M&i Week 2

Front 1

Sepsis

Back 1

-heterogenous infectious syndrome that is mediated by uncontrolled inflammatory responses.
-"blood poisoning"
- Host responses triggered during sepsis initiated by recognition of pathogen-associated molecular patterns that trigger pro-inflammatory immune responses that are meant to clear the pathogen, as well as anti-inflammatory responses that result in immune suppression during the later stages of sepsis
-similar uncontrolled inflammatory state can occur following massive tissue injury, exposure to certain drugs, in absence of infectious agents

Front 2

Provide a definition of sepsis (progression)

Back 2

Classification scheme characterizing progressive stages of generalized inflammatory responses involved in sepsis:
1. Systematic inflammatory response syndrome (SIRS)
2. Sepsis
3. Severe sepsis
4. septic shock
5. multiorgan dysfunction syndrome (MODS)

Front 3

SIRS- systematic inflammatory response syndrome

Back 3

Defined as having 2 or more of following:
-fever
-elevated or low WBC (leukocytosis or leukopenia) [why low WBC? cells sticking to vasculature, through selectins. Chemokine will activate, integrins bind Ig receptors, stick to endothelium]
-elevated heart rate (tachycardia)
-elevated respiratory rate (tachypnea)
*SIRS can be infection-mediated or not

Front 4

Sepsis (as progressive stage)

Back 4

SIRS PLUS confirmed infection

Front 5

Severe sepsis

Back 5

Sepsis + low blood pressure (hypotension).
-this can be corrected by administration of fluids

Front 6

Septic shock

Back 6

Severe sepsis, persistent hypotension (aggressive fluid resuscitation not effective. Drugs to increase vascular tone required).

Front 7

Multiorgan dysfunction syndrome

Back 7

Septic shock, plus persistent hypotension leading to:
-evidence of clotting disorders or failure of renal, hepatic, cardiac, or cognitive function
-accumulation of products of metabolism that cannot be cleared (e.g. lactic acidosis)
*not enough oxygen to organs, failure

Front 8

What are the main organisms associated with septic shock

Back 8

-bacterial infections (most common cause)
--gram (-) sepsis-mediated by LPS, endotoxin- "endotoxic shock"
--gram (+) sepsis has increased due to increased incidence in pneumonia, use of intravascular devices
-viruses, fungi, parasites

Front 9

What are presumed sites of infection in patients with culture-positive severe sepsis

Back 9

Primary infections of chest, abdomen, genitourinary system, and bloodstream account for more than 80% of all cases of sepsis
--major location is lung
--But in 30% cases, cultures are negative because patients treated with antibiotics before hospital admission or because infectious agents not involved (sepsis-like syndrome can occur following tissue injury)

Front 10

Discuss the epidemiology of sepsis

Back 10

In US- 750,000 cases per year, about a third die
-young (<1 y/o) or old
-underlying diseases (for example neutropenia- fewer neutrophils, more susceptible to infection. Neutrophils don't produce that much cytokines so not as involved in proinflammatory septic response)
-surgery or instrumentation
-prior drug therapy (immunosupressants, corticosteroids)
-other: childbirth, abortion, trauma, burns, intestinal ulceration

Front 11

General sepsis pathogenesis

Back 11

Sepsis initiated by a variety of pathogen-associated molecular patterns that induce a cytokine storm and numerous other inflammatory mediators resulting in acute vascular injury and activation of multiple plasma protein cascades. During late stages of sepsis, anti-inflammatory cytokines and antagonists of pro-inflammatory cytokines gain upper hand, resulting in immune suppression and secondary infection

Front 12

Lipopolysaccharide (LPS) as a pathogen-associated molecular pattern (PAMP) that initiates systemic inflammatory response

Back 12

-LPS is integral part of outer membrane of gram-negative cell wall; made up of repeating O antigen subunits, core polysaccharide, and Lipid A.
- Lipid A is toxic
-LPS activates macrophages (as well as DC, B cells)
-binds with CD14, signals through TLR4 (toll-like receptor) to activate NF-kB- inducing inflammatory mediators

Front 13

Limulus amebocyte lysate assay

Back 13

LPS causes coagulation of amebocytes (blood cells) of horse shoe crabs. So blood of these crabs used as test for presence of LPS

Front 14

Other pathogen-associated molecular patterns (PAMPs)

Back 14

In addition to receptors for LPS, macrophages also express receptors like other TLRs, NLRs, RIG-I-like receptors.

These can bind ligands on surface, in endosomes, cytoplasm

Also recognize endogenous proteins, uric acids-sometimes ultra-activation, leading to SIRS

Front 15

Superantigen

Back 15

-produced by some Gram (+) bacteria like pathogenic streptococci, staphylococci (toxic shock syndrome)
-are EXOTOXINS

*SAg binds to T-cell receptor, MHC class II. This binds to antigen/peptide. The T cell receptor only recognizes certain peptides. SAg binds to outside, on entire molecule complex, crosslinks MHC class 2 molecules and Beta chain of T cell receptor. Binds to a good number of T-cells. 2-3% of all T-cells. 2% of t-cells will be activated! Huge. T-cells produce cytokines (IL-2, TNF-a)- activate antigen presenting cells (macrophages)- IL-6, 12 production. Downstream effect similar to what LPS induces. Massive cytokine response. Confuses immune system, pathogen can then do its thing!

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*Why is superantigen response not useful?

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*T-cells will only be useful if they know what respond to, but this APC doesn’t contain the pathogen (not infected cell)… superantigen spread through your body- activate T-cells and produce cytokines, not effective immune response against pathogen-misdirect

Front 17

DAMPs- damage-associated molecular patterns

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*Damage-associated
-Stress induced
-Present in environment
Ex. Gout- uric acid- will be damaging molecular pathways.
Ex. Necrosis- mitochondria damage- protein synthesis is in mitochondria, From n-formylmethionine! Peptides from mt- contain n-formylmethionine on terminus- act like bacteria! Dna also looks like bacteria dna. TLR-9- can recognize it
Ex. CpG-containing DNA- recognized by neutrophils- cause SIRS

Front 18

Release of sepsis mediators following LPS injection

Back 18

This is measuring cytokines.
*see waves of cytokine production. IL1, IL6 important for inflammatory response...on vascular system

C5a-mast cells, histamines, prostaglandins leukotrienes, recruitment of neutrophils

TNF-a: inject in mouse, septic shock!
*Shock doesn't have to be from sepsis

Front 19

Pro-inflammatory cytokine storm causing variety of effects on vascular endothelium?

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1. systemic increase in vascular diameter, leading to vasodilation and drop in bp; in part this is due to induction of NO in endothelial cells, which induces smooth muscle cell relaxation

2. increase in vascular permeability leading to vascular leak and loss of plasma volume

3. increase in expression of adhesion molecules, which bind to the surface of phagocytes (which also have increased expression of adhesion molecules) and now can enter tissues

Front 20

Effects of LPS on vasculature

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*effects on endothelial cells, junctions. Move away from each other. Edges looser, induce cell adhesion molecules. Platelets leak out much quicker. Liquid leaks out. Reduction in bp locally. Activation of coagulation system. Compartmentalize infection. In small capillaries...

*Happen throughout body. WBC sticking to tissue, cells. Clotting in many capillaries (systemic edema). Hypoproteinmia, disseminated intravascular coagulation (DIC)

Front 21

Inflammation-induced activation of plasma protein cascades

Back 21

1. Kinin-kallikrein cascade- endothelial injury will activate kallikrein to generate kinins such as bradykinin, which lowers blood pressure
2. complement cascade- microbes can activate complement cascade (LPS-alternative complement) resulting in production of C5a which has chemotactive activities and more importantly, causes mast cell activation and production of histamine (C5a is an anaphylatoxin) which induces further vasodilation
3. Coagulation cascade- endothelial damage results in binding of platelets to collagen, initiation of coagulation cascade through activation of Hageman factor (Factor XII), leading to activation of thrombin and formation of fibrin strands. results in systemic clot formation in small blood vessels- DISSEMINATED INTRAVASCULAR COAGULATION, a process that prevents blood flow and delivery of oxygen to viral organs thus leading to failure of multiple organs. consumption of clotting factors and platelets during this process also puts the patient at risk for bleeding in the skin and mucous membranes.
4. Fibrinolysis cascade- endothelial damage activates the function of tissue plasminogen activator thus promoting plasmin formation, fibrin degradation and clot resolution. During sepsis, this pathway is inhibited (via the coagulation and complement pathways) and therefore insufficient to suppress DIC.

