Concept of Immunity & Body Defenses

The Concept of Immunity

• Susceptibility is the lack of resistance to a disease.
• Immunity is the ability to ward off disease.
• Innate immunity provides defenses against any pathogen and is present from birth.
• Adaptive immunity is resistance to a specific pathogen and is a slower response.

Overview of Body's Defenses

• Innate immunity includes the first and second lines of defense.
• The first line of defense includes:
  • Intact skin.
  • Mucous membranes and their secretions, including the ciliary mechanism and lacrimal apparatus.
  • Normal microbiota.
• The second line of defense includes:
  • Phagocytosis by neutrophils, eosinophils, dendritic cells, and macrophages.
  • Inflammation.
  • Fever.
  • Antimicrobial substances, such as the complement system.
• Adaptive immunity is the third line of defense and includes:
  • Specialized lymphocytes: T cells and B cells.
  • Antibodies.

Physical Factors of First Line of Defense

• The epidermis consists of tightly packed cells and contains keratin for protection.
• Mucous membranes trap microbes that are dispelled through coughing.
• The ciliary escalator transports microbes trapped in mucus away from the lungs.
• The lacrimal apparatus washes the eyes.
• Saliva washes microbes off.
• Urine flows out.
• Vaginal secretions flow out.

Chemical Factors of First Line of Defense

• Fungistatic fatty acids in sebum inhibit fungal growth.
• The skin has a low pH of 3-5.
• Lysozyme in perspiration, tears, saliva, and urine breaks down peptidoglycan.
• Gastric juice has a low pH of 1.2-3.0.
• Vaginal secretions have a low pH of 3-5.

Normal Microbiota and Innate Immunity

• Microbial antagonism/competitive exclusion occurs when normal microbiota compete with pathogens or alter the environment.
• Mutualistic microbiota provides benefits to both the host and the microbes.

Formed Elements in Blood

• Red blood cells transport O2 and CO2 for cellular respiration and ATP production.
• White blood cells include:
  • Granulocytes:
    • Neutrophils perform phagocytosis.
    • Basophils and mast cells release histamine.
    • Eosinophils kill parasites (helminths, protozoa).
  • Agranulocytes:
    • Monocytes perform phagocytosis.
    • Dendritic cells perform phagocytosis.
    • Natural killer (NK) cells destroy target cells.
    • T cells (lymphocytes) mediate cell-mediated immunity.
    • B cells (lymphocytes) produce antibodies.
• Platelets are involved in blood clotting.

Natural Killer Cells

• NK cells recognize "nonself" cells by detecting the absence of MHC (Major Histocompatibility Complex) markers.
• When NK cells identify a cell as "nonself", they release cytotoxic perforins and granzymes to kill the cell.

Differential White Cell Count

• A differential white cell count is the percentage of each type of white cell in a sample of 100 white blood cells.
• The order from most to least numerous is: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils (Never Let Monkeys Eat Bananas).

Lymphatic System

• Lymphatic fluid flows from interstitial fluid, gets absorbed, and moves up to the heart.
• Blood vessels leak fluid into tissues, forming interstitial fluid, which is absorbed by lymphatic capillaries.
• Lymph nodes filter the fluid and contain white blood cells that ingest pathogens.
• The filtered fluid is then returned to the circulating bloodstream.
• If interstitial fluid is not reabsorbed by the lymphatic capillaries, edema (swelling) can occur.

Phagocytosis

• Phagocytosis is the ingestion of microbes or particles by cells called phagocytes.
• Phagocytes include neutrophils, fixed macrophages, and wandering macrophages.
• Steps of phagocytosis:
  • Chemotaxis and adherence of microbe to phagocyte.
  • Ingestion of microbe by phagocyte.
  • Formation of a phagosome.
  • Fusion of the phagosome with a lysosome to form a phagolysosome.
  • Digestion of ingested microbe by enzymes.
  • Formation of residual body containing indigestible material.
  • Discharge of waste material through exocytosis.

Microbial Evasion of Phagocytosis

• Damaged cells release chemicals that can lead to inflammation if a bacterial infection is involved.

