Immunology Unit EXAM 2 Flashcards
Antibodies are bifunctional with the goal of- explain
- Antibodies bind to foreign antigens
- Antibodies interact with components of host defense system to facilitate destruction, removal, or neutralization of foreign organisms or substances.
Why is the diversity of the antibody repertoire enormous?
We can produce up a BILLION (10^7 to 10^9) structurally different antibodies to recognize different foreign antigens.
Where do foreign antigens bind and where do interactions with host defense components occur?
- One foreign antigen binds to the two highly variable domains (light and heavy chain). Thus an antibody can bind one or two EPITOPES.
- The conserved (constant) domain can bind the Fc receptor of an effector cell and complement proteins.
What is the result of having a bifunctional antibody.
Bifunctional antibody links antigen recognition with specific host response.
Discuss antibody cross-reactivity with autoimmune conditions.
Antibodies can sometimes cross react with a foreign antigen and self antigens.
Antibodies are glycoproteins produced by what type of cell?
Activated B cells in response to specific antigens
5 classes of Igs and what differentiates each as a physical factor?
GMADE. Each have a distinct heavy chain.
Antibody structure has two identical heavy and light polypeptide chains. What are the light/heavy chains physical factors?
50:50 ; kappa : lambda light chains.
Heavy are alpha, delta, gamma, epsilon, or mu
How are the light and heavy chains of an antibody linked? What regions do they have?
Disulfide bonds. Both have a variable and constant region.
How many hypervariable regions are on each chain (heavy & light)?
3 about 10 a.a long
The variable region contains the complementarity defining regions (CDR). Define what a CDR is composed of.
The CDR are hypervariable segments on both heavy and light chains.
What type of bonding exists between the antibody Fc region and either Fc receptor or complement proteins?
Noncovalent bonding.
- Hydrogen bonds
- Electrostatic interactions
- Van Der Waals interactions (induced dipoles)
- Hydrophobic interactions (exclusion of water)
In what case can an antibody serve as an integral membrane protein? How does it differ from the secreted form?
- They are found only on unstimulated B cells.
- Membrane form differs at the heavy chain C’; tm helix; secreted has hydrophilic sequence.
Note these differences are achieved by posttranslational modification (change in polyadenylation site use)
Carbohydrates on heavy chains provide (2)?
solubility and protection against proteases
Discuss the basic structure of the constant and variable domains. And where else can we find this generic structure?
- They both have anti-parallel beta sheet structures held together by disulfide bonds.
- MHC and proteins of the immunoglobulin superfamily of proteins.
- The loops at the ends pointing out into solution of the beta strands are the hypervariable regions aka CDR.
Flexible hinge region exists where in the antibody. Unfortunate the hinge region is the target of?
- Exists in the heavy chain between the 1st and 2nd constant domains. It allows for the binding of two antigenic determinants.
- Proteases
What determines the class and subclass of immunoglobulins?
The heavy chain type.
Which immunoglobulin makes up 70-75% of total circulation Ig? How many subclasses does it have? What’s special in terms of pregnancy?
IgG has 4 subclasses and the only antibodies to cross the placenta and passively protect the fetus.
What is the primary antibody used for diagnostics, research, Tx?
IgGs
Which Igs are the principle antibody mediator of the secondary immune response.?
IgGs
The IgG (150K kDa)subclasses contains what unique? Describe the heavy and light chain specifics.
The GAMMA heavy chain with different numbers and arrangements of disulfide bonds.
- A heavy chain has 1 variable, 3 constant regions, 1 hinge region (helical segment). The 2nd constant region has the carb and region for FcR and complement components.
- A light chain has 1 variable, 1 constant region.
what makes up 10% of total Ig pool and found almost exclusively in the circulation?
IgM
Why does it make sense for the IgM (970K kDa) to be the principal antibody to mediate primary immune responses?
- It’s huge. A pentameric structure that highlights to immune and inflammatory defense mechanisms because it tends to cause AGGLUTINATION of antigens.
- Proficient at complement fixation.
Which Ig is known to be involved with peripheral neuropathy?
IgM. Reconginizes carb epitope on bacteria (glycosphinolipid) and corss-reacts with epitopes on myelin associated glycoproteins –> complement fixation.
- Causing demyelinazation and axonal degeneration.
What type of heavy chain does IgM have and list the specifics. Also it has an odd quirk due to the size of the Ig,- describe.
- IgM contain mu heavy chains; one variable and four constant domains. The mu heavy chain is glycosylated and contain a cystein-rich tail piece involved in the pentamer formation.
- Has a cysteine- rich J (joining) peptide to aid in the pentamer formation.
IgA has two forms which both are glycosylated. Where are they found and which one has a similarity to IgM and what’s its defining physical factor?
- IgA1 (monomer) is found in circulation
- IgA2 (dimer) is the predominant antibody in mucosal secretions
- IgA2 similarly to IgM uses a J chain to stabilize the dimer
- Alpha heavy chains
Explain the process of IgA2 being made and becoming the secretory IgA2 (with the secretory component)
The submucosal plasma cell secretes IgA2 dimers which bind to the polyIg receptor on the luminal side of mucosal epitheilial cells. The bound complex gets endocytosed and the receptor is later cleaved while keeping the secretory component (SC) associated with IgA2. The SC stays bound after traversing the mucosal epithelial cell and is secreted. SC provides the IgA2 protection from proteolysis.
Describe Berger’s Disease and the incidence among transplant patients.
