Noor M. Abu-Zaid

Diapedesis

Lymphatics in Inflammation:

y Lymphatics are responsible for draining edema.y Edema: An excess of fluid in the interstitial tissue or serous

cavities; either a transudate or an exudatey Transudate: An ultrafiltrate of blood plasma

y permeability of endothelium is usually normal.y low protein content (mostly albumin)

y Exudate: A filtrate of blood plasma mixed with inflammatory cells and cellular debris. y permeability of endothelium is usually alteredy high protein content.

y Pus: A purulent exudate: an inflammatory exudate rich in leukocytes (mostly neutrophils) and parenchymal cell debris.

Increased permeability

Balance between hydrostatic pressure and the colloidal osmotic pressure, which keeps the proteins and fluids inside the vessels

Increased fluids content

Imbalance between the 2 pressures, leads to the efflux of the fluids from inside the vessels to the interstitial cavity. Leaking fluids are not rich in proteins also there’s no increase in the endothelial space.

High proteins + endothelial space is

Function of Inflammatory Exudates

• Dilute the invading microorganism and its toxins.

• Bring antibodies through the plasma to the inflamed area.

• Bring leukocytes that engulf the invading microorganisms.

• Bring fibrinogen through the plasma, which is converted, to fibrin mesh, helping in trapping the microorganism and localize the infection

Are inflammatory exudates something beneficial in the inflammatory process? Yes it is.

And prevent the spread of infection to other parts of the body

Inflammatory Mediators:• Chemical substances synthesised or released and

mediate the changes in inflammation.1. Histamine by mast cells - vasodilatation.2. Prostaglandins – vasodilation, fever, and pain.3. Bradykinin - induce vasodilation, increase vascular

permeability, cause smooth muscle contraction, and induce pain.

• Cytokines including TNF, IL1, IL6, IL8• Lipid mediators: prostaglandins, leukotirns, and

platelet activation factor

Its role in initiating the expression of selectins within minutes

Their role in the production of both selectins, P and E selectins

It plays a role in recovery stage of the inflammation, once the infection source has been contained, there will be increasing in lipid mediators, which helps in the healing process

Cytokines and Inflammation

• Macrophages or DCs stimulated via innate immune receptors make pro-inflammatory cytokines, especially TNF (Tumor necrosis factor), IL-1, and IL-6

• TNF and IL-1 signal to endothelial cells to make them:– Leaky to fluid (influx of plasma; containing antibodies,

complement components, etc.)– Sticky for leukocytes, leading to influx of first neutrophils,

later monocytes, lymphocytes– IL-6 promotes fever and adaptive immune responses and

has systemic effects (“acute phase response” of liver, including C-reactive protein or CRP; levels used clinically as an indication of systemic inflammation)

The role of the inflammatory process in the extravasation of leukocytes

It’s the first one which reaches the site of infection

It works as ,In the circulation for up to 8 hoursدوريات متنقلة في الجسم once it enters the tissue (so leaves the blood) through endothelial cell is being expressed to the ICAM2 at a very low levels, so monocytes can bind to them here it goes to the tissues as macrophages.

It’s a test for people which gives an indication of inflammation, which is induced by IL6

While leukocytes enters the tissues and leaves the blood through extravasation process, and by the role of selectins, ICAM1, CD31, cross basement membrane and goes through chemo attractants into the site of

Inflammation Outcomes

1. Abscess formation2. Progression to chronic inflammation3. Resolution--tissue goes back to normal4. Repair--healing by scarring or fibrosis5. Spread through lymphatics or blood stream

If the infection becomes localized, it might lead to the abscess formation

Usually

Sometimes

Suppurative or Purulent Inflammation

y Pus: thick fluid containing viable and necrotic polymorph and necrotic tissue

y 1. Localized: ex. Abscess: Abscess is the localized collection of pus, commonly seen solid block of tissue -Example: dermis, liver, kidney, brain etc. Pus consists of partly or completely liquefied dead tissue mixed with dead or dying neutrophils and living or dead bacteria, formed of 3 zones y Small abscess is called boil or furuncle y Large one carbuncley Fistula

y 2. Diffused: Spreading of pus to adjacent areas e.g.cellulites occurring in subcutaneous tissue . Usually caused by streptococci.

