Week 2 Flashcards
o Defensins
• Highly concentrated in granules of PMNs and Paneth cells (found in small intestine)
• Production is regulated by pro-inflammatory cytokines
o Human beta defensin (HBD) 1-4 mainly expressed in various epithelial tissues; also expressed by monocytes, macrophages, and dendritic cells
• HBD4 limited to testes and epididymis epithelial cells
• Defensins are secreted (~5 g/day) and integrate themselves into membranes, dimerize and pull the membrane apart making holes (antimicrobial function)
o TLR
are transmembrane proteins that control innate immunity as well as AP differentiation; bind to and are activated by PAMPs and DAMPs which results in signaling cascades that lead to activation of AP-1, NF-κB, and interferon regulatory factors (IRFs).
o NF-κB
“the Mother of all immune system transcription factors” – signaling results in:
• Production of IFNs, pro-inflammatory cytokines (prostaglandins, leukotrienes, interleukins, other cytokines), and effector cytokines that direct the adaptive immune response
• Increased phagocytosis and synthesis of ROS and RNOS in macrophages and neutrophils
• Increased efficiency of antigen presentation
o TLR Signaling: three distinct pathways
) MyD88-dependent pathway (TLR: 1,2,4,5,6)→ production of inflammatory cytokines (IC)
• 2) TLR 7/8 (ssRNA) & TLR 9 (CpG DNA) → Production of IFNα
• 3) MyD88-independent pathway (TLR 3 &4)→ stimulates IFNβ, maturation of dendritic cells
o MyD88-dependent pathway is common to all TLRs (except TLR3). Upon activation by PAMPs or DAMPs, TLRs homo- or heterodimerize including the recruitment of adaptor proteins via cytoplasmic TIR domain
o Triggers of Inflammation
- Complement C5a stimulation of basophil and mast cell degranulation and activation
- Histamine, prostaglandin E2, leukotriene D2 and D4 all increase vascular permeability
- Macrophages release:
- TNFα – can cause fever, stimulates expression of E-selectin (diapedesis)
- IL-1 (endogenous pyrogen) – stimulates expression of E-selectin (diapedesis)
- IL-8 – chemotaxis
- NK cells release IFNγ which activates phagocytic cells
• Cells of acute inflammation
neutrophils, activated T helper cells
• Cells of chronic inflammation:
macrophages, cytotoxic T cells, B cells
• Important inflammatory cytokines:
TNFα, IL-1, and IFNγ
• TNFα
o Produced by macrophages and other mononuclear phagocytes
o Upon binding to TNFR it activates AP-1 and NFkB genes as well as activation of caspase 8 which is involved in apoptosis
o Additional functions: induction of other cytokines, regulates hematopoiesis, co-mitogen for T and B cells, causes induction of endothelial adhesion molecules used in diapedesis, has prothrombotic action, etc.
• Note, monocytes/macrophages express lots of CD15 (adhesion molecule) which increases its likelihood of completing diapedesis; binds to E-selectin
o Neutrophils
phagocytize bacteria and viruses when phagosome fuses with granules to create a phagolysosome
• Have Fc receptors to bind antibodies → able to kill by antibody-dependent cell cytotoxicity
• Cytokines used for synthesis:
• 1 & 2: IL-3 + GM-CSF
• 3: GM-CSF, G-CSF, M-CSF, IL-3
o Eosinophils
capable of phagocytosing and killing ingested microorganisms; activated by complement C5a and C3a to degranulate releasing major basic protein (MBP) which: • Is a potent toxin to helminth worms • Induces histamine release from mast cells • Activates neutrophils and platelets • Can provoke bronchospasm • Cytokines: • 1 & 2: IL-3 + GM-CSF • 3: GM-CSF, IL-3, IL-5
Basophils
have IgE on surface (express FcɛR1), release histamine when IgE is cross-linked by antigen; activated by complement C5a and C3a to release basophilic granules – mediators of delayed allergic response
• Cytokines:
• 1 & 2: IL-3 + GM-CSF
• 3: GM-CSF, IL-3, IL-4
o Mast Cells
found in tissues; release pro-inflammatory cytokines upon complement C5a and C3a activation; release histamine when surface IgE is cross-linked by antigen
o Monocytes
: form macrophages in peripheral tissues, act as first line of defense against microbe invasion
• Cytokines:
• 1 & 2: IL-3 + GM-CSF
• 3: GM-CSF, G-CSF, M-CSF, IL-3
o Macrophages
effector cells of chronic inflammation
• Functions: phagocytosis (highly activated by IFNs), present antigen to adaptive immune system, produce cytokines and lymphokines:
• IFNα (antiviral)
• IL-1β, IL-6, and TNF-α: mediators of fever
• CXCL8 (IL-8): chemotactic factor for PMNs, basophils, and T cells
• IL-12: activation of NK cells and CD4+ Th1 T cells
• Cytokines:
• 1 & 2: IL-3 + GM-CSF
• 3: GM-CSF, G-CSF, M-CSF, IL-3
• 4: M-CSF, GM-CSF
o NK Cells
- CD16 (FcγRIII) and CD56 (NCAM) are important markers of NK cells
- Recognize damaged cells by deficiency in MHC antigens
- Exposure to IFNs activates NK cell killing
- IL-12 and TNFα activate NK cells to secrete cytokines, primarily IFNγ
- Cytokines for Differentiation:
- 1: IL-3
- 2: SCF, IL-2
• Name the cells that form the bridge between innate & adaptive immunity.