Is when the IgA complexes deposit in the mesangial region of the kidney –> activating complement and leading to kidney failure “glomerulonephritis” “IgA nephropathy”.
Incidence is 50%
Which Ig is used as cell surface marker of B cell maturation?
IgD is relatively rare and has delta heavy chains. Found almost exclusively on various stages of B cell maturation.
It has a monomeric immunoglobulin structure and is glycosylated.
What’s the function of IgD?
Data suggests IgD functions as a co-stimulator in antigen presentation and acts in B cell development, and may also be something of an evolutionary relic.
What’s a possible Tx for patients with chronic idiopathic thrombocytopenia (often associated with SLE and other autoimmune diseases) in which platelet destruction results in a bleeding disorder.
- anti-IgD increased PLT count.
-
IgE contains epsilon heavy chains which, like IgM heavy chains, contain __ domains.
5
Unlike IgM, IgE is monomeric and is present only in trace quantities in serum as free antibody. It is primarily found bound to ____?
high affinity Fc receptors on the surface of basophils and mast cells and to moderate affinity receptors on the surface of lymphocytes and monocytes.
IgE is the primary antibody mediator of ___? Anti-IgE significantly reduces the symptoms of __?
- hypersensitivity, allergies, and asthma, due to histamine release caused by antigen binding to mast cell or basophil-associated IgE.
- asthma and allergies.
-
What is the primary antiparasitic antibody, and sensitizes worms and other parasites for destruction by eosinophils?
IgE
Papain cleaves
cleaves IgG in the hinge region between the first two heavy chain constant domains, yielding two antibody-binding (Fab) fragments and one fragment containing the constant region (Fc).
Pepsin cleaves
heavy chain beyond interchain disulfide bonds, yielding one fragment which can bind two antibody molecules ( F(ab’)2 ) and cleaves the remainder of the heavy chain into small peptides
Fab and F(ab’)2 bind how many antigenic determinants?
1, 2 respectively
Define isotype and where are the markers found?
- present in all healthy members of a species - represent separate genes
1. Light chain isotypes - Kappa or Lambda. Markers present in the constant domains of the light chains.
2. Heavy chain isotypes - markers present in the constant domains of heavy chains, distinguishing classes and subclasses
Note: the “immunoglobulin isotype” refers to the phenotypic variations in the constant regions of the heavy and light chains.
Define allotype and where the markers are found.
- variations within a species involving different alleles at a given locus. Not all members of the same species have a particular allotype. Markers are found in heavy chain constant regions
Note: allotype refers to the idea that each immunoglobin has unique sequences particular to the individual’s genome that manifest in its constant region (normally).
Define idiotype
variations associated with the antibody binding site, especially the hypervariable region within the variable domain specific for the individual antibody clone (private idiotype) or may be shared (public idiotype)
Note: idiotype describes the distinctive sequence and region that makes any immunoglobing/TCR unique from others of the same type which is its variable region.
Monoclonal antibodies are derived from a single clone and is specific for __?
Specific for an epitope on an antigen.
Describe process of generating antibodies in mice.
Mice are introduced to an antigen and we ensure that antibodies are being produced. Then we take the spleen cells and fuse it with plasmacytoma cells. Fusion is rare so we start with tens of millions of cells to only get hundreds to thousands fused cells. To select for the hyrbidomas the plate has HAT medium (Hypoxanthine, Aminopterin and Thymidine; start up for de novo synthesis pathways). Lastly screen the remaining hybridomas for the relevant antibodies.
Note: the spleen cells die in culture and plasmacytoma cells have mutant salvage pathway and the aminopterin blocks the de novo synthetic pathways.
Define antigenicity and immunogenicity.
A. Antigenicity = ability to bind an antibody
B. Immunogenicity = ability to stimulate an immune response (many small molecules are antigenic but not immunogenic)
Discuss factors of immunogenicity.
Properties of antigens that contribute to immunogenicity – foreigness, molecular size, chemical complexity, degradability
Other factors in immunogenicity – method of antigen administration, genetic constitution of immunized animal, amount of antigen, presence of adjuvant
Define paratope and epitope
A molecular surface on the antibody (paratope) interacts with a molecular surface on the antigen (epitope)
Binding is the interaction of two 3 dimensional surfaces.
Define affinity.
sum of all attractive and repulsive forces within one epitope-paratope association
Affinity = monovalent binding (one epitope + one paratope)
Affinity calculations: K = [AbAg] / [Ab][Ag]
Antibodies recognize two properties of epitopes which are?
composition and configuration. Change either and you loose affinity.
define aviditiy. How many epitopes can IgA2 and IgM bind?
- Avidity = multivalent binding (increases stability of complex)
- multimeric antibodies such as IgA2 and IgM can bind up to 4 and 10, respectively.
Define specificity and antiserum specificity
- Specificity: the relationship between the affinity of an antibody towards the protein to which it was raised and the affinity of that antibody towards other nonrelated proteins. A highly specific antibody will bind very tightly to one protein and not very tightly to most all other proteins.
- Antiserum specificity: an antiserum is composed of many different antibodies, and the specificity of the antiserum towards any one antigen is determined by how many antibodies recognize that antigen with high affinity, as compared to any other antigens that are also recognized by the different antibodies in the antisera
define cross-reactivity and the sources to it (2)
an antibody raised against one protein binding to another protein.
Antigens may share the same or similar epitope or the antibody has polyfunctional sites where the variable region can bind more than one epitope.
