Immunity in Health and Disease

An antigen is any substance that stimulates the immune system. An antigen leads to ANTIGEN RECOGNITION and ANTIGEN PRESENTATION.

Antigen recognition is carried out by the immune system which is split into two parts:

• The innate immune system
• The adaptive immune system

In the case of ADAPTIVE IMMUNITY the antigen enters the body through either an injection, or a pathogenic infection. This is called CELL-MEDIATED IMMUNITY.

Cell mediated immunity leads to the production of cytokines and antibodies (the humoral response) as well as 'acute phaseproteins' and COMPLEMENT PROTEINS.

• INTRAPERITONEAL injections. Go into the posterior quadrant of theabdomen
• SUBCUTANEOUS injections. These go into the scruff of the neck.
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• INTRAMUSCULAR injections. Go into the quadriceps muscle group on the anterior of the thigh.
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1  The degree of 'DANGER' of the antigen (its nature)

2  How close the PHYLOGENETIC RELATIONSHIP is between the antigen and the host (its distance)

3 The ACCESSIBILITY OF antigenic EPITOPES for 'recognition'

(The epitope/antigenic determinant is the part of the antigen recognised by the immune system and specifically where the antibody binds to)

4 The ROUTE of antigen administration into the host

5 The DOSE of antigen administered into the host

6 The FREQUENCY of antigen administration (is it a repeated exposure) or the PERSISTENCE of the antigen stimulus (the bacterium/virus/parasite etc.)

7 The presence of CONCOMITANT infections (other infections)

8 The presence of an adjuvant

(An adjuvant is a substance enhancing the body's immune response to an antigen)

9 The IMMUNE STATUS of the host (its genetic constitution)

10 The AGE of the host (immunosenescence)

(Immunosenescence refers to the gradual and natural deterioration of the body's immune system due to ageing)

ANTIGEN Source - links to phylogenetic distance to host ; Purity - links to specificity of response ; Form - links to complexity ;Mode of administration - skin-DTH (delayed type hypersensitivity), systemic-humoral, iv ; Dose - links to tolerance ;Time - links to titer (a titer is a measurement of concentration, usually referring to the amount of antibodies found in a patient's blood) ;No. of Boosters - the primary-secondary response

RESPONDER Genetic constitution - high responders and low responders are determined somewhat genetically ; Host's physiological status - concominant infections, illness, viral infections, pregnancy all influence immune status

CHEMICAL NATURE OF ANTIGEN Metals - nickel ; Organic compounds - dinitrophenol ;Proteins - serum proteins, bacterial toxins, enzymes ; Lipoproteins - cell membrane proteins ;Glycoproteins - blood group antigens ;Polypeptides - hormones (insulin)

PHYSICAL FACTORS  Molecular mass>10kDa ; Conformation (polymer - co - polymer) ; Rigidity (gelatin-tyrosin) ; Localization of antigenic determinants/epitopes within the molecule; Tertiary structure ; Degradability

An antigenic challenege often leads to the activation of complement proteins and acute-phase proteins.

Complement proteins consist of around 25 PROTEINS PRESENT IN BLOOD PLASMA in an INACTIVE FORM. They work together to 'complement' the action of antibodies in destroyng bacteria.

They form a COMPLEMENT SERIES. An antibody will typically lock or bind to an antigen (such as bacteria) activating the first protein in the complement series. This causes a cascade effect called a COMPLEMENT CASCADE.

There are 3 distinct ways:

1 CLASSICAL complement pathway

2 ALTERNATIVE complement pathway

3 MANNOSE-BINDING LECTIN pathway

Classical pathway

Is triggered by the activation of the Cl- complex binding to antibodies from classes M and G, complexed with antigens or by its binding Clq to the surface of the pathogen

Alternative pathway

Is triggered by C3 hydrolysis directly on the surface of the pathogen. It does not rely on a pathogen-binding protein like the other pathways. The protein C3 is produced in the liver, and is then cleaved into C3a and C3b by enzymes in the blood. If there is no pathogen in the blood, the C3a and C3b protein fragments will be deactivated. 