Inflammation

• Activated proteins (complement, cytokine, and kinins) lead to inflammation.
• Vasodilation (histamine, kinins, prostaglandins, and leukotrienes) causes redness, swelling (edema), pain, and heat.
• The steps of inflammation:
  • Chemicals like histamine, kinins, prostaglandins, leukotrienes, and cytokines are released by damaged cells.
  • Blood clots form.
  • An abscess starts to form.
  • Phagocytes stick to the endothelium (margination).
  • Phagocytes squeeze between endothelial cells (diapedesis).
  • Phagocytosis of invading bacteria occurs.

Tissue Repair

• Tissue repair occurs once damaged cells are gone, chemical release stops, and blood clots form.

Fever

• Fever is an abnormally high body temperature.
• The hypothalamus is normally set at 37 degrees C.
• Gram-negative endotoxins cause phagocytes to release interleukin-1 (IL-1).
• The hypothalamus releases prostaglandins, resetting the body to a higher temperature.
• Advantages of fever:
  • Increases transferrins.
  • Increases IL-1 activity.
  • Produces interferon.
• Disadvantages of fever:
  • Tachycardia.
  • Acidosis.
  • Dehydration.
  • Temperatures of 44-46 degrees C are fatal.

Complement System

• Inactivated C3 splits into activated C3a and C3b.
• C3b binds to the microbe -> opsonization.
• C3b splits C5 into C5a and C5b.
• C5b, C6, C7, and C8 bind together and insert into the microbial plasma membrane, creating a receptor to attract C9.
• Multiple C9 fragments form a channel -> membrane attack complex, which causes cytolysis (MAC).
• C3a and C5a cause mast cells to release histamine, resulting in inflammation, and C5a attracts phagocytes.

Classical Pathway of Complement Activation

• C1 is activated by binding to antigen-antibody complexes.
• Activated C1 splits C2 into C2a and C2b, and C4 into C4a and C4b.
• C2a and C4b combine and activate C3, splitting it into C3a and C3b.

Alternative Pathway of Complement Activation

• C3 combines with factors B, D, and P on the surface of a microbe.
• This causes C3 to split into fragments C3a and C3b.

Lectin Pathway of Complement Activation

• Lectin binds to an invading cell.
• Bound lectin splits C2 and C4.
• C2a and C4b combine and activate C3.

Bacterial Evasion of Complement

• Capsules prevent Complement activation.
• Surface lipid-carbohydrates prevent membrane attack complex (MAC) formation.
• Enzymatic digestion of C5a.

Overview of Complement System

• The complement system results in opsonization, inflammation, and cytolysis.
• Step 1: Activation pathways:
  • Classical: Ab + Ag + C1
  • Alternative: Protein factors
  • Lectin: Lectin + pathogen
• Step 2: General Cascade: C3-C9
• Step 3: The three outcomes.

Immunity

• Innate immunity is defense against any pathogen present at birth or soon after.
• Adaptive immunity is induced resistance to a specific pathogen.
• An antigen-antibody complex results when antigens and antibodies bind together, causing clumping and agglutination.
• Adaptive immunity is slower because it takes time to create the memory cells of resistance.

Dual Nature of Adaptive Immunity

• B cells mediate humoral immunity.
• T cells mediate cell-mediated immunity.
• T cells mature and differentiate in the thymus.
• B cells mature and differentiate in the red bone marrow.
• Humoral immunity:
  • B cells mature in the bone marrow.
  • Clonal selection and plasma cells make antibodies.
  • Antibodies circulate in the blood.
  • Active against free antigens.
• Cellular immunity:
  • T cells mature in the thymus.
  • Mobilizes cells (phagocytes and T cells).
  • Active against bound antigens inside of cells.

Nature of Antigens

• An antigen (Ag) is a substance that causes the body to produce specific antibodies or sensitized T cells.
• Antigens are found on the surface of cells and are seen as nonself.
• Often a protein.
• Antibodies (Ab) interact with epitopes or antigenic determinants.
• Each antigen can have multiple antibodies that can react to it.
• Binding sites of antigens are called epitopes or antigenic determinants.
• High specificity and affinity for binding.