o APCs:
• Dendritic cells: critical in uptake and presentation of antigen to T cells
• Macrophages: specialized for degradation and presentation of particulate antigens to T cells
• B cells: immunoglobulin functions as a receptor; antigen is internalized, degraded, and presented to T cells
o Congenital neutropenia:
Lack of GM-CSF; Frequent bacterial infections
o Chronic granulomatous disease
• Inability to produce H2O2 and HOCl; Inability to kill phagocytosed bacteria
o Leukocyte adhesion deficiency (LAD
- Lack of integrin subunit, the common β chain
- Inability to recruit innate immune cells to site of inflammation
- Increased susceptibility to bacterial, fungal, and viral infections.
o Complement defects
• ↑ susceptibility to bacterial infections; ↓ ability to remove immunocomplexes
o Chediak-Higashi Syndrome
- Defect in gene LYST (CHS1), a lysosomal trafficking gene that affects lysosomes and melanosomes
- Increased susceptibility to bacterial infection
o T cells
survey the surfaces of body’s cells looking for ones that have parasites within them or that are dangerously changed/mutated (cell-mediated immunity)
• Starting in lymphoid tissues, T helper cells recognize antigens (epitopes) with their surface receptors which bind antigens presented by dendritic cells
• T helper cell becomes activated, proliferates, and the daughters travel throughout the body until they reach the place where antigen has invaded. The T helper cell needs to come into contact with the antigen in order to elicit this response
• T helper cells can be re-stimulated by local APCs and release a family of short range mediators called lymphokines which attract and activate monocytes/macrophages that phagocytose and destruct pathogens, and eventually repair affected tissues
• Cytotoxic T cells (CTL) also examine surfaces of incoming dendritic cells for antigen presentation. In particular they are looking for MHC I surface molecules as this is expressed on all cells (except for RBCs)
• The clone of the CTL gets expanded and the daughters circulate in large numbers throughout the body. When a daughter cell binds to a foreign/abnormal epitope it delivers a ‘lethal hit’ signaling the cell to undergo apoptosis.
• CTL is now free to find more targets
CD4+ Helper T cells, Th1
recognize antigen and produce lymphokines that attract thousands of macrophages to area of infection to eliminate the microorganism
• This response can wipe out a serious infection… or a transplanted kidney
Type 2 Helper T cells, Th2
stimulate macrophages to become ‘alternatively activated.’ Th2 cells are able to function in walling-off pathogens and promote healing. These are very important in parasite immunity.
