascades.

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Complement receptors

surface

membrane glycoproteins

react

with one or more of the fragments of C3 produced during complement activation and degradation

Effects of complement activation

Production of inflammatory mediators

Cell membrane lysis of antibody-coated targets

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Opsonization

Enhances phagocytosis

Neutrophils

Macrophages

Antibody Dependent Cellular Cytotoxicity

Natural killer cells

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Classic Pathway

Recognition: C1 recognizes the antigen-antibody reaction

Amplification of Proteolytic Complement Cascade

Membrane Attack Complex

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Alternate PathwayNon antibody initiated pathway

Can be activated by microbial and mammalian cell surfaces

Factor H is a major controlling factor: blocks C3bBb complex

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Mannose-Binding Lectin Pathway

Family of Calcium dependant lectins

Bacteria

Deficiency: mutation reduces level of

lectin

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Biologic Functions of Complement Proteins

Two categories:

Cell lysis by the membrane attack complex (MAC)

Biologic effects of proteolytic fragments of complement

C3a and C5a:

chemotaxins

,

anaphylatoxins

C3b:

opsonization

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Alterations in Complement Proteins

Elevated Complement Levels

Increased in many inflammatory conditions.

Limited clinical significance because separate complement components are acute-phase proteins.

C3, C4 levels

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Alterations in Complement Proteins

Decreased Complement Levels

Decreased because of excessive activation, currently being consumed, or a single complement component is absent because of a

genetic defect

.

C1, C2 and C4 deficiencies assoc. with Systemic Lupus Erythematosus

C1q associated with Hereditary Angioedema (HAE)

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Three types of complement deficiency can cause increased susceptibility to pyogenic infections:

Deficiency of opsonic activities of complement.

Any deficiency that compromises the lytic activity of complement.

Deficient function of mannose-binding lectin pathway.

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C3 deficiencies

Homozygous C3 deficiency: lifelong life threatening infections.

Hereditary C3 deficiency: pneumococcal septicemia.

Encapsulated bacterial infections

C5 associated with bacterial infections

C6, C7 and C8 assoc. with infections caused by

Neisseria

spp.

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Assessment of Complement component levels:Radial Immunodiffusion

Nephelometry

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Other Soluble Mediators of Immune Response

Cytokines are made by leukocytes (T cells, B cells, Monocytes)and act on other leukocytes.

Interleukins is alternate term for cytokines.

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Interleukins

IL-1 APC signal to T-helper cell

IL-2 T-cell growth factor

IL-3 Cell growth factor from

thymic

epithelial cells, helps T cell differentiation

IL-4 produced by T-helper2 cells involved in Ig class switching to IgE. Also

downregulates

T-helper1 cells

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Interleukins

IL-5 produced by T-helper2 cells

 involved in IgA

production

IL-10 produced by T-helper2 cells, it

downregulates

the cell-mediated immune system

IL-12 associated with the development of cell-mediated immunity

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Common Properties of Cytokines

Secreted in response to cell activation.

Bind to specific membrane receptors on target cells.

Regulate receptor expression in T and B cells.

Act on different cell types

Excite same functional effects with multiple cytokines.

Act close to the site of synthesis.

Influence synthesis and actions of other cytokines.

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Immunoregulatory Activity of Other Cytokines

Interferons

: group of cytokines discovered in virally infected cultured cells; name derived from interference with viral replication.

Tumor necrosis factor (TN

F): principal mediator of acute inflammatory response to gram-negative bacteria and other infectious microbes.

Stimulates recruitment of neutrophils and monocytes to sites of infection.

Activates neutrophils and monocytes to eradicate microbes.

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Hematopoietic Stimulators

Stem cell factor (

c-kit

ligand)

Colony-stimulating factors: GM-CSF, M-CSF, G-CSF

Transforming growth factor beta (TGF-

)

Chemokines: Chemotactic Cytokines

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Assessment of Cytokines

Traditional

: bioassays, enzyme-linked immunosorbent assay (ELISA), intracellular staining, ribonuclease protection assay, polymerase chain reaction (PCR)

Newer methods

:

multiplexed assay, flow cytometry, cord blood cell stimulation, real-time PCR, ELISPOT assays, enhanced immunoassays for cytokines, high-sensitivity ELISA

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Let’s go back to the Immune Response

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First Line of Defense

Intact skin, mucosal membranes

Tears

Saliva

Skin secretions

Mucosal secretions

Normal flora

G.I. tract

Urinary tract

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Body Defenses—Resistance to Microbial Disease

Natural Immunity: Innate or inborn resistance to infection; nonspecific mechanism.

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Monocyte (blood)

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Monocytes and Macrophages

(From Barrett JT:

Textbook of immunology,

ed 5, St Louis, 1988, Mosby.)

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Monocytes and Macrophages

(Modified from Roitt IM:

Essential immunology,

ed 5, Oxford, 1984, Blackwell Scientific.)

