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VTMC 330 Immunology

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NUTRITION

PROLIFERATION
Immune cell proliferation is highest in body

Requires:
- Protein
- Glucose
- Fatty acids
- Fat soluble vitamins
- trace minerals
NUTRITIONAL ROLES

SEVEN
Intestinal Lumen

Epithelial and Mucosal Barries
- Respiratory
- Intestinal
- Skin

Development of Lymphoid Tissue

Synthesis of Active Immunologic Sustances

Cellular Activation, Proliferation and Movement

Intracellular Killing

Modulation and Regulation of Immune process
NUTRITION

PROTEIN
Amino Acids are building blocks for:

Acute phase proteins

Cell
- proliferation
- maturation,
- secretions

Tissue repari

Restoration of normal immune function
NUTRITION

GLUCOSE
Energy for cellular metabolism
NUTRITION

FATTY ACIDS
Essential long chained polyunsaturated fatty acids;

Omega-6
- linolenic acid and byproducts gamma-linolenic acid and arachidonic acid
- proinflammatory

Omega-3
- Alpha linolenic acid and by products eicosapentaenoic acid, docosahecaenoic acid
- anti inflammatory

Optimum Balance Omega-6:Omega-3
- Dogs 6:1
- Humans 4:1

C2 - C4 Shortchain Fatty Acids
- fermentation of soluble fibre
- prefered energy source of colon mucosa
NUTRITION

VITAMIN C
Quench freed radicals in extra and intracellular fluid
NUTRITION

ZINC
Decreased barrier function

Decreased circulating thymic hormone

Essential for T lymphocytes and cell mediated immunity
NUTRITION

IRON
Balance between:

- sequester to prevent microbe growth

- release for host metabolism and oxygen transport
NUTRITION

SELENIM / VITAMIN E
Antioxidants quench free radicals

Prevents lipid peroxidation of cell membranes
NUTRITION

COPPER
Acute phase proteins
- ceruloplasmin

Quench free radicals
- super oxide dismutase
NUTRITION

6 NUTRIENTS FOR

EPITHELIAL AND MUCOSAL BARRIERS
Protein

Vitamin A

Vitamin D

Zinc

Omega 3 and 6 Fatty acids

Short Chain Fatty Acids
NUTRITION

7 NUTRIENTS FOR

NEUTROPHIL AND MACROPHAGE FUNCTION
Iodine

Copper

Iron

Vitamin D

Vitamin C

Vitamin E

Selenium
NUTRITION

6 NUTRIENTS FOR

ANTIBODY RESPONSE
Protein

Arginen

Glutamine

Chromium

Iron

Vitamin B6
NUTRITION

2 NUTRIENTS FOR

KILLER T CELLS
Vitamain A

Arginine
NUTRITION

3 NUTRIENTS FOR

CELL MEDIATED IMMUNITY
Protein

Zinc

Chromium
MEASUREMENT OF B CELLS AND B CELL RESPONSES

2 GENERAL METHODS
Measurements of Immunoglobiulin
- Antgen Specific Ig
- Total Ig (Class/Subclass Ig)

Measurement of B Cells
IMMUNE COMPLEX
Interation between antigen and immuoglobin

Forms compound upon which assay is based

Assay can be used to detect either antigen or immunoglobin

Not visible tf require detection system to make visible
ANTI-SERA
Serum containing antibodies to immunoglobulin

Ig from one species create antibodies in the serum (anti-sera) of another

Anti-sera can be used in assays to detect presence of Ig of first species
5 DETECTION METHODS FOR

VISUALIZING IMMUNE COMPLEXS
Percipitation

Agglutination

Immunohistochemistry
- immunofluorescence
- immunoenzyme

ELISA
- enzyme linked immunosorbent assay

Western Blot
PERCIPITAION
Antibody and Antigen are placed in seperated wells of clear agar gell

Antigen and antibody diffuse towards each other meeting along straight line

If antigen and antibody are specific for each other immune complexes form

Immumne complexs form a lattice which percipitates forming an opaque line in gel

Not quantitative
COGGINS TEST
Percipitin based assay for EIA (Equine Infectious Anemia) Virus antigen specific immunoglobulin
AGGLUTINATION
Antigen coated particles (ie latex beads or red blood cells) are mixed with antibodies

If antigen and antibodies are specific for each other immune complexes form on the surface of the beads

Bivalency (think Y shape) of antibodies allows for cross linking between immunce complexes which results in visible clumping of particles

Quasi quantitative via titer
TITER
Highest (last) dilution of test serum that agglutinates antigen coated particles

Titer is proportional to the levels of antibodies in the serum
ASSAY FOR TOXOPLASMA GONDII
Agglutination based assay for T.gondii antigen specific Ig

Patient serum is incrementally diluted and placed in wells containing red blood cells with surface T.gondii antigen

Negative
- redd cells settle into bottom of well forming small dot

Positive
- immunocomplexes form and cross link froming an agglutination that fans out over bottom of well
IMMUNOHISTOCHEMISTRY

IMMUNOFLUORESCENCE
Mainly used to detect antigen

Immune complexes are formed on tissues on a microscope slide

Immune complexes are made visble via:

IMMUNOFLUORESCENCE
- antibody is labled with ultra-violet visible fluorescein dye

- Direct: the anitbody forming the immune complex with the target antigen is labeled

- Indirect (most commonly used): antibody forming immune complex is unlabeled. Second labled antibody from anti-sera to first specific antibody binds to constant domain of first antibody
- tf only need one type of labled anti-sera antibody for a range of antigen specific antibodies from a given species
- also will get multible binding of labled anti-sera antibody tf more visible

IMMUNOENZYME
- Antibody is labled with an enzyme ie peroxidase
- if immune complexes form enzyme will be bound to tissue
- addition of substrate for the enzyme results in coloured reaction product which is visble under light microsope
IMMUNOHISTOCHEMISTRY

IMMUNOENZYMES
Antibody is labled with an enzyme ie peroxidase

If immune complexes form enzyme will be bound to tissue

Addition of substrate for the enzyme results in permenant coloured reaction product which is visble under light microsope

Can be direct or indirect
IMMUNOHISTOCHEMISTRY

ABC
Avidin-Biotin Complex Immunoenzyme Method

Specific antibody forms immune complex with tissue

Second Anti-srea bound with biotin is added

Avidin, a large molecule with multiple binding sites for biotin, is added and triggered with peroxidase

Resuling complexes are large providing upto 1000 x MAGNIFICATION

tf used for detection of sparse antigens or when antigen has been damaged by fixatives such as formalin

Can be used for detection of antigen or antibodies
ASSAY FOR

LEPTOSIRA
Indirect immunoperoxidase test using peroxidase labled antisera to leptospira antigen specific rabbit IgG
ASSAY FOR

NEOSPORA
Indirect immunoperoxidase assay for ANTIBODIES specific to Neospora

Determines if primary IgG or ongoing IgM response via:
- add serial dilutions of patient serum to TWO SETS of wells containing neospora antigen
- add antisera to IgG to one set of wells and anti-sera to IgM to another set of wells
ASSAY FOR

INSULIN ANTIBODIES
ABC Avidin-Biotin Complex Immunoenzyme Method

- add patient serum to normal pancrease which will contain normal insulin

- add biotin labeled anti- sera to patient antibodies

- add avidin and peroxide to from visible complexes
ASSAY FOR

RABIES
ABC Avidin-Biotin Complex Immunoenzyme Method

- primary antibodies to rabies virus protein

- secontary anti-sera antibody, labeled with biotin, to specific species antibody

- avidin plus peroxidase from visible complexes aka Negri bodies
ELISA
Enyme-Linked ImmunoSorbent Assay

Antibody or Antigen detection

Immune complexes form on a SOLID SURFACE

- antigen or antibody is coated on polyvinyl chloride plate or nitrocellulose membrane

- Patient serum/tissue containing antibody or antigen of interest is added

Immune complexes are labled with an enzyme coated secondary antibody that is species specific to antibody of interest (anti-sera)or is specific to antigen of interest

Immune complexes are made visible with an enzyme substrate which produces a soluble coloured reaction product

Quantitative
- color of reaction product is proportional to antibody or antigen in serum/tissue
ASSAY FOR

BHV-1
Bovine Herpes Virus Type 1

Antibody detection ELISA
ASSAY FOR

BORRELIA BURGDORFERI
Lyme Disease

Antibody detection Rapid ELISA
ASSAY FOR

CANINE HEARTWORM
Antigen CAPTURE ELISA

- form of thin layer chromatography (TLC)

- labled antibody binds directly to antigen then imobilized with second antibody

- read location of colour reaction on strip
MONOCOLONAL ANTIBODIES
Identicle Antibodies that derive from a single HYBRIDOMA and are specific to a single epitope

Hybridomas are formed by fusing immortal non secreting plasma cells (myelomas) with mortal antibody producing (ie from inmmunized animal spleen) B cells

Note polyclonal anti bodies dervive from a hetergenous population of B-cells ie the normal situation that exists in an animal
ANTIGENIC RELATIONSHIPS
Antigneically-related organsims share some but not all antigens

Monoclonal antibodies can distinguish amoung antigenically related organisms by detection of the unique epitopes
COMPETITIVE ELISA
Detection and measurement of antibodies binding a single epitope

- antigen of interest is bound to wall of well

- both monoclonal antibody to specific epitope of and patient serum are placed in well

- wash and add enzyme conjugated secondary antibody specific to MONOCLONAL antibody

- wash and add substrate

- if patient has ployclonal antibody specific to epitope then it will out COMPETE the monoclonal antibody and there will be no enzyme for substrate to react with

- tf NEGATIVE test means patient HAS polyclonal antibody for epitope

- POSITIVE test means that patient has no polyclonal antibody to specific epitope so the enyzme labled monolonal antibody can bind and there will be a colour reaction
WESTERN BLOT
Quasi percipitation based assay

Separates antigens by size and charge via electrophoresis

Antigens are blotted and then washed with enzyme labeled antibody

Useful to identify which antigens or which species/strain of microrganism (based on size and charge characterstics) is generating immune response
ASSAY FOR

STAPH BACTERIA
Western blot to determine strains based on characteistic proteins
MEASUREMENT OF ANTIGEN SPECIFIC IMMUNOGLOBULINS

4 REQUIREMENTS
Pateint serum and source of target antigen

Method to allow the formation of immune complexes

Detection system for making the interaction visible
- percipitation
- agglutination
- immunohistochemical
- ELISA

Means of quantifying the amounts of anitbody detected
- titer
- spectrometry
MEASUREMENT OF TOTAL IMMUNOGLOBULIN LEVELS IN SERUM

4 REQUIREMENTS
Antisera to immunoglobulin
- generate by injecting purified Ig into a heterologous species

Method to allow the formation of immune complexes

Detection system for making the interaction visible
- percipitation
- agglutination
- immunohistochemical
- ELISA

Means of quantifying the amounts of anitbody detected
- Immunoelectrophoresis
- Radial Immunodiffusion
IMMUNOELECTROPHRESIS
Perciptiation based assay