Cross reactivity and disease (3)
1.myocarditis, 2. MS, 3. Mayasthenia gravis
- Chlamydia outer membrane protein contains an epitope similar to cardiac myosin heavy chain; antibodies raised against this epitope during chlaymidia infection can also bind cardiac muscle and lead to myocarditis
- hepatitis B viral protein contains an epitope similar to myelin basic protein; antibodies raised against this epitope during viral infection can also bind myelin membranes and can lead to demyelinating diseases such as multiple sclerosis
- a herpesvirus protein contains an epitope similar to an epitope on the nicotinic acetylcholine receptor; antibodies raised against this epitope during viral infection can also bind to nicotinic acetylcholine receptors at the neuromuscular junctions and cause neuromuscular diseases such as Myasthenia gravis
The immunological elements of the innate respond to ___?
broad recognition of molecular patterns associated with pathogens, while the adaptive components (antibody and T cell responses) are highly antigen specific.
The inducible first line of defense works how?
- Pattern recognition key: unique pathogen-associated molecular patterns (PAMPs) exhibited by microorganisms are recognized by pattern recognition receptors (PRRs) of the innate response.
- the innate response can recognize danger associated molecular patterns (DAMPS) that represent sterile (non-infectious) evidence of cellular stress.
Adaptive immunity is clonal and characterized by rearranged ____ and products of the adaptive immune system stimulate ____.
- antigen receptors (TCR and Ig)
- the innate response.
note: to maintain homeostasis the innate and adaptive immune system need to interact. Innate response also activates the adaptive response.
Differentiate between innate non-induced and induced mechanisms.
The non-induced innate mechanisms include physical and chemical barriers to infection that are always present. The induced innate mechanisms involve the activation of components through the interactions of PAMPs with PRRs.
What are the Barriers (2) of non induced innate?- anatomic barriers to prevent penetration and invasion by infectious agents.
Skin: Keratinized layers with tight junctions between cells. Intact skin is the most important barrier to infection -barrier compromise (e.g. burns) leads to severe systemic infection.
- Mucous membranes - less efficient barriers than skin
a. Intact membranes can be colonized
b. Can be penetrated by some organisms
c. Integrity of the membranes easily disrupted
d. Major site of entry of infectious agents
What are the Physical of non-induced innate- protective surface phenomena?
Washing action of body fluids: tears, saliva, mucus, urine
2. Surface motion: peristalsis, mucociliary escalator, swallowing
What are the Biochemical and biological defense of non-induced innate?
Enzymes - digestive tract, lysozomal, tissue secretions (e.g. lysozyme)
- Competition and interference - glycoproteins, lactoferrin, transferrin, interferon, normal microflora
- pH - lactic acid, fatty acids lower pH
- Defensins: cysteine-rich peptides present at epithelial surfaces and in neutrophil granules. Broad-spectrum antibiotics that kill wide variety of microorganisms.
What the induced innate acute phase proteins (APP) (4)? Serum concentrations of these proteins rise rapidly with infection.
Examples:
- CRP (C-Reactive protein): binds the C protein of Pneumococci, facilitating uptake by phagocytes
- Complement components: certain complement components increase during infection; a number of micro-organisms activate the classical or alternate complement pathway
- α-2 macroglobulin and other anti-proteinases increased
- IL-6 regulation of fibrinogen: increased fibrinogen leads to increased erythrocyte sedimentation rate. “Black bile” - thought to be dangerous and was ancient rationale for bleeding patients.
What are the cellular components of the innate response?
monocytes, macrophages, natural killer cells, dendritic cells, eosinophils, neutrophils. Some of these play important part in the adaptive responses as well. A new category of innate cells called “innate lymphoid cells”, of which NK cells is the prototype
the innate response, pathogens are recognized by these cells via “pattern recognition receptors” (PRRS). Examples are?
PRRs include C-type lectin receptors of which the mannose receptor is the prototype. These C-type lectin receptors recognize repeating carbohydrates on pathogens. The Toll-like receptors (TLR 1-10) are important pattern recognition receptors. Some of the TLR are expressed on the cell surface, while others (TLR 3,7,8,9) are expressed in the endosomes. These highly conserved receptors are expressed selectively on different cell types and recognize structures/components of pathogens
TLR 4, 9, 2, 3 explain what they recognize.
TLR-4 is the receptor for bacterial LPS while TLR-9 is the receptor for bacterial and viral DNA sequences (CpG) that are non-methylated. TLR2 recognizes a variety of bacterial components and TLR3 recognizes double-stranded RNA, a replicative intermediate for many viruses.
What can recognize viruses replicating in the cytoplasm.
DNA and RNA sensors.
The DAMPS are (define). DAMPS activate the production of IL-1β through a family of protein complexes known as _____.
- endogenous factors associated with molecular stress such as ATP released from dying (necrotic cells) and uric acid crystals.
- inflammasomes
Types of phagocytes are (2). Which come from what lineage?
a. Neutrophils - differentiate in bone marrow; short lived. Function early in innate response.
b. Macrophages
i. Bone marrow precursors, long-lived
ii. Differentiate from blood monocytes to fixed or mobile cells
iii. Heterogeneous in range and scope of function
iv. Function later in immune response and are important bridge to adaptive immunity
-myeloid lineage.
With the chemotaxis to get phagocytes to moves how are chemical signals recognized and subsequent movement?