Lectin pathway

Is homologous to the classical pathway, but with the opsonin, mannan-binding lectin (MBL) and ficolins instead of Clq. Is activated by binding mannan-binding lectin to mannose residues on the pathogen surface, which activatres the MBL - associated serine proteases, MASP-1, MASP-2, MASP-3 which can then split C4 and C2 into C4b and C2a then bind together to form C3 convertase - as in the classical pathway.

Jules Bordet did an experiment on this winning him a nobel prize in 1919. He began his career in 1892 whilst a student in med school.

• Animals exposed to bacteria develop antibodies which specifically AGGLUTINATE the same bacteria in the presence of a complement
• Bordet concluded that the bacteria-killing phenomenon was due to the combined action of two distinct substances: AN ANTIBODY IN THE ANTISERUM WHICH SPECIFICALLY ACTED AGAINST THE BACTERIUM and A NON-SPECIFIC SUBSTANCE, SENSITIVE TO HEAT, FOUND IN ALL ANIMAL SERUMS WHICH WAS LATER NAMED COMPLEMENT
• Complement proteins affect both innate and acquired immune systems however, A COMPLEMENT RESPONSE TO AN ANTIGEN IS CONSIDERED PART OF THE INNATE IMMUNE SYSTEM
• Elements of complement cascade can be found in species earlier than vertebrates in the evolutionary scale; most recently in the protostome horseshoe crab species, putting the origins of the complement system as an ANCIENT DEFENCE and part of a PRIMITIVE IMMUNE DEFENCE MECHANISM
• Complement proteins are synthesisied mainly by HEPATOCYTES. Complement is also produced by MONOCYTES, MACROPHAGES AND EPITHELIAL CELLS IN THE GASTROINTESTINAL AND GENITOURINARY TRACTS

• C3 is at the centre of the complement system. C3 IS A MAJOR SERUM PROTEIN (1g/litre)
• C3 Convertases (enzymes) activate C3 by limited proteolysis
• There are 2 C3 Convertases
• One is produced by the activation of the classical pathway (antibody dependent) either IgM or IgG
• The other is produced by the activation of the alternative pathway, in the absence of an antibody
• On the surface of the bacteria, C3 is converted into C3b and prepares the bacteria for phagocytosis
• C3b also acts as a focus for other complement components (C5,C6,C7,C8&C9) for lysis

Complement activation leads to: 

1 PHAGOCYTOSIS                                       Neutrophils and monocytes/macrophages

2 LYSIS                                                        are the 2 most important                                 3 INFLAMMATION                                        phagocytic cells of the immune system

Complement kills microorgansims in 2 ways:

1 OPONISATION FOLLOWED BY PHAGOCYTOSIS AND INTRACELLULAR KILLING (indirect)

2 ASSEMBLY OF THE CYTOLYTIC C5b-9 MEMBRANE ATTACK COMPLEX (direct)

OPONISATION IS THE PROCESS OF COATING MICROORGANISMS WITH PLASMA PROTEINS TO INCREASE THEIR ADHERENCE TO PHAGOCYTIC CELLS IN PREP FOR PHAGOCYTOSIS

2 Main Opsonins: IgG antibody AND Third component of complement (C3) which bind to the surfaces of microorganisms

REGULATION OF COMPLEMENT SYSTEM - Complement regulation proteins found on self-cells' surfaces, These prevent self-cells being targeted by complement proteins. Deficiencies in regulation of complement system lead to disease with an immunecomponent such as:ALZHEIMERS (neurodegenerative), BARRAQUER-SIMONSSYNDROME (lipodystrophy), MS (neurodegenerative), ASTHMA (type 1 hypersensitivity),SYSTEMIC LUPUS ERYTHEMATOSUS (type III hypersensitivity), VARIOUS FORMS OF ARTHIRITIS, AUTOIMMUNE HEART DISEASE and DEFICIENCIES OF THE TERMINALPATHWAY PREDISPOSE TO BOTH AUTOIMMUNE DISEASE AND INFECTIONS E.G. MENINGITIS

First comes INFECTION or INJURY --> then INFLAMMATION --> and then ACUTE PHASE PROTEINS ARE FORMED.