Serum of patien applied to wells in an agar gel

Immunoglobulins diffuse into gel and separate based on size and charge via electrophoresis

various antisera applied at seperate locations along the gel and allowed to diffuse into the gel where it binds to the Ig forming an immune complex which percipitates forming arcs around the anti-sera spots

Location and size of arcs provides semi qualitative (size of arc) and qualitative estimate of differnet classes of immunoflobulin present in patients serum
ASSAY FOR

PLASMA TUMORS
aka monoclonal gammopathies
- B cell tumor

Immunoelectrophoresis

- shows extremely thick and skewed arc
RADIAL IMMUNODIFFUSION
Gold Standard

Antisera to immunoglobulin incorporated into agar gel

Serum samples from patient and standards with known Ig levels placed in wells

Ig in serum diffuses into gel forming immune complexes which percipitate froming rings around wells

Diameter of rings is proportional to serum Ig levels
- tf determine patient levels via standard curve

Requires 24 hrs for diffusion
MEASUREMENT OF B/T CELL NUMBERS AND SUBSET NUMBERS

Fluorescent Activated Cell Sorter (FACS) or Flow Cytometry
Fluorescent Activated Cell Sorter (FACS) or Flow Cytometry

Expose suspension of cells to primary monoclonal antibodies to specific CD (cluster of differentiation) cell surface molecule specific to the cell type/subtype of interest ie CD4 or CD8

Expose suspension of cells to differently fluoro labeled secondary antibodies

Count cell numbers via flow xytometry of fluorescent activated cell sorter (FACS)

Ratio of CD4:CD8 provides indication of humoral or cellular immunity
MEASUREMENT OF B/T CELL NUMBERS AND SUBSET NUMBERS

IMMUNOHISTOCHEMICAL STAINING
Stain for CD (cluster of differentiation) molecules or other cell surface markers

ie show distribution of B and T cells in tissues such as lymph nodes or skin
T LYMPHOCYTE RESPONSE

3 MEASUREABLE CHARACTERISTICS
Proliferation Responses

Cytokine Secretion Responses

Cytotoxicity to target cells
- CD8
PROLIFERATION RESPONSES
aka Clonal Expansion

Measurement of Blastogenesis which is a hall mark of T cell function

Mitogen Response
- mitogens are generally lectins that bind to CHO residues on cell surface glycoproteins stimulating division

- expose extracted patient lympocytes to mitogens and radioactive thymidine

- incorporation of H3thymidine in specific time period is proportional to proliferation
CYTOKINE PRODUCTION RESPONSES
BIO-ASSAY
- expose extracted patient lympocytes to mitogens or antigens that will stimulate proliferation and cytokine release
- extract supernatent and add, along with H3tymidine to cells that are targeted by specific cytokine to proliferate

ELISA
- antigen capture method using surface impregneated with antibody specific for the cytokine and an enzyme conjugated antibody that is ALSO specific for the cytokine
MEASUREMENT OF T CELL CYTOXICITY RESPONSES
CYTOTOXIC ASSAY
- radio labled target cells release radio activity when killed
- antigen specific
- MHC I restricted

IN VIVO
- expose antigen to skin
- measure (subjective) Delayed Type Hypersensitivity (DHT) response
- provides measure of T-Cell memory
NEONATAL IMMUNITY

3 BASIC FACTOIDS
Domestic animals become immunocompetent prior to birth

Partuition is associated with immunosuppressive levels of stress
- high levels of corticosteroids

Colostral immunoglobulin is critical for the survival of newborns
- except primates
DEVELOMENT OF IMMUNE RESPONSE IN FETUS
Primary lymphoid organs develop in the first trimester

Secondary lymphoid organs follow shortly thereafter

Lymphoid organs and phagocytic capability functional by mid gestation

Immune responses including B and T Cells to foreign invadors are detected soon after the development of the lymphoid organs

Animals can respond to vaccine antigens in utero

ie chick embryos vaccinated for Mareks Disease (herpes virus) at 18 days
FETAL IMMUNE RESPONSES TO INTRAUTERINE INFECTIONS
Consequence depends on the pathogen and stage of development of the fetus

ie BVD Virus
- early abortion
- early to mid tolerance and persistent infection because perceives virus as self
- mid to late malformations
- late normal

ie BHV-1 can result in death at any time because virus is highly lytic

Most pathogens do not cross placenta

Overall fetus is less capable than adult at mounting effective response

Fetal immune responses are PRIMARY (mainly IgM)

Elevated fetal serum Igs and pathogen specific IgM can be used to diagnose intrauterine infections
EFFECT OF TIME OF GESTATION

BY THE TRIMESTER
First
- more likely to result in death because no effective immune response

Second
- result in variable damage to fetus but fetus likely to remain viable

Third
- infections can provoke effective immune responses tf fetus often undamaged
NEONATAL IMMUNOSUPPRESION
Parturition is triggered by fetal stress (high steroid levels)

Steroid levels are immunosuppresive resulting in:
- decreased phagocytosis
- decreased T Cell blastogensis and cytokine secretion

For the first 3 - 5 days of life:
- susceptible to infections which would not cause serious problems in older animals
- disease can be caused by organsisms and MODIFIED LIVE VIRUS VACCINES which are safe in older animals

Dam is also immunosuppressed
TRANSFER OF MATERNAL IMMUNITY

IN UTERO
Route determined by the nature of placenta

- hemochorial ie primates allows full transfer of IgG but not other Ig classes

- Endothelialchorial ie dogs and cats allows transfer of only 5 - 10% of IgG

- Syndesmochorial ie ruminants no transfer of maternal Ig prior to birth

- Epitheliochorial ie pigs and horses no transfer of maternal Ig prior to birth
COLOSTRUM
first milk/secretion produced by mammary gland after parturition

Vehicle for transfer of maternal Ig to neonate
- Mainly IgG
- also IgM, IgA, IgE

Receptors for Ig in mammary gland upregulated results in transfer from plasma to milk
- tf can vaccinate dam to protect neonate
- also high levels of lactoferring
- also high levels of gowth factors for development of GI and other organs

Thick yellow and viscous ie 50 - 100 g IgG/l vs 1 - 2 g/l normally

Normal milk Igs are locally produced in mammary gland not sourced from serum
- ruminants IgG is major milk Ig
- non ruminants IgA is major milk Ig
TRANSFER OF COLOSTRAL Ig TO NEONATE
Igs reach small intestine intact and functional
- low proteolytic activity in new born
- high typsin injibitor levels in colostrum reduces degradation of Igs

Intestinal epithelium of neonate allows absorption of intact Igs DIRECTLY to blood stream
- binding of Fx receptors on epi cells results in uptake by pinocytosis
- Ig is passed into intestinal lacteals and capillaries then RESECRECTED back to mucosal surfaces
- transfer phenomina only lasts for a few hours
PROTECTION OF COLOSTRAL Ig
Serum Ig protects from viremia and bacteremia

Re-excretion of Igs on mucosal surface blocks pathogen invasion MOST IMPORTANT

Half life of IgG is 3 weeks. Neonate antibody production begins at 5 weeks
- tf period of vunerabilitity is 5 - 12 weeks
FAILURE OF PASSIVE TRANSFER
FPT newborns are those with indadequete levels of colostral Ig to protect from disease

Adequate passive transfer results in 10g/l of IgG in serum at 24 hrs

Immunoglobulin production varies widely amongst dams
FAILURE OF PASSIVE TRANSFER

3 MECHANISMS
Too little or to late

Indaequate colostrum production because young or premature (leaking) lactation

Inadequate colostrum ingestion via poor mothering or suckling

Inadequate If absorbtion via low Ig [] or acidosis because of difficult birth
- requires 100 - 120 g IgG to be injested by calf by 6 hrs
- calf usually only injests 2 l tf concentration must be high enough
FAILURE OF PASSIVE TRANSFER

DIAGNOSIS
Colostral Ig peaks in serum of newborns at 24 - 36 hours after suckling

Measure via
- Radial Immunodiffusion
- antigen capture ELISA cows and horses
FAILURE OF PASSIVE TRANSFER

CONSEQUENCES
Less than half normal levels results in high morbidity or mortaliity due to bacterial septicemia

More than half normal levels results in variable morbidity/mortality most often due to diarrheas

Amount of disease and losses highly dependent on management
- reduce challenge level
- suppportive care
- serum tranfusion if over 12 hours
IMMUNE RESPONSES IN YOUNG
Primary (IgM) responses can occur in utero

Neonates are unresponsive for 3 - 5days

Colostral Ig interfers with development of active immunity:
- sequestration of antigen
- negative feedback on specific B cells. Binding of antibody to FcReceptor blocks BCR activity

- tf time to effective vaccination of the young is inversely correleated to the titer of antibody found in the mother
ANTIGENS AND PATHOGENS TRANSFERED WITH COLOSTRUM
Transfer of antibodies to blood group associated antigens
- neonatal iserthrolysis and thrombocytopenia

Transfer of cell associated infectious disease agents in macropages and neutrophils which directly enter the blood stream through "open gut"
- Johnes Disease
PASSIVE IMMUNITY IN BIRDS
Naternal serum IgG is concentrated in the egg in the ovary and enter the bloodstream of the chick

IgM and IgA enter the abumin in the oviduct and are swallowed by the chick
INTENT OF NEONATAL IMMUNITY
Fetus and newborn are immuo-compentent but immunologically naive

Colostral antibodies designed to provide initial high levels of passive protection from pathogens at time of birth

Gradual decline in the passive protection at the same time as exposure to low levels of pathogens

Young gradually replace the passive maternal protection with active responses

ie NATURAL CONDITIONS ARE GRADUAL EXPOSURE TO PATHOGENS otherwise system would not work this way
TOLERANCE
Central Tolerance (Thymus)
- deletion of T cells that have nonfunctional TCRS ie no ability to bind self MHC or too strong binding
- All individuals have self reactive T cells which escape deletion in thymus

Peripheral Tolerance - other regulatory mechanisms that prevent self-reactive lymphocytes from damaging self
- anergy lack of co-stimulatory signals
- Suppression
- Immunological Ignorance ie self antigens are hidden (CNS) or cryptic
PHYSIOLOGICAL AUTOIMMUNITY
Normal mechanism for removal of aged cells
- new immunogenic epitopes are formed as cells age ie band 3 on degenerating surface proteins of red blood cells and others
AUTOIMMUNE DISEASES
Pathological conditions associated with ongoing tissue damage from self reactive antibodies (T Cell help) and or cytotoxic T Cells

Develop spontaneously

Idiopathic

Partial or complete failure of tolerance

Result of either and immune response directed to single self antigen or general defect such that may self antigens are recognized

Results from either:
- normal immune response to an abnormal self-antigen
- abnormal immune response to a normal self-antigen (worst situation)

Autoimmune conditions are failures of both tolerance and immunoregulation
- tolerance to self is broken
- normal regulatory mechanisms do not function to pervent or down regulate response to self antigens

Can be specific to one cell type or general to many cell types
4 CAUSES OF LOSS OF TOLERANCE
Exposure of HIDDEN antigens