Chemotaxis - movement toward a concentration gradient of a chemical signal
b. The signals involved are of diverse nature and are derived from a number of sources, e.g.: bacterial factors, tissue injury factors, cytokines, chemokines.
c. Chemical signals are recognized and responded to by means of receptors at the cell surface.
d. Chemotactic signals induce biochemical events leading to the polymerization of microtubules and the contraction and relaxation of microfilaments which in turn allow for the formation of pseudopodia and locomotion.
How does the phagocyte move from blood vessel into the site of tissue inflammation?
Phagocytes adhere to the endothelium, first loosely, then more tightly and dissolve the basement membrane, allowing them to pass from the vessel toward the site of inflammation (diapedesis or extravasation). Adherence to the epithelium mediated through adhesion molecules that are upregulated on epithelium in response to infection/cytokines
Describe the events of phagocytosis at the point of adherence to foreign agent.
At the point of adherence, microtubules are formed and micro-filaments begin contraction-relaxation cycle
b. Pseudopods draw agent into intracellular, membrane-bound vesicle called a phagosome
c. Not all phagocyte/micro-organism binding leads to phagocytosis
Degranulation define
primary lysosomal granules of the cell migrate toward and fuse with phagosome, discharging their contents into the vesicle, now called a phagolysosome.
While degranulation is taking place, the cell is also undergoing a metabolic burst -explain
Increase in oxygen consumption
b. Activation of the hexose-monophosphate shunt
c. Activation of the nicotinamide adenine dinucleotide system (NADP)
d. Increase in glycolysis
Within lysosomes what types of enzymes are their and they function in what environment?
These include a wide array of hydrolytic enzymes which can act on a wide range of substrates, thus inactivating and digesting them down to component residues.
Most of these enzymes act at acid pH (3.5 -4.5) and this pH is generated in the phagolysosome by the formation of lactic acid during the “respiratory burst”.
Explain oxygen-dependent system of the phagocyte oxidase system.
phagocyte oxidase system. Phagocyte oxidase is upregulated by IFN-gamma, signaling through TLRs, which leads to the generation of toxic free radicals.
Explain oxygen-independent system (4). cationic proteins, acid, lactoferrin, lysozyme
(a) Acid: lactic acid via glycolytic activity
(b) Lysozyme - an enzyme which hydrolyzes beta 1,4 linkages between carbohydrate residues in the cell walls of many bacteria. Active against gram-positive organisms.
(c) Lactoferrin - competes with microbial cells for iron which is essential to many bacteria.
(d) Cationic proteins - “Defensins” also active here. These are a diverse family of basic proteins such a phagocytin and leukin which have antimicrobial activity. Some of these act as detergent-like agents at the cell membranes by forming ion-permeable channels. Most active at pH 7.0 before acidification. Active against diverse organisms
Nitric Oxide (.N=O) and Host Defense (5). Synthesis and secretion by what cell type, porduction, major cytoxic effect against what, mechanism of action, evasion of streptomyces.
a. Synthesis and secretion of nitrogen oxides carried out by activated macrophages induced by LPS and IFN-γ. Unstable free radicals involved in prevention of platelet aggregation, and defense against parasites, tumor cells and intracellular bacteria.
b. Produced through the action of inducible NO synthetase. Converts L-arginine to citrulline + nitric oxide.
c. Major cytotoxic effector against tumor cells, parasitic fungi, protozoa, helminths, mycobacteria bacteria. May also be mediator of autoimmunity through destruction of healthy tissue.
d. Mechanism of action: inhibition of Krebs cycle, electron transport, DNA synthesis.
e. Evasion: streptomyces make N-iminoethyl -L- ornithine which is an irreversible inhibitor of NO synthase in macrophages.
Host damaged by release of ROIs, NO, lysomal enzymes. Specifically how can host be damaged by neutrophils?
Neutrophil activation can lead to absess, which is liquified tissue, neutrophils, cell debris - “pus”
Do NK cells rearrange antigen-specific receptors?
Unlike T and B cells, they don’t
Provide a description of the origin of NK cells and what they are capable of killing.
Cells of the lymphoid lineage capable of recognizing and killing certain virus infected cells, tumor cells, some normal cells and certain bacteria, fungi, parasites.
Note: Belong to the innate immune system in that they are relatively non-specific and don’t exhibit classical memory, but recent study show a modified type of memory.
What cytokines do NK cells produce?
NK in addition to killing, produce IFN-γ, GM-CSF, TNF as well as other cytokines. IFN-γ, for example activates macrophage killing.
Describe dependent cellular cytotoxicity (ADCC) with NK cells.
NK cells have receptors for the Fc region of IgG antibody (FcR) allowing them to bind Ab and mediate antibody - dependent cellular cytotoxicity (ADCC). This FCR receptor is called “CD16”.
Explain “Missing self” paradigm of NK cells.
NK cells recognize lack of MHC Class I molecules. Target cells that have MHC I engage killer inhibitory receptors (KIR) and negatively signal NK —> No kill. Killing results from engagement of killer activating receptors (KAR) and failure to negatively signal through KIR.
The lysis that occurs via NK cells is induced by ?
perforins, proteases; very similar to lysis by CTL (cytotoxic T cell)
discuss interferons
Released from several cell types in response to virus infection, DS RNA, endotoxin,mitogenic and antigenic stimuli. Can augment immune response (NK, B, macrophage); lead to an antiviral effect.
- Type I IFN: IFN-α, β —> signal through the IFNAR. Made by leukocytes, dendritic cells, monocytes, fibroblasts. IFN-γ —-> signals through IFNGR. Made primarily by CD4+ T cells, NK cells. Type III IFN: IFN-λ; active at mucosal surfaces, in pDC
- IFNs act as links between innate, adaptive response. IFN production by a specialized dendritic cell subtype (the plasmacytoid dendritic cell, PDC) primes for development of Th1 response.