It’s an exudate

الدملAbscess: it’s a pus containing lesions, usually needs to be drained and the patient must be treated with antibiotics.

It starts as: In the skin, it’s called hair follicles.Once it

grows larger:

When boils or furuncles accumulate.

It’s an abnormal connection between 2 cavities or 2 body parts, which is normally a not connected tissue. It occurs through the inflammatory process, when it starts as an infection then, it’s going to lead to cellular necrosis.

Anti-Inflammatory Therapeutics

• NSAIDs: inhibitors of inflammation and fever (block prostaglandin synthesis)

• Glucocorticoids are also potent anti-inflammatory drugs

• Agents that block TNF are effective in treating rheumatoid arthritis, Crohn’s disease, etc.

• Agents that block IL-1 are less effective for these diseases but are useful for some genetic inflammatory diseases (and are currently in clinical trials for more common conditions)

What’s their mechanism of action?

By the inhibition of both, COX1 and COX2 pathways. Homework: what’s the difference between the 2 pathways and what’s the function of each of them?

Ex: hydrocortisone, doxymethaxone, betamethaxone.

1- Normally, WBCs moves in the center of blood vessels and the blood surrounded them, once you get infected or damaged, endothelium starts to prepare for the presence of invading microorganisms or inflammation, firstly by:

Mast cell is the first cell to respond by producing histamine, histamine is going to lead to the preparation of the endothelial of •the vessels by producing the P selectins (it appears at the inner surface of the endothelial surface of the vessels within minutes). Macrophages will produce tumor necrotic factor (TNF) and interluekins (IL), in response to the presence of the microbes. •

Once this occurs, whipple bodies (they’re molecules which are present within the endothelial cell, and they have preformed P selectin, once the endothelial cell is triggered via TNF or IL, these bodies are going to externalised and express the P selectins on the inner surface of the endothelial cell within the vessels)

P and E selectins are the first step of the “Weak process or weak interaction” between WBCs and the endothelial cell of blood •vessels, these selectins bind with sialyl-Lewis X modified glycoprotein, this leads to slow the movement of WBCs by vasodilation and let the WBCs to leave the vessels and transport into the site of infection, then rolling of WBCs occurs. ICAm-1 (integrin ligand) is going to bind with integrin in the WBC, this is the “Strong process”, •

Then, expression of new receptor “CD31/PECAM-1” in the surface of leukocytes and in the surface of endothelial cell which facilitates and leads to “diapedesis”: let the WBCs to penetrate through the endothelium and penetrate the basement membrane in the interstitium., this journey is guided by the chemo attractants which is a cytokines produced to guide the WBCs to the location of the damaged or inflammed region. Once it reaches the location, firstly, neutrophils which do phagocytosis try to lysis and destruct the infecting bacteria or virus and produce cytokines and oxygen based at the site of infection which leads to invading the pathogen. Macrophages phagocytosis the bacteria, also, once the neutrophils finished their role, they’re going to be lysis and killed, so it targets both: the invading bacteria and the dead neutrophils. Macrophages mainly, also, the neutrophils considered antigen presenting cell, immune system will be stimulated by: *exudation of the tissue due to vasodilation and diapedesis through the basement membrane, leads to loose connection between the endothelial cells, then, fluids move to the interstitial and to the site of infection. Due to the fluid pressure this leads to fluids and substances returning back through the lymphatic system, some of them are the macrophage , once it return back to the lymph node, it goes to the B and T zone within the lymph node. Here, it’s going to concentrate and present the foreign antigen to the B and T cells, also, stimulate the immune system for further humoral and cellular immunity. According to the concentration gradient of the cytokines, the leukocytes know the target cell and do phagocytosis

Related to the previous lecture and slide number 12.