New epitopes or antigens on self proteins

Cross Reactive antigens

Changes in antigen processing or presentation
EXPOSURE OF HIDDEN ANTIGEN
antigens not normally exposed to cells of the immune system
- CNS, Testes, Cytoplasm
- cryptic epitopes normally hidden by conformation of antigen

These antigens are normally protected from exposure to lymphocytes tf no active tolerance

Inflammation can cause exposure of antigens leading to triggering of immune response and AID Auto immune disease
- antibodies t cardiac mitochondria after heart attack
- antibodies to sperm after inflammation
- antibodies to liver membrane proteins
NEW EPITOPES OR ANTIGENS
Generated on self proteins by molecular changes

- rhumatoid factor: auto-antibodies to the Fc portion of IgG in immune complexes via conformation change when bound

- Immunoconglutinins: auto-antibodies to complement components created dureing complement activation esp C3
CROSS REACTIVE ANTIGENS
Molecular Mimicry

Immune response to an infectious agent which shares epitopes to self antigens
- T helper cells become activated to epitope
NAME 5 DISEASES WHICH CROSS REACT WITH SELF ANTIGEN
Gp A Streptocacci (rheumatic fever
- AID of heart, kidney, joints

Mycoplasma cattle and pigs
- antibodies to normal lung antigens

Trypansomes
- mammalian cardia muscle and neureons

Epstein Barr Virus (MS)
- myelin basic protein

Leptospira interrogans Moon Blindness horses
- immune responses to cornea / uvea
CHANGES IN ANTIGEN PROCESSING OR PRESENTATION
Once immune response is triggered the antigen presenting cells in the area becaume activated and have enhanced interactions with T helpers

Enhanced antigenprocessing results in better ability to present self antigens

High levels of cytokines in the environment results in supplying the co-stimulatory signals needed to trigger self reactive cells

Both bacteria and viruses have been implicated in inducing auto immunity this way
AID

2 BIG THINGS
Auto Immune Disease

Failure of Tolerance and Immunoregulation
- Autoreactive lymphocyte loses tolerance and starts to react to self tissues

Regulatory Control Fails
- mechanisms not understood
- low level auto immune responses are normal
- usually response is regulated
- disease develops when ther is a failure of regulatory control of self antigens
PREDISPOSITION TO LOSS OF TOLERNANCE AND IMMUNOREGULATION

6 FACTORS
Genetics
- AIDs are familial

Age
- middle age or older

Gender
- females more affected

Infections
- bacteria, viruses

Vaccination
- adjuvanted vaccines

Altered Immune System
- lymphoid neoplasia
- immunosupressive therapy
- immunodeficiency states
AUTO IMMUNITY

GENETICS
MHC II genes are associated with development of AIDs

MHC II fundamentally controls immune response
- regulate the ability to respond to antigen
- onset of AIDs after reproductive age has resulted in lack of selection pressure against MHC antigen binding grooves that present self antigen

Most diseases are associated with particular combinations of MHC genes not a single MHC gene

AIDs tend to be familial in all species

Inbreeding can result in high levels of AIDs
AUTOIMMUNITY

GENDER
Hormones
- estrogen increases AID prevlaence if females 10x

Micro-Chimerism
- exchange of fetal and maternal cells during pregnancy
- cells survive for years
- fetus lymphocytes view dam as foreign
- dam lymphocytes view fetus cells as foreign
AUTOIMMUNITY

INFECTIOUS AGENTS
Cross Reactivity

Bystander Activation
- upregulation of immune response in area of infection activates local self reactive cells
AUTOIMMUNITY

VACCINATION
Administration of potent adjuvanted vaccine may trigger transient production of a variety of auto-antibodies
- ie via macrophage activation

Rare Events
AUTOIMMUNITY

ALTERED IMMUNE SYSTEM AND LOSS OF IMMUNOREGULATION
Neoplasia of lymphoid tissues associated with AIDs
- Thymoma: autoantibodies to Ach receptors block Ach binding

Immuno-deficent individuals have increased AIDs
- IgA defecient dogs have weakened mucosal barries which allows increased numbers of micro invadors to enter resulting in increased stimulation
DETERMINANT SPREADING
The increase with time in numbers of epitopes recognized in an autoimmune response

Response starts out confined to a one or oa fuw immunodominant peptide epitopes

With time immune response spreads to recognize othe epitopes on the same protein and then on other proteins

ie antibodies to heterlogous species insulin is induced by the foreign epitopes but rapidly recognizes all sites on host species insulin and foreign insulin
VACCINATION

2 CRITEREA

2 METHODS
Will immune response from vaccination actually protect against disease

Do benefits of vaccination outweigh risks and does risk of contacting disease outweigh risk of vaccination

PASSIVE
- temporary resitance achieved by transferring antibodies from a resistannt to a susceptable individual

ACTIVE
- adminsistering antigen to an animal so that it responds by producing a protective immune response
5 ADVANTAGES OF LIVE VACCINES
Few inoculateing doses required
- risk that non pathogenic altered agent could revert or be contaminated with other virus

Adjuvants unnecessary

Less chance of hypersensitivity
- because of low amount of antigen used

Induction of interferon
- better cell mediated response because of accurate epitopes

Relatively cheap
- low amounts of antigen required because animal will amplify response
3 ADVANTAGES OF INACTIVATED VACCINES
Stable during storage

Unlikely to cause disease through residual virulence

Unlikely to contain contaminating organisms
3 WAYS OF GENETICALLY ENGINEERING ANTIGENS
Inactivated recombinant organisms or purified antigens derived from recombinant organisms
- ie isolate AG gene from virus and insert into bacterial plasmid aka Subunit vaccine
- antibody response only
- can be used for disease eradication by vaccinating for specific antigen subunit and then culling any animal with other subunits of pathogen

Live organisms that have virlulence genes deleted or contain heterologous marker genes

Vaccines that contain live expression vectors expressing heterlogous genes for immunizing antigens or other stimulantes
- canary pox, modified to carry rabies surface glycoportein gene, as vector. Has limited replication in mammals
- risk that when booster vaccination occurs body has developed response to canary pox vector
- advantage is cell mediated response
PARENTERALLY
Out of GI tract
ie
- Sub Q
- IM
- Intranasally
- orally
DEPOT ADJUVANTS
Aluminum Salts
- aluminum phospate
- aluminum hydroxide

Emulsify antigen
- sequester antigen (depot effect) slowing release which prolongs immune response

Inert and safe but not highly effective
- only adjuvant used in human vaccines in NA
IMMUNOSTIMULATORY ADJUVANTS
Anaerobic corynebacteria, BCG, LPS
- All MACROPHAGE STIMULATORS
- not neccesarily inert
- LPS stimulates specific response but also IL-1 proinflamatory cytokines

Saponin
- STIMULATES ANIGEN PROCESSING
- plant material used in veterinary vaccination
- fools system into processing it ENDOGENOUSLY
- tf cellular response but safe because not live agent
PARTICULATE ADJUVANTS
Liposomes
- STIMULATES ANTIGEN PROCESSING
- vesicles resemble cell membranes
- induce both antibody and cellular response (via endogenous processing)

Micro Particles
- DEPOT effect and ANTIGEN PROCESSING
- tiny solid particles degrade slowly
- antibody and cellular response
MIXED ADJUVANTS
Freund's Complete Adjuvant
- water in oil emulsion plus mycobacterium protein
- depot effect
ANTIVIRAL VACCINES

5 TYPES
Mixed parenteral vaccines
- modified live
- inactivated

Subunit Vaccines

Genetically Engeineered vaccines

Vector Vaccines

Intranasal Vaccines
ANTI BACTERIAL VACCINES

3 TYPES
TOXIODS
- inactivated (formaldehyde)
- given with adjuvant
- ie Tetanus, blackleg

BACTERINS
- killed bacteria (formaldehye)
- adjuvant
- Ananculture bacteria 9esp membranes + toxoid
- Autogenous Bacterins made from bacteria isolated from farm to get specific strain
- CORE antige J5 from LPS

LIVING BACTERIAL VACCINES
- Attenuated virus ie brucella 19, capsulless anthrax, raough salmonell dublin
- requires lower dose
- Th1 response
PROTOZOA VACCINES
Very Few Successful

Theileria Parva
- infection with low dose of sprozoites and treatement with tetracycline
- must get cellular response because part of life cycle is intracellular

Toxoplasma gondi
- live vaccine
HELMITH VACCINES
Very Few Successful
- large size
- diversity of life style

Dictyocaulus viviparus
- irradiated larva
EXPERIMENTAL CHALLENGE STUDIES
Naive vs Seronegative (no antibodies but has memory cells)

Randomization

Blinding

Relevance of model(ie IV) to natural disease (aereosolised)

Preventable Fraction
- PF = (%controls dying - %vaccinates dying)/%controls dying
FIELD TRIALS
Pen effect vs herd immunity effect or dilution with vaccinates

Randomization

Blinding

Outcomes

Murphy's law in reverse
IMMUNOLOGICAL STUDIES
Agents used in assays

difficuly in comparing serological results amoun laboratories

Tests of cell mediated immunity

Corelative challenge data

Human medicine can work inductively via serological studies because high $ and #

Veterinary medicine should work deductively via challenge studies and history
VACCINE FAILURE MODES
CORRECT ADMINISTRATION

Animal Responds
- vaccine too late already infected
- wrong strain or organism
- non portective antigens

Animal Fails to Respond
- prior passive immunixation
- immunosuppressed
- biological variation
- inadequate vaccine

INCORRECT ADMINISTRATION

- inappropriate route of administration
- death of live vaccine (dashboard)
- administration to passively protected animal
ADVERSE REACTIONS TO VACCINE
NORMAL TOXICITY
- Fever, Inflammation, Malaise, Pain

ERRORS
- manufacture/administrion
- Bacterial or Viral contamination
- abnormal toxicity
- residual virulence

INAPPROPRIATE RESPONSES
- Hypersensitivites
- Neuologic reactions
- Foreign Body reactions
- biological variance
IMMUNOTOXICOLOGY
Study of immunododulatory effects of chmicals on the immune system

Aimed at detection subclinical problems before they become clinical

Enviromentally realistic exposure ie normal paths of exposure are not IV

Requires a wide range of tests because immune system is compelex and highly redundant
IMMUNOTOXILOGICAL ENDPOINTS
Act as BIOMARKERS of
- exposure
- effects of exposure

Are used to evaluate risks to wildlife

Animal serv as sentineld for human risk from chemical exposure
XENOBIOTICS
Foreign compound
- effects on biological system
- if in body must be detoxified and excreted
- may cause immunosuppression or immunostimulation
- may cause disregulation or immunododulation
IMMUNOTOXICITY TESTS
TIER I

Post-Mortem
- immunopathology
- organ weights, spleen, thymus, bursa

TIER II

In Vivo
- PHA (mitogen) skin test
- Antibody response (DNP-KLH (keyhole lympet antigen), foreign RBCs)
- DTH (delayed type hypersensitivity) GOLD STANDARD

In Vitro
- wbc countand differential
- plasma proteins
- phagocytosis assay (macrophages, splenocytes)
- respiratory burst (macrophages, PMNs/heterophils)
- NK assay
TYPE I HYPERSENSITIVITY
Immediate Hypersensitivity