Tumor necrosis factor-α is produced by what cells and what role does it have?
Produced mostly by activated macrophages but also NK cells. Roles in endotoxic shock, inflammation, immunoregulation, antiviral responses, fever.
IL-6 - discuss is produced by what cells and what role does it have?
Produced by macrophages, T cells, endothelial cells causes increases in bone marrow production of neutrophils and stimulates production of fibrinogen, CRP (acute phase proteins), induces fever.
IL-1β is produced when and define it.
Produced upon activation of the inflammasome. Along with TNF-α and IL-6, it is an “endogenous” pyogen or fever-inducing agent through the activation of prostaglandin E2. Induces acute phase proteins, mobilizes neutrophils. [In fact, TNF, IL-1 and IL-6 have a lot of overlapping functions, demonstrating the concept of cytokine pleiotropy and redundancy.
IL-12 - discuss is produced by what cells and what role does it have?
Produced by monocytes and other antigen presenting cells, particularly the type I dendritic cell populations. Stimulates cytotoxic cell activity and proliferation of T cells and NK cells. One of the major links between innate and adaptive immunity, leading to development of TH1 responses and cell mediated immunity.
Discuss what the complement system is and besides mediating a killing with an antibody how else can it come into play?
- The Complement system is an important collection of serum proteins that work in concert with other elements of the immune system to initiate effector mechanisms that result in the destruction of extracellular infectious organisms.
- the complement system can also act in the absence of specific antibodies based on binding of complement proteins to certain surface molecular patterns expressed by pathogens.
Complement plasma proteins are normally found in an active or inactive form?
in an inactive form, in the absence of infection. In the presence of a pathogen, these proteins are “activated” in a sequential manner to non-specifically complement the action of antibodies in both opsonization and lysis of red blood cells and bacteria.
Along with complement proteins being activated what is released and describe its effect?
In the process of being activated, many of the complement proteins release low molecular weight peptides (split products) which themselves have powerful activating effects on inflammatory cells.
What’s is the primary in vivo source of complement components, except for one? Oddly in vitro what can make most of the complement components?
Hepatic parenchyma cells.
- C1 ; in the epithelium of the gastrointestinal and urogenital tracts.
- monocytes or macrophages
What are the three pathways of complement activation?
Complement system performs four vital functions
- classical, alternative, lectin
- Opsonization and phagocytosis
- Stimulation of inflammatory reactions
- Complement-mediated cytolysis (osmotic lysis)
- Clearance of immune complexes
Complement system has a sequence of two events for the activation and one event to inhibit the system from self.
- Activation of complement involves the sequential proteolysis of proteins to generate enzymes with proteolytic activity.
- The products of complement activation become covalently attached to microbial cell surfaces or to antibodies bound to microbes and to other antigens.
- Complement activation is inhibited by regulatory proteins that are present on normal host cells and absent from microbes.
Discuss the nomenclature of Complement proteins. Which letter suffix gen. is the larger complement protein? And discuss the two exceptions to the nomenclature
A. The first proteins discovered belong to the classical pathway and are designated by the letter C followed by a number.
B. Products of cleavage reactions are designated by adding a lowercase letter as a suffix. Cleavage of C3 produces a small protein fragment called C3a and a larger fragment C3b. In general, the smaller cleavage fragment is designated by the suffix a, and the larger fragment by the suffix b.
C. Some exceptions, C2, larger fragment is C2a. C1q, C1r and C1s are not cleavage products of C1 but are distinct proteins that comprise C1.
D. Components of the Alternative Pathway are called “factors” and are distinguished by capital letters (e.g., Factor B; Factor D). Cleavage fragments are designated by adding a lowercase letter as a suffix.
Define a ficollin and lectin.
Is an oligomeric lectin with collagen and fibrinogen subunits. Lectin are carbohydrate binding proteins.
Discuss all three complement system cascades.
A. Lectin pathway – initiated by soluble carbohydrate-binding proteins – mannose binding lectin and the ficolins that bind to carbohydrates on microbial surfaces.
B. Classical pathway – initiated when the complement component C1 either recognizes a microbial surface directly or binds to antibodies bound to a pathogen.
C. Alternative pathway – initiated by spontaneous hydrolysis and activation of complement component C3 which can bind directly to microbial surfaces.
All three complement system during activation converge at ___?
most important step in complement activation – the generation of C3 convertase.
What’s a safegaurd against the activation of complement system?
One regulatory safeguard is that the key activated complement components are rapidly inactivated unless they bind to the pathogen surface on which their activation was initiated.
In the lectin pathway what are the four different pattern recognition receptors?
1 lectin and 3 ficollins
The lectin pathway can be triggered by any of the four different pattern recognition receptors circulating in the blood and extracellular fluids that recognize carbohydrates on microbial surfaces.
Discuss the cascade once MBL complexes with MASP-1 and MAPS-2. What is the C3 convertase of this pathway?
Mannose binding lectin complexes with Mannose assoc. serine protease 1/2. MBL binding to a pathogen surface induces a conformational change in MASP-2 which can then cleave complement components C4 and C2. Cleavage of C4 releases C4a and C4b, whereas C2 is cleaved producing C2a, an active serine protease which binds C4b, forming C4b2a. C4b2a is the C3 convertase of the lectin pathway.