Th17 Helper T cells, Th17 –
similar role to Th1 cells (focus on inflammation) although Th17 cells are more powerful than Th1 cells and have been implicated in many serious forms of autoimmunity
Follicular Helper T cells, Tfh
stimulated by antigen and migrate from T cell areas of lymph nodes into the B cell follicles where they help B cells become activated to make IgM, IgG, IgE, and IgA antibody subclasses
Regulatory T cell, Treg
make cytokines that suppress the activation and function of Th1, Th2, and Th17 cells (immune homeostasis)
CD8+ Cytotoxic (Killer) Cells, CTL
destroy any body cell they identify as bearing a foreign or abnormal antigen on its surface; if a cell does not have an MHC I surface marker (as MHC I are expressed on all cells, except RBCs) the cell is destroyed
• Note, all T cells express CD3+ (part of TCR)
• All T helper cells (Th1, Th2, Th17, Tfh, Treg) express CD4+ in addition; CTLs express CD8+ in addition
• Differentiation of T cells:
Naïve T cell →
- IL-12, IFNγ → Th1
- IL-4 → Th2
- TGFβ, IL-6 → Th17
• Thymic selection of T cells:
Positive selection – T cells must recognize MHC I or MHC II molecules in order to be stimulated to mature (self-restricted)
Negative selection – T cells that recognize self-antigens bound to MHC II on thymic epithelial cells are driven to apoptosis (tolerant to self-antigens)
• Mature T cells that are self-MHC restricted and tolerant to self-antigens leave the thymus to settle in lymph nodes or the spleen
B cells
protect the extracellular spaces of the body (tissue fluids, blood, secretions) by releasing antibodies into these fluids (humoral immunity)
• B cells also recognize antigens via surface receptors, become activated, proliferate (with help from a Tfh cell), and arrange for phagocytosis and destruction of foreign materials
• Fully differentiated B cells, aka plasma cells, release/secrete soluble versions of their receptors (antibodies) which correspond to a specific antigen which may be enough to neutralize a toxin or prevent a microorganism from binding to its target cell
B cells always express CD79a and b (part of BCR)
o IgA:
• Most important antibody class
• Found in secretions such as saliva, tears, GU and intestinal fluids, and milk
• These secretions are associated with secretory component which the IgA acquires from epithelial cells during the process of secretion
Secretory component makes the IgA resistant to digestive enzymes and is the first line of defense against microorganisms trying to gain access to the body through mucous membranes
o IgD:
• Main form of antibody inserted into B cell membranes and acts as their antigen receptor when they are naïve B cells; only has biological role
o IgE:
- Designated to attach to mast cells and upon antigen binding results in production of prostaglandins, leukotrienes, and cytokines within the mast cell as well as release of powerful mediators of inflammation such as histamine from the mast cell
- These mediators produce allergy symptoms (hay fever – anaphylactic shock) depending on site of antigen entry and dose
- True role = parasite resistance
o IgG
• Most abundant antibody in the blood
• At least two adjacent IgG molecules binding an antigen cooperate to activate complement (system of proteins that enhances inflammation and pathogen destruction)
Note, complement is very important in disease resistance
• Some IgG’s can lyse a bacterium by making holes in its membrane. Others diffuse away from site and attract phagocytic cells (predominately PMNs)
• Useful in disposing many kinds of antigens
• Only antibody class that passes from the mother to fetus through the placenta; important for newborn acquired maternal immunity
o IgM:
- Large polymeric Ig that is better at activating complement than IgG
- First antibody to appear in the blood after exposure to new antigen; replaced by IgG in 1-2 weeks
o Antibody Function in Disease
- Antigen enters body through mucosal membranes and can penetrate to lymphoid tissues where B and T cells are located. IgA is produced; in some people IgE too.
- IgA is secreted and local immunity is established
- If antigen penetrates further into the body (reaches lymph nodes or spleen) IgM is produced (if environment favorable) and then IgG binds up pathogens as they circulate
- When most antigens enter the body, there will be both T and B cell responses. Some will be more important than others for that particular antigen
- Antibody is important for combating extracellular pathogens like staph, strep, and hemophilus; neutralizing toxins such as tetanus and blocking the spread of virus in the blood (once virus enters cells, CD8 killer cells are needed to rid the infection)
antigen and epitope
o Antigen = substance that when introduced into the body stimulates the production of an antibody
o Epitope = regions on an antigen that can be recognized by an antibody or by T cell receptors; aka antigenic determinants; usually 10-20 amino acids in length
o CD4+ Helper T cell Activation
requires both binding of antigen PLUS costimulation
• First Signal: antigen binding
T cell receptor recognition of MHC (HLA) bound antigen
CD4+ bind to MHC II; CD8+ bind to MHC I
• Second Signal: costimulation
B7-1 or B7-2 on APC binds to T cell surface protein CD28
ICAM on APC binds to LFA-1 on T cell
CD2 T cell surface protein binds to leukocyte functional antigen-3 (CD58)
Cytokine signals:
• General activators: IL-2 and IL-15
• Make Th1: IL-12 and IFN-γ; Down regulates Th1: IL-10, TGF-β
• Make Th2: IL-4; Down regulates Th2: TGF-β
o CD8+ Cytotoxic T cell activation
• T cells expressing FAS ligand bind to FAS (a protein on the target cell) which induces caspase activation and apoptosis
• T cells then secrete different toxic agents to kill cells:
TNFα → induces apoptosis
Perforin → pore-forming protein (similar to MAC)
Granzymes → induces apoptosis (activate capases)
o MHC Molecules
• Class I: antigens synthesized within the cell. Expressed on all cells except RBCs. Recognized by CD8+ Killer Cells
Note, lack of MHC I on RBCs may play a role in persistence of malarial parasite (plasmodium)
• Class II: antigens are products of phagocytosis. Expressed on monocytes/macrophages, dendritic cells, B cells, and epithelial cells of thymus. Recognized by CD4+ helper T cells
• Define the different types of hypersensitivity reactions.