This is known as the Acute Phase Reaction

The best known acute phase protein is C-reactive protein which is synthesised in the LIVER and plays an important role in INNATE IMMUNITY and early-defence against infections. C- Reactive protein can be abbreviated to CRP.

C- Reactive Protein

Is a PENTAMERIC molecule with a molecular weight of 120 kDa (larger than albumin but smaller than antibody). Albumin is a simple form of protein, soluble in water, and coagulable by heat such as that found in egg white, milk and particularly blood serum. CRP has been conserved through evolution and a similar protein has been found in crabs. IT BINDS STRONGLY TO PHOSPHORLYL CHOLINE, A COMPONENT OF MANY BACTERIAL CELL WALLS. IT ACTIVATES THE COMPLEMENT AND PROMOTES PHAGOCYTOSIS. IT IS PRODUCED BY THE LIVER BUT ONLY PRESENT DURING ACUTE INFLAMMATION. ITS SINGLE MOST IMPORTANT ROLE IS ITS INTERACTION WITH THE COMPLEMENT SYSTEM.

The CRP Test is a new clinical test.

Uses of a CRP test:

• Check for Rheumatoid Arthiritis
• Check for Reheumatic Fever Flare-Ups
• Monitor response to therapy

High levels of circulating CRP in patients has recently been linked with increased risk of myocardial infarction.

Conditions that Cause a Rise in CRP Levels:

• Rheumatic Fever - a disease characterized by the inflammation of many connective tissues throughout the body, particularly in the heart, joints and central nervous system (brain and spinal cord). It is the result of a streptococcal infection or 'strep throat' which has been left untreated with antibiotics
• Rheumatoid Arthiritis - a degenerative autoimmune disease marked by pain, stiffness and/or swelling of the joints that eventually results in destruction of the joint. Rheumatoid arthiritis may also have systemic effects, (affecting the whole body) and is characterized by high levels of inflammation and activation of the immune system
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CRP TEST Is a BLOOD TEST that measures the level of an INFLAMMATORY MARKER ( a substance the body releases in response to inflammation) 

• High CRP levels in the blood mean there is inflammation
• Atheosclerosisor 'hardening of the arteries' is an INFLAMMATORY PROCESS whereby immune system cells are activated in response to a LOCAL INJURY INSIDE THE ARTERIES. White blood cells release inflammatory chemicals that contribute to the atherosclerotic process and are one component of arterial plaque deposits. When a plaque deposit ruptures, a heart attack or stroke can result. In response to this inflammation, CRP and other inflammatory markers in the blood rise.

SYSTEMATIC LUPUS ERYTHEMATOSUS - An autoimmune in which the body produces antibodies or immune system cells that are targeted at its own tissue. Lupus can affect the heart, lungs, kidneys, joints and nervous system. Like arthirits, lupus is an inflammatory condition characterized by activation of the body's immune system against itself. 

CANCER - A fatal disease marked by malignant tumors in the body.

POSTOPERATIVE INFECTION - CRP tests are perfromed following surgery; high levels may indicate infection. While an erythrocyte sedimentation rate test is also used for this purpose, CRP levels may be better tools for EARLY DETECTION because they will show an elevation more quickly.

Blood serum = blood plasma without the clotting elements 

Blood

• 55% = plasma
• <1% = buffy coat (platelets and leukocytes)
• 45% = erythrocytes

Clinical Tests

Blood tests are actually conducted on the blood SERUM, not the blood cells. Common serum tests are for CHOLESTEROL; TRIGLYCERIDES; LIPOPROTEINS; SUGAR/GLUCOSE LEVELS. Human Chorionic Gonadotropin (HCG) is a hormone produced during pregnancy, so a high level of this protein usually indicates the patient is pregnant in tests. MALNUTRITION may be indicated by a decrease in proteins (in the blood). Inflammatory diseases such as rheumatoid arthiritis can be indicated by an increase in alpha-1 globulin proteins. An increase/elevation in C reactive proteins is also a sign of disease. 