Front 22

Release of sepsis mediators following LPS injection

Back 22

*Immune system has own way of self-regulation. Makes it come down even in sepsis- start producing immunosuppressant cytokines. Endogenous proteins that are meant to counteract TNF and IL-1.

But no clear mechanism to see what timepoint a patient is at

Front 23

The sepsis seesaw

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*Burst followed by immunesupressant (compensatory anti-inflammatory response syndrome)

-too much of response in first stage, then too little immune function in last stage: the viruses like EBV can come out again, sensitive to reinfection

Front 24

Clinical manifestations of sepsis

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1. non-specific syndromes: fevers, chills, fatigue, malaise, anxiety, confusion, increased heart rate, increased respiration

2. multi-organ dysfunction (*many due to vascular coagulation!): lungs, kidneys, liver, digestive tract, heart, skin, brain, systemic effects (lactic acidosis, edema, bleeding, stress hormones)

3. virus reactivation and secondary infection

4. death

Front 25

Why see blue-purple lesions?

Back 25

*Vascular coagulations-mediated by the dysfunction. Fever-skin is red, flushed. Later, once multiple organ dysfunction, try to compensate, pumping more blood. Direct oxygen to brain, lungs, central organs. Shut down oxygen to skin. Now skin can become white cold. (warm shock initially, then redirect oxygen from skin (shut down capillaries) to vital organs, suddenly cold). Fever overall but not at skin.

Clotting of local capillaries-look purple on skin.

Front 26

Sepsis treatment

Back 26

1. Immediate stabilization of the patient: protection of airway (intubation, mechanical ventilation), blood pressure and tissue perfusion (fluids, vasopressors), cardiac output, kidney function (dialysis)
2. Clearing of microorganisms from blood (antimicrobials-empirical)
3. Removal of the original focus of infection (drain exudate, remove foreign body/organ, debride or amputate gangrenous tissue)

Front 27

Sepsis treatment: other strategies with limited success

Back 27

1. inhibit LPS (anti-LPS)
2. anti-TNF, other cytokines
3. inhibit inflammation (NSAIDs, corticosteroids)
4. Inhibit coagulation (activated protein C)

Front 28

B lymphocytes

Back 28

-derived from pluripotent stem cells in the bone marrow
-undergo genetic program of DNA rearrangement that results in the generation of a wide variety of antigen-recognition molecules (receptors) on B cells
-initially expressed on SURFACE (BCR), then later secreted as antibodies by terminally differentiated B cells (plasma cells). Immunoglobulins are secreted or surface form. Each B cell ( a clone produces antibodies with a single specificity)

Front 29

What are the constant and variable regions of an antibody? (also miscellaneous information on antibodies)

Back 29

*Y-shaped
*Look at secreted version mostly
*Monoclonal antibody therapies, for diagnosis of many diseases using ELISAs, Western Blots
*Secreted form referred to as antibody or soluble immunoglobulin
*IgG most important functional antibody
*In this diagram-two identical binding sites

Front 30

Activation of B cell by T-Cell

Back 30

Most antibody response is T-cell mediated. CD4 T-cells - cytokines that help B-cells produce antibodies and what isotypes to start producing.

*2 signals (cell surface)-antigen with surface Ig receptor-signal transduction.
-second signal is from T-cell

Some B-cells are T-cell independent (pneumococcal polysaccharide). Ex. Digeorge- can still produce antibodies (some IgM, but not IgG and IgA)

Front 31

What are the heavy chain and light chain components?

Back 31

Two green chains-exactly the same, linked by disulfide bond. Heavy chains

Two yellow shorter chains- light chains

*Heavy chains will be different. Different light chains for different B-cells. Variable regions!

Front 32

Antibody isotypes and chains

Back 32

Humans have 4 different IgGs
IgAs all have same function
Heavy chain determines class, isotype
Humans typically use kappa for light chain, lambda is minority

Front 33

IgM and IgA can form polymers

Back 33

B-cells born with IgM on surface can have 10 different binding sites as shown in diagram.

IgA is dimeric, involved with mucosal immunity. But in the bloodstream it is a monomore

J-chain helps to generate pentamers, dimers

Front 34

Affinity vs Avidity

Back 34

Affinity has to do with one binding site
Avidity is the sum total of all binding sites

Front 35

General structure of antibodies

Back 35

-Antibodies part of immunoglobulin family. Domains of antibodies contain similar structures
-Ig domain:
Antiparallel beta pleated sheets, two attached by disulfide bond. Has a beta barrel structure. The fold is the immunoglobulin superfamily

Front 36

Antibody structure -cleavage into distinct fragments by Papain

Back 36

-This protease cleaves antibodies just above disulfide bonds that connect 2 heavy chains together, resulting in two Fab fragments (fragment antigen binding)-these components can bind with antigen, and Fc fragment (fragment crystallizable-can precipitate out of solution)- which mediates effector functions of the antibody meaning it elicits the immune response.

Front 37

The hinge region of antibodies

Back 37

-enhances its flexibility
-this hinge region links two Fab fragments and Fc portion.
-Other portions of antibody are also flexible (region between variable and constant domains)
-Allow antibodies to interact with multiple identical antigens that are spaced at variable distance on a surface.
-Most often the antibody is IgG. But can also be IgA, IgE, IgM

Front 38

Antigen

Back 38

any substance recognized by an antibody (protein, sugar, lipid, nucleic acid, small molecule, etc)

Front 39

Immunogen

Back 39

Antigen that can elicit an antibody response when injected into animals or humans in the context of inflammation (an adjuvant)

Front 40

Hapten

Back 40

Antigen that can be recognized by an antibody but cannot by itself elicit an antibody response (i.e., a hapten is an antigen but not an immunogen)

Front 41

Hypervariable domains

Back 41

Areas of increased variability on the variable domains (HV1, HV2, HV3) on each heavy chain or light chain region.

-These HV domains form the loops (total 6 regions)- each referred to as CDR region (complementary determining region) that bind to antigens

Front 42

Epitope

Back 42

portion of antigen that is bound by antibody. This epitope can be conformational, each antibody may recognize different epitopes on the same antigen

Front 43

Antiserum

Back 43

mixture of antibodies

Front 44

ELISA

Back 44

Enzyme-linked ImmunoSorbent Assay:
-Used to detect antigen-antibody reactions (attach antibody or antigen to fixed surface, detect colorimetrically)
-ELISA good screening tool for diagnosis of infection. Can be used to screen for HIV infection
-Low false-negative rate (negative test considered inconclusive), but false-positives possible (thus confirm with Western blot)

Front 45

Western Blot

Back 45

-Combines specific antibody-antigen interaction with procedure to separate HIV antigens, allowing identification of specific antibody reactions with various viral proteins present in viral particle
-Adds specificity, requirement for additional interpretation by clinical laboratory
-Viral proteins separated by SDS-PAGE (gel electrophoresis): negatively charged proteins pulled through, larger proteins move slower, giving size-separation differential
-Viral proteins transferred to nitrocellulose membrane, then cut into strips. THEN PATIENT SERUM is added, allow patient antibodies to bind to antigens on membrane
-Wash is done to remove unbound human antigens, then second antibody (anti-human immunoglobulin) with reporter enzyme (alkaline phosphatase)
-Substrate for enzyme is added, color reaction occurs. Color on strip corresponds to position of antigen-antibody reaction, seen as band of particular molecular weight

Front 46

B-cell signaling

Back 46

-Cross-linking of membrane Ig by antigen
-tyrosine phosphorylation events
-biochemical intermediates
-active enzymes
-transcription factors
*These events are required for the development of B cells in the bone marrow as well as for activation of mature B cells in response to antigens in peripheral lymphoid organs.