Results of Ag-Ab Binding

• Protective mechanisms:
  • Agglutination: reduces number of infectious units.
  • Opsonization: coating antigen with antibody enhances phagocytosis.
  • Activation of complement: causes inflammation and cell lysis.
  • Antibody-dependent cell-mediated cytotoxicity: antibodies attached to target cell cause destruction by macrophages, eosinophils, and NK cells.
  • Neutralization: Blocks adhesion of bacteria and viruses to mucosa.
• Haptens are molecules that can link to a carrier cell or a molecule and appear as a pathogen.

Terminology of Adaptive Immunity

• Serology is the study of reactions between antibodies and antigens.
• Antiserum is the generic term for serum containing Ab.
• Globulins are serum proteins.
• Immunoglobulins are antibodies.
• Gamma (y) globulin is the serum fraction containing Ab.
• Antibodies = globular proteins called immunoglobulins.

IgG Antibodies

• Monomer.
• 80% of serum Abs.
• Fix complement.
• In blood, lymph, and intestine.
• Cross placenta.
• Enhance phagocytosis; neutralize toxins and viruses; protect fetus and newborn.

IgM Antibodies

• Pentamer.
• 5-10% of serum Abs.
• Fix complement.
• In blood, in lymph, and on B cells.
• Agglutinates microbes; first Ab produced in response to infection.

IgA Antibodies

• Dimer.
• 10-15% of serum Abs.
• In secretions.
• Mucosal protection.

IgD Antibodies

• Monomer.
• 0.2% of serum Abs.
• In blood, in lymph, and on B cells.
• On B cells, initiate immune response.

IgE Antibodies

• Monomer.
• 0.002% of serum Abs.
• On mast cells, on basophils, and in blood.
• Allergic reactions; lysis of parasitic worms.

Immunological Memory

• Antibody titer is the amount of Ab in serum.
• Primary response occurs after initial contact with Ag.
• Secondary (memory or anamnestic) response occurs after second exposure.

Types of Adaptive Immunity

• Naturally acquired active immunity results from infection.
• Naturally acquired passive immunity is transplacental or via colostrum.
• Artificially acquired active immunity is the injection of Ag (vaccination).
• Artificially acquired passive immunity is the injection of Ab.

Activation of B Cells

• T-independent antigens stimulate B cells to make Abs without T cell help.
• Major histocompatibility complex (MHC) expressed on mammalian cells.
• T-dependent antigens need T cells to stimulate help become plasma cells.
  • Ag presented with (self) MHC to TH cell.
  • TH cell produces cytokines that activate the B cell.
• B cells differentiate into:
  • Antibody-producing plasma cells.
  • Memory cells.
• Clonal deletion eliminates harmful B cells.

Clonal Selection

• Stem cells differentiate into mature B cells, each bearing surface immunoglobulins against a specific antigen.
• B cell III complexes with its specific antigen and proliferates.
• Some B cells proliferate into long-lived memory cells, which at a later date can be stimulated to become antibody-producing plasma cells.
• Other B cells proliferate into antibody-producing plasma cells.
• Plasma cells secrete antibodies into circulation.

T Cytotoxic Cells

• A normal cell will not trigger a response by a cytotoxic T lymphocyte (CTL).
• A virus-infected cell or a cancer cell produces abnormal endogenous antigens.
• The abnormal antigen is presented on the cell surface in association with MHC class I molecules.
• CD8+ T cells with receptors for the antigen are transformed into CTLs.
• The CTL induces destruction of the virus-infected cell by apoptosis (programmed cell death).

Natural Killer (NK) Cells

• Granular leukocytes destroy cells that don’t express MHC I.
• Kill virus-infected and tumor cells.
• Attack parasites.

Types of Vaccines

• Whole agent (killed cell) vaccines.
• Subunit vaccines.

Vaccine Administration

• Most vaccines are administered:
  • Subcutaneously.
  • Intramuscularly.
  • Intradermally.
• Nasal and oral vaccines:
  • Available for only a few diseases.
  • Stimulates IgA protection on mucous membranes.
• Adjuvant: special binding substance required by some vaccines.
  • Enhances immunogenicity.
  • Prolongs antigen retention at the injection site.
  • Most common adjuvant: alum (aluminum hydroxide salts).