Antibodies are the most important protein in the human body and also the most sophisticated as it displays protein-protein interactions of a high affinity. ANTIBODIES RETAIN THEIR BIOLOGICAL ACTIVITY in vitro AND OUTSIDE THE BODY. Without antibodies we would die of infections.

Antibody uses in the pharmaceutical industry has seen an EXPONENTIAL RISE since 1975 in the use and approval of monoclonal antibodies for clinical therapeutic use (around 10 approves ones in 2007) with main targets being CANCER, AIDS, TRANSPLANTATION and INFECTION. They are also a powerful research tool. The binding of a tagged monoclonal antibody to an antigen receptor on a cell surface is a reaction commonly expolited in immuno-based tests. 

Their DISCOVERY

1900 Paul Ehrlich ~ there is something in the blood that provides protection. In 1908 he received the nobel prize for physiology of medicine for his contributions to immunology. 

1890 - Robert Koch 

Injected a sheep antiserum against the DIPTHERIA TOXIN into a girl who was dying from diptheria. She MIRACULOUSLY recovered just hours later!

1907

Sera from survivors/those recovering from RUBEOLA (measles) was injected into the general pop to prevent infection from this HIGHLY CONTAGIOUS VIRUS

Administering sera, sometimes URINE from survivors of viral epidemics for the treatment of PATIENTS was WIDELY USED. Particularly for ACUTE cytopathic viral diseases :) 

Today many IgG immunoglobulin preparations are licensed for INTRAVENOUS USE to prevent and treat viral diseases

EXPERIMENTS LEADING UP TO ANTIBODY DISCOVERY ARE CONSIDERED CLASSICAL IN MEDICAL HISTORY AND WERE CARRIED OUT BY TISELIUS AND KABAT IN 1939!

• were the techniques used to prove the antibody fraction was present in the blood
• in particular, in a fraction of proteins called gamma fraction

EXTRACORPOREAL IMMUNOADSORPTION

• used today in therapy
• is a blood purification technique
• it ELIMINATES PATHOGENIC ANTIBODIES IN AUTOIMMUNE PATIENTS
• good results; rare side effects; maximum elimination of pathogenic immunoglobulins from the blood

• Step 1: HYPERIMMUNISE RABBITS WITH OVALBUMIN (a protein found in egg white)
• Step 2: ISOLATE SERUM FROM RABBIT BLOOD AND SEPARATE INTO TWO ALIQUOTS
• Step 3: ABSORB ONE ALIQUOT OF THE SERUM WITH OVALBUMIN
• Step 4: ELECTROPHORESE SAMPLES TO SEPARATE PROTEIN FRACTIONS

The heterogenicity of the gamma fraction was an obstacle UNTIL MYELOMA TUMOURS WERE IDENTIFIED! 

A myeloma is a MALIGNANT PROLIFERATION OF PLASMA CELLS SECRETING A SINGLE IMMUNOGLOBULIN ISOTYPE. THE CELLS PRODUCE A MONOCLONAL ANTIBODY (IgG). This is called MONOCLONAL GAMMAPATHY.

Patients w. myeloma produce an EXCESS OF LIGHT CHAINS but since they have a small molecular weight can be excreted in urine. :) Called BENCE-JONES proteins.

The peak of the myeloma proteins is SHARP suggesting MONOCLONALITY of the proteins

The myelomas become a HOMOGENOUS SOURCE OF ANTIBODIES FOR RESEARCH!