Front 47

X-linked agammaglobulinemia (XLA)

Back 47

-Genetic mutation in an X-linked kinase called Bruton's tyrosine kinase (Btk)
-lack B cells, suffer multiple bacteria and viral infections
-significant immunodeficency
-Treat with serum (antibodies) from other people
-Btk is a kniase that normally activates PLC-y

Front 48

Transcription factors activated by B-cell signalling

Back 48

NFAT
NF-kB
AP-1

Front 49

What are the first antibodies to be generated?

Back 49

-IgM and IgD
-IgM form pentamers, present in high concentrations in blood
-IgG, IgA, IgE generated in secondary immune response (have underwent affinity maturation)

Front 50

IgM distribution and function

Back 50

-form pentamers, present in high concentrations in the blood, lower concentrations in lymph, low concentrations in tissue
-Adept at activating the complement system

Front 51

IgG distribution and function

Back 51

-most important antibody class in blood and tissues, confers immunity to neonates because it can cross the placenta (half-life is 2-3 weeks, protects neonate for up to 6 months)
-Effective at activating complement and opsonizing pathogens for uptake by phagocytes
-Humans have 4 different IgG subclasses with specialized functions

Front 52

IgA distribution and function

Back 52

-main antibody found in secretions, including mucosal secretions in the lung, gut, saliva, and tear glands, and in breast milk
-Humans have 2 different IgA subtypes

Front 53

IgE distribution and function

Back 53

-Play key roles in allergic reactions, present in very low levels in blood and tissues, typically bound with receptors on mast cells found beneath the skin and mucosal surfaces, and, and around blood vessels in connective tissue
-also basophils in blood

Front 54

Effector functions of antibodies

Back 54

(1) neutralization of pathogens and toxins
(2) complement activation
(3) opsinization
(4) Induce ADCC in NK cells
(5) activate mast cells, basophils and eosinophils

Front 55

Antibody function
-neutralization of pathogens and toxins

Back 55

-Antibodies can neutralize the pathogen or toxin by blocking binding of toxin to cellular receptors
-this interruption can be mediated by IgG or IgA classes (or sometimes IgM)
-therapies being developed to elicit antibodies against snake venoms in species like horses, this immune sera can that be used passively to treat individuals exposed to toxins

Front 56

Antibody function
-complement activation

Back 56

-Activation of complement
-Occurs by binding of IgM or IgG to antigens on the surface of bacteria, eliciting conformational change in Fc portion, permitting binding of C1q of classical complement pathway
-Lead to activation of membrane attack complex, inflammation, opsonization of pathogens, clearance of immune complexes

Front 57

Antibody function
-Opsonization

Back 57

-IgG antibodies bound with antigens on pathogen surfaces can bind with Fc-gamma receptors on phagocytes
-engagement of these receptors leads to ingestion, destruction of pathogens

Front 58

Antibody function
-induce ADCC in NK cells

Back 58

-IgG binds pathogen-derived proteins on the surface of infected cells
-Become targets for recognition by Fc-gamma receptors on NK cells, trigger cell destruction
*remember this doesn't work when antibodies are attached to virus, bacteria. Since NK cells can only target own cells (like tumor cells or an autoimmune response)

Front 59

Antibody function
-Activate mast cells, basophils, eosinophils

Back 59

-IgE antibodies can interact with Fc-epsilon receptors on mast cells IN THE ABSENCE of antigen
-then engagement of antigen with IgE on mast cells leads to crosslinking of Fc-epsilon receptors that then trigger release of inflammatory mediators like histamine.
-Antigens triggering IgE include allergens, parasitic worms

Front 60

What hypothesis explains the level of receptor diversity?

Back 60

-somatic diversification theory called somatic recombination

Front 61

What determines the variable domain of immunoglobulin?

Back 61

-For light chain, the variable domain is encoded by a V (variable) gene segment and a shorter J (joining) segment.
-For heavy chain, there is a V, short diversity (D) segment, and J. Assembly involves two joining steps: first D to J then V to D-J.
-After rearrangement, genes transcribed and spliced
-V region genes encode areas of heavy and light chains that include CDR1, CDR2. Joints between V, D, and J elements encompass CDR3

Front 62

VDJ recombination

Back 62

-due to hypermutation

Front 63

How are DNA fragments rearranged in a highly ordered manner during VDJ recombination?

Back 63

-First, rearragement of one allele at the heavy chain occurs first, if this is successful, rearrangement at the light chain locus is initiated
-Guided by DNA sequences called recombination signal sequences (RSS) flanking individual gene sequences
--> recognition sequences with 12-nt spacers can only recombine with 23-nt spacers: 12/23 rule
--> Recombination process involves variety of proteins specialized for process or play ubiquitous roles in DNA modification or repair. RAG-1, -2 essential for VDJ recombination process

Front 64

Omenn syndrome

Back 64

-RAG genes partially disrupted, leading to leaky phenotype, involving the non-physiological expansion of small populations of T cells, leading to inflammatory reactions similar to those seen in graft-versus-host disease
-more severel genetic disruptions in RAG genes-no VDJ recombination, lack mature B, T cells completely- severe immune deficiency

Front 65

deoxynucleotidal transferase (TdT)

Back 65

-Enzyme does random addition of N-nucleotides
-dramatically increases antigen receptor diversity, but often (2/3 of times) results in generation of non-functional protein.
-VDJ rearrangement can be either productive or non-productive

Front 66

How can erroneous recombination lead to tumor formation?

Back 66

-Can lead to chromosomal translocations, which are present in a large number of B-cell (and sometimes T-cell) tumors.
-ex. Burkitt lymphoma

Front 67

Burkitt lymphoma

Back 67

translocation of immunoglobulin heavy or light chain promoter/enhancer to the myc oncogene, resulting in uncontrolled myc expression
-the development also involves Epstein-Barr virus (EBV) infection of B cells, together with immune suppression, most commonly associated with malaria

Front 68

What are the general mechanisms that generate antibody diversity?

Back 68

1. Combinatorial diversity- multiple different copies of distinct gene segments (V,D, J) that can be combined in random manner. In addition, heavy and light chains can pair in may different combinations
2. Junctional diversity- introduced at joints between gene segments as a result of removal and addition of nucleotides during recombination process

Front 69

Regulation of VDJ recombination and antigen receptor expression

Back 69

-Allelic exclusion is a process where productive rearrangement on one allele of heavy chain locus prevents rearrangement of other heavy chain allele. Then rearrangement is initiated at one allele of kappa light chain locus.
--if rearrangement fails on first heavy chain locus, initiates rearrangement on other allele, if fails again, immature B cell targeted for apoptosis.
-initially IgM and IgD isotypes produced, co-expressed on cell surface
--Co-expression of IgM and IgD is accomplished by differential RNA processing
-Processes are in place in bone marrow to weed out many self-reactive B cells, also to prevent activation of autoreactive B cells that escaped weeding out process

Front 70

Understand the molecular mechanisms employed by activated B cells to secrete antibodies

Back 70

-some activated B cells will differentiate into plasma cells that secrete large amounts of antibodies. Plasma cells migrate to bone marrow, or mucosal immune system.
-Following infection or vaccination, small numbers of antigen-specific B cells will continuously differentiate into plasma cells even after pathogen or immunizing antigens has been long cleared. This process is critical for the generation of neutralizing antibodies against pathogens, and the effectiveness of most vaccines.
-Switching of immunoglobulins from membrane to secreted form accomplished by differential RNA processing

