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Review chapters 15,16,17,18

Chapter 15

Nonspecific Lines of Defense

Vocabulary

Interferons Interleukin pyrogens defensins Interferon

Complement Properdin Pathway Lysozyme phagocytosis

Eosinophils Langerhans cells microglia

Küpffer cells Basophils Eosinophils Neutrophils

Innate Resistance

" " Resistance to most plant and animal pathogens

" " Resistance due to physiological processes of humans that are incompatible with those of the pathogen

" " Correct chemical receptors not present on human cells

" " Temperature and pH may be incompatible with those necessary for the pathogens survival

" " When humans don’t have innate resistance to a pathogen, that organism may cause disease

First Line of Defense

" " Structures, chemicals, processes that work to prevent pathogens entering the body

" " Nonspecific defenses

" " Includes the skin and mucous membranes of the respiratory, digestive, urinary, and reproductive systems

Skin – Physical Components of Defense

" " Two major layers

" " Epidermis

" " Outer layer composed of multiple layers of tightly packed cells

" " Few pathogens can penetrate these layers

" " Shedding of dead skin cells removes attached microorganisms

" " Epidermal dendritic cells

" " Also termed Langerhans cells

" " Phagocytize pathogens

Skin – Physical Components of Defense

" " Dermis

" " Contains protein fibers called collagen

" " Give skin strength and pliability to resist abrasions that could introduce microorganisms

Skin – Chemical Components of Defense

" " Perspiration secreted by sweat glands

" " Salt- inhibits growth of pathogen by drawing water from their cells

" " Lysozyme- destroys cell wall of bacteria

" " Sebum secreted by sebaceous (oil) glands

" " Helps keep skin pliable and less likely to break or tear

" " Lowers the pH of the skin to a level inhibitory to many bacteria

Mucous Membranes

" " Line all body cavities open to the outside environment

" " Two distinct layers

" " Epithelium

" " Deeper connective layer that supports the epithelium

Epithelium

" " Thin, outer covering of the mucous membranes

" " Unlike surface epidermal cells, epithelial cells are living

" " Tightly packed to prevent entry of pathogens

" " Continual shedding of cells carries attached microorganisms away

Microbial Antagonism

" " Normal microbiota help protect the body by competing with potential pathogens

" " Various activities of the normal microbiota make it hard for pathogens to compete

" " Secrete antimicrobial substances that limit pathogen growth

" " Consumption of nutrients makes them unavailable to pathogens

" " Create an environment unfavorable to other microorganisms by changing pH

Microbial Antagonism

" " Helps stimulate the body’s second line of defense

" " Promote overall health by providing vitamins to host

Other First-Line Defenses

" " Many body organs secrete chemicals with antimicrobial properties

" " Lacrimal glands that bathe the eye

Second Line of Defenses

" " Operates when pathogens succeed in penetrating the skin or mucous membranes

" " Nonspecific defense

" " Composed of cells, antimicrobial chemicals, and processes but no physical barriers

" " Many of these components are contained or originate in the blood

Blood

" " Composed of cells and portions of cells within a fluid called plasma

" " Plasma is mostly water containing electrolytes, dissolved gases, nutrients, and proteins

" " When the clotting factors, a group of plasma proteins, are removed from plasma, the remaining fluid is called serum

" " Other plasma proteins include complement proteins and antibodies

" " The cells and cell fragments in plasma are called formed elements

Formed Elements

" " Three types of formed elements

" " Erythrocytes- carry oxygen and carbon dioxide in the blood

" " Platelets- involved in blood clotting

" " Leukocytes- involved in defending the body against invaders

" " 2 groups

" " Granulocytes

" " Agranulocytes

Granulocytes

" " Contain large granules that stain different colors based on the dye used

" " 3 types

" " Basophils- stain blue with the basic dye methylene blue

" " Eosinophils- stain red/orange with the acidic dye eosin

" " Neutrophils- stain lilac with a mixture of acidic and basic dyes

" " Neutrophils and eosinophils can phagocytize pathogens

" " Neutrophils and eosinophils are capable of diapedesis

Agranulocytes

" " Cytoplasm appears uniform under a light microscope

" " 2 types

" " lymphocytes- most involved in specific immunity

" " monocytes- leave the blood and mature into macrophages

Macrophages

" " Phagocytic cells of the second line of defense

" " Wandering macrophages leave the blood via diapedesis and phagocytize throughout the body