Extravasation means: leukocytes leave the blood vessels into the interstitial tissue

Antigen Antibody Reaction

Immunology Lecture 6Ashraf KhasawnehFaculty of Medicine

The Hashemite University

Objectives

• Discussion of general principles of antigen-antibody interactions

• Definition and importance of affinity, avidity, and cross reactivity

• Laboratory methods used for visualizing antigen-Antibody Reactions

DefinitionsyAntigen: Any chemical that creates immune

response, most are proteins or large polysaccharidesyMicrobes: Capsules, cell walls, toxins, viral capsidsyNon microbes: Pollen, egg white

yAntibodies: Immunoglobulines that recognize and bind to a particular antigen with high specificity and is made in response to exposure to the antigen

yEpitope: Small part of an antigen that interacts with an antibody (10-12 amino acids). Any given antigen may have several epitopes. Each epitope is recognized by a different antibody

It consists of polysaccharides

Remember the difference between: immunogen: humoral and cellular •response Antigen: humoral response, and it •mostly being a protein

One antigen can have more than one epitope.

Paratope: it’s the equivalent region in the antibody which properly binds with the epitope in the antigen.

Antibody-Antigen Interaction

• The interaction of the antibody with an antigen causes a change in shape of the antibody

• May cause the exposure of another site which then is responsible for the various reactions elicited by the antibody to destroy the foreign substance.

• The interaction of antibodies and antigens may produce a network type complex

Once there’s binding between the antigen and the antibody, there will be a structural changes, which expose more part of the antigen, which is going to be targeted by the immune system.

Nature of Antigen Antibody Reaction

• Lock and Key Concept: The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains

• Non-covalent Bonds: The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds.

• Reversibility: Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible

If we supposed that the antigen represents the رأس المثلث Then, the antibody will be موازي إله So they can bind to each other

The Ag-Ab interaction is due to lots of non-covalent interactions-lock and key!

bondingتفاصيل الـمش مطلوبة بالتفصيل

Antigen-antibody binding site

• The Fab portion of the antibody has the complementarity-determining regions (red) providing specificity for binding an epitope of an antigen.

• The Fc portion (purple) directs the biological activity of the antibody.

• (S-S = disulfide bond; N = amino terminal of glycoprotein; C = carboxy terminal of glycoprotein; CHO = carbohydrate.)

Between the heavy and light chains.

The red region represents the variable region, while the purple region represents the constant region

Homework: what’s the difference between -Isotype, -Allotype, -Idiotype according to: 1- it’s being in the constant or variable region. 2- species. 3- in the same individual

Antibody Binding Variations

• The various genes the cell splices together determine the order of amino acids of the Fab portion of both the light and heavy chain; the amino acid sequence determines the final 3-dimensional shape.

• Therefore, different antibody molecules produced by different B-lymphocytes will have different orders of amino acids at the tips of the Fab to give them unique shapes for binding epitope.

• The antigen-binding site is large enough to hold an epitope of about 5-7 amino acids or 3-4 sugar residues.

Variable region can bind to:B cell can encode for one antigen or one epitope, it has a single antigen, once the immune system has encountered this antigen, then, will occur a clonal expansion differentiation of this b cell, to produce more and enough antibodies (which is unique for this antigen) against this antigen. Whereas the variable region for Fab, has different shapes to accommodate other epitopes for the antigen.

Antigen Antibody Binding Equilibrium

Window zone or window period of Hepatitis B: when you examine the patient, no antibodies have been formed against the hepatitis B, and you cannot even (if you do assay for the present of antigens) find it, because it’s in the equivalent zone where all antibodies are bound to the antigens, so there’s no detected level of the antibodies. You need to do further tests or examinations to in case of to distinguish if this patient has developed immunity against the virus or he is in the window period.

Affinity

• Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody

• Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated. Most antibodies have a high affinity for their antigens.

Avidity

• Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies.

• Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

• Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities. This is illustrated in the on the next page.

It represents a multiple points of affinity or multiple interactions between the antigen or the epitope and the antibody. Or it can also be defined as binding between multiple epitopes in a single antigen with multiple antibodies.

Specificity

• Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen.