Front 71

Affinity maturation

Back 71

-undergo point mutations within the variable region gene segments, called somatic hypermutation
-somatic hypermutation involves enzyme called activation-induced deaminase (AID)
-Mutations can result in decreased, similar, or increased affinity of antibody with cognate antigen
-B cells expressing surface Ig with increased affinity for antigen will bind more avidly with FOLLICULAR DENDRITIC CELLS in germinal centers, have proliferation advantage
-selective survival of B cells expressing antigen receptors with highest affinity for antigen
-Repeated immunization results in enrichment of mutations within CDRs
-Germinal center reaction causes antibody responses to improve over time

Front 72

Class-switch recombination

Back 72

-first antigen receptors expressed by B cells are IgM and IgD. First antibody secreted is always IgM
-Later in immune response, antibodies with identical antigen-specifity may be of IgG, IgA, or IgE.. this process is known as class or isotype switching
-EAch Ig heavy chain constant region gene fragment (except for Ig delta) is preceeded by a stretch of reptitive DNA, called switch region, that guides recombination process, termed class switch recombination.
-DNA recombination occurs between switch region upstream of Ig mu to another Ig gene.
-AID plays role in class switch recombination. Selection of switch region is guided by cytokines produced by other immune cells, like CD4-expressing antigen-experienced producing cytokines.
-antibody response can often become biased towards production of a particular antibody class (IgE in allergic reaction)

Front 73

[Essay Question] DiGeorge syndrome- what types of abnormalities do you expect to find?

Back 73

Bone marrow- normal. defect is at a step after development of T cell precursors in bone marrow

Thymus- is absent. genetic defect causes defective embryonic development of 3rd and 4th pharyngeal pouches. Thymus derived from 3rd pharyngeal pouch. In complete DiGeorge syndrome it would be hard to find any thymus tissue

Spleen-defective development of PALS. The T cell zone in the splenic white pulp. The B cell zone in white pulp will also contain few secondary lymphoid follicles containing germinal centers (because T cells also control B cell responses)

Lymph nodes- T cell zone in paracortex would be absent. B cell center in cortex will contain few secondary lymphoid follicles with germinal centers. Few plasma cells will be present in medulla

Blood flow cytometry- normal prevalence of B cells (CD3-negative, CD19-positive) and absent T cells (CD3-positive, CD-19 negative)

Serum Ig isotype levels: IgM will be suppressed although not absent largely because some B cells (especially B-1 B cells and marginal zone B cells) in patients with DiGeorge syndrome can still generate T cell-independent antibody responses such as those directed against pneumococcal polysaccharides. T cell-independent immune responses are largely of IgM class, because B cell class switch recombination in the absence of T cells is inefficient. Thus, IgG levels will be very low, IgA levels will likely be undetectable and IgE levels will be undetectable. Patients with DiGeorge syndrome are unable to generate T cell-dependent antibody responses, which are critical for the generation of antibody responses of the IgG, IgA, and IgE classes. Although IgG antibodies can cross the placenta from mother to fetus, the half-life is approximately 3 weeks, and will therefore be turned over by the time the child is 6 months of age. IgA antibodies, which are present in breast milk, may have provided some neonatal passive immunity to the child, but would not have entered the circulation.

Front 74

MHC class I structure

Back 74

-polymorphic heavy chain and an invariant light chain (b2m). The alpha chain is encoded by region in genome referred to as major histocompatibility complex (MHC), while beta chain is not.

-peptide binding region are formed by the two homologous segments of a1 and a2 domains.
-Ig-like domain is a3 and b2m, and a transmembrane and cytoplasmic region

-cleft allows for 8-9 residue peptide binding. the cleft is cloed

Front 75

MHC class II structure

Back 75

Two polymorphic chains called alpha and beta. Both encoded by MHC. a1 and b1 make up the peptide region, and a2 and b2 make up the Ig-like region.

The three-dimensional structure of the MHC class I and class II molecules are very similar

-cleft allows for 13-20 residue peptide binding, the cleft is open at both ends (longer)

Front 76

Antigen binding pockets of MHC

Back 76

-are areas of the MHC groove that interact with antigenic peptide. These bind with specific amino acid residues of peptide, called anchor residues. Based on these anchor residues, a peptide motif can be assigned for each MHC molecule

Front 77

Superantigen binding!

Back 77

-some bacteria like Staphylococcus and some viruses produce toxins called superantigens that can stimulate large number of T lymphocytes. They bind directly to MHC molecules without need for antigen processing. Bind to outer surface of specific MHC class II molecules and the Vb region of T cell receptor

-each superantigen can bind one or a few of the different Vb segments, irrespective of the associated Va chain. Can stimulate between 2-10% of all T cells. This induces massive production of cytokines and the functional inactivation of responding T cells. Cytokines are toxic, cause a general immunosuppression of host. Can cause toxic shock (TSST-1 antigen of Staphylococcus aureus)

*Superantigens DONT bind to antigen pocket. Bind outside and crosslinks with T-cell receptor

Front 78

Discovery of major histocompatibility complex (MHC)

Back 78

MHC genes and products were originally defined by studying genetic basis of skin graft rejection. Genes responsible for causing grafted tissue to be rejected

Differences between MHC genes of different strains were attributed to genetic polymorphisms

Humans exposed to tissues from other individuals often had antibodies reacting with blood leukocytes of donor. The antigens recognized by these alloantisera were named Human Leukocyte Antigens (HLA)

Front 79

MHC Genetic Organization

Back 79

-on chromosome 6
-Class I genes encoded by HLA-A, B, C loci
-Class II genes encoded by HLA-DP, -DQ, -DR loci
-Class III genes encode components of complement system, cytokines, heat shock proteins, antigen processing involved products
-inherited as block, haplotype

Front 80

Polymorphic

Back 80

-two different phenotypes exhibited, loci have differences

Front 81

Polygenic

Back 81

-multiple genes for same functions

Front 82

Polymorphism of MHC molecules

Back 82

genes encoding MHC class I a and MHC class II a and b chains are the most polymorphic of all known genes. These polymorphic residues are clustered in the peptide-binding groove, mainly in positions that make contact with the peptide. Thus different MHC alleles bind different sets of peptides.

Front 83

What contributes to diversity of MHC molecules expressed by an individual?

Back 83

-Individual heterozygous for particular allele can present a more diverse set of peptides to T cells
-MHC expression co-dominant, polymorphism
-MHC polygenic

Class I: as many as 6 molecules
Class II: as many as 15 molecules

Front 84

Inheritance of MHC molecules

Back 84

-as a block, haplotype

Front 85

Outline the cellular distribution of MHC molecules

Back 85

-Class I on all nucleated cells (except RBC)
-Class II only expressed by subset of hematopoietic cells, including B cells, monocytes/macrophages, dendritic cells, epithelial cells of thymus

* remember that thymic expression of class II is required for the intrathymic selection of CD4+ T cells

Front 86

Where do exogenous antigens come from?

Back 86

- derived from bacteria, viruses that are endocytosed and presented

Front 87

Where do endogenous antigens come from?

Back 87

- proteins synthesized in cytoplasm, that include virus infected the cell and own host material

Front 88

Endogenous and exogenous pathways of antigen processing and presentation

Back 88

Front 89

Functional implications of two pathways of antigen processing and presentation

Back 89

-The best way to deal with extracellular versions of microorganisms or toxins is to generate neutralizing antibodies (CLASS II)

-Once microorganisms enter cells (for all viruses and some bacteria), antibodies are not very effective (CLASS I)

-Many intracellular bacteria reside in endosomal compartments of monocytes and macrophages (CLASS II)

Front 90

2 pathways of antigen processing and presentation (BASIC)

Back 90

extracellular--> class II MHC--> CD4+ --> help B cells --> neutralizing antibodies

intracellular (cytoplasm) --> class I MHC --> CD8+ --> kill

Front 91

Steps in MHC Class I antigen presentation

Back 91

1. microbe or virus enters cell, genome transcribed and translated into protein
2. cytoplasmic viral protein degraded by proteasome.
3. Peptides transported to ER lumen by TAP transporter. (transporter associated with antigen processing)
4a. Calnexin, tapasin, calreticulin, ERp57 (attach disulfide bond from b2m subunit to alpha chains) are accessory proteins
4. peptide binds with newly synthesized class I molecules in ER.
5. class I/peptide exocytosed to cell surface, presented to CD8 T cells

*processing -- foreign antigens, self proteins both degraded and presented on cell surface. T cells in body do not recognize self antigens complexed with MHC because T cells learn to ignore self-antigens during selection in thymus.