o Type I: patients make too much IgE to an environmental antigen (often innocuous pollen or food)
• Pathogenesis: IgE bound to mast cells and basophils causes degranulation and results in release of histamine → synthesis of prostaglandins and leukotrienes. Recruitment of Th1 cells and basophils occurs in late phase.
• Epidemiology: > 10% of population have allergic symptoms
• Comorbidities: asthma, anaphylactic shock (due to sudden degranulation of mast cells throughout the body), bee sting, certain foods
o Type II: autoimmunity due to antibodies which can react against self
• Pathogenesis: many different mechanisms
If an antigen binds to certain cells in the body, the immune response may destroy those innocent bystander cells
If foreign antigen happens to look like self-molecule, the response to antigen may accidentally ‘cross-react’ with self
• Disease examples:
Hemolytic disease of newborns (maternal Ab to fetal blood antigens cross placenta and destroy fetal RBCs)
Myasthenia gravis (Ab to ACh receptors → nerve conduction problems)
Good pasture’s syndrome (Ab to basement membranes causes nephritis)
o Type III: antibody is made against soluble antigen
• Pathogenesis:
Antigen and antibodies are usually eaten by phagocytes (if too small they may be trapped in basement membrane of capillaries).The trapped complexes activate complement and the inflammatory response occurs (tissue is damaged as an innocent bystander). Symptoms manifest as: arthritis, glomerulonephritis, pleurisy, rash
Drugs (i.e. penicillin) and foreign serums (i.e. rattlesnake venom → serum sickness) can cause type III reactions in large doses
Most detrimental when antigen is internal as part of autoimmune process:
• SLE → antibody to their own DNA
• RA → make antibody to antibody
o Type IV: cell-mediated hypersensitivity caused by activated CD4+ helper T cells
• Can be autoimmune or innocent bystander injury
• Examples:
TB → lung destruction occurs due to T cell mediated degradation (not bacteria)
Acute viral hepatitis → liver destruction occurs due to CD8+ killer T cells
Contact hypersensitivity (to nickel) → dendritic Langerhans cells react recruiting CD4+ cells
Tuberculin reaction → macrophages react recruiting CD4+ and CD8+ cells
Granulomatous hypersensitivity → macrophages wall off mycobacterium and undergo changes to become epithelioid, recruit CD4+ cells
• Crohn’s disease = granulomas containing macrophages and CD4+ cells in ileum and colon
o Fc
constant region of an antibody (stem of the Y); composed of only heavy chain constant regions
o Valence
number of epitopes an antibody can theoretically bind
• i.e. IgA = 4, IgM = 10, IgG, D, E = 2; isolated VH or VL = 0; Fab = 1, F(ab’)2 = 2
o Allotype
inherited minor allelic differences in the sequence of immunoglobulins between individuals
• Allotypes are useful in genetics to determine relatedness
• Immunodeficient patients receiving immunoglobulin treatments will make Ab to someone else’s allotype
• Some people are more susceptible to infections than other people, possibly due to different allotypes
o Idiotype
a unique combining region within an antibody made up of the CDR amino acids in the H and L chains
• Anti-idiotypes = antibodies that recognize the unique sequence of the combining site
• In other words, it is correct to say that an idiotype is an antibody’s unique combining site considered as an antigen
IgA
- Have J chains which hold Ab together
- Secretory component protects IgA from proteolysis
- Secreted in blood as dimer → 4 binding sites
IgD
- Embedded into B cell surface as a BCR
- Binds with CD79a and b (aka Ig α and β)
IgE
- Ab that causes type I hypersensitivity reactions
- Gives resistance to worms and other parasites
IgG
- Binds bacteria; activates complement
- Main Ab in blood and tissue fluids
- Neutralizes toxins and blood-borne viruses
IgM
- Pentamer → 10 binding sites
- First Ab to appear in serum following immunization
- Very efficient at activating complement
- Does not go into tissue fluids, nor bound by phagocytic cells (size, J chain)
• Distinguish the immunoglobulin classes IgG, IgA, and IgM in terms of size and approximate concentration in serum.