Vaccine Side Effects

• Common reactions:
  • Local reactions at the injection site.
  • Fever. -Allergies.
• Rare reactions:
  • Panencephalitis (measles vaccine).
  • Back-mutation to a virulent strain (polio vaccine).
  • Disease due to contamination with dangerous viruses or chemicals.
  • Neurological effects of unknown cause (pertussis and swine flu vaccines).
  • Allergic reactions to the medium rather than vaccine antigens (egg or tissue culture).

Immunopathology

• Immunopathology is the study of disease associated with the overreactivity or underreactivity of the immune response.
• Hypersensitivity is allergy and autoimmunity.
• Hyposensitivity or immunodeficiency means the immune system is incompletely developed, suppressed, or destroyed.

Type I Reactions: Atopy and Anaphylaxis

• Allergens are innocuous substances that induce allergy in sensitive individuals.
• Atopy is chronic local allergy such as hay fever or asthma.
• Anaphylaxis is a systemic, sometimes fatal reaction that involves airway obstruction and circulatory collapse.

Allergy Susceptibility

• Type I allergies are usually relatively mild.
• Some last for a lifetime, some are "outgrown", others develop later in life.
• Generalized susceptibility to allergens is inherited, not the allergy to a specific substance.
• Genetic basis for atopy: increased IgE production, increased reactivity of mast cells.

Allergens Portals of Entry

• Inhalants: airborne environmental allergens such as pollens, house dust, dander, or fungal spores.
• Ingestants: allergens that enter by mouth that cause food allergies.
• Injectants: allergies triggered by drugs, vaccines, or hymenopteran (bee) venom.
• Contactants: allergies that enter through the skin – many are type IV (delayed) hypersensitivities.

Treatment and Prevention of Allergy

• Corticosteroids: inhibit the activity of lymphocytes and reduce the production of IgE.
  • Have dangerous side effects and should not be taken for long periods.
• Antihistamines:
  • Most widely used medications for preventing symptoms of atopic allergy.
• Epinephrine (adrenaline): reverses constriction of airways and slows the release of allergic mediators.

Type II Hypersensitivities

• Reactions that lyse foreign cells
• Complement-assisted destruction (lysis) of cells by antibodies directed against those cells’ surface antigens
  • Transfusion reactions
  • Some types of autoimmunities
• Alloantigens
  • Molecules that differ in the same species that are recognized by recipient lymphocytes

Preventing Hemolytic Disease of the Newborn

• RhoGAM antiserum
  • Passive immunization for an Rh- mother with an Rh+ fetus
  • Injected at 28-32 weeks and again immediately after delivery
  • Sequesters fetal RBCs that have escaped into maternal circulation and prevents sensitization

Type III Hypersensitivities

• Soluble antigen reacts with antibody and forms large complexes
• Complexes are deposited in various tissues in the body
• Neutrophils release lysosomal granules that digest tissues and cause a destructive inflammatory condition

Type IV Hypersensitivities

• Cell-Mediated (Delayed) Reactions
• Result when T cells respond to antigen displayed on self tissues or transplanted foreign cells
• Symptoms arise from one to several days following the second contact with an antigen
  • Tuberculin reaction
  • Contact Dermatitis

Autoimmunity

• Autoimmune diseases: individuals actually develop hypersensitivity to themselves
  • Autoantibodies, T cells, or both mount an abnormal attack against self antigens
  • Systemic: involves several major organs
  • Organ specific: involves only one organ or tissue

Immunodeficiency Diseases

• Primary immunodeficiencies: present at birth (congenital), usually stemming from genetic errors
• Secondary immunodeficiencies: acquired after birth and caused by natural or artificial agents
  • Examples: HIV

Severe Combined Immunodeficiencies (SCIDs)

• Dysfunction in B and T cells
• Most serious form of immunodeficiency
  • Involve dysfunction in both lymphocyte Systems
• SCID children require rigorous aseptic techniques to protect them from opportunistic infections