* A subset of Dendritic cells can present exogenous antigens on class I molecules- cross-presentation- appears crucial for initiating CD8 T cell responses against many viruses and tumor cells

Front 92

Steps in MHC Class II antigen presentation

Back 92

1. antigens ENDOCYTOSED into cell. this can happen nonspecifically by dendritic cells, via pattern recognition, complement and Fc receptors on macrophages and via surface antibody on B cells

-Newly synthesized class II molecules in ER complexed with invariant Ii chain. This Ii contains signals to transport class II complex to correct intracellular compartment (MHC class II compartment -MIIC). This Ii has a region called CLIP that is embedded in antigen binding groove of class II avoiding peptide binding in ER and Golgi

2. endosomes with antigens fuse with lysosomes. antigens are degraded by lysosomal proteases (cathepsins) in acidic environment

3. The class II/Ii complex targeted to endocytic pathway, and endosomes fuse with these vesicles

4. invariant chain degraded by proteases until only CLIP there

5. DM, and MHC class II-related protein, binds with class II, catalyzes the release of CLIP, and loading of class II with high-affinity proteins.

6. MIIC fuses with plasma membrane, allowing peptide presentation to CD4+ T cells

*every protein present in endocytic compartments, including extracellular and cell surface molecules will be presented on class II

Front 93

T cell receptor molecular structure

Back 93

-resembles that of antibody molecule
-Differences in T cell receptors never secreted, have only a single antigen-binding domain, and recognize antigen in the context of self-MHC molecules. no somatic hypermutation (dangerous), only one isotypes

Front 94

T cell receptor chains interacts with CD3 and zeta

Back 94

-CD3 necessary for T cell receptor transduction of signals to cytoplasm of cell after engagement of T cell receptor with ligand
-TCR alpha and beta, CD3 chains, and zeta chains interact with each other via charged residues in membrane spanning regions
-cytoplasmic CD3 and zeta- signal with protein tyrosine kinases....

Front 95

CD4 and CD8 co-receptors contribute to T cell activation

Back 95

Two functions:
1. cell-cell adhesion between APC and T-cell, mediated by MHC class 1 --- CD8 and MHC class 2---- CD4
2. transduction of a signal to cytoplasm, and subsequently to nucleus

Front 96

Intracellular signal transduction events that result in T cell activation

Back 96

1. early signalling events that result in the activation of protein tyrosine kinases and adapter proteins. (ctyoplasmic portions of CD3 and zeta components of TCR contain ITAMs that are targets for intracellular protein tyrosine kinases (PTKs) that catalyze phosphorylation of tyrosine residues in various protein substrates. Lck and CD4/8 comes in proximity with ITAMs, then ITAMS are phosphorylated by Lck and ZAP70 phosphorylated. ZAP 70 activates multiple signalling pathways)

2. Activation of biochemical intermediates (secondary messengers) of signaling (PLC-gamma--> calcium release, MAPK pathway)

3. Activation of cellular enzymes (Ca2+ activates PKC, induces MAP kinases, SAP kinases, JNK )

4. Activation of transcription factors..NFAT, NF-kB, AP-1 for T cell activation

Front 97

Transplant drugs Cyclosporin

Back 97

-used to prevent allograft rejection.
-inhibits calcineurin, preventing translocation of NFAT to nucleus

Front 98

self-MHC restriction

Back 98

T cells from one individual only recognize foreign antigens when they are presented by MHC molecules from that individual, but not someone else's MHC molecules.

*My T cells have own MHC. Another person's T cells have other MHC

Front 99

T cell development, selection in thymus

Back 99

Progenitor cells derived from stem cells in bone marrow enter thymus, develop into mature T lymphocytes.
-during maturation, T cells rearrange their T cell receptor genes to produce functional T cell receptor complex, and undergo process of positive and negative cellular selection

Front 100

Importance of thymus in T cell development

Back 100

Immature T cells enter thymus in subcapsular region
-do not express TCR, CD3, CD4, CD8
-Immature thymocytes referred to as double negatives
-rearrangement of TCR genes, TCR complexes with CD3 at cell surface
-Express both CD4 and CD8 (double positives)
-Subsequently double positive thymocytes differentiate into single positive thymocytes, allowed to leave thymus

Front 101

Generation of T cell receptor diversity

Back 101

-Immature T lymphocytes rearrange their T cell receptor genes during their development in thymus
-T cell receptor locus has V, J, C region genes, beta chain locus also contains diversity D gene segments. The VDJ segments encode the variable domain of the TCR

-Rearrangement of the TCR chain genes similar to that of Ig genes and involves the products of RAG genes. B locus rearranges prior to the alpha locus. Productive rearrangement of the beta chain locus inhibits rearrangement of the corresponding allelic locus on the other chromosome by allelic exclusion!

-T cells express only 1 type of T cell receptor at the surface. Functional rearrangement at the TCR B locus induces rearrangement at the alpha locus

Front 102

Sources of T cell receptor diversity

Back 102

1. combinatorial diversity- many V, D, and J gene segments can be combined in different ways. Different alpha chains can be combined with different beta chains

2. Junctional diversity- random addition (N sequences) and deletion of nucleotides

*Diversity of T cell receptors is even larger than that of immunoglobulins.

TCR genes do not undergo somatic mutation, no affinity maturation

Front 103

T cell repertoire selection: positive and negative selection

Back 103

Immature T lymphocytes undergo a cellular selection process that eliminates useless and harmful T cells
1. immature T cell with TCR with very low affinity for any MHC will undergo DEATH BY NEGLECT because it does not receive survival signal. Useless T cell
2. immature T cell with TCR with intermediate affinity will receive survival signal, in positive selection process....T cells with MHC molecules on thymic epithelial cells. Becomes CD8 or CD4 depending on what affinity it has for class I or class II molecule
3. immature T cell with TCR with strong affinity goes through negative selection (death signal). Autoreactive T cells are eliminated

Front 104

bare lymphocyte syndrome

Back 104

type II- individuals have genetic mutation in TF that control MHC class II expression.... So lack CD4 T cells due to defective positive selection

Front 105

autoimmune polyendocrine syndrome (APS-1)

Back 105

deficient in AIRE gene-defects in negative selection.. no negative selection, leads to autoimmunity

Front 106

5 phases of antigen-specific T cell response

Back 106

1. cognitive phase
2. activation phase
3. effector phase
4. decline phase
5. memory phase

Front 107

Naive T cells encounter antigen in lymphoid organs

Back 107

-naive t cells see antigen in lymph nodes, spleen
-pathogens trapped in lymph nodes, pathogens in bloodstream trapped in spleen
-T cells enter lymph nodes through high endothelial venules ... L-selectins home naive T cells to lymph nodes (CD34, GLYCAM-1).
-adhesion molecules (integrins, Ig superfamily) and chemokines required for entry across epithelial barrier

Front 108

Entry of lymphocytes from blood into lymphoid tissue

Back 108

Front 109

Naive T cells interact with what professional antigen presenting cells?

Back 109

1. B-cells
2. macrophages
3. dendritic cells (most important APC for initiation of T cell response)

Front 110

Interaction between naive T cells and professional APCs mediated by?