o 10 subclasses/isotypes of antibodies
o Serum concentrations: IgG > IgA > IgM > IgD > IgE
o IgG, IgD, IgE = monomer, IgA = dimer, IgM = pentamer
• Describe the antigen-antibody interaction
o When an IgG or IgM antibody binds antigen with at least one of its binding sites, there may be a change in the angle between the two Fab parts, so that the molecule may be more Y or T shaped than before (reason for hinge)
o This causes bulging of Fc part so that one or two biological activities are initiated:
• 1) Binding to phagocytic cells (especially PMNs, eosinophils, macrophages) which have FcRs for the altered Fc of IgG (but not IgM), and
• 2) Cross-link two antigens, bind to phagocytic cell, or binding of C1q (first component of complement system) to two adjacent Fcs and becomes activated
Note, IgGs will bind close together on same (bacterial) surface; IgM can do it alone because it carries 5 Fcs at all times (reason why IgM is better at activating complement than IgG)
• How to make Heavy Chains:
Developing B cell first brings one random D segment close to one J, the DNA is cut and the intervening DNA is discarded and the ends are joined
Then the V segment is brought to the recombined DJ segment and the process of cutting and joining occurs again
• Note, there are splice acceptor and donor sites adjacent to each segment
The entire region from the assembled VDJ unit through the end of the delta (of IgD) constant region gene can be transcribed into RNA
These primary mRNA transcripts are alternatively processed using alternative polyA sites and splicing:
• First, only make VDJ-mu (stop at polyA site 2), later make both VDJ-mu and VDJ-delta
RAG recombinases 1 & 2 are the enzymes that carry out the recombination of antibody and T cell receptor DNA. They first bind to splice signals to the right of a D segment and the left of a J segment, pull them together, and then cut and splice. Then they look for a splice sequence to the right of the V segment and complete this process again
• Follow 12/23 rule – need to have 12 or 23 base pair spacer in between the different domains in order to be recombined
• Note, if RAGs are knocked out, neither B nor T cells can be made (Omenn Syndrome)
• How to make Light Chains:
Gene rearrangement is similar to the heavy chain method, but light chains have only V and J segments and only one C domain
C domains = kappa and lambda; kappa is the preferred C domain, but can have this gene be defective so lambda is used
RAG recombinases 1 & 2 are used in this process as well. See above for more details
• Production of V-D and D-J joints are ‘sloppy’ due to randomizing mechanisms:
1) Exonucleases chew away a few nucleotides after the DNA is cut but before two gene segments (D to J, V to DJ) are joined
2) The cell can add a few nucleotides thru the use of terminal deoxynucleotidyl transferase (TdT) which does not use a template so the additions are random
• Thus, the sequence at the joining area (called region ‘N’) cannot be predicted
These processes allow extensive random antibody diversity
However, sloppy recombination often results in a frame-shift mutation which can be a nonsense codon that results in transcription termination
• When this happens, the cell tries again with the other allele (remember, two alleles – 1 dad, 1 mom) → productive rearrangements
• If things work, the cell goes on to become a B cell
• If things don’t work, complete antibody cannot be made and the cell dies
Heavy chains are made first, then light chains are produced → usually kappa first, then lambda
• Describe the process of class switching.
o Mature naïve B cells initially express IgD and IgM on their surfaces (BCRs)
• The choice of IgD and IgM occurs at level of mRNA processing, thus a given B cell can express both IgD and IgM
o The Mature naïve B cell is activated upon antigen binding which causes it to divide and differentiate. They then switch from membrane-bound IgD and IgM to secretory IgM due to differential splicing.