Acute inflammatory reactions

Mediated by IgE antibody responses

Effector cells are mast cells, basophils and eosinophils

ANAPHYLAXIS
- systemic and severe
- immediately life threatening

ALLERGY
- localized
- usually not immediately life threatening
TYPE I HYPERSENSITIVITY

MECHANISM
Antigen binds (crosslinks) IgE antibody bound to FcRI on mast cells, basophils triggers cell degranulation

Pathogenesi of tissue damage is due to the effects of released granules
- vasodilation
- puritis (itching)
- bronchoconstriction
TYPE I HYPERSENSITIVITY

IgE
200KdA Dimer ala IgG

Produced primarily at body surfaces (skin, gi, lung)
- Th2 Cytokines (IL-4 esp, 5, 10, 13) elicit B cell Class Switch to IgE
- Th1 Cytokines (gamma IFN, IL12)inhibit IL4 production

Half life of 11 - 12 days

Induced by parasites

Normally produced in low levels

Allergies result from IgE production to an antigen tf aka ALLERGEN
ATOPY
Condition in which IgE produced at abnormally high levels to many antigens
IgE RECEPTORS
FcRI
- receptor with very high affinity binding for IgE
- mainly found on mast cells and basophils and some on eosinophils
- also found ondendritic cells and monocytes of ATOPIC patients (ie differential)

FcRII
- receptor with much lower affinity fo rIgE
- found on NK cells, macrophages, dentritic cells, eosinophils, platelets and some B cells

ALLERY LOOP
- dendritic cells bind antigen via FcRI and IgE
- processing stimulates cytokine release which stimulates Th2 responses (IL4, 5, 10, 13)
- stimulation of B Cells produces more IgE
- more IgE binding and crosslinking on Mast cells causes them to release Th2 activating cytokines (IL-4, 5, 13) which further amplifies IgE production
- IgE bound and cross linked mast cells release allergic mediators
ALLERY LOOP
- dendritic cells bind antigen via FcRI and IgE
- processing stimulates cytokine release which stimulates Th2 responses (IL4, 5, 10, 13)
- stimulation of B Cells produces more IgE
- more IgE binding and crosslinking on Mast cells causes them to release Th2 activating cytokines (IL-4, 5, 13) which further amplifies IgE production
- IgE bound and cross linked mast cells release allergic mediators
MAST CELL RESPONSES

ANTIGEN CROSSLINKED TO SPECIFIC IgE
Cytokine Synthesis and Secretion
- positive feedback

Granule Exocytosis
- rapid within 60 seconds

Prostaglandin and Leukotriene synthesis
- pro inflammatory
SOLUBLE MEDIATIORS RELEASED BY MAST CELLS
SECONDS TO MINUTES Gronule Exocytosis

Histamine
- smooth muscle contraction
- broncho constriction
- exocrine secretions mucus, tears, saliva
Serotonin
- vasodilation, edema
Proteases
- trypsin, chymotrypsin
Kalikreins, Proteoglycans

MINUTES Eicosanoid sythesis and secretion

Arachidonic acid, protaglandins, thromboxanes, Leulotriene- dramitic effects on vasculature tone and permeability

HOURS Cytokine sythesis and secretion

IL-4, 5, 6, 13, TNF-alpha, MIP-1alpha

Animal dies of low Blood Pressure due to acute inflamatory response
TYPE I HYPERSENSITIVITY

ROLE OF EOSINOPHILS
Mobilizes and activated preferentially in Type I Hypersensitivity reactions
- IL5 form Th2 mobilize marrow esosinophils
- Mast cell degranulation, inflammation increases eosinophil chemotaxis

Peiecemeal Degranulation
- induced by binding of IgE/antigen to FcRII and by Th2 cytokines (IL5)
- small vesicles of the large secondary granules (crystaloid) move to plasma membrane and release granule contents
- very damaging ie peroxidase, neurotoxin etc
- keep inflammation going in chronic allergic reaction
TYPE I HYPERSENSITIVITY

DEVELOPMENT
Sensitization
- can't be allergic on first encounter with antigen
- phase of exposure such that IgE immune response is induced
- nature, site, dose (high) result in Th2 dominated immune response

IgE is produced and will bind to the high affinity FcI receptors on mast cells

Re-Exposure
- to antigen results in CROSS-LINKING of the IgE on mast cells causing release of granules initiating response

Sensitivity should tail off if no ongoing exposure for more that 11 days due to 1/2 life of IgE
TYPE I HYPERSENSITIVITY

CLINICAL MANIFISTATIONS

ANAPHYLAXIS
MAIN SPECIES VARIATION IS TARGET ORGAN

Symptoms depend on number and location of mast cells affected

Drugs (penecillin), Milk allergy, parasites arble pupae disruption, blackflies

Cattle, sheep, humans
- serotonin, kinins, leukotriens
- lung
- dyspnea via bronchoconstriciton and pulmonary edema

Horse
- seotonin, histamine
- lungs and intestine
- dispnea, diarrhea

Dog
- histamin, prostoglandin, leukotrienes
- liver
- weakness, collapse hypotension

Pigs
- histamine
- lungs
- syspnea

Cats
- histamine leukotrines
- lungs, skin
- dyspnea, puritis
TYPE I HYPERSENSITIVITY

CLINICAL MANIFISTATIONS

ALLERGIES
Site of symptoms do not always relate to site of exposure
- most allergens manifest in skin
- histological lesions edema and infiltration of mast cells, eosinophils results in acute inflammation

SKIN
- puritis via eosinophil granules impacting nerves
- 30% of K9 skin desease
- Urticaria (Hives), Angioedema sub Q
- Atopic Dermatitis genetic, percutaneous
- Flea Allery Dermatitis may also involve Type IV HS
- Eosinophilic granuloma complex cat high eosinophils, nasty lesions

FOOD ALLERY
- 2% of food absorbed as peptides large enough to be allergens
- most cases puritic dematitis, 15% GI, vomiting, diarrhea
- 4 -4 24 hrs of reexposure
- diagnose via exclusion diets
- May be associated with IgA which acts at mucosal surfaces
- tf if deficient get high peptide entry

RHINITIS/ASTHMA
- one of components in COPD horse
- dogs and cats but not common
- genetic
TYPE I HYPERSENSITIVITY

TESTING
ELISA for specific IgE
TYPE I HYPERSENSITIVITY

TREATMENT
Avoid allergens

Treat secondary infections

Antiinflamatory agents to prevent mast cell degranulation
- 2 types of catecholamine receptors block or stimulate

HYPOSENSITIZATION
- repeated injection of allergen sub Q (macrophages)at increasing doses
- promotes swtich to Th1 and IgG response
- maintenance every 2 - 4 weeks
TYPE II HYPERSENSITIVITY
Destruction of CELLS due to binding of antibodies (IgG, IgM) to cell surface

Mediated by:
- NK CELLS
- Complement
- Phagocytic Cells

PHAGOCYTOSIS
- occurs mainly in the spleen
- Extra vascular hemolysis

COMPLEMENT
- lyssis cccurs mainly in the bloodstream
- Intra-vascular hemolysis

Damage does not occur in isolation from other immune mediated damage II and IV

Damage also occurs during beneficial immune inflammatory conditions
- destruction of virus infected cells
TYPE II HYPERSENSITIVITY

IMMUNE MEDIATED HEMOLYTIC ANEMIA
Antibodies directed to
- self RBC antigen ie cross reactivity
- altered self RBC antigen ie drugs creating novel epitopes
- exposure of cryptic self antigens ie drugs, microbes, toxins
- adsorbed foreign antigens ie immune response destroys the RBC in the process of attacking the foreigner ie penicilling, ASA, EIA
- Alloantigens ie transfusions, neonatal isoerythrolysis

Direct Coombs Test
- agglutination test for IgG, IgM antibodies binding to rbcs and for fixation of C3
-
TYPE II HYPERSENSITIVITY

NEONATAL ISOERYTHROLYSIS
Alloantigens inherited from sire are expressed in the fetus

Late in gestation the placenta develops small tears which allow the feta tissues (aalo antigens) into dam

Dam mounts immune response ro foreign bllod group antigens

Antibodies concentrate in colostrum

Colostral antibodies caus RBC lysis in newborn

Diagnose via Indirect Coombs test
- stallion blood and mare serum plus anti sera to mare Ig
TYPE II HYPERSENSITIVITY

DISEASES
Cold Agglutinin Disease
- necrosis of the tips, tail ears, toes
- associated with cold reactive IgM (bigger) to RBCs
- formation of micro thrombi in small blood vessels

Immune Mediated Thrombocytopenia
- petechial hemmorrhages on skin and mucus membranes
- can be transferred by maternal antibodies, piglets or sheep via bovine colostrum (heterophile antibodies)

Skin Diseases
- Pemphigus and Pemphigoid target antigens in epithelium
- severity depends on depth of target antigen ie basement membrane is severe

Addisons Disease
- antibodies target adrenal cortex
- low levels of corticoids

Diabetes Mellitus
- target cells are insulin producing beta cells

Myasthenia Gravis
- antibodies to AchR block and initiate complement mediated injury to the post synaptic membrane

Immune mediated destruction of the thyroid
- antibodies to thyroid follicular cells and thyroglobulin
- thyroid high conserved tf antigens are the same across individuals and species
TYPE III HYPERSENSITIVITY
Immune Complex Disease

Immune complexes are formed with SOLUBLE ANTIGEN

Complement is fixed by the CLASSICAL pathway

Chemotaxic and activation substances (C3a, C5a) for NEUTROPHILS are generated from complement pathway

Neutrophil DEGRANUALATION is EXTRACELLULAR
- because the immune complexes formed from soluble antigen are on sufraces the neutrophil cant phagocytise

Effort to clear persisten antigen may trigger Type III

Clears if antigen can be cleared
TYPE III HYPERSENSITIVITY

ARTHUS REACTION
Antigen inject sub Q into animal with high levels of antibody

- 6 - 8 hrs peak of thrombosed blood vessels
- antigen diffuese into blood vessel walls
- antigen forms immune complexes with between and beneath vascular endothelial cells
- immune complexes fix complement in blood vessel walls
- neutrophil chemotaxis and degranulation
- problematic lesion is Vasculitis ie immune complexes and inflammation generated CLOT FROMATION and ISCHEMIA
TYPE III HYPERSENSITIVITY

LOCAL
Blue Eye
- anterior uvetis and crneal edema in dogs following vaccination with modified live adenovirus

Feline Infectious Peritonitis uveitis
- immune complexes

Hypersensitivity Pneumonitis
- lungs of immune animals after inhaling antigens
- mold spores encounter antibody and form immune complexes in alveolar walls

Tissue damage involved in immune responses to many pathogens may also be mediated through the formation of immune complexes
- Staphlococcal Hypersensitivity
- Neutrophilic dermal vasculitis
TYPE III HYPERSENSITIVITY

GENERALIZED
Large immune complexes or those bound to RBCs or platelets are removed by proffesional phagocytes