Deficient in MBL or MASP-2 are prone to have more?
have more respiratory infections by common extracellular bacteria during early childhood.
Discuss the structure of MBL, it’s nickname, what it binds to really well (spec. microbes), and it’s levels in the blood before and during an infection.
Note: Surfactant proteins A and D (SP-A and SP-D) are collectins that are found in the fluid that bathes the epithelial surfaces of the lung.
Mannose-binding lectin (MBL) is an oligomeric protein built up from a monomer that contains an amino-terminal collagen-like domain and a carboxy-terminal C-type lectin domain. Collectins is another name for these proteins. MBL in the blood is composed of 2-6 trimers that bind with high avidity to repetitive carbohydrate structures on a variety of microbial surfaces, such as Gram-positive and Gram-negative bacteria, mycobacteria, yeasts and some viruses and parasites. MBL is present in low concentrations in the plasma but in the presence of an infection its production is increased.
Discuss the structure of ficollins and its specificity containing _____ sugars.
They contain a fibrinogen-like domain attached to the collagen-like stalk. Ficollins have specificity for oligosaccharides containing acetylated sugars.
Classical pathway of complement activation. What is used as the pathogen sensor and discuss the cascade.
This pathway uses a pathogen sensor known as the C1 complex. The C1 complex is composed of the C1q subunit, which acts as the pathogen sensor and two serine proteases C1r and C1s. C1r and C1s are closely related to MASP-2. C1q interacts with a ligand, which leads to activation of C1r that cleaves C1s to generate an active serine protease. Activated C1s cleaves C4 to produce C4b which binds to the pathogen surface. C4b and cleaves C2 to produce C2a. C4b binds to C2a that produces the active C3 convertase C4bC2a.
How were the components of the Classical C pathway numbered?
The components of the Classical C pathway are numbered C1-C9, and were so named in order of their discovery rather than their activation sequence (C1, 4, 2, 3, 5, 6, 7, 8, 9) which became apparent later.
Classical C pathway : C1q can attach itself to the surface of pathogens in different ways (3)
- Can bind directly to surface components on some bacteria and to polyanionic structures such as lipoteichoic acid.
- Can bind to C-reactive protein, an acute phase protein in human plasma that bind phosphocholine residues in bacteria surface molecules.
- Can bind to the constant regions of antibodies (Fc region) that have bound pathogen via their antigen-binding sites.
Human — IgGl, IgG3 and IgM
Mice — IgG2a and IgM
Classical C pathway : What’s the quirk of the If Fc region and C1q molecule?
Each Ig Fc region has a single Clq-binding site, and each Clq molecule must bind to two Ig heavy chains to be activated. This requirement explains why antibodies bound to antigens and not free circulating antibodies can initiate classical pathway activation.
What are some key feature of the alternative pathway (3).
Key features of this pathway are
- its ability to be spontaneously activated
- its unique C3 convertase, the alternative pathway C3 convertase. The cleavage fragment of Factor B is Bb which binds to C3b to form C3bBb.
- Once C3b has been formed by any pathway, the alternative pathway can acts as an amplification loop to increase C3b production.
Discuss the cascade of the alternative pathway activation (2)
- by the action of either the lectin or classical pathway. C3b generated by either pathway and covalently linked to a microbial surface can bind factor B. This binding enables factor D to cleave it into Ba or Bb. Bb remains associate with C3b, forming the C3bBb convertase.
- The second way involves the spontaneous hydrolysis (“tickover”) of the thioester bond in C3 to form C3(H2O). C3(H2O) binds factor B, which is cleaved by factor D producing fluid-phase C3 convertase, C3(H2O)Bb.
Properdin-deficient patients are very susceptible to infections with ___? What is Properdin’s fxn with the alternative pathway C3 convertases?
Alternative pathway C3 convertases are very short-lived. They are stabilized by the plasma protein properdin (factor P), which binds to C3 and C3(H2O). Properdin is made by neutrophils.
Properdin-deficient patients are very susceptible to infections with Neisseria meningitides, the main agent of bacterial meningitis.
Discuss complement pathway following formation of C3 convertase.
A. C3b binds to C4b2a and C3bBb forming the active C5 convertases C4b2a3b and C3b2Bb.
B. C5 binds to the C3b component of the C5 converting enzyme.
C. C5 is cleaved by C2a or Bb to form C5a and C5b.
D. Main effect of complement activation is to deposit large quantities of C3 on the surface of the pathogen that signals the destruction of the pathogen.
What is the most important action of complement and how is this accomplished?
Most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytic cells.
This occurs by the specific recognition of bound complement components by complement receptors (CRs).
how does CRl, or CD35 function? How does C5a get an effect?
CRl, or CD35 functions mainly to promote phagocytosis of C3b- and C4b-coated particles and clearance of immune complexes from the circulation. Expressed on many types of immune cells including macrophages and neutrophils. C5a activates macrophages to ingest bacteria bound to their CR1 receptors.
Note: C5a receptor transducer their signals via intracellular guanine-nucleotide-binding proteins called G proteins.
How does CR2, or CD21, CR3 (CD11b/CD18), CR4 (CD11c/CD18), and CRIg (complement receptor of the immunoglobulin family) function?
They bind to inactivated forms of C3b that remain attached to the pathogen surface. Binding of iC3b to CR3 stimulates phagocytosis.
What (discuss) reaction is termed anaphylactic shock and the complement components ___ and ___ are referred to as anaphylotoxins.