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Monocytes and Macrophages

Functions of Monocytes and Macrophages in Host Defense

(Modified from Johnston RB:

Monocytes and macrophages,

N Engl J Med

318[12]:749, 1988

.)

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Monocytes and Macrophages

Antigen presentation: induction of the immune response

Secretion of biologically active

molecules:cytokines

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Acute Inflammation

(From Delves PJ, Roitt, IM: The immune system. Part I,

N Engl J Med

343[1]:37-49, 2000.)

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Sepsis: is a systemic inflammatory response syndrome (SIRS)

Sepsis is an infection-induced syndrome defined as the presence of two or more of the following:

Fever or hypothermia

Leukocytosis or leukopenia

Tachycardia: increased heart rate

Increased respiratory rate

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Process of Phagocytosis

Chemotaxis

Adherence

Engulfment

Digestion

Subsequent phagocytic activity

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Process of Phagocytosis

(Redrawn from Turgeon ML:

Clinical hematology: theory and procedures,

ed 3, Philadelphia, 2000, Lippincott–Williams & Wilkins.)

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Monocytes and Macrophages

(From Barrett JT:

Textbook of immunology,

ed 5, St Louis, 1988, Mosby.)

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Monocytes and Macrophages

Antigen presentation: induction of the immune response

Secretion of biologically active

molecules:cytokines

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Acute-Phase Proteins

Produced as an innate body defense or response to tissue injury. (inflammation, infection, etc.)

Group of glycoproteins

ESR, CRP used as indicators

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Synthesis and Catabolism of Acute-Phase Proteins

Level increases 2 to 5 fold during inflammation

C-Reactive Protein: is the first one to appear

Significance of Other Acute-Phase Reactants: C3, C4,

Haptoglobin

, coagulation factors

Acute-Phase Reactant Assessment Methods: ESR, a1-antitrypsin,

ceruloplasmin

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Acute phase proteins increase or decrease at different rates.

Most last 2 to 4 days

CRP half life of 5 to 7 hrs.

They are inflammation indicators, no diagnostic capability, no specificity

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Adaptive Immunity- organized around the T and B lymphocytes

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Comparison of Innate and Adaptive Immunity

Innate immune system is an ancient form of host defense that appeared before the adaptive immune system.

It may not recognize every possible antigen;

it focuses on a few large groups of microorganisms called pathogen-associated molecular patterns (PAMPs).

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Comparison of Innate and Adaptive Immunity

Receptors of innate immune system that recognize these PAMPs are called pattern-recognition receptors (e.g., toll-like receptors).

Mechanisms of innate immunity (e.g., phagocytes, complement pathways) are activated immediately after infection.

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Cross-Talk among Immune Cells

(From Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis,

N Engl J Med

48[2]:138 150, 2003.)

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Origin and Development of

Blood Cells

2-8 weeks of life: erythroblasts formed in islets of yolk sac.

2-5 months of gestation: liver and spleen are major sites of hematopoiesis.

Granular leukocytes also appear.

Fourth month of gestation: bone marrow begins to produce blood cells.

Fifth month of gestation: bone marrow assumes ultimate role as primary site of hematopoiesis

.

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Granulocytic Cells

Neutrophils provide host defense against bacterial and fungal infections.

Eosinophils are homeostatic regulators of inflammation; play role in host defense because of ability to kill certain parasites.

Basophils play role in acute, systemic hypersensitivity reactions.

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Cell Surface Receptors

Three protein families are associated in a network of cellular interactions:

Immunoglobulin family

Integrin family

Selectin family

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T-Lymphocyte Subsets

Helper T (Th) lymphocytes

Th1: cell-mediated effector mechanisms.

Th2: regulation of antibody production.

Treg (regulatory T cells): an immunoregulatory type of Th

cell

Cytotoxic T (Tc) lymphocytes

:

capable of directly destroying virally infected target cells.

Suppressor T (Ts) lym

phocytes:

down-regulate actions of other T and B cells.

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T Lymphocytes

Antigen Processing and Antigen Presentation to T Cells

Endogenous pathway: MHC class I, CD8

Exogenous

pathway:MHC

class II, CD4

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Disorders of Neutrophils

Neutrophils as Mediators of Noninfectious Inflammatory Diseases

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Disorders of Neutrophils

Signs and Symptoms of Abnormal Neutrophil Function

Quantitative problem (neutropenia)

Qualitative defect (leukocyte mobility)

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Disorders of Neutrophils

Congenital Abnormalities of Neutrophil Structure and Function

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Chediak

-Higashi Syndrome: Abnormal granulation, large granules impair phagocytosis

Chronic Granulomatous Disease: There are variants of CGD diseases. Disorders in oxidative metabolism leading to the production of H

2

O

2

.

Complement Receptor 3 Deficiency: Affects leukocyte adhesion, can’t bind to opsonized bacteria

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Myeloperoxidase Deficiency: MPO is present in

azurophilic

granules in the neutrophils. Deficiency in MPO leads to ineffective bacterial killing

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Monocyte-Macrophage Disorders

Gaucher’s disease represents a deficiency of beta-glucocerebrosidase (enzyme).