Small circulating immune complexes that cant be bound by phagocytes become deposited in blood vessels
- especially medium sized arteries of glomeruli, synovia, skin
- areas of high volume low pressure

Serum Sickness
- soluble immune complexes with horse serum

Systemic Lupus Erythematosus (SLE)
- development of autoantibodies to many self antigens
- mmune complexes deposited in basement membrane zone of skin, glomeruli of kidney, small vessels
- diagnose via anti nuclear antibodies
- implies failure of regulation to produce antibody to DNA (poor antigen)

Immune Mediated Arthritis
- rheumatoid arthritis
- synovial membrane of joint
- Aglutination test for rheumatoid factor (RF)
- RF is an IgM auto antibody against immune complexed IgG

Purpura Haemorrhagiica
- horses recovering from Srep. equi (strangles)
TYPE IV HYPERSENSITIVITY
Delayed Type Hypersensitivity (DTH)

CELL MEDIATED
- Activated Macrophages (main effect) and Cytotoxic T Lymphocytes
- Antigen via antigen presenting cell activates Th1 cells
- cytokine release activates macrophages and sometimes CD8 T Cells

Differs from I, II, III because cell mediated not antigen mediated

Type IV reactions can be transfered by Th1 lymphocytes

Normal mechanism for recovery from intracellular infections
-
TYPE IV HYPERSENSITIVITY

DELAYED TYPE HYPERSENSITIVITY SKIN TEST

DTH
Intradermal injection of antigen
- because immunitiy via Th1 cells no reaction if not previously exposed
- reaction if Th1 memory cells present

Antigen phagocytosised by APC

Presentation of antigen by MHC Class II to memory Th1 cells
- preferential recognition by Th1 cells because expanded specific population due to prior exposure
- Th1 binding along with IL12 prompts release of of Th1 cytokines (IFN-gamma, TNF, IL2,8)

Th1 cytokines
- activate macrophages
- increase expression of adherence molecules on local blood vessel endothelium
- chemotactic for neutrophils
- extravascularize neutrophils, lymphocytes, basophils

Tissue damage and inflammation peaks at 24 - 72 hours when lymphocytes show up
TYPE IV HYPERSENSITIVITY

OUTCOMES
Antigen is cleared
- nomral course of events
- repilcation and spread of virus is limited
- means of killing intracellular bacteria and protozoa
- classic test for cell mediated immunity

Antigen Persists
- granuloma formation
- accumulations of macrophages
- giant fused cells
- fibrosis and pathological consequences
TYPE IV HYPERSENSITIVITY

PATHOLOGICAL CONSEQUENCES
Feline Leprosy
- cutaneous mycobacteria causes grnuloma formation

Johnes Disease
- mycobacteria paratuberculosis
- macrophages in lamina propria of GI tract

Leishmaniasis
- protozoa in dermal macrophages resulting in grnulomatous dermatitis

Contact Hypersensitivity
- dermatits resuling from contact with wide range of chemical or biological substances
- contact irritant substance causes direct tissue inflammation
- Type IV hypersensitiviy from prolonged exposure to smarll reactive molecusle ie drucgs neomycin, dyes, plastic food bowles, metal etc
- occurs via haptens

Graft Rejection
TYPE IV HYPERSENSITIVITY

ALLOGRAFT REJECTION

3 TARGETS
Targets of Graft Rejection are the Histocompatibility Antigens

MHC I on all nucleated cells
- looks self MHC I + foreign peptide
- cytotoxic T cells triggered to kill all cells epxpressing foreign MHC I
- Helper CD4 T cells recognize a complex of MHC I and foreign antigen
- foreign MHC I antigens are interpreted as self + foreign
- Th cell is triggered to release helper cytokines

MHC II on APCs and B cells
- Helper CD4 T cells recognize a complex of MHC II and foreign antigen
- foreign MHC II antigens are interpreted as self + foreign
- Th cell is triggered to release helper cytokines


Blood goroup antigens
- occur on all nucleated cells as well as on RBCS
- associated with antibody responses
- trigger type II destruction of the graft
TYPE IV HYPERSENSITIVITY

GRAFT VS HOST DISEASE
Lymphocytes in graf develop and immune response to host tissues

- Recipient is usually immunosuppressed

- donor passenger lymphocytes with the graft mount a type IV response towards recipient cells
TYPE IV HYPERSENSITIVITY

ALLOGRAFTS AND PREGNANCY
Fetus is an allograft

Mechanism which prevent rejection:
- no expression of immunogenic MHC I or II molecules on the cell layer of the trophoblast in contact with maternal tissues
- fetal antigens entering the maternal cirulation initiate an antibody response which limits cell mediated immunity via destruction of antibodies in liver
- placenta nd fetus are source of many immunosuppressive factors ie estradiol, progesterone, alpha fetoprotein, indolamine
- Mild immunosuppression and kewing to anitbody response is feature of pregnancy
PRIMARY IMMUNE DEFICIENCEIES

3 TYPES
Inherited or Congemital

Defects in Phagocytosis

Defects in Antibody Production

Defects in T Cell Functions

Rare in outbred ie people, cats

More common in inbred ie dogs, dairy cattle , horses

Increased susceptibility of infections at young age
- especially opportunistic invaders
- pathogens
- increased IgE response
- Increeased autoimmune diseases
- increased malignancies


SCIDs Severe Combined Immuno Deficiency
PRIMARY IMMUNE DEFICIENCEIES

DEFECTS IN PHAGOCYTOSIS
Phagocytic cell function requires
- normal neutrophil cell numbers and morphology
- response to chemotctic factors
- Phagocytosis ie opsonization, engulfment, formation phgolysosome
- oxidative burst
- killing of micro organisms

Failure of Opsonization

Defect in formation of intracellular granules
- abnormally large granules associated with grey coat colour

BOVINE LEUKOCYTE ADESHION DEFICIENCY (BLAD)
- defect in the adhesion proteins that enable neutrophils to leave the bloodstream and enter the tissues in response to chemtaxic factors
- Integrin on surface of neutrophils defective
- neutophils cannot squeeze through vasculature
- results in uncontrolable bacterial infection in tissue
- pneumonia
- NO NEUTROPHILS IN TISSUE BUT BLOOD VESSELS PACKED FULL
- Holstein cattle (14% of bulls, 5% cows)
- Irish setters
PRIMARY IMMUNE DEFICIENCEIES

LYMPHOCYTE DEFECTS
B Cell and or T Cell Defects

Test via:
- numbers and morphology of cells
- Expression of CD markers
- cell function assays ie blastogenesis, cytokine release
- DTH or antibody response in vivo

SEVER COMBINED IMMUNODEFICIENCY (SCID)
- people cattle dogs, horses
- 8% of Arabs carriers in NA
- molecular test
- absence of a component of a DNA dependen torptein kinase which is required to rejjoin the ends of DNA when V, D, J genes are cut and rejoinded to produce BCRs and TCRs
- no production of functional T or B cells
- have NK cells
- undeveloped lymph nodes
- low circulating lymphocytes
- no IgM before suckling
- survive as long as colostral antibodies are present (t1/2 3 weeks)
- die from first opportunistic invader ie adenovirus or pheumocystis
- X linked SCID defect in gene that codes for the gamma chain of IL-2 Receptor and component of many cytokines tf lymphocytes csnt proliferate
PRIMARY IMMUNE DEFICIENCEIES

B CELL DEFECTS
Usually result in lowered levels of serum Ig
- measure via radial diffusion

Agammaglobinemia
- no Bcells, no serum Igs

Trasient Hypogammaglobinemia
- production of Igs delayed for 2 - 3 months

Selective IgA Deficiency
- infections at surfaces ie respiratory, skin, eyes
- most clinically relevant ie survivable
- increased autoimmunity, cancers
- German Shepards

Selctive IgM Deficiency
- IgA, IgG levels normal or elevated
- recurrent bacterial infections
- mortality after a few months from pneumonia

Selective IgG Deficiency
- IgA, IgM levels may be normal
PRIMARY IMMUNE DEFICIENCEIES

T CELL DEFECTS
T Cell defects tend to be incompatible with life
- tf fewer conditions perptuated

NUDE CONGENITAL HYPOTRICHOSIS AND THYMIC APLASIA
- cats, rats, pidgs, calves, guinea pigs, MICE
- NO THYMUS because of deficiency in epithelial cells
- tf animals have no hair
- used as living test tubes
- animals die once maternal antibody declines

Parvo virus
- breed susceptabilitiy suspected due to T cell defficency
SECONDARY IMMUNE DEFICIENCEIES

VIRUS INDUCED
Virus Induced Immunoduppression

- usually associated with infection of the lymphocytes
- damage/loss of lymphocytes results in decreased cell numbers and functions

- less often vuruses stimulate lymphoid cell activity ot patholgical extent ie unable to respond to other pathogens

- other viruses cause lymphoid neoplasia
- tf neoplastic cells replace normal lymphocytes

Infectious Bursal Disease Virus
- chickens
- targets bursa and secondayr lymphoid organs

Primate Retroviuses
- HIV I and HIV II
- targets CD4 cells damaging receptor
- tf failure of antibodiy and cell mediated response

FeLV (Feline Leukemia Virus)
- infectsts lymphocytes
- various effects including neoplasia

Feline Immunodeficiency Virus (FIV)
- ralated to HIV
- infects T cell, mega karyocytes, macrophages, dendritic cells, neurons
- inital reponse to virus is follicular hperplasia
- followed by gradual depletion of T Cells

Canine Distemper Virus (CDV)
- infects and destroys secondary lymphoid organs
- depresses activities of lymphocytes and macrophages via IL1,2

Bovine Viral Diarrhea Virus
- infects and can destroy lymphocytes
- may preferentially target Peyers Patches tf if infected early will have life long immunodeficiency
- acute infections result in long term decrease in immune responses
SECONDARY IMMUNE DEFICIENCEIES

OTHER CAUSES OF IMMUNOSUPPRESSION
Lymphoid Neoplasia

Nicrobial and Parasitic Infections
- chronic disease results in diminished immune reponses
- decreased IL2 production
- decreased proliferative response

Malnutrition
- first affects T Cell functions, complement, neutrophill funtion
- driven by protein loss
- zinc deficiency is especially immmono suppressive
- also vit A, Bqw folic acid, copper selenium, taurine

Exercise
- regular moderate excercise increases immune response
- strenuous excercise has opposite effect possibly via increased steroid levels

Trauma
- severe injuries rusultin immmunodeficiency via corticosteroids, prostoglandins, protein loss

Age
- B and T Cell response decrease with age
- Thymic involution lowers T cell production
- tf majority of cells in secondary lymphoid organs are memory cells
- loss of T cell division and response to IL2
- Bone marrow ok but B cell repitoire diminished because of lower Th cells

Stess
- many management situations are stressfull
SECONDARY IMMUNE DEFICIENCEIES

DRUGS
Nonspecific Immunosuppression
- drugs or radiation that have general effects on rapidly dividing celss
- GIT, skin, blood and immune system cells