• In general, there is a high degree of specificity in Ag-Ab reactions. Antibodies can distinguish differences in – the primary structure of an antigen – isomeric forms of an antigen– secondary and tertiary structure of an antigen

When we say that there’s antibody against SARS COV-2 then this is specific for the antigen of the SARS COV-2.

Antigens might have different shapes depending on the folding of the antigen or the protein, and the antibody can distinguish the differences between all these structures.

Cross Reactivity

• Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen.

• Cross reactions arise because the cross reactingantigen shares an epitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen (multispecificity).

Ex: 1- people who developed immunity against cowpox they also developed immunity for smallpox, this means that the antibody which has been formed against the antigen of cowpox it can also binds to other antigen like that of smallpox.

2- people who are sensitive against pencillin, they also may be sensitive against cephalosporin. 3- when the epitope is located in the normal cell, they are similar, like in fever .4- SARS COV-2 and SARS COV-1 similarity structure of the epitope, so antibody might recognize and partially be effective against this antigen. This means that people may developed immunity against COV-2 if they have been recovered from COV-1, so it deals with the similarity of the antigen or epitope structure, so antibody might recognize partially be effective against this

Visualizing Antigen-Antibody Reactions

– Agglutination– Precipitation– Complement fixation– Fluorescent antibody tests– ELISA and RIA– Western Blot

1. Agglutination Testing

• Agglutination: antigens are whole cells such as red blood cells or bacteria with determinant groups on the surface

• Antibodies cross-link the antigens to form visible clumps

• Performed routinely to determine ABO and Rhblood types

• Widal test: tube agglutination test for diagnosing salmonella and undulant fever

• Latex agglutination tests: tiny latex beads with antigens affixed

There’s no agglutination, so this person has no B antigen in his blood.

There’s agglutination, so this person has A antigen in his blood

Rh+, there’s agglutination, so this person has D antigen in his blood.

This test contains only blood sample of the person without adding the Anti A or Anti B. This test is done to assure that the clumping or the agglutination occurs due to the reaction between the Ag and the Ab, not due to thrombosis. If agglutination occurs in this control test so this give an idea that something is wrong and this results of agglutination are not valid,

So this agglutination occurs as a result of the presence of A antigen with Anti A, and D antigen with anti D. And once we add these antibodies to the antigen, mesh work are formed which represents the agglutination in the slide.

2. Precipitation Tests

• Precipitation is the interaction of a soluble Ag with a soluble Ab to form an insoluble complex.

• The complex formed is an aggregate of Ag and Ab

• Reaction is observable as a cloudy or opaque zone at the point of contact

• Example: VDRL (Veneral Disease Research Lab) test streptococcal group antigens testing

Ex: syphilis

Differentiate between agglutination and precipitation: Agglutination: the whole cells and there’s expression of the antigen in the surface, whereas precipitation: soluble antigen with soluble antibody

May appear as Mesh work, and the test tube will be cloudy and clear. Or as an aggregation. Mesh work = شبكة Aggregation = مترسبة

3. Complement Fixation

• Lysin or cytolysin: an antibody that requires complement to complete the lysis of its antigenic target cell

It’s a part of the innate immunity, classical pathway 1 which is triggered firstly by Ag Ab complex, leads of the activation of the complement

A sample from an infected patient (with HIV for ex), and it contains Ab.

A sample from the same patient but does not infected so there’s no any Ab.

When Adding the Ag of HIV, then, Ag/Ab complex is formed.

When adding the Ag, no Ag/Ab complex has been formed, because there’s no Ab.

Adding the complement, but no complex has been formed.

Adding the RBCs (it’s an antigen antibody), which is going to precipitate, because the complement was all used up with the original antibody (it has already been bounded with Ag/Ab complex), so it doesn’t react with RBCs.

Adding the complement, then binding of this complement with the Ag/Ab complex .

Adding RBCs, does not precipitate, rather, it’s going to react with the complement and be lysis. So it gives negative result of the presence of the antigen specific antigen.