" " Fixed macrophages do not move throughout the body and often phagocytize within a specific organ

" " Include Langerhans cells (epidermis), alveolar macrophages (lungs), microglia (central nervous system), Küpffer cells (liver)

" " All macrophages, plus monocytes attached to endothelial cells, constitute the mononuclear phagocytic system

Lab Analysis of Leukocytes

" " The differential white blood cell count test can signal signs of disease

" " Increased eosinophils can indicate allergies or parasitic worm infection

" " Bacterial diseases often show increase in leukocytes and in neutrophils

" " Viral infections show increase in lymphocytes

Components of the Second Line of Defense

" " Phagocytosis

" " Extracellular killing by leukocytes

" " Nonspecific chemical defenses

" " Inflammation

" " Fever

Phagocytosis

" " Cells capable of phagocytosis (certain leukocytes or their derivatives) are called phagocytes

" " Phagocytosis is not completely understood

" " Can be divided into 5 stages

Host Cell Protection

" " The host’s cells are protected from destruction by the phagocytes

" " Some phagocytes have receptors for bacterial surface components, such as flagellar proteins or cell wall components, that are lacking on the body’s cells

" " Opsonins such as complement and antibody provide a signal to the phagocyte

Extracellular Killing by Leukocytes

" " 2 Cell types that kill extracellularly

" " Eosinophils

" " Mainly attack parasitic helminths (worms) by attaching to their surface

" " Secrete toxins that weaken or kill the helminth

" " Eosinophilia, or elevated eosinophil levels, is often indicative of a helminth infection

Extracellular Killing by Leukocytes

" " Natural killer lymphocytes (NK cells)

" " Secrete toxins onto the surface of virally infected cells and tumors

" " Differentiate normal body cells because they have membrane proteins similar to the NK cells

Nonspecific Chemical Defenses

" " Augment phagocytosis

" " Some attack pathogens directly

" " Some enhance other features of nonspecific resistance

" " Includes various chemicals

" " Lysozyme

" " Complement

" " Interferon

" " Defensins

Complement System

" " Set of serum proteins designated numerically according to the order of their discovery

" " Complement activation results in lysis of the foreign cell

" " Complement can be activated in several ways

" " Classical Pathway

" " Alternate Pathway

The Classical Pathway

" " Complement named for the events of this originally discovered pathway

" " Various complement proteins act nonspecifically to “complement” the action of antibodies

The Alternate (Properdin) Pathway

" " Activation occurs independent of antibodies

" " Less efficient than the classical pathway

" " Useful in early stages of infection before antibodies have been made

Inactivation of Complement

" " Body’s own cells withstand complement cascade

" " Membrane-bound proteins on many cells bind with and break down activated complement proteins

" " High turnover rate for cell membranes means any bound membrane attack complexes are shed or endocytosed before any damage occurs

Interferons

" " Protein molecules released by infected host cells to nonspecifically inhibit the spread of viral infections

" " Particularly effective against viruses with RNA genomes

" " Cause many symptoms typically associated with viral infections-chills,fever, muscle aches

" " 3 Classes

" " Alpha

" " Beta

" " Gamma

Interferons

" " Alpha and beta interferons are present early in the infection

" " Gamma interferon appears later in the course of infection; produced by activated T-lymphocytes

" " Stimulates macrophages to migrate and phagocytize

Interferon Therapy

" " It was thought that this might be a good antiviral treatment

" " Many viral infections don’t respond to interferon therapy at all

" " Only a slight effect is seen with those viral infections that do respond

Defensins

" " Small peptides that function nonspecifically to protect against a broad range of pathogens

" " Act against pathogens in various ways

" " Punch holes in cytoplasmic membranes

" " Interfere with internal signaling and other metabolic processes

" " Interfere with the protein that preserves the shape of bacterial proteins under heat stress