CORTICOSTEROIDS
- steroids: wide variety of suppressive effects
- mechanisms: poorly understood
- Steroids dircetly absorbed and transported to nucleus
- block cytokine synthesis
- humans and lab animals more susceptable than domestics
- lymphopenia ie reduced emigration of neutrophils to tissues
- decreased chemotaxis and phagocytosis
- decreased cytokine production tf redueced T cell response
- inhibits acute inflammatory response via decrased macrophage production of protaglandins and IL1

CYTOTOXIC DRUGS
- designed to inhibit cell deivison
- inhibit immune response by reducing cell proliferation
- toxic effects on bone marrow tf thrombocytopenis, anemia, leukopenia

DRUGS THAT TARGET T CELLS
- developed from fungi
- blocks signal transduction oin Th1 cells tf lower production of IL2 and ganna INF
- VERY IMPORTANT IN GRAFT SURVIVAL
- ie renal graft cats can last more than a year
TUMOR IMMUNITY AND IMMUNOTHERAPY

TUMORS
Tumor = Cancer = Neoplasms

Benign
- remain in a single location
- not highly invasive

Malignant
- mestastic via blood or lymph
- highly invasive

Carcinoma
- arises from epithelial cells

Sarcoma
- arise from mesenchymal cells

Leukemia
- tumor cells of haemopoietic cells in blood
TUMOR IMMUNITY AND IMMUNOTHERAPY

TUMOR SURVEILLANCE
Although tumors arise after reproductive age immuno suppression results in increased rates of some cancers indicating some role for the immune system in the prevention of some cancers

But most tumors are not a consequence of falure of the immune responses
- nude mice are not at increased risk for most cancers
TUMOR IMMUNITY AND IMMUNOTHERAPY

ANTIGENS
Tumor cells are genetically unstable
- tf gain and lose surface antigens
- loss of MHC and or blood group antigens
- expression of developmental proteins
- new antigens from incomplete or altered synthesis of normal proteins
- virus related antigens in virus induced proteins
- expression of antigens normal to other tissues
- expression of antigens in unsusual quantities

Most structures that differ are CHO and lipids which are poor antigens
TUMOR IMMUNITY AND IMMUNOTHERAPY

NATURAL KILLER NK CELLS
Main Effector Cells in tumor immunity
- 15% of lymphocytes are NK
- found in secondary lymphocytes do not recirculate
- No T or B cell markers
- Not MHC restricted ie no specificity
- kill virus infected and tumor celss without pervious sensitization ie no memory
- serial killers not damaged in process of killing
- kill via perforins and ganule enzymes ala CD8 T cells

NK cells recognize Tumor cells via:
- loss of MHC I expression
- recognition of antibody via CD16 Fc receptor
- binding CD 16 triggers release of INF gamma and TNF
- recognition of MICA and MICB proteins expressed by stressed cells
TUMOR IMMUNITY AND IMMUNOTHERAPY

OTHER EFFECTOR MECHANISMS
Cytotoxic T cell
- most important in virus induced tumors that express viral antigens

Activated Macrophages
- via IFN gamma
- release TFN

Antibody
- may have effects through complement mediated lysis and NK mediated killing
TUMOR IMMUNITY AND IMMUNOTHERAPY

FAILURE OF IMMUNITY
NK cells are capable of killing oly certain types of tumors
- ineffective against carcinomas (70% of tumors)
- better against sarcomas

Tumors are poorly immunogenic
- tf other effector mechanisms are not activated

Tumor population is gentically unstable
- antigenic heterogenity tf only some tumor cells targeted

Failure to express Class I MHC antigens
- tf best killers, CD8 T cells not active

Immunosuppression
- relese of immunosuppressive factors ie prostaglandins and cytokines form tumor cells
- catabolic state tf protein drain
- Suppressor cells: regulatory poplulations of T cells result in AB mediated response which is not as effective

Blocking Antibodies
- antibodies bind tumor cell without harm but suppress cell mediated immunity

Tumor Cell Selection
- tumor cells appear normal
- do not trigger immune response until large size
- immune systme must kill every tumor cell
- if large high heterogenity tf hard to do
TUMOR IMMUNITY AND IMMUNOTHERAPY

IMMUNOTHERAPY
Nonspecific Immune Stimulation
- attenuated mycobacteruim bovis (BCG) activates macrophages tf increases cytokine release

Active Specific Immunization

Vaccination to prevent tumors
- difficult because of heterogenity
- used for viral induce tumores ie FeLV, Mareks Disease

Vaccination to TREAT tumors
- harvest tumor
- use to produce vaccine via adding adjuvant or treating tumor cells to increase immunogenicity
- papillomas, ocular squamous cell carcinomas in cattle
- equine lymphsorcoma via infecting tumor cells with vaccina virus which produces antigen for NK cells
- some results but NOT SUSTAINED

Monoclonal Antibodies
- antibody and complement dependent cytotoxicity
- coupled to a toxin
- coupled to a radioisotope
- coupled to an enzyme which specifically activates a cytotoxic drug
- heterogenity is problem
- low proportion of dose reaches tumor cells
- production of immune reponses to monoclonal Igs ie usually from mouse
- can build recombinant Ig
- anti tumor effects present but not clinical or sustained in most patients

Cytokine Tumor Immunotherapy
- Interferons successful in some tumors esp hhuman hiry cell leukemia and kaposi sarcomas
- cytokines are very toxic
- tf local application best ie injection inotp papillomas or carcinomas of cattle

Cytokine Activated Cell Therapy
- lymphokin Activated Killers (LAK) cells:
- blood lymphocytes harvested and incubated with IL2 then reinfused into patient
- mainly expands NK population
- Tumor Infliltrating Lymphocytes (TIL):
- lymphocytes harvested from tumor tissue
- cultured with IL2 and reinfused
- killers are both NK and T cells
- can produce remission
IMMUNE MEMORY

CLONAL SELECTION
Once a lymphocyte binds antigen it DIFFERENTIATES into either:

- Effector Cell
or
- Memory Cells

both then proliferate
IMMUNE MEMORY

MECHANISMS OF STRONGER MEMORY RESPONSE

4 KEY WORDS
Increased number of antigen specific RESPONDER cells
- ie the memory cells

Increased responsiveness to antigen presenting cells
- naive cells require more costimulatory bonds and more time to become activated
- memory cells have lower activation threshold and require less costimulatioN
- tf more effective RECOGNITION

Better expression of costimulatory molecules

Phenotypic commitment

More effective MIGRATION
- naive T cells recirculate searching for novel antigen
- memory cells located in tissues at invasion sites

More effective FUNCTION
- class switch has already occured for B cells
- broader range of cytokines
IMMUNE MEMORY

KINETICS OF T AND B CELL RESPONSE
IMMEDIATE 12 - 24 HRS
Primary - Induction
Secondary - Amplification, Differentiation

EARLY 24 - 96 HRS
Primary - Amplification, Differentiation
Secondary - Effector

LATE > 96 HRS
Primary - Amplification, Differentiation, Effector
Secondary - Effector
IMMUNE MEMORY

CLINICAL DISEASE
Race between pathogen's ablility to replicate and/or cause disease and the speed with wchich to body can mount a protective immune response
IMMUNE MEMORY

IMMUNITY
Ability to mount MEMORY response
IMMUNE MEMORY

PROTECTION
Clinical disease does not Develop

- ie difficult to have for rota virus because would need to have antibody in GIT

-ie easy to have for small pox because it has a long incubation period
IMMUNE MEMORY

DURATION OF MEMORY

5 WAYS
Memory cells PERSIST in lymph node follicle in presence of antigen

Dendritic cells in lymph nodes SEQUESTER antigen
- months to years

RE-EXPOSURE to low levels of endemic antigen

Exposure to related antigen via CROSS REACTIVITY

Physiological MAINTENANCE of memory cells via CYTOKINES
- non antigen dependent mechanism
HERD IMMUNITY
The disease sparing effect on susceptible individuals within a group of individuals that are immune

Immune individuals REDUCE the PROBABILITY of an ADEQUATE CONTACT with susceptable individuals
HERD IMMUNITY

IMMUNITY IN POPULATIONS

3 FACTORS
Persitence and Spread of disease within a population depends on:

Immunity of INDIVIDUALS
- ie innate, T-cells, B-cells, antibodies, cytokines

Nature of CAUSAL AGENT
- How contagious
- host specificity
- Duration of infection
- carrier status

Structure and Dynamics of Population

aka Disease Triangle or Ecology of disease
HERD IMMUNITY

REED - FROST MODEL

3 CATAGORIES

3 ASSUMPTIONS
Individules categorized as:
- Infected
- Susceptable
- Immune

Assumptions
- infection is only spread from infected individuals to others by adequate contact
- if an individual is susceptable and adequately contacted it will develop disease
- there is a fixed probability of adequate contact between individuals
HERD IMMUNITY

5 EFFECTS

OF INCREASING # OF IMMUNE ANIMALS IN POPULATION
Delays Epidemic

Increases duration of epidemic

Reduces Magnitude of Epidemic

Reduces total number of infected individuals

Can prevent epidemic
HERD IMMUNITY

EFFECTS OF ADEQUATE CONTACT
The rate of contact of cases (infecteds and contagious) with susceptable animals determines if an epidemic can start

- decrease contact disease dies out

- increase contact maintenace of disease in populationn or epidemic
HERD IMMUNITY

BASIC REPRODUCTION RATE

HERD IMMUNITY THRESHOLD
Basic reproduction rate (Ro)is the spreading potential of an infection in a population
- function of biological mechanism of transmission
- function of rate of contact between members of the host population

Herd Immunity Threshold (H) is the proportion of immune individuals which must be exceeded if disease incidence is to decrease

- ie a disease with lower transmissiblity requres a lower number of immune individuals to avoid disease

- many common contagious diseases have an immune threshold of 80 - 85%
HERD IMMUNITY

2 WAYS OF ENHANCEMENT
Increase the proportion of immune animals in a population
- increase exposure to disease when appropriate
- increase vaccination

Reduce the probability of adequate contact
- identify and isolate infected animals
- pervent clusters of increased contact
HERD IMMUNITY

POPULATION DYNAMICS
Clusters of Susceptable Animals
- age cohort
- sex cohort
- emigrant populations
- management groups
- house holds

Clusters of Adequeate Contact
- Pounds, kennels
- racetracks, horse shows
- auction markets, trucks
- vet clinics, treatment areas
EVOLUTION OF IMMUNE RESPONSE

INVERTEBRATES
PHYSICAL BARRIERS

PHAGOCYTOSIS
- remember Metchnikov and Starfish larvae
- universal defense mechanism
- hemocytes, coelomocytes
- chemotaxis, adherence, ingestion, digestion (proteases and oxidants)
- enhanced activity via cytokine like (TNF, IL1,6) molecules

PROPHENOL OXIDASE activating system

ANTIMICROBIAL PEPTIDES

COMPLEMENT
- ancient system
- alternate and lectin pathways (note lytic pathway (C6 and MAC) does not appear until lancets)
- C like proteins occur in echinoderms, sea urchins have C3, C2/factor B