C3a, like C5a signals through G-protein-coupled receptors. In large amounts or if injected systemically they produce a shock-like syndrome similar to a systemic allergic reaction involving IgE antibodies. This reaction is termed anaphylactic shock and the complement components C3a and C5a are referred to as anaphylotoxins. During this reaction, there is smooth muscle contraction, increased vascular permeability, upregulation of adhesion molecules and release of inflammatory molecules such as histamine and TNF-α.
What specifically is the end goal of Complement activation?
- One of the most important effects of complement activation is the assembly of the terminal complement components to form a membrane-attack complex.
- End result is a pore in the lipid bilayer membrane that destroys membrane integrity.
Discuss the cascade of the terminal end of complement activation.
C5 is cleaved by a C5 convertase to release C5b. One molecule of C5b binds to one molecule of C6 and the C5b6 complex binds to C7. This results in a conformational change allowing C7 to insert itself into the lipid bilayer. C8 binds to the C5b6 complex and inserts into the lipid bilayer. C8 molecule binding to the complex induces the polymerization of 10-16 molecules of C9. The 10-16 molecules form a pore in the membrane and constitute the membrane-attack complex.
What can happen if pt. is deficient in complement components C5-C9.
There’s an association with susceptibility only to Neisseria species, the bacteria that causes the sexually transmitted disease gonorrhea and a common form of bacterial meningitis.
Several mechanisms ensure that complement activation occurs where ?
that complement activation occurs only on the surface of a pathogen or on damaged host cells and not on normal host cells and tissues and to limit the duration of complement activation even on microbial cells and antigen-antibody complexes.
Extent of complement amplification is dependent on? And what may amplify complement activation?
Complement activation by any pathway and may amplified via the alternative pathway. Extent of amplification is dependent on the stability of C3 convertase C3bBb.
What controls the stability of C3 convertase C3bBb?
What protects host cells from complement damage.
Positive and negative regulatory proteins control the stability of C3 convertase C3bBb. Negative regulatory proteins protect host cells from complement damage. Complement regulatory proteins interact with C3b and either prevent the convertase from forming or promote its rapid dissociation.
What’s the action of properdin?
Properdin acts as a positive regulatory protein and stabilizes C3bBb.
What’s the action of Decay-accelerating factor (DAF or CD55)?
Decay-accelerating factor (DAF or CD55) competes with factor B for binding to C3b and can displace Bb from a convertase that has already formed.
Factor I and membrane cofactor of proteolysis (MCP or CD46) action is ?
C3b can be cleaved to an inactive form iC3b by a plasma protease, factor I and membrane cofactor of proteolysis (MCP or CD46).
What has has similar activities as DAF and MCP.
Cell-surface complement receptor type 1 (CR1)
What’s the action of Factor H?
Factor H binds C3b and competes with factor B to displace Bb from the convertase.
What’s the action of C1 inhibitor (C1INH) and what happens if deficient in it?
- C1 inhibitor (C1INH) is a plasma serine protease inhibitor that binds the active enzymes C1r:C1s and causes dissociation from C1q. Limits the time during which active C1s is able to cleave C4 and C2.
- C1INH deficiency disease – hereditary angioedema (HAE) causes extensive swelling in the larynx and can lead to suffocation.
What’s the action of Factor 1, when is it only active in the presence of what, and deficiency can lead to?
Cell-associated C3b (and C4b) are proteolytically degraded by a plasma serine protease called Factor I, which is active only in the presence of regulatory proteins, such as CR1 and Membrane Cofactor Protein (MCP). Factor I deficiency – uncontrolled complement activation resulting in repeated bacterial infections, especially pyogenic bacteria.
What’s the action of CD59 and S-protein in terms of the formation of the MAC?
Formation of the MAC is inhibited by a membrane protein called protectin (CD59), which works by inhibiting addition of C9 molecules; and by plasma proteins, such as S-protein, that binds to soluble C5b, 6, 7 complexes and hereby prevents their insertion into the cell membrane.
The complement system promotes (3)?
promote phagocytosis of microbes on which complement is activated, stimulate inflammation, and induce the lysis of these microbes.
Discuss opsonization and phagocytosis of microbes via complement activation.
Opsonization and Phagocytosis: Microbes on which complement is activated by the alternative or classical pathway become coated with C3b, iC3b, or C4b and are phagocytosed by the binding of these proteins to specific receptors on macrophages and neutrophils.
Discuss complement: Stimulation of Inflammatory Responses
Stimulation of Inflammatory Responses: The proteolytic complement fragments C5a and C3a, and C4a are known as anaphylatoxins because they induce acute inflammation (C5a > C3a > C4a). Myeloid cells are attracted to areas where these C split products are released, and increased adhesion and activation of neutrophils, and mast cell degranulation also occurs. Such reactions form the basis for the pathology that is observed in some of the hypersensitivity states associated with many diseases.
Discuss complement-mediated cytolysis.
Complement-Mediated Cytolysis: Complement-mediated lysis of foreign organisms is mediated by the MAC.
Discuss complement system: solubilization of immune complexes.
Solubilization of immune complexes: During an antibody response, antigen-antibody (i.e., immune) complexes are formed. If these complexes accumulate in the blood, they may deposit in blood vessel walls, leading to inflammatory reactions that can damage surrounding tissue. Complement activation on Antibody molecules can promote dissolution of immune complexes to prevent such damage from occurring.
The number of tests used to asses immune function can be used in two ways.