" " In humans, inflammation increases the production of defensins

Inflammation

" " Nonspecific response to tissue damage resulting from various causes

" " Characterized by redness, heat, swelling, and pain

" " Two types

" " Acute

" " Chronic

Acute Versus Chronic Inflammation

" " Acute inflammation

" " Develops quickly and is short lived

" " Is usually beneficial

" " Important in the second line of defense

" " Dilation and increased permeability of the blood vessels

" " Migration of phagocytes

" " Tissue repair

" " Chronic inflammation

" " Develops slowly and lasts a long time

" " Can cause damage to tissues

Chemical Mediators of Inflammation

Fever

" " A body temperature over 37°C

" " Results when chemicals called pyrogens trigger the hypothalamus to increase the body’s core temperature

" " Various types of pyrogens:

" " Bacterial toxins

" " Cytoplasmic contents of bacteria released by lysis

" " Antibody-antigen complexes

" " Interleukin-I (IL-1)

Fever Production

" " IL-1 production causes the hypothalamus to secrete prostaglandin which resets the hypothalamic “thermostat”

" " Communication with the brain initiates muscle contractions, increased metabolic activity, and constriction of blood vessels which raises the body’s temperature

" " Chills associated with fever are due to the reduced blood flow of constricted vessels

" " Decrease in IL-1 production results in the body’s temperature returning to normal

Benefits of Fever

" " Enhances the effects of interferons

" " Inhibits growth of some microorganisms

" " May enhance the performance of phagocytes, cells of specific immunity, and the process of tissue repair

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CHAP 16 &17

Vaccination, Specific Immunity & Testing

Specific Immunity

• • Is acquired over time

• • Antigens trigger specific immune responses

• • Various cells, tissues, and organs are part of specific immunity

– – Includes B and T lymphocytes

B Lymphocytes

• • Arise and mature in the red bone marrow

• • Found primarily in the spleen & lymph nodes.

• • Small percentage of B cells circulate in the blood

• • Major function is the secretion of antibodies

T Lymphocytes

• • Produced in the red bone marrow and mature in the thymus

• • Circulate in the lymph and blood and migrate to the lymph nodes, spleen

• • Part of the cell-mediated immune response because they act directly against various antigens

Antigens

• • Molecules that trigger a specific immune response

• • Include components of bacterial cell walls, capsules, pili, and flagella, as well as proteins of viruses, fungi, and protozoa

• • Food and dust can also contain antigenic particles

• • Enter the body by various methods

– – Through breaks in the skin and mucous membranes

– – Direct injection, as with a bite or needle

– – Through organ transplants and skin grafts

Antibodies

• • Also called immunoglobulins (Ig)

• • Soluble, proteinaceous molecules that bind antigen

• • Secreted by plasma cells, which are B cells actively fighting exogenous antigen

• • Considered part of the humoral immune response since bodily fluids such as lymph and blood were once called humors

Antibody Function

• • Antigen-binding sites are complementary to antigenic determinants (epitopes)

– – Due to the close match can form strong, noncovalent interactions

– – Hydrogen bonds and other attractions may also be involved

Antibody Function

• • Function in several ways

– – Activation of complement

– – Stimulation of inflammation

– – Agglutination

– – Neutralization

– – Opsonization

Classes of Antibodies

• • A single type of antibody is not sufficient for the multiple types of invaders to the body

• • The class involved in the immune response depends on the type of foreign antigen, the portal of entry, and the antibody function needed

• • 5 different classes of antibodies: IgG, IgM, IgA, IgE, IgD

Direct and Indirect Testing

• • Direct: demonstration of the presence of an infectious agent

– – Culture

– – Microscopy

– – Molecular methods such as PCR

• • Indirect: demonstration of presence of antibodies to a particular infectious agent

– – Serology

Immunization and Immune Testing

Immunizations

• • 2 artificial methods to make an individual immune to a disease

– – Active immunization-administration of a vaccine so that the patient actively mounts a protective immune response

– – Passive immunization-individual acquires immunity through the transfer of antibodies formed by an immune individual or animal

History of Immunization

• • The Chinese noticed that children who recovered from smallpox did not contract the disease a second time

• • They infected young children with material from a smallpox scab to induce immunity in these children, a process known as variolation

• • The use of variolation spread to England and America but was eventually stopped due to the risk of death

• • Edward Jenner found that protection against smallpox could be induced by inoculation with material from an individual infected with cowpox, a similar but much milder disease

History of Immunization

• • Since cowpox was also called vaccinia this process was called vaccination, and the inoculum was termed a vaccine