• This switch occurs at the level of mRNA processing (polyA site 1 is chosen instead of 2)
o As the B cells continue to divide and differentiate, they may undergo additional class switching to produce IgG at the level of DNA rearrangements
o Summary:
• Thus, a single B cell starts by making both IgM and IgD, which it puts into its membrane as receptors, then later it may switch to making IgG, IgE, or IgA
• In all cases, the L chain and VH domain remains the same, but the C region of the H chain changes
o What happens is that the B cell (which has put its particular H-chain VDJ combination together with its mu and delta genes goes back to its DNA, does a loop-out of mu and delta, and then puts VDJ next to the C region gene of γ or ɛ or α which excises and discards intervening DNA
o The new mRNA then may be VDJα, VDJγ, or VDJɛ
o Thus a cell that is making IgM can go on to make IgG, but a cell making IgG cannot go back to making IgM as this part of the transcript is now excised and gone
o ‘M to G’, ‘M to A’, or ‘M to E’ switches are common in antibody responses and require T cell help (without the T cell, only IgM responses are possible as the T cells release cytokines to guide class switching)
o Cytokines involved in class switching, proliferation, and differentiation:
• Proliferation: IL-2, IL-4, IL-5
• Differentiation: IL-2, IL-4, IL-5, IFN-γ, and TGF-β
• Class switching = IFN-γ → IgG2a, IL-4 → IgG1,or IgE; IL-5 → IgE
o Somatic mutation
antibody diversity is generated by variation incorporated at the joining site for the various segments of the heavy and light chains; (exonuclease and TdT)
o Somatic hypermutation
= antibody diversity is generated via DNA recombination after the B cell divides following antigenic stimulation; there is a “good” change on the daughter B cells that will make a slightly different antibody; (AID, DNA polymerase)
• Selection of the best mutants after antigenic stimulation allows for a gradual increase of affinity during an immune response = affinity maturation
Note, T cells do NOT have somatic mutation following antigen contact
• How somatic hypermutation works:
Activation-Induced (Cytidine) Deaminase (AID) converts random cytosines in the CDR region to uracils.
Thus a C:G pair becomes a mismatched U:G pair which is excised by uracil-DNA glycosylase and the error-prone DNA polymerases fill in the gap, creating mostly single-base substitution mutations.
At the end of B cell division, the daughter cell may be making a different (either worse or better) antibody than the parent B cell
o Antibody diversity is generated by mixing and matching heavy and light chains in a combinatorial manner
• Burkitt’s Lymphoma
Genetics: c-myc gene (chromosome 8), affects B-cells; t(8,14)
Fastest human growing tumor—rapidly fatal if not treated quickly
LT survival in >50% if intense chemo is used
Most common >40 yrs, past exposure to EBV/malaria
• Acute Lymphocytic Leukemia
Starts in lymphocytes, least common type leukemia in adults
Can be in T or B cells
Without treatment, can be fatal in months (ACUTE)
• Acute Myelogenous Leukemia
Spreads very quickly, needs to be treated quickly
Affects myeloid cells
• Chronic Lymphocytic Leukemia
Adult disease—RARELY in kids
Slow growing
Abnormal lymphocytes
• Chronic Myelogenous Leukemia
Adult disease—rarely in kids
Slow growing
Too many cells become granulocytes
Philadelphia chromosome translocation (9:22)
• Identify the disadvantages and common side effects that arise during cytokine therapy.