Niemann-Pick disease represents a deficiency of sphingomyelinase (enzyme).

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Disease States Involving the Leukocyte Integrins

Leukocyte adhesion deficiency (LAD) is characterized by impaired leukocyte adhesion that leads to recurrent and often fatal bacterial and fungal infections.

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Chapter 8

Basic Serologic Laboratory Techniques

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Procedure Manual

Must be a complete record of current techniques and policies that are available at all times.

New pages must be dated and initialed.

Retired procedures must be retained for five years, with date and reason for removal.

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Blood Specimen Preparation

Serum preferred- plas

ma (coagulation factors

Clot retraction- at room temp, pour serum to be used

Unused serum- store in freezer

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Types of Specimens Tested

Most of immunoassays use serum

Lipemia, hemolysis, icteric or bacterial contamination-

Other specimen types- body fluids. urine

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Inactivation of Complement

Purpose- Complement proteins may interfere with reaction, C1q can agglutinate latex particles

Procedure- heat serum at 56

o

C for 30 min,

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Pipettes

Graduated Pipettes

Serologic Pipettes

Volumetric Pipettes

Inspection and Use

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Figure 08-01.   Types of manual pipettes. TD,

To deliver.

(From Turgeon ML:

Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques,

ed 5, St Louis, 2007, Mosby.)

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Figure 08-02.

   Pipetting technique.

(From Turgeon ML:

Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques,

ed 5, St Louis, 2007, Mosby

.)

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Figure 08-03.

   Reading the meniscus.

(From Turgeon ML:

Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques,

ed 5, St Louis, 2007, Mosby.)

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Figure 08-04.

   Steps in using piston-type automatic micropipette.

A,

Attaching proper tip size for range of pipette volume, and twisting tip as it is pushed onto pipette to give an airtight, continuous seal.

(From Kaplan LA, Pesce A:

Clinical chemistry: theory, analysis, and correlation,

ed 4, St

Louis, 2003, Mosb

Figure 08-04.

   Steps in using piston-type automatic micropipette.

B,

Holding pipette before use.

(From Kaplan LA, Pesce A:

Clinical chemistry: theory, analysis, and correlation,

ed 4, St Louis, 2003, Mosby.)

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Figure 08-04.

   Steps in using piston-type automatic micropipette.

C,

Follow instructions for filling and emptying pipette tip.

(From Kaplan LA, Pesce A:

Clinical chemistry: theory, analysis, and correlation,

ed 4, St Louis, 2003, Mosby

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Pipetting Techniques Using Manual Pipettes

Automatic micropipettors-

Automatic dispensers or syringes-

Diluter-Dispensers-

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Automatic Pipettors

Faster

Deliver the equal volume repeatedly

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Dilutions

Diluting Specimens-

Dilution Factor-

Single Dilution:

Calculations of concentration of a single dilution

Use of dilution factors

Serial Dilutions-

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Principles of Antibody Levels

Testing for Antibody Levels-

Antibody Titer-

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Chapter 9

Point-of-Care Testing

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Waived Testing

Waived tests: simple.

Moderately complex tests: usually automated.

Highly complex tests: require considerable judgment.

Provider-performed microscopy tests: slide examinations.

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Point-of-Care Policies and Procedures

Written policies and procedures must be available for:

Patient preparation

Specimen collection and preservation

Instrument calibration

Policies for quality control and remedial actions

Equipment performance evaluations

Procedures for test performance

Result report and recording

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Non–Instrument-Based Point of Care

Largest POC groups- HCG and Quik Strep

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Chapter 10

Agglutination Methods

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Principles of Agglutination

Precipitation-

Agglutination-

Artificial Carriers-

Quality of Test Results

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Latex Agglutination

Coated Latex-

Coagglutination and liposome enhanced tests-

Agglutination inhibition-

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Figure 10-01.

   

Agglutination patterns. A,

Slide agglutination of bacteria with known antisera or known bacteria.

Left,

Positive reaction;

right,

negative reaction

.

Figure 10-01.

   

Agglutination patterns. B,

Tube agglutination.

Left,

Positive reaction;

right,

negative reaction.

(From Barrett JT:

Textbook of immunology,

ed 5, St Louis, 1988, Mosby

.)

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Flocculation Tests

Based on interaction of soluble antigen and antibody.

RPR-

VDRL-

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Hemagglutination

Mechanisms of Agglutination-

Methods of Enhancing Agglutination-

Graded Agglutination Reactions-

Microplate Agglutination Reactions-

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Pregnancy Testing

Biochemical Detected by Testing-

Physiology of Human Chorionic Gonadotropic (hCG) Hormone-

Principle of Agglutination Inhibition-

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Figure 10-05.

   Reading red blood cell agglutination reactions.

(From Lehman CA:

Saunders Manual of Clinical Laboratory Science,

Philadelphia, 1998, Saunders, pp 394-395.)