Limulus Amoebocyte Lystate (LAL) Assay
- use of horseshoe crab clotting defense mechanism to detect endotoxin (LPS)
EVOLUTION OF IMMUNE RESPONSE

IMMUNOLOGICAL BIG BANG
Development of Specific Aquired Immunity via genes for B and T receptors

- started with jawed fish (sharks and above)
- inclusion of bacterial TRANSPOSON via integrase gene
- BCR and TCR require rearangement of V, D, J genes
EVOLUTION OF IMMUNE RESPONSE

JAWED FISH
INNATE
- phagocytosis similar to mammals
- granulocytes enter inflamatory sites first
- originate from head kidney (lympohid function)
- macrophages (blood) and lymphocytes
- melanin prevents oxidation
- lysozyme, defensisns, C, acute phase proteins
- classical (antibody dependent), alternate and lectin pathways
- MAC

ACQUIRED IMMUNITY
- full set of lymphoid organs except bone marrow
- pronephros (head kidney) has antibody forming cells and macrophages
- Immunoglobulins Ig cluster genes, IgG, NO EVIDENCE OF OPSONIZATION

Cell Mediated Immunity
- TCR, MHC I, II
EVOLUTION OF IMMUNE RESPONSE

AMPHIBIANS
Potent Antimicrobial Peptides in SKIN

C system similar to mammals
- works best at 16 deg C

Uordeles (newts and salamanders more primitive)
- lack bone marrow
- monomeric IgM
- do not react to soluble portein antigens ie toxins

Anurans (frogs and toads)
- IgM pentamers or hexameres
- IgY
- IgX
- secretory antibody (IgY, IgX)
- Functional TCR ie graft rejection
- Cytokines IL1,2, INFs
- MHC II larva
- MHC I adults
- immunosuppresed during metamorphis
EVOLUTION OF IMMUNE RESPONSE

REPTILES
Age and seasonal involution of Thymus

Splenic structure similar to mammals

Primative lymph nodes
- no germinal centres

C3 in venom of some snakes

Immunoglobulins
- IgM primary, IgY secondary
- Respond to BSA
- T cell dependent response
- no secondary response to LPS ie T independent via CHO ala mammals

Cell Mediated Immunity
- Graft rejection (temperature dependent)
- mixed luekocyte reactions
- Delayed Type Hypersensitivity (DTH)
EVOLUTION OF IMMUNE RESPONSE

BIRDS
Organs
- Thymus
- Bursa
- primative lymph nodes
- well developed germinal centres

Immunoglobulins
- IgM primary
- IgY secondary main and involved in opsonization
- truncated reduces lethal hypersensitivity
- IgA including secretion
- Diversity generated in ovo via one V and J gene tf less than mammals

Cell Mediated Immunity
- Graft Rejection
- DTH
- alpha,beta and gamma,delta T cells
- Th1,2 helper cells with appropriate cytokines
IMMUNE GENETICS

MHC
MHC I
- A, B , C genes

MHC II
- DP, DQ, DR genes

Homos 3 genes, Heteros 6 genes

MHC is DEGENERATE
- recognizes many epitopes
- ie has a peptide pocket on size fits most
- Specificity is deterimined by T cell recognizing specific bound epitope in context of MHC
IMMUNE GENETICS

BCR
GENES
- V = variable region
- J = joining
- D = diversity
- C = constant region

HEAVY CHAIN
- coded for by two gene clusters, one cluster for constant domain and one cluster for variable domain

VARIABLE REGION
- coded for by muliple genes
- gene rearrangement via looping out, deletion and splicing

CONSTANT REGION
- coded for by a single gene for each class

CLASS SWITCHING
- via combining any variable region with a constant region (class)
- recombination for class swtich occurs via excision (Rcombinase) and splicing (DNA PK repair enzymes - defect = Equine SCID)
- induced via Th signaling and cytokines

Light Chain Exclusion
- only one of kappa or lambda light chain is transcribed

HYPERVARIABLE REGION
- aka Complemntarity Determining Region (CDR)
- 6 - 10 ammino acids
- shape deterimines specificity of epitope binding
IMMUNE GENETICS

TCR
GENES
- V = variable region
- J = joining
- D = diversity
- C = constant region
- fewer VDJ genes than BCR but greater diversity via junctional diversity

TCR TYPES
- alpha/beta or gamma/kappa types are created by 3 gene clusters

HYPERVARIABLE REGION
- aka Complemntarity Determining Region (CDR)
- CDRs 1,2,3 in V region and V(D)J region
- gene rearrangement via looping out, deletion and splicing
IMMUNE GENETICS

MECHANISMS TO GENERATE DIVERSITY

7 FOR BCR
4 FOR TCR

LOCATION AND IMPORTANT DIFFERENCE
BCR
- occurs in Bone Marrow, Bural Equivalent ie Peyers Patches, Lymph node ie Germinal Centre
- VJ and VDJ gene recombination
- Base Deletion
- Base Insertion
- Combinatorial association ie combination of different K or L light chains wtih different heavy chains
- Gene Conversion ie segments of psuedo genes inserted into Variable region
- Receptor Editing ie rearrangement of VDJ genes after antigen exposure in Germinal Centres. Note if after 4 attempts B cell cant bind antigen targeted for apoptosis
- SOMATIC MUTATION ie after antigen exposure, mediated by cytosine deaminase converting cytosines to uracils which are then repaired as thymadines

Note if after 4 attempts via Receptor editing or somatic mutation B cell cant imporve antigen binding it is targeted for apoptosis ie cell selection

TCR
- occurs ONLY in the thymus
- VJ, VDJ and VDDJ gene recombination
- Base Deletion
- Base Insertion
- Combinatorial association ie combination of different K or L light chains wtih different heavy chains

Note the only thing worse than having somatic T cell Mutation in your body (would get mondo B cell response to self via Th not to mention killer T cells) would be to neglect to point out that SOMATIC MUTATION DOES NOT OCCUR IN T-CELLs on the exam
IMMUNE GENETICS

HERIDITY AND DISEASE RESISTANCE
Immune responsiveness is inherited via multiple genes

Immune system is one of most variable physiological systems

Primary Genes:
- BCR, TCR and MHC

Secondary genes:
- immune regulatory, innate

Parameters
- Ig []
- antibody response
- T cell proliferation
- DTH
- Macrophage function

Identify resitant individuals and characterized response

Select for appropriate parameters
MUCOSAL SURFACES

3 FAST FACTS

3 FUNCTI0NS
Large surface between body and evironment

More than 95% of pathogens enter body via mucosal surfaces

Mucosa-associated lymphoid tissue (MALT) represents LARGEST IMMUNE COMPARTMENT WITHIN THE BODY

Physical BARRIER

TOLERATES Antigens of commensal flora, nutritional and normal enviromental elements

Mounts rapid and psecific IMMUNE RESPONSE against invading pathogens
3
DEFENSE MECHNAISMS
AT
BODY SURFACES
Non Immunological
- Mucus, saliva tears
- Normal bacterial flora ie outcompete pathogens
- Enzymes ie lysozyme
- Fatty acids on skin
- mucocillary escalator
- vomiting
- diarrhea

Innate Immunity
- Antimicrobial Peptides
- Phagocytes ie eosinophilic granulocytes
- TLRs + PAMPs = activation of monocytes, macrophages and dendritic cells results in INDUCTION of innate and psecific immune repsonses

Specific Immunity
- Antibodies IgA monogastrics, IgG ruminants
- Cytotoxic T Cells
DENDRITIC CELLS
Link Innate and Specific Immunity

Immature dendritic cells take up antigen/pathogen at mucosal surface via TLRs

Binding TLRs activates NFkB intracellular system which upregulates cytokine production

- TNF alpha, IL12, INF alpha attracts/activates NK cells
- TNF, IL6 attracts B cells
- TNF, attracts T cells

ie Activates Innate immune system and ALERTS Specific immune system

Dentritic cell migrates to lymph nodes and becomes mature via changing surface receptors from antigen sensing to ANTIGEN PRESENTING
- up regulates costimulatory molecules stimulating T-cell proliferation
- ie ACTIVATES Specific immune system
ANTIMICROBIAL PEPTIDES

FAST FACTS AND FUNCTIONS
aka HOST DEFENSE PEPTIDES
- do much more than just make sieves out of microbes

Small peptides secreted by
- epithelial cells
- granulocytes
- macrophages

Two Families
- Defensins
- Cathelicidins

FUNCTIONS

Antimicrobial Activity
- several defensins insinuate into outer leaflet of cell membrane via cationic peptides then join to create pore

Chemmoattracts for Immune Cells

Activate antigen presenting cells

Immunomodulate
- specific peptides stimulate either Th1 or Th2
- tf can be used as adjuvants

Promote wound healing and angiognesis

Anti-endotoxic (LPS) effects

Increase inflammation

In short don't leave home without them safely stowed in your epithelium because they could just be the most important molecules in the whole immune system
SPECIFIC IMMUNITY

MUCOSAL BASICS
Cytotoxic TCD8 cells reside in the epithelium amongst the epithelial cells
- gamma-delta T cells prevelant in intestinal epithelium

Plasma Cells (B cells stimulated by Th cells) reside in Lamina Propria where the secrete antibody
MALT

COMPARTEMENTS AND SIGNIFICANCE
Mucosa Associated Lymphoid Tissues

Form the COMMON MUCOSAL IMMUNE SYSTEM
- composed of compartments
- GALT Gut associated lymphoid tissues
- BALT Bronchus associated lymphoid tissues
- NALT Nasal associated lymphoid tissues
- Genitlal associated lymphoid tissues

Compartements are connected by permenant but varied exchange of immune cells
- lymphocyte trafficking and homing

INDUCTIVE TISSUES
- afferent limb
- Peyer's patches, Tonsils, Solitary lymph nodules
- M-cells, Dendritic cells, Epthelial cells

EFFECTORS
- efferent limb
- lamina propria
- Intraepithelial Lymphocytes (IELs), Lamina proprial Lymphocytes (LPLs)

Allow infection in one mucosal location to generate memory response (immunity) in another
MUCOSAL IMMUNITY

ANTIGEN UPTAKE

3 WAYS
M CELLS
- reside in epithelium above Peyers patchs
- shorter micrvilli creates a pocket that traps particles under 5 micrometers
- antigen is passed through M cell to its basal surface
- basal surface is involuted forming large pocket which is full of lymphocytes and antigen presenting cells which go to town with the antigen
- actually the dendritic cells take the antigen to the nearest germinal center in the Peyers patch

DENDRITIC CELLS
- dendritic cells open up tight junctions between epithelial cells
- extend dendrites (imagine that!) into lumen and fish for antigen
- once bound with antigen they head to a germinal centre to make men and women out of the naive B cells and the T cells join in on the fun

EPITHELIAL CELLS
- not wanting to miss out on the fun epithelial cells can also take up antigen
- however being weekend warriors they are not very effective
- low or no secretion of costimulatory molecules so they are a bit like wall flowers at the dance
- more important in chronic disease situations with severe inflamations
MUCOSAL IMMUNITY