• variable region of antibody is amine group side, constant is side with carboxyl group
• Hypervariable region is antigen binding site

Immunoglobulin Fragments Structure/Function

• Fab region : antigen binding; valence=1; specificity is determined by VH and VL
• Fc region : functions as an effector

2 Types of digestion - papain and pepsin

Mercaptoethanol reduction produces 2 straight H chains (53,000 daltons each) and 2 straight L chains (22,000 daltons each) 

Treatment of antibodies with a reducing agent like 2-mercaptoethanol as mentioned above splits the antibody into 4 fragments

H fractions have a higher absorbance when separated with chromatography

Light Chains

• 2 different types called κ and λ
• Antibodies are always κ2, λ2, (not κλ)
• Functional unit of all immunoglobulin superfamily molecules = formation of disulphide bonds that create loops of compact structure
• loops have a size of 100-110 amino acide residues & confer a global structure to the members of the family

FORCES INVOLVED IN ANTIGEN BINDING

• Van der waals (generated by the interaction between electron clouds, oscillating dipoles)
• hydrogen bonds (hydrogen bridges form between appropriate atoms)
• hydrophobic interactions (non-polar hydrophobic groups associate to minimise contact with water, contributes up to half the strength of the bond between antigen-antibody)
• electrostatic interactions (the attraction of oppositely charged groups on two protein side chains)

ANTIGEN BINDING DOES NOT INVOLVE COVALENT BONDING OR IT WOULD NOT BE REVERSIBLE!

Antibody's may bind to LINEAR or CONFORMATIONAL epitopes.

Linear epitopes = are composed of amino acids in a continuous sequence (the antigen protein amino acid sequence)

Conformational epitopes = is formed by the folding of the protein antigen. The amino acids forming the structural epitope do not occur in a linear sequence (in the amino acid sequence of the antigen protein)

Antigenic epitopes are usually formed by 5-15 amino acids

Hyper Variable Regions of Antibody Chains

• fold to form the antigen binding site
• they may be spaced apart in the linear sequence of the amino acid chain but when protein folding occurs they come together to form a single antigen binding site
• occurs for bith H and L chains so that 3 from H and 3 from L usually form an antigen binding site composed of SIX hyper variable regions

HIGH AFFINITY BINDING MEANS ANTIBODY FITS BINDING SITE MORE SNUGLY

• Multivalent antigen = multiple DIFFERENT antigenic determinants
• Polyvalent antigen = multiple IDENTICAL antigenic determinants
• CDR stands for complementary determining regions; CDR1, CDR2 & CDR3 are the three regions of hypervariablity which occur on both H and L chains
• The affinity with which an antibody binds an antigen is a result of the balance between attractive and repulsive forces - a higher affinity means a good fit and a lower affinity gives a poor fit and lower affinity constant
• Ab + Ag <=> AbAg
• The four types of intermolecular attractive forces between Ag-Ab are: van der waals; electrostatic interactions; hydrophobic interactions; hydrogen bonding
• antibodies are the MAIN proteins of the immune system and found in blood serum, and embedded in cell membrane of B cells as BCR (B cell receptors)
• produced in response to infection but always present in the body
• Lots is known about their molecular structure, genetics and how they interact with antigens
• antibodies are not however infallible as non-neutralizing antibodies directed against pathogens  fail to prevent pathogen replication, as seen with anti-HIV antibodies 

• Antibodies neutralise pathogens
• Some antibodies however , are non-neutralising - e.g. anti-HIV antibodies are ineffective so infection continues despite large amounts of antibodies specific to the HIV virus present in patients blood plasma - one of the main reasons the infected individual cannot rid themselves of the virus

THREE KEY FUNCTIONAL PROPERTIES OF ANTIBODIES

1) Pathogen neutralisation

2) agglutination

3) opsonisation ( a means of identifying the invading particle to the phagocyte)

FIVE ISOTYPES OF ANTIBODIES ARE KNOWN

IgG  IgA IgM IgE IgD

Neutralisation by antibody

Antibody binding to toxins or molecules on the surfaces of viruses or bacteria prevents their binding to cellular receptors. This prevents internalisation of toxins or viruses and prevents adhesion of bacteria to cell surface. 

Agglutination

Antibody binds to antigens on bacteria, causing them to agglutinate or clump, which inhibits the bacteria from leaving the site and also makes the bacteria much more susceptible to phagocytosis. 

Opsonisation

Antibody binds to antigen on microbial surfaces via the antigen-binding site. Phagocytes have FcRs that bind to the Fc part of the antibody molecule and the antibody/microbe