• • Louis Pasteur developed a vaccine against Pasteurella multocida

• • Practice of transferring protective antibodies was developed when it was discovered that vaccines protected through the action of antibodies

Vaccination Problems

• • Socioeconomic and political problems prevent many developing nations from receiving vaccines

• • Inability to develop effective vaccines for some pathogens

• • Vaccine-associated risks discourage investment in developing new vaccines

Vaccine Types

• • 3 general types of vaccines

– – Attenuated (live)

– – Killed (inactivated)

• • Whole cell

• • Sub-unit (bioengineered)

– – Toxoid

Attenuated Vaccines

• • Also called modified live vaccines

• • Uses pathogens that are living but have reduced virulence so they don’t cause disease

• • Attenuation is the process of reducing virulence

– – Viruses often attenuated by raising them in tissue culture cells for which they aren’t adapted until they lose the ability to produce disease

– – Bacteria can be made avirulent by culturing under unusual conditions or through genetic manipulation

Attenuated Vaccines

• • Can result in mild infections but no disease

• • Contain replicating microbes that can stimulate a strong immune response due to the large number of antigen molecules

• • Vaccinated individuals can infect those around them, providing herd immunity

Problems with Attenuated Vaccines

• • Attenuated microbes may retain enough virulence to cause disease, especially in immunosuppressed individuals

• • Pregnant women should not receive live vaccines due to the risk of the modified pathogen crossing the placenta

• • Modified viruses may occasionally revert to wild type or mutate to a virulent form

Inactivated Vaccines

• • Can be either whole agent vaccines produced with deactivated but whole microbes, or subunit vaccines produced with antigenic fragments of microbes

• • Both types are safer than live vaccines since they cannot replicate or mutate to a virulent form

• • When microbes are killed must not alter the antigens responsible for stimulating protective immunity

• • Formaldehyde is commonly used to inactivate microbes by cross-linking their proteins and nucleic acids

Problems with Inactivated Vaccines

• • Do not stimulate herd immunity

• • Whole agent vaccines may stimulate a inflammatory response due to nonantigenic portions of the microbe

• • Antigenically weak since the microbes don’t reproduce and don’t provide many antigenic molecules to stimulate the immune response

Problems with Inactivated Vaccines

• • Administration in high or multiple doses, or the incorporation of an adjuvant, can make the vaccine more effective

– – Adjuvants are substances that increase the antigenicity of the vaccine

– – Adjuvants may also stimulate local inflammation

– – High and multiple vaccine doses may produce allergic reactions

Toxoid Vaccines

• • Chemically or thermally modified toxins used to stimulate active immunity

• • Useful for some bacterial diseases

• • Stimulate antibody-mediated immunity

• • Require multiple doses because they possess few antigenic determinants

Modern Vaccine Technology

• • Research attempts to make vaccines that are more effective, cheaper, and safer

• • A variety of recombinant DNA techniques can be used to make improved vaccines

Vaccine Safety

• • Problems associated with vaccination

– – Mild toxicity is the most common problem

• • Especially seen with whole agent vaccines that contain adjuvants

• • May cause pain at the injection site and in rare cases can cause general malaise or fever high enough to induce seizures

– – Anaphylactic shock

• • Is an allergic reaction that may develop to a component of the vaccine

Vaccine Safety

– – Residual virulence

• • Attenuated viruses occasionally cause disease in healthy children or adults

– – Allegations that certain vaccines against childhood diseases cause or trigger autism, diabetes, and asthma

• • Research has not substantiated these allegations

Passive Immunity

• • Administration of preformed antibodies to a patient

• • Used when protection against a recent infection or an ongoing disease is needed quickly

• • Immunologists remove the serum from human or animal donors that have been infected with the disease or immunized against it

• • Serum used for passive immunizations is called antiserum

Limitations of Antisera

• • Contain antibodies against many different antigens not just the ones of interest

• • Repeated injections of antisera collected from a different species can trigger allergic reactions

• • Antisera may be contaminated with viral pathogens

• • Antibodies of antisera are degraded relatively quickly

Serologic Terms

• • Antigen (Ag): anything that induces a specific immune response

• • Antibody (Ab): a large protein molecule produced in response to an antigen that interacts specifically with that antigen, found in serum