Back 110

-cell adhesion molecules
-allow transient binding, search MHC molecules of professional APCs for the presence of cognate peptide antigens

Front 111

Professional APCs have co-stimulatory activity

Back 111

-T cell activation requires "second signal"
-co-stimulatory signal is B7, interacting with CD28 expressed by T cells
-APCs need to receive "danger signals" to induce B7 expression.
-Avoid erroneous autoreactivity

Front 112

Activated T cells produce IL-2 and its receptor

Back 112

-Cytokine interleukin 2 (IL-2) and counterreceptor produced by professional APC activation of T-cells.
-Binding of IL-2 to its receptor induces signaling program, involving JAK/STAT signaling pathway, promoting T cell growth in autocrine fashion.

Front 113

Activated T cells change expression of cell surface molecules

Back 113

-change homing receptors. L-selectin expression lost, VLA-4 increased.
-VLA-4 interacts with VCAM-1 on vascular endothelium. VCAM-1 induced by proinflammatory cytokines produced by infected tissue. Activated T cells then enter infected tissue site
-CD40L also expressed on CD4-activated T cells. This stimulates macrophages and B cells.

Front 114

Know the prototypic glucocorticoid (hydrocortisone/cortisol) and 3 commonly used drugs in this class: prednisone, methylprednisone, and dexamethasone

Back 114

Adrenocorticosteroids- vital steroid hormones produced in adrenal cortex

--cortisol/hydrocortisone (glucocorticoid)- modulator of metabolic activity. can increase with stress. Are anti-inflammatory and immunosupressive at physiological concentrations
--aldosterone (mineralocorticoid)- modulates electrolyte balance.
--Synthetic glucocorticosteroid- chemical modification of cortisol that have altered potency, degree of mineralocorticoid effect, and halflife

*can't separate anti-inflammatory activity from metabolic processes (gluconeogensis, protein breakdown, permissive effects , fluid retention-mineralocorticoid, hypertension, polycythemia, decrease of WBC, increase in neutrophils)

Front 115

Anti-inflammatory role of glucocorticosteroids

Back 115

-supress immune moedulators
-elaboration of vasoactive and chemoattractive forces, cytokines are released

Front 116

Molecular mechanisms of glucocorticosteroid action

Back 116

-Bind to glucocorticosteroid receptors (GR) predominantly in nucleus in inactive form bound to other proteins
-Binding to GR activates GR, these GR bind glucocorticoid-responsive elements (GREs) which induce gene transcription.
-GR can bind and decrease nuclear transcription factor AP-1 (activator protein-1). Decrease production of AP-1 dependent inflammatory cytokines, other proteins
-Steroids decrease stability of mRNA.. COX-2, iNOS mRNA.

Front 117

Macro effects of corticosteroids

Back 117

-Depress cytokine production (IL-1, TNFa, IL-6, IL-2, 3, 4, 6, Gm-CSF, INF-gamma)
- decrease adhesion molecule expression
-decrease eicosanoid produciton
-decrease degranulation of PMNs, Eos, Basos
-Resdistribute leukocytes. Neutrophils demarginate into circulation. Lymphocytes leave circulation

Front 118

Bio-availability of corticosteroids

Back 118

oral: glucocorticosteroids well absorbed
IV: as esters to enhance water solubility, rapidly hydrolyzed in vivo
IM: prolonged effects obtained by suspensions
Topical: works at skin, not absorb systemically
Steroids protein bound (CBG, albumin)

Front 119

Recognize the relative glucocorticoid (GC) potency of the agents above (cortisol, prenisone, methylprenisone, dexamethasone)

Back 119

-Dexamethasone > 6a-methylprednisolone>PPrednisone>Cortisol

-6g-methylprednisolone most common IV
-Dexamethasone most potent but no sodium retaining effect

Front 120

Metabolism, Understand that 11-keto glucocorticoids must be bioactivated to work, thus are not topically active

Back 120

-Phase I metabolism in liver followed by glucuronidation (conjugation) and making more soluble
-Bioactivation is 11-keto to 11-OH. This occurs in liver. Prednisolone does not need activation.
-Double bond slows down metabolism, extends half-life, increases activity. Flourination enhances activity.

Front 121

CS: Immune uses

Back 121

-Low doses
-Moderate doses (asthma-take prednisolone)
-High doses (transplant)
-Variable responsiveness
-BUT increased rate of infection, masking of inflammatory processes, risk of peptic ulcer disease with NSAIDs, do risk-benefit analysis

Front 122

Cytotoxic immunosupressives

Back 122

-originally anticancer drugs
-prevents clonal expansion of B, T cells
-Are steroid sparing, calcineurin inhibitor sparing
--Acathioprine and 6-mercaptopurine
--mycophenolate mofetil
--methotrexate

Front 123

Azathioprine, 6-MP

Back 123

--purine analogs, prodrug. azathioprine cleaved to 6-MP
--6-MP further activated to become operative agent
--prevents clonal expansion of B and T cells
---inhibits de novo purine syntehsis
---interferes with salvage pathway
---false nucleotide (thio-IMP) inserted in DNA
--Toxic as myelosupression. pancreatitis, hepatitis, nausea, myalgia, rashes. malignancy?

Front 124

Understand the competing pathways for 6--MP and the effects of genetics. toxicity

Back 124

-low bioavailability
-people with TPMT deficency - extremely likely to have drug toxicity
-allopurinol use for gout (inhibits XO)-- needs dose reduction to avoid toxicity
--too much drug-WBC drugs

Front 125

mycophenolate mofetil

Back 125

--MPA potently inhibits inosine monophosphate dehydrogenase, critical enzyme for de novo purine synthesis
-selective for B, T lymphocytes because cells rely on this pathway rather than salvage pathway
-MMF suppresses lymphocyte proliferation, antibody formation by B cells
-inhibit leukocytes to inflammatory sites by depleting leukocyte guanine nucleotides, inhibits glycosylation of lymphocyte glycoproteins involved in adhesion to endothelial cells

-high bioavailability. used when AZA intolerant. more immediate response. Allows for dose reduction of CSA or tacrolimus. decreased supression of platelet, RBC. Possibly be used for RA, psoriasis, pancreatitis, hepatitis

-causes GI upset, used to be very expensive

Front 126

Methotrexate

Back 126

-Inhibits dihydrofolate reductase.
-inhibits folate-dependent enzymes
-similar in structure to tehtrahydrofolate
-->inhibits cell proliferation (WBC), cell-mediated immune reactions, cytokine production (LTB4, IL-1)

-used for psoriasis, RA.

-toxicity is decreased with folate, increased with renal dysfunction
-nausea, anorexia, stomatitis, diarrhea, fibrosis, pulmonary fibrosis, pneumonitis, myelosupression

Front 127

cyclophosphamide

Back 127

-alkylating agent for chemotherapy
-back up agent for transplant immunosupression
-treat vasculitic syndromes like Wegener's Granulomatosis, lupus
-toxicity: myelosupression, alopecia, etc

Front 128

cyclosporine and tacrolimus

Back 128

-potent immunosupressants, used in transplantation
-structurally dissimilar
-selective inhibitory effect on T lymphocytes.
--suppressing early cellular response to antigenic and regulatory stimulus
--decrease calcineurin function (cyclosporine binds to cyclophilin, tacrolimus binds to FK binding protein)
--affects Ca2+ dependent T cell activation
prevents dephosphorylation of NF-AT. thus preventing transcription of cytokines IL-2, TNFa, Gm-CSF, IFN-gamma

Front 129

cyclosporine and tacrolimus drug interactions, toxicity

Back 129

-cyclosporine- microemulsion form.
-tacrolimus used today, more potent but has side effects like hyperkalemia, hypertension, renal compromise

Front 130

mTOR inhibitors

Back 130

-Sirolimus (rapamycin) is macrolide.
-Everolimus approved for transplantation in 2012
-binds to mTOR TO INHIBIT THE SECOND PHASE OF T CELL ACTIVATION.. involving signal transduction, clonal proliferation of T-cells

-metabolized by CYP3A4, transported by P-glycoprotein
-binds to FK binding protein but does not affect calcineurin

-Issues: adjunct to calcineurin inhibitors, but increases their toxicity. inhibits wound healing. drug interactions. possible calcineurin sparing agent

Front 131

Overview of anti-inflammatory and immunosupressive agents

Back 131

Front 132

Therapeutic antibodies

Back 132

--immunoglobulin replacement
--antimicrobial antibodies
---HepA, HepB, CMV hyperimmune globulin
---anti-Rh, anti-lymphocyte, anti-TNF, anti-tumor antibodies
-->anti-lymphocyte is anti-CD3
--> anti-IL-2 receptor binds on activated T cells, block IL-2 and T-cell mediated activation effects
-->anti-TNF alpha antibodies

Front 133

How can some tumors be recognized and rejected by immune system?