Methods of Microbial Identification

• Phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry
• Immunologic: serological analysis
• Genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria

Phenotypic Methods: Microscopic Morphology

• Light microscopy aids in the observation of
  • Cell shape, size, and arrangement
  • Gram stain reaction, acid-fast reaction
  • Endospores, granules, and capsules
• Electron microscopy can pinpoint additional structural features such as
  • Cell wall
  • Flagella
  • Pili
  • Fimbriae

Phenotypic Methods: Macroscopic Morphology

• Traits that can be assessed with the naked eye
  • Appearance of colonies: texture, size, shape, and pigment
  • Patterns of growth in broth and gelatin media

Phenotypic Methods: Physiological/ Biochemical Characteristics

• Assess nutritional and metabolic activities
  • Fermentation of sugars
  • Capacity to digest complex polymers
  • Production of gas
  • Sensitivity to antibodies Rapid tests – Enterotube II

Immunologic Methods

• Serology
  • Involves in vitro testing of serum
  • Based on the principle that antibodies have an extreme specificity for antigen
  • Urine, cerebrospinal fluid, whole tissues, and saliva can be analyzed for the presence of specific antibodies Latex agglutination test – Agglutination Involves the Clumping of Antigens
• Hemagglutination involves agglutination of RBCs
• Some viruses agglutinate RBCs in vitro Precipitation reactions – Require the Formation of a Lattice Between Soluble Antigen and Antibody
  • In fluid, the molecules diffuse until they reach the ideal concentration (the zone of equivalence)

Direct ELISA

• Antibody is absorbed to well
• Patient sample is added: complementary antigens binds to antibody
• Enzyme-linked antibody specific for test antigen is added and binds to antigen, forming sandwich
• Enzyme's substrate is added, and reaction produces a product that causes a visible color change Direct ELISA – Enzyme Linked immunosorbent assay
  • Primary antibody binds to well
  • Blocking agent is added
  • Sample added; if antigen is present, it binds to the antibody
  • Unbound sample is washed away
  • Antibody-enzyme conjugate is added
  • Unbound secondary antibody-enzyme conjugate is washed away
  • Substrate is added; if present, enzyme converts substrate to colored product

Indirect ELISA

• Antigen is absorbed to well
• Patient serum is added; complementary antibody binds to antigen
• Enzyme-linked anti-HISG is added and binds to bound antibody
• Enzyme’s substrate is added, and reaction produces a product that causes a visible color change Indirect ELISA – Enzyme linked immunosorbent assay
  • Antigen is bound to well
  • Blocking agent is added
  • Sample added; if antibody is present, it binds to the antigen
  • Unbound sample is washed away
  • Antihuman enzyme-linked antibody is added
  • Unbound antihuman antibody is washed away
  • Substrate is added; if present, enzyme converts substrate to colored product

Complement Fixation Test

• Patient A’s serum contains antibodies to the suspected antigen. Patient B’s serum does not. Both patients have complement, but different amounts.
• Heating the serum destroys all of the complement in the patients serum. Antibodies remain in Patient A’s serum.
• An equal amount of complement is then added to the serum for both patients. Antigens are also added. In patient A’s serum, antibodies bind to antigens and complement fixation occurs. Patient B’s serum lacks antibodies, so complement fixation does not occur.
• Sheep RBCs and antibodies to sheep RBCs are added to both samples.
• In patient A, complement is already fixed and cannot lyse RBCs. The antibodies bind to RBCs and settle to the bottom. In patient B, antibodies bind to the RBCs and complement lyses the RBCs. Serum turns pink.

Fluorescent-Antibody (FA) Techniques

• Direct FA
  • Group A: streptococci from patients throat + Fluorescent dye—labeled antibodies to group A streptococci --> Fluorescent streptococci

Indirect Fluorescent-Antibody Test

• Antigen is fixed to a surface
• Patient serum is added; if antibodies are present, they bind to the antigen
• Secondary antibody (with fluorescent label) is added; if patient antibodies are present, the secondary antibody binds to the patient antibodies