• • Antiserum: Serum containing specific antibodies

Serologic Testing

• • Used to detect Ab to infectious agents for diagnosis

• • Gold standard is isolation of infectious agent:

– – Can have low sensitivity

– – Comparatively expensive

– – Can be slow (days vs. hours)

– – Can be less safe than serology

Serologic Testing (2)

• • Antibody can also be used to detect Antigen

– – Salmonella serotypes

– – Neisseria meningitidis serogroups

– – Streptococcus groups

Serologic Reactions

• • We need to demonstrate when Ag+Ab reaction has occurred

– – Agglutination, or clumping

– – “Sandwich” techniques

• • IFA (Indirect Fluorescent Antibody)

• • ELISA (Enzyme-Linked Immunosorbent Assay)

Serologic Testing

• • Usually quantitative

• • Serial dilutions indicate how much Ab can still be demonstrated

• • Titer is reciprocal of dilution factor

– – Dilution of 1:1280 = titer of 1280

Immune Testing

• • Numerous types of serologic test

– – Precipitation tests

– – Agglutination tests

– – Neutralization tests

– – Complement fixation test

– – Various tagged antibody tests

Virus Neutralization

• • Mix dilutions of antiserum with known virus

• • Incubate 1 hour

• • Add cells of any cell line that is sensitive to this virus

• • Incubate

• • Observe for CPE (Cytopathic Effect)

Complement Fixation Test

• • Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes

• • Used to detect the presence of specific antibodies in an individual’s serum

• • Can detect antibody amounts too small to be detected by agglutination

Labeled Antibody Test

• • Use antibody molecules that are linked to some molecular “label” that enables them to be easily detected

• • Used to detect either antigens or antibodies

• • 3 examples

– – Fluorescent antibody tests

– – ELISA

– – Western blot test

Indirect Fluorescent Antibody Test (IFA)

• • Known antigen (can be viral, bacterial, or eukaryotic) is placed on microscope slides to dry

• • Serial dilutions of patient serum added to individual antigen spots on slides

• • Washed, antihuman serum tagged with fluorescent dye (FITC) applied, incubated, washed

• • Read slides with UV microscope, look for fluorescence.

Uses fluorescent dyes as labels

• • Fluorescein is the most important dye used in these test

– – Chemically linked to an antibody without affecting antibody’s ability to bind antigen

– – Glows bright green when exposed to fluorescent light

• • Fluorescein-labeled antibodies used in 2 types of tests

– – Direct fluorescent antibody test

– – Indirect fluorescent antibody tests

Direct Fluorescent Antibody Tests

• • Identifies the presence of antigen in tissue

– – Tissue sample flooded with labeled antibody

– – Antibody and antigen are allowed to bind for a short period

– – Unbound antibody washed from the preparation

– – Results observed under a fluorescent microscope

• • Used to identify small numbers of bacteria in patient tissues

• • Not a quantitative test- the amount of fluorescence observed is not directly related to the amount of antigen present

ELISA

• • Stands for enzyme-linked immunosorbent assay

• • Uses an enzyme as the label

– – Reaction of the enzyme with its substrate produces a colored product indicative of a positive test

• • Most common form of ELISA is used to detect the presence of antibodies in serum

Advantages of The ELISA

• • Can detect either antibody or antigen

• • Can quantify amounts of antigen or antibody

• • Easy to perform, inexpensive, and can test many samples quickly

• • Plates coated with antigen and gelatin can be stored for later testing

• • Technique for detecting antibodies against multiple antigens in a complex mixture

• • Can detect more types of antibodies and are less subject to misinterpretation than other tests

Recent Developments in Immune Testing

• • Development of simple immunoassays that give results in minutes

• • Generally not quantitative but are useful in determining a preliminary diagnosis

• • Most common are the immunofiltration and immunochromotography assays

– – Immunofiltration

• • Rapid ELISA that uses antibodies bound to membrane filters rather than polystyrene plates

• • Membrane filters have a large surface area making the assay quicker to complete

Recent Developments in Immune Testing

– – Immunochromatography

• • Very rapid and easy to read ELISAs

• • Antigen solution flows through a porous strip where it encounters antibody labeled with either pink colloidal gold or blue colloidal selenium

• • Antigen-Antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible pink or blue line