Back 133

-Tumor immunity against chemically induced sarcomas
1. tumor resected, and transplanted in mouse of same strain (same MHC)- tumor will grow
2. but this same tumor resected is placed back into original host, tumor is rejected (no growth)
3. injection of killed cells from same tumor into a mouse induces protective immunity to that tumor, but injection of killed cells from unrelated tumor B does not.
4. immunity to transplanted tumor could be adoptively ttransferred to tumor-free animal by CD8+ cytotoxic T lymphocytes

Front 134

What is the nature of tumor antigens?

Back 134

1. mutated versions of normal cellular proteins that are unrelated to the malignant phenotype
2. mutated versions of normal cellular proteins that are related (oncogenes or tumor suppressor genes) to the malignant phenotype
3. products of genes that are normally expressed at low levels and to which immune responses are present but that are overexpressed (differentiation antigens) or aberrantly expressed (embryonic antigens)
4. proteins encoded by oncogenic viruses

Front 135

Different immune mechanisms directed against tumors

Back 135

1. cytotoxic T lymphocytes (CTL) - directed against MHC class I bound antigens expressed by tumors. CTLs are main effector cells that mediate the elimination of tumors
2. NK cells- kill tumor cells indiscriminately of their origin. NK cells most efficient at killing tumor cells that express low levels of MHC class I molecules, and can kill cells that are coated with IgG, via ADCC
3. antibodies- directed against tumor antigens
4. macrophages- phagocytose opsonized tumor cells, can kill tumor cells by production of TNF. cytokines produced by Th1 cells may recruit macrophages to the tumor

Front 136

How do tumors escape recognition by immune system?

Back 136

1. low immunogenicity: tumor cells often do not express adhesion molecules, MHC class I or class II molecules, tumor-specific antigens, or co-stimulatory molecules
2. Antigenic modulation: tumor cells may lose antigens that the immune system can recognize due to mutations. Antigen-loss variants may not be recognized by the immune system
3. immunosupression: tumors often secrete factors, like transforming growth factor B (TGF-B), which suppress immune responses

Front 137

Immunotherapy for cancer: non-specific enhancement of tumor immunity

Back 137

A. pro-inflammatory stimuli (BCG vaccine to treat bladder cancer, TLR7 agonist imiquimod to treat skin cancers)
B. recombinant cytokines- can non-specifically augment immune responses against tumors. A lot of side effects (toxicity, fever, vascular leakage, edema). IL-2 approved for treatment of advanced melanoma, renal cell carcinoma. IFN-alpha used in combination with chemotherapy for treatment of melanoma
C. blocking inhibitory pathways (CTLA-4, PD-1)

Front 138

Immunotherapy for cancer: antibody-based approaches

Back 138

-require availability of monoclonal antibodies directed against tumors. tumor-specific antibodies may elicit lysis of tumor cells by NK cells, complement fixation.

-can have inefficient killing, so use alternative strategies like TOXINS, magic bullets

Front 139

Immunotherapy for cancer: cell-based approach

Back 139

1. Lymphokine activated killer (LAK) cells- peripheral blood lymphocytes isolated, cultured in vitro with cytokine IL-2. NK cells proliferate, are activated. These LAK cells then re-introduced back in patient together with IL-2

2. Tumor infiltrating lymphocytes (TIL)- lymphoctes (mainly cytotoxic T cells) isolated from tissue derived from the tumor are cultured in vitro in the presence of tumor cells (which are necessary for presentation of the tumor antigen to the T cells) and IL-2. These TIL cells are then re-introduced into the patient together with IL-2.

Front 140

immunotherapy for cancer: approaches based on vaccination

Back 140

-anti-viral vaccines
-for therapeutic, only for common antigens molecular defined
-vaccinate cancer patients with killed tumor cells together with non-specific adjuvants (cytokines), augmenting immune response against tumor (largely unsuccessful, because tumor cells do not effectively activate specific CTL responses

recent approaches:
1. transfection of tumor cells with genes encoding co-stimulatory moecules like B7, or genes encoding cytokines. This can then stimulate naive T cells, elicity, cytotoxic T lymphocyte responses

2. Provenge- pulsing dendritic cells with prostate-derived antigens

Front 141

How does Provenge work?

Back 141

Front 142

Hypersensitivity: Type I

Back 142

mediated by IgE antibodies and mast cell activation (occurs within 2-30 min in sensitized individuals)

Front 143

Hypersensitivity: Type II

Back 143

mediated by IgG (or IgM) antibodies that bind with cells or matrix proteins (5-8 hrs in sensitized individuals)

Front 144

Hypersensitivity Type III

Back 144

mediated by IGG (orIgM) antibodies that form immune complexes (2-8 hrs in sensitized individuals)

Front 145

Hypersensitivity Type IV

Back 145

mediated by T cells (Th1 cells, Th2 cells or CTL) (24-72 hours in sensitized individuals)

Front 146

Hypersensitivity reactions and immunological diseases

Back 146

Front 147

What immunological mechanisms favor the production of IgE antibodies?

Back 147

-first exposure to antigen will "prime" the individual, will generate strong allergic reaction next time. Often the reaction becomes worse with each exposure
-IgE-mediated allergic reactions- first exposure leads to strong TH2 cell response with production with IL-4.
-IL-4 induces B cells to switch to IgE.
-IgE will bind to Fc-epsilon receptors expressed by mast cells, basophils.
-When antigen encountered later on, it will bind with IgE molecules at surface of mast cells, cross-linking of Fc receptors.
-Mast cell releases immune mediators
*Individuals with high IgE in circulation (atopic individuals) more susceptible to allergic diseases.

Front 148

What are effector mechanisms involved in generating allergic reactions?

Back 148

-Immediate and late response
-immediate reaction (histamine, leukotrienes released by mast cells).
-mediators recruit inflammatory cells (eosinophils, basophils)
-late phase response 8-12 hrs after exposure -long term pathology and morbidity
-Mast cells in high conc beneath epithelia of skin, resp, GI tracts, CT around blood vessels.
-Mast cells, basophils have Fc-epsilon receptors at surface, which can bind with IgE antibodies. Release granules when IgE crosslink-bind to antigen
-histamine causes local incr in blood flow, vascular permeability-fluid accumulation in surrounding tissue, influx of granulocytes. Leukotrienes extend this response. Other cytokines also contribute
-eosinophils can also be activated, degranulated at site of allergic reaction during late phase response

Front 149

What is the clinical presentation of IgE-mediated immune response?

Back 149

1. local administration of allergen in skin will lead to local wheal, flare reaction. This is also assay to test allergies
2. inhalation of allergens will lead to activation of mast cells beneath nasal mucous membrane (sneezing, runny nose, allergic rhinitis, hay fever) or activation of mast cells in lower airways, leading to bronchial constriction (allergic asthma)
3. ingestion of allergens leads to activation of mast cells associated with GI tract. Smooth muscle contraction-vomiting, diarrhea. Mast cells in skin also activated, binding ingested and absorbed allergens in blood, disseminated swelling under the skin occurs (urticaria or hives)
4. When allergen give systematically, mast cells associated with all blood vessels will be activated, leading to systemic anaphylaxis (bee sting)

Front 150

What are treatment strategies for allergic reactions???