EFFECTOR CELL POPULATIONS

WHO ARE THEY AND WHAT DO THEY DO
LPL lAMINAL PROPRIA LYMPHOCYTES
- mainly B Cells and T helpers
- Secretors of Antigen
- IgA MOST IMPORTANT Ig on mucosal surfaces
- IgG also present on mucosal surfaces in ruminants
- IgE also secreted ie allergies and parasites

T HELPER CELLS
- provide micro environment (a satisfying little swirl of cytokines, interleukins and costimulatory embraces)

INTRAEPITHELIAL LYMPHOCYTES (IEL)
- shoulder to shoulder with epithelial cells
- make up to 40% of total peripheral T cells
- mainly of the gamma-delta receptor CD8 cytotoxic types
- express alpha/alpha CD8 homo dimers
- bind MHC Class Ib which is uniquely expressed by enterocytes
- uniquely use Vgamma and Vdelta genes to form TCR antigen binding site
- secrete IFN-g
- regulatory function ie oral tolerance to dietary antigens
MUCOSAL IMMUNITY

THE IgA STORY

HOW DOES IT GET TO THE SURFACE
SYNTHESIS

IgA secreted in lamina proprial by plasma cell
- forms dimer via J-chain

Taken into epithelial cell via POLY Ig RECEPTOR (pIgR)and transported to luminal surface where it is SECRECTED and forever known as SIgA
- being a good citizen, the epithelial cell recycle the pIgR rather than let it float around in the lumen
- however there is always someone willing to abuse the system as some pathogens use the recycled pIgR as route through the epithelial cell to the basal side

FUNCTIONS
MUCOSAL IMMUNITY

THE IgA STORY

3 FUNCTIONS
FUNCTIONS

Blocks Bacterial Adhesion
- bacteria use adhesins to bind to host cell surfaces
- IgA binds adhesins on pathogen so the bacteria slide on by
- tf E-coli vaccine uses bacterial fibril protein as antigen to stimulate IgA response

NEUTRALIZATION OF VIRUSES
- viruses like to use cell surface receptors (ie AIDS virus uses CCR5 on Th cell)to bind host cell
- IgA via its handy hypervariable region can bind the receptor binding sites on the virus and whisk it off the dance floor
- tf when making vaccines handy to know which proteins viruses use to bind host cell surface receptors

NEUTRALIZATION OF BACTERIAL TOXINS
- most toxins need to be internalized to be effective
- bind cell surface receptors to enter cell
- IgG in this case binds toxin keeping the receptors clear for friendly physiological molecules
COMMON MUCOSAL IMMUNE SYSTEM

CELL TRAFFICKING
Hallmark of immune response

NAIVE T CELLS
- remember they already have their specific antigen receptors
- migrated only to secondary lymphoid organs via L-selectin

ACTIVATED EFFECTOR/MEMORY T/B CELLS
Migrate preferentially to site of antigen encounter
- ie HOMING

Mediated via mucosal addressins
Effector cells leave lymph node through efferent lymph
Each tissue expresses different chemokines which are upregulated and expressed during inflammation
- ie CC secondary lymphoid tissue chemokine
- ie alpha4beta7 integrin on effector T cell binds MAdCam on Mucosa
- ie CCR9 is a receptor for TECK/CCL25
- TECK/CCL25 is expressed on endothelial cells of High Endothelial Venules (HEV), site of lymphocyte extravasation, of small small intestine but not the HEV of the colon
- CCR9 is highly expressed on Lamina Propria Lymphocytes (LPL), Intra Epithelial Lymphocytes (IEL) and IgA secreting B cells
- tf SPECIFIC HOMING of LPL, IEL and IgA B cells to small intestine but not colon via SPECIFIC receptor and chemokine EXPRESSION

DENDRITIC CELLS
- specific receptors for lymph nodes once bound with antigen
COMMON MUCOSAL IMMUNE SYSTEM

HOMING EXTRAVASATION

I SAY MUCOSAL ADDRESSINS

YOU SAY
Integrins and Selections expressed on surface of lymphocytes

Lymphocytes travel at speed in blood vessels

TETHERING via SELECTINS binding with chemokines expressed on endothelial tissue induces ROLLING which slows them down

ADHESION via INTEGRINS binding with chemokines expressed on endothelial tissue causes ADHESION which brings the lymphocytes to a stop

Once stopped, the lymphocytes can TRANSMIGRATE through the endothelium and follow the immflammatory chemokines to party central
COMMON MUCOSAL IMMUNE SYSTEM

SUMMARY

FIVE KEY EXAM FRIENDLY POINTS
Homing of primed lymphocytes is high specific

Mediated by mucosal adressins ie the homing molecules

Primed (Activated/Effector) lymphocytes home selectively to certain compartments of the immunce system

Chemokines are important in orchestratiing migration patterns

Important for development and application of vaccines
COMMON MUCOSAL IMMUNE SYSTEM

ORAL VACINES

EFFECTIVE AND INEFFECTIVE RESPONSE SITES
EFFECTIVE

Proximal Small Inestine
- site of e-Coli infections
Ascending colon
Mammary Glands
Salivary Glands

INEFFECTIVE

Distal large intestine
Genital mucosa
Tonsils
COMMON MUCOSAL IMMUNE SYSTEM

RECTAL VACINES

EFFECTIVE AND INEFFECTIVE RESPONSE SITES
EFFECTIVE

Rectum

INEFFECTIVE

Small intestine
Proximal Colon
COMMON MUCOSAL IMMUNE SYSTEM

NASAL OR TONSILAR VACINES

EFFECTIVE AND INEFFECTIVE RESPONSE SITES
EFFECTIVE

Upper airway,
Regional secretions
Genital Mucosa

INEFFECTIVE

Gut
COMMON MUCOSAL IMMUNE SYSTEM

VAGINAL VACINES

EFFECTIVE AND INEFFECTIVE RESPONSE SITES
EFFECTIVE

Genital mucosa

INEFFECTIVE

None tf highly focused
MUCOSAL VACCINES

THE GOOD AND THE BAD
ADAVANTAGES

Immune response where needed

Effective immune response

Needle-free injection

No injection site reactions

DISADVANTAGES

Inneffective Delivery Routes
- ie if oral have to contend with the effects of acid and enzymes on the delicate little antigen peptides
- difficult to identify as antigen not nutrition
- tf target M Cells via antibody mediated targeting
- tf use liposomes, microparticles, viral vectors
- tf us

Require large amounts of antigen to make up for inneffective delivery routes

Stimulation of mucosal immune response difficult due to complexity of inductive sites
MUCOSAL IMMUNITY

REGULATION
TOLERANCE

Tissue resident dendritic cells and macrophages take in antigen
Release TGF beta attracting other dendritic cells and neutrophils
- ie positive feed back

Regulatory T Cells in laminal propria down regulate immune response in effort to minimize energy expenditure
- ie negative feedback
MUCOSAL IMMUNITY

SUMMARY

6 POINTS
Cell Trafficking is essential for effective mucosal immune response

Naive lymphocytes permanently circulate through secondary lymphoid tissues in order to encounter antigen

Effector (activated) lymphocytes home to the mucosal effector tissues

Common mucosal immune system but site specific trafficking

Mucosal immune system is effectively divided into compartments by specificity of trafficking

Important for vaccine delivery
VASOACTIVE MOLECULES
HISTAMINE
- released from mast cells, basophils, platelets
- increase vascular
permeability

KININS
- released from plasma kninogens and tissues
- vasodilation, permeablilty

PROSTAGLANDINS
- arachidonic acid
- vasodilation, permeablilty

C3a AND C5a
- serum complement
- mast cell degranulation, sm contraction, Neutrophil chemotaxis (C5a)
MAST CELLS

ACTIVATORS

SECRETIONS
ACTIVATORS
- IgG or IgE and antigen
- LPS
- ethdothelins
- adenosin
- neuropeptides

SECRETIONS
- histamine

- IL 4,5,6,13,16
- TNF alpha
- CCL3 chemokine

Proinflammatory or promote Th2 responses
TNF alpha
SECRECTED BY
- macrophages
- mast cells
- lymphocytes
- endothelial cells
- fibroblasts

FUNCTIONS

Activates
- mast cells
- dendothelium
- macrphages
- lymphocytes
- neutrophils

INFLAMMATION
- adhesion
- procoagulants
- induces cute phase proteins
- promotes ganuloma formation

ENHANCES
- fibroblast growth
- collagen syntesis
- bone resoption

TOXIC
- kills tumor cells
- septic shock
- sickness behavior
- altered lipid metabolism
IL 1
SECRESTED BY
- MAINLY macrophages
- dentritic cells
- lymphocytes T, B, NK
- endothelium

FUNTIONS
- proinflammatory
- mucus production
- induces acute phase proteins
- tissue growth
- IFN gamma synthesis, integrin expression
- fever, drowsines loss of appetite
- Th2 cytokine production
- eosinophil and basophil degranulation
- cytotoxic to tumor cells
MACROPHAGES

ACTIVATORS

SECRETIONS
ACTIVATORS
- IL 2, 4, 13
- C 1, 3
- antibody
- PAMPS
- INF gamma

FUNCTIONS
- phagocytosis
- MHC II antigen presentation
- bactercidal activity
- tissue repair IL 4, 13
- IL 1 costimulates Th2, stimulates acute phase response
- IL 6 promotes B cell differentiation, stimulates acute phase response
- IL 12 costimulates Th1 cells
- IL 18 promotes INF gamma production by Th1 cells
- TNF alpha stimulates T cell growth, acute phase response, triggers inflamation
Th1

FUNCTIONS

ACTIVATORS

SECRETIONS
FUNCTIONS
- T cell cytotoxicity
- macrophage activation
- some IgG responses

ACTIVATORS
- CD80 costimulation
- IL 12 (dendritic cells or macrophages

INHIBITORS
- IL 10

SECRETIONS
- IL 2 activates T cells, B cells, NK cells and macrophages
- INF gamma inhibits Th2 cells, stimulates Th1 cells, activates NK cells and macrophages
- TNF alpha and beta
Th2

FUNCTIONS

ACTIVATORS

SECRETIONS
FUNCTIONS
- costimulation of B cells
- tf IgG response
- tf IgA and IgE production

ACTIVATORS
- CD86 costimulation
- IL 1, 4 (dendritic cells)

INHIBITORS
- INF gamma

SECRETIONS
- IL 4 simulates B cell growth and differentiation, activates mast cells
- IL 5 simulates B cell growth, activates eosinophils
- IL 9 T cell growth factor
- IL 10 inhibits Th1 and macrophages
- IL 13 stimulates B cell growth, inhibits macrophages
IMMUNOGLOBULINS

NAME EM

SIZE EM

SOURCE EM
IgM
- primary response
- pentamere
- spleen and lymph nodes

IgG
- major IgG, class switch to in secondary response
- monomere, smallest tf most mobile
- spleen and lymph nodes

IgA
- epithelial surfaces tf mucosal immunity
- dimer via a jazzy J chain
- intestinal and respiratory surfaces

IgE
- parasites, allergies
- monomere
- intestinal and respiratory tracts

IgD
- not in horses or rabbits
- monomere
- spleen and lymph nodes

Deck Info

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