Detection or identification of antigens and specific antibodies are used for DIAGNOSIS OF INFECTION and quantitative assays are used to measure the SEVERITY OF INFECTION or HOST IMMUNE RESPONSE.
note: A large number of these tests are based on antigen-antibody interactions, while others measure cellular competence.
In analyzing the immune status what measurement mechanism is valuable for the detection and measurement of cells?
Cell flow cytometry.
When analyzing blood counts, it is important to analyze them as two counts.
percentages and absolute.
What is the objective of cell flow cytometry and how is it accomplished?
Flow cytometry enumerates different cell types. It measures characteristics of cells as they flow in a single file past a laser beam. Immunologists use flow cytometry to determine the types of markers and receptors on the surface of a cell. A fluorescently labeled antibody specific for a surface marker or intracellular protein is used to identify the cell.
Using flow cytometry how are unlabeled cells measured? Compare side vs. forward scatter (What they measure).
Unlabeled cells scatter the light as they pass through the laser in the nozzle of the flow cytometer. Information on the size and complexity (granularity) of a cell is obtained by the way the light is scattered.
side = complexity/granularity forward = size
Flow cytometry: How many labels can be measured on a scattergram? How many different dyes can be analyzed
- 2 labels ==> scattergram
- 6 to 8 different dyes
detecting a decrease in CD4+ T cells and abnormal CD4/CD8 ratios can be done how?
flow results on patients via flow cytometry
Name 4 of the 7 applications of flow cytometry
quantitative analysis of lymphocyte subsets, using surface markers CD antigens
• immunophenotyping of leukemia’s and lymphomas
• defines early stages in the development and lineage of T AND B Lymphocytes
• monitoring of HIV infected and immunocompromised patients
• DNA analysis (ploidy)
• Intracellular flow
• Fluorescence-activated cell sorting (FACS) for isolating different cell populations
reaction that becomes visible as precipitate marks a(n) __?
immune complex
What ratio is required for an antigen-antibody reaction?
Almost equivalent concentrations of antigen and antibody
in a precipitin reaction the antigen is present as soluble or insoluble?
solube
when observing an antigen-antibody reaction what causes agglutination? How is hemagglutination particular?
- particulate antibody coated on surface of latex beads or cells.
- uses RBC as an endpoint of detection
What’s more sensitive precipitin reaction or hemagglutination?
hemagglutination
Hemagglutination: what does it entail, application, and what is it used to measure?
entails clumping of beads/cells
more sensitive than precipitin reactions
Applications: blood typing, coombs direct antiglobulin test (for fetal Rh reactions)
measurement of Rheumatoid factor, C reactive protein, antibodies to Treponema pallidum (reagin)
Immunoassys (Radiolabeledimmunoassay and Enzymeimmunoassay) - Discuss its sensitivity, use of mono/poly clonal antibodies, applications.
Used currently in immunology
Much more sensitive – detect pg. levels
Uses mostly monoclonal antibodies
Can be used to detect antigen or antibody or cells that make these molecules
Applications: measure antibodies to HIV & Hepatitis and other viruses
ELISA - what types of enzymes are used and what’s the fxn of their reactions. Discuss the procedure of ELISA, the use of different labels, and ELISPOT
Label is an enzyme e.g. peroxidase or alkaline phosphatase which reacts with a substrate to generate a signal – signal is amplified – increases sensitivity
Immunoassay for antibody or antigen
Bind antigen to plate
Add first antibody (labeled – direct)
Add secondary antibody (with label - indirect)
Different labels – radioisotopes (RadioImmuno Assay – RIA)
ELISPOT – Elisa around cells – forms spot ; Can measure antibody secreting cell but more widely used now for the single cell detection of cytokine secretion.
Immunoblotting/ Western blotting : discuss the how to perform this, and application.
Electrophoretic separation of antigen (protein) by molecular weight
Transfer to a nitrocellulose membrane
Antibodies to specific proteins bind & are detected colorimetrically
Applications: antibodies to HIV
Tests for the measurement of Cell-mediated Immunity : measure what types of things? (4) And what’s an application of it?
Measure delayed type hypersensitivity responses
Antigens (recall) against which people are exposed and develop memory cells
Lymphocyte proliferation in response to specific antigens
Cytokine production by CD4+ T cells
-Applications: Quantiferon Gold test for TB; measurement of IFN-γ due to specific stimulation of T cells sensitized to TB
Lymphocyte proliferation for (immunosuppresed) transplant recipients
Serum electrophoresis : how does it work, what does it detect, and applications
Separation of serum proteins based on charge
Albumin most negatively charged, followed by α, β and γ globulins
Used to detect Ig spikes – myelomas
IgG, IgA myeloma proteins
IgM Waldenstrom’s macroglobulinaemia
Applications: Monoclonal gammopathies
Immunoelectrophoresis or Immunofixation uses antibodies to identify the type of immunoglobulin and the light chain utilized
Define MHC
The major histocompatibility complex (MHC) is a large genetic region that encodes class I and II molecules and other proteins. The MHC genes are extremely polymorphic - many alleles existing for each gene and polygenic - many different MHC genes exist (cluster of genes).
What’s the role of MHCs? CD8+ Tcyt cells and CD4+ Th cells recognize which MHCs types?
physiological role is to bind peptide antigens and present them for recognition by antigen-specific T lymphocytes. Peptide antigens associated with MHC class I molecules are recognized by CD8+ cytotoxic T cells, whereas class II-associated peptide antigens are recognized by CD4+ helper T cells.