The same patient and the same sample but we get 2 different results of the present of the Ab (positive and negative), why? Due to the serial dilution, this means that we are adding specific amounts of the complement and the RBCs, whereas we dilute the concentration of the Ab. When we get positive result, this is due to the high concentration of the Ab, while we get a negative result when the concentration of the Ab is low. Dilution is important for the measurement of Ab titer.

4. Flurorescent Antibodies and Immunofluorescence Testing

• Direct testing: an unknown test specimen or antigen is fixed to a slide and exposed to a fluorescent antibody solution of known composition

• Indirect testing: the fluorescent antibodies are antibodies made to react with the Fc region of another antibody

It’s a test looking for the antigen itself. If the antigen is present, it’s going to react and bind to the antigen, then fluorescent. It has been done on the cell culture, to detect if the cells are expressing certain antigen, then adding the Ab and looking for that under the light microscope.

Antigen is targeted by Ab labeled with fluorescent dye.

Detect the presence of Ab against certain Ag, like the detection of the presence of specific virus.

This is specific for not the Ag but rather for the primary Ab, we’re looking or detecting the presence of the Ab against certain Ag. Ex: detect the presence of HIV patient, detect the Ab against the HIV Ag.No binding with

anything, because of..

The primary is targeted with the secondary

No binding with Ag.

This known Ag is targeted by the primary Ab

5. Radioimmunoassay (RIA)Enzyme-Linked Immunosorbent Assay (ELISA)

• Antibodies or antigens labeled with a radioactive isotope (RIA) or Enzyme (ELISA) used to pinpoint minute amounts of a corresponding antigen or antibody

• Compare the amount of radioactivity present in a sample before and after incubation with a known, labeled antigen or antibody

Types of ELISA: direct, indirect, sandwich, competitive. They all use the same principles.

The indirect test: We can bind the plates for the ELISA either coated with Ag or with Ab. 1- coats with Ab, 2- adding the sample test of the patient to look for the Ag, 3- if Ag is binding then washing step is done to eliminate from the non bound Ag, 4- adding the primary Ab and the secondary Ab.

The direct test: Ag is bound with the plate ( جاهز من الشركة ), adding the sample from the patient to look for the Ab wich will directly bound with Ag. Also it’s called sandwich test because of the using of Ag between the Ab and other Ab. Competitive test: a test that have to do with concentration of different components.

6. The Western Blot for Detecting Proteins

• Test material is electrophoresed in a gel to separate out particular bands

• Gel transferred to a special blotter that binds the reactants in place

• Blot developed by incubating it with a solution of antigen or antibody labeled with radioactive, fluorescent, or luminescent labels

1- the sample contains the Ag, 2- then you have to run the sample on the gel, 3- transfer the Ag from the gel into the nitrocellulose membrane (the arrangement of antigen will be according to their size, here we deals with proteins so we use the kilodalton unit), 4- blockage with 3-5% milk, 5- incubation with primary and secondary Ab, 6- scanning on a special machine.

1- looking for specific proteins or antigen, 2- Ab specific to this Ag, 3- primary Ab then a secondary Ab which contains the fluorescent

7. Flow Cytometry

• The flow cytometer was designed to automate the analysis and separation of cells stained with fluorescent antibody

• The flow cytometer uses a laser beam and light detector to count single intact cells in suspension

• Every time a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded

• Those cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a second detector system located at a right angle to the laser beam

• It has large number of medical application for example in classification and treatment of leukemias

Cell sorting: 1- separation of the cells, and get a single cell. 2- labels this cell with specific Ab. 3- run on the flow cytometry. This test is used in case like cancer and leukemia..

Can differentiate between cells, بتنزل الخلايا بخط رفيع خلية خلية ، يتم التمييز بين الخلايا بأكثر من لون This colors is going to be displayed in the chart.

Why we don’t get Ab against the foods like proteins, etc? Because that immune system doesn’t recognize them as foreign bodies so will not activate the immune system and the B and T cells to react against.

Once these plates are detected by the laser, then they can guide the cells into different location. Sorting is based on the direction of the label cell by the laser, and chart appears in the state of the flow