o Disadvantages in Cytokine Therapy:
• Extremely short half-lives (minutes); can change delivery/administration to SQ or IA
• Extremely potent (can be good) and activate complicated cascades, invoke other cytokines, and can result in unpredicted/undesirable side effects
o Common Side Effects:
• Anorexia, fever, flu-like symptoms, fatigue → general malaise
• Unique Life threatening SE: IL-2 (as a drug) → diarrhea, thrombocytopenia, shock, respiratory distress, coma, fatal hypertension
darbepoeitin
• MOA: EPO – produce RBCs; works via JAK/STAT activation
o Inverse relationship between [EPO] and hematocrit due to chronic renal failure
• Pharmacokinetics: short half-life (admin 3-4x/week)
o Darbepoeitin: admin every week
o MPEG-EPO: admin weekly/biweekly
• Uses:
o Anemia (esp. chronic kidney disease + iron/folate)
o Given in anticipation of blood loss during high-risk surgery
• DO NOT use in athletes; MPEG-EPO should not be administered when RBCs are reduced 2⁰ to chemo
• SE:
o Life-threatening: thrombus
o Serious:
• Common = hypertension
• Rare = increased tumor growth (head and neck cancers); allergic reactions
erythropoietin
• MOA: EPO – produce RBCs; works via JAK/STAT activation
o Inverse relationship between [EPO] and hematocrit due to chronic renal failure
• Pharmacokinetics: short half-life (admin 3-4x/week)
o Darbepoeitin: admin every week
o MPEG-EPO: admin weekly/biweekly
• Uses:
o Anemia (esp. chronic kidney disease + iron/folate)
o Given in anticipation of blood loss during high-risk surgery
• DO NOT use in athletes; MPEG-EPO should not be administered when RBCs are reduced 2⁰ to chemo
• SE:
o Life-threatening: thrombus
o Serious:
• Common = hypertension
• Rare = increased tumor growth (head and neck cancers); allergic reactions
methoxy polyethylene glycol protein
• MOA: EPO – produce RBCs; works via JAK/STAT activation
o Inverse relationship between [EPO] and hematocrit due to chronic renal failure
• Pharmacokinetics: short half-life (admin 3-4x/week)
o Darbepoeitin: admin every week
o MPEG-EPO: admin weekly/biweekly
• Uses:
o Anemia (esp. chronic kidney disease + iron/folate)
o Given in anticipation of blood loss during high-risk surgery
• DO NOT use in athletes; MPEG-EPO should not be administered when RBCs are reduced 2⁰ to chemo
• SE:
o Life-threatening: thrombus
o Serious:
• Common = hypertension
• Rare = increased tumor growth (head and neck cancers); allergic reactions
filgrastim, pegfilagrastim,
(3, 5) sargramostim
• MOA:
o Filgrastim = produce neutrophils
o Sargramostim = produce any myeloid cells
• Pharmacokinetics: PEG-Fil has longer half-life due to PEG!
• Uses: cancer treatment
• SE:
o Filgrastim, Pegfilgrastim
• Innocuous, common: mild/moderate bone pain
• Rare, serious: allergic reactions; splenic rupture
o Sargramostim
• Common:
• Serious – capillary leak syndrome → peripheral edema
• Innocuous – moderate to severe bone pain, fever, malaise, arthralgias, myalgia
• Rare, serious: allergic reactions
IL-11
• MOA: increased platelet production (DO NOT use thrombopoietin → generation of autoantibodies)
• Pharmacokinetics: remains in use 3-4 days
• Uses: adjunct cancer chemotherapy; thrombocytopenia
• SE: IL-11 (does NOT cause fever)
o Common:
• Serious – Atrial fibrillation
• Innocuous – fatigue, headache, dizziness, mild edema → fluid dyspnea, anemia due to hemodilution
o Rare, serious: hypokalemia
romiplostim
• MOA: increased platelet production (DO NOT use thrombopoietin → generation of autoantibodies)
• Pharmacokinetics: remains in use 3-4 days
• Uses: adjunct cancer chemotherapy; thrombocytopenia
• SE: IL-11 (does NOT cause fever)
o Common:
• Serious – Atrial fibrillation
• Innocuous – fatigue, headache, dizziness, mild edema → fluid dyspnea, anemia due to hemodilution
o Rare, serious: hypokalemia
Functions of Complement System
• Generally, the complement system is a group of plasma proteins that acts as an auxiliary system in immunity, both on its own and in conjunction with humoral immunity
o It is a primitive surveillance system used to detect microbes independent of T cells and Ab
• Specific Functions:
o Lysis of many microorganisms, viruses, and nucleated cells
o Opsonization of antigen – uptake of particulate antigen by phagocytes
• Opsonin = molecule that binds to foreign surface and enhances phagocytosis by phagocyte
o Source of mediators of the inflammatory response
o Solubilization and clearance of immune complexes
o Clearance of apoptotic cells
o Augments stimulation of the B cell through CR2 receptor to increase the humoral immune response
• Ultimately, complement is good at fighting bacteria and for immune complex control
Location of Complement
• Complement is a plasma protein primarily found in plasma, but can also be found interstitially and in secretions (bronchoalveolar lavage fluid, etc.) Present at portals of entry.