Back 150

1. epinephrine (adrenaline) and related B-adrenergic agents: promote relaxation of constricted bronchial smooth muscle, stimulate heart (used for anaphylactic shock and asthma)
2. corticosteroids to suppress inflammation (used for asthma)
3. anti-histamines- bind with and function as antagonists of histamine receptors (allergic reactions besides asthma)
4. leukotriene receptor antagonists ("Singulair") inhibiting action of leukotrienes

Other ways:
1. desensitization "allergy shots"- small but increasing quantities of allergen administered subq over period of hrs, weeks, months. Specific IgE levels decrease, IgG levels rise (suppress Th2 and promote Th1 or induce Treg)
2. antibodies ("xolair") directed against IgE (to prevent asthma)

Front 151

What is normal physiological role of Ig-E mediated immune responses?

Back 151

-Mast cells are located near to body surfaces, allow recruitment of specific and non-specific effectors of immune system to site of infection
-In gut, mast cell degranulation leads to diarrhea or vomiting
-degranulation in lung leads to mucus secretion, bronchial contraction.
-these mechanisms are aimed at expulsion of infectious agents from hollow organs
-eosinophils through Fc-epsilon receptors, are capable of ADCC. Granules contain proteins toxic to microogranisms, parasites

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What are the two types of antibody-mediated disease?

Back 152

1. Specific antibodies (IgG or IgM) directed against antigens expressed by specific cells or tissues (Type II hypersensitivity). Bound antibodies activate complement system (MAC) and Fc-receptor bearing cells (macrophages, neutrophils, NK cells)
2. Immunological diseases mediated by specific antibodies (IgG or IgM) directed against soluble antigens (Type III). Complexes formed in circulation, can deposit in blood vessel walls virtually anywhere in the body. Complexes can activate complement, creating inflammatory response, systemic usually. Mast cell activation and histamine production are also common

Front 153

What are diseases caused by antibodies against fixed cell or tissue antigens (Type II reactions)?

Back 153

-occurs mainly in autoimmune diseases
-autoantibodies can be directed against cell surface receptors like hormone receptors, disease can be cause by aberrations in cellular physiology without inflammation or tissue injury
-or antibodies are produced against foreign antigens that cross-react with self proteins

Example 1: Graves' disease-autoimmune disease of thyroid-hyperthyroidism. autoantibody produced against thyroid stimulating hormone. leads to unregulated stimulation and excess thyroid hormone production

Example 2: Myasthenia gravis- autoimmune disease of muscle characterized by progressive muscle weakness, caused autoantibodies reactive with acetylcholine receptors at neuromuscular junctions. binding of antibodies interferes with transmission

Example 3: acute rheumatic fever- arthritis, endocarditis, lesions of heart valves, myocarditis, neuro problems. antibody against streptococcal cell wall protein that binds to cross-reactive antigen in cardiac muscle cells

Front 154

What are diseases caused by immune complex-mediated mechanisms? (Type III)

Back 154

Soluble immune complexes trigger activation of complement, activated components C4b, C3b bind covalently to complex. These are then cleared by binding of C4b and C3b to CR1 on surface of RBC. These are transported to liver, spleen where macrophages strip off complexes and degrade them. Can deposit in basement membrane of small blood vessels, renal glomerulus
Ex. 1: Serum sickness: large amounts of foreign antigens (serum, drugs) injected into blood of individual, systemic response develops. Immune horse serum for example, These complexes deposit in arteries, renal glomeruli, synovia of joints, clinical manifestations are fever, vasculitis, nephritis, arthritis.
Ex. 2: Post-streptococcal glomerulonephritis: kidney disease developing 1-3 weeks after streptococcal skin and throat infections. Due to glomerular deposits of immune complexes composed of streptococcal antigen and anti-streptococcal antibodies. binding of antigen to glomeruli first, followed by deposition of antibody

Front 155

What are the pathogenic mechanisms of T cells?

Back 155

1. delayed type hypertensivity (DTH) reactions caused by Th1 cells: Secretion of cytokines IFN-y and TNF-a inducing increase in permeability of local blood vessles, recruitment of T cells, phagocytes, local tissue destruction
2. chronic inflammation caused by Th2 cells: cytokines (IL-4, IL-5, IL-13) produced by Th2 cells can contribute to inflammatory responses
3. cytotoxic T lymphocytes: these directly lyse target cells bearing MHC class I plus specific peptide

Front 156

What are diseases caused by DTH (delayed type hypersensitivity)?

Back 156

Example: Tuberculin test

-Injection with purified protein derivative (PPD) in individuals that have been in contact with M. tuberculosis or vaccinated against TB with BCG. A local T cell-mediated immune response occurs leading to local swelling (Mantoux). Inflammatory Th1 T cells that produce cytokines, chemokines, cytokines.

Front 157

What are diseases caused by Th2 cells?

Back 157

-Chronic allergic ashtma.
-many of the chronic symptoms of allergic (atopic) asthma are due to chronic production of cytokines (IL-4, IL-13) by Th2 mcells, mast cells, eosinophils.

Front 158

What are diseases caused by cytotoxic T lymphocytes?

Back 158

Insulin-dependent diabetes mellitus (IDDM) or type 1 diabetes.

-destruction of insulin-producing B cells in pancreatic islets of Langerhans. Destruction caused by autoantigen-specific cytotoxic T lymphocytes. some Th1 cells play role as well

Front 159

Activated T cells differentiate into effector T cells with distinct functions

Back 159

-MHC class I-restricted CD8 T cells differentiate into cytotoxic T lymphocytes (CTL).
-MHC class II-restricted T cells differentiate into cytokine-producing helper T (Th) cells. Th cells can then further differentiate into T helper 1 (Th1) or Th2 cells.
-cytokine signal in T cell activation is sometimes referred to as "signal 3".
-IL-12 and IFN-gamma predominantly produced by activated macrophages and dendritic cells promotes Th1 cells.
-IL-4 predominantly produced by activated macrophages and dendritic cells, CD4+, , promotes Th1 differentiation.

-CD4 T cells can differentiate into Th17 cells (protective in some extracellular infections and pathogenic in autoimmune diseases) and regulatory T cells (Tregs suppress the function of all other effector T cells, Th3, Th9, Th22, or T follicular helper (Tfh) cells.

Front 160

What are the functions of the three types of armed effector T cells (effector phase)?

Back 160

1. cytotoxic T cells-recognize cytoplasmic pathogens (viruses, some bacteria) and tumor antigens
2. T helper 1 (Th1) cells focus their effector function on bacteria that grow in intracellular vesicles
3. T helper 2 (Th2) cells are most important for humoral immune responses against extracellular microorganisms and toxins, and in immune responses against parasitic worms

Front 161

CD8+ cytotoxic T lymphocytes

Back 161

Two distinct pathways:
1. perforin/granzyme pathway:
-contents of secretory granules released in space between CTL and APC. Contents of granules includes perforin, granzymes
2. perforin polymerizes, forms membrane pore through which enzymes (granzymes) can be delivered
3. target cells undergo osmotic swelling
4. granzymes that entered cell via perforin activate suicide program in target cell (apoptosis)

1. Fas/Fas ligand (FasL) pathway: activation of FasL on surface of CTL triggers itneraction with Fas expressed by target cell
2. this interaction transduces a signal to target cell to undergo apoptosis

Front 162

CD4+ Th1 cells and macrophage activation

Back 162

Th1 cells important as MHC class II+peptide/TCR interaction, interaction of CD40 (on macrophages) with CD40L on T cells. Th1 cells will produce cytokines, IFN-y and TNF. Further activation of macrophages. These activated macrophages can kill intracellular pathogens using lysosomal proteases, oxygen radicals, nitric oxide. Can also influence class switch recombination in B cells.