• Synthesized mainly by liver hepatocytes and by tissue macrophages, but also by epithelial cells, fibroblasts, and monocytes
• Activator Proteins according to specific pathways:
o Classical: C1q, C1r, C1s, C4, C2
o Mannose binding Lectin (MBL): MBL, MASP-1/2 (MBL-associated serine protease)
o Alternative: Factor B, D, P (properdin)
• All pathways have C3
o Terminal Lytic Pathway: C5, C6, C7, C8, C9; MAC
• Concentrations in plasma:
o MBL and Factor D = 1-2 ug/mL
o C4 = 300 ug/mL
o C3 = 1200 ug/mL (LOTS, difficult to run out!!)
Activation of the Complement System → Lysis
• Classical Pathway:
requires antibody activation; triggered by antigen binding to either 2 IgG or IgM
o Activation of C1:
• Present in plasma as inactive C1qr2s2 complex
• Binding of C1qr2s2 complex to either 2 IgG or IgM causes conformational change in C1q → C1 active enzyme → C1r enzymatic cleavage → C1r active enzyme → C1s enzymatic cleavage → C1s active enzyme → C4 cleavage → C4a (leaves, anaphylotoxin), C4b binds to bacterial/foreign surface via a thioester bond and acts as an opsonin and interacts with complement receptors on white blood cells (CR1)
• C2 floats around and binds to C4b and is cleaved by C1s into C2a which binds to C4b and C2b which floats away.
• C4bC2a = C3 convertase and is covalently bound to the bacterial surface (permanent)
• C3 floats around and has a thioester which C3 convertase recognizes; C3 convertase cleaves C3 into C3a (floats away as an anaphylotoxin) and C3b which becomes covalently bound via free hydroxyl or amino groups to the bacterial surface adjacent to C4bC2a (C3 convertase)
• Together, C4bC2aC3b = C5 convertase
• Amplification: 1 C1 esterase can cleave 30 C4 molecules → 6 C4b2a molecules → 1200 C3b molecules
• Summary:
• Activation occurs in conjunction with specific antibody (2 IgG or IgM)
• C3b and C4b covalently bind via thioester bonds to bacterial surfaces
• C4b2a = C3 convertase; C4b2a3b = C5 convertase
requires antibody activation; triggered by antigen binding to either 2 IgG or IgM
o Activation of C1:
• Present in plasma as inactive C1qr2s2 complex
• Binding of C1qr2s2 complex to either 2 IgG or IgM causes conformational change in C1q → C1 active enzyme → C1r enzymatic cleavage → C1r active enzyme → C1s enzymatic cleavage → C1s active enzyme → C4 cleavage → C4a (leaves, anaphylotoxin), C4b binds to bacterial/foreign surface via a thioester bond and acts as an opsonin and interacts with complement receptors on white blood cells (CR1)
• C2 floats around and binds to C4b and is cleaved by C1s into C2a which binds to C4b and C2b which floats away.
• C4bC2a = C3 convertase and is covalently bound to the bacterial surface (permanent)
• C3 floats around and has a thioester which C3 convertase recognizes; C3 convertase cleaves C3 into C3a (floats away as an anaphylotoxin) and C3b which becomes covalently bound via free hydroxyl or amino groups to the bacterial surface adjacent to C4bC2a (C3 convertase)
• Together, C4bC2aC3b = C5 convertase
• Amplification: 1 C1 esterase can cleave 30 C4 molecules → 6 C4b2a molecules → 1200 C3b molecules
• Summary:
• Activation occurs in conjunction with specific antibody (2 IgG or IgM)
• C3b and C4b covalently bind via thioester bonds to bacterial surfaces
• C4b2a = C3 convertase; C4b2a3b = C5 convertase
• MBL Pathway
o Activation does NOT require antibody; primary constituent is the plasma protein mannose binding lectin (MBL) which is triggered by polysaccharide found on microbes such as Salmonella, Listeria, Neisseria, Candida, etc.
o The MBL-polysaccharide complex then binds to sugar residues like N-acetyl glucosamine or mannose which leads to the C1 independent formation of the classical pathway C3 convertase (C4b2a)
o MBL interacting with MASP-1 and MASP-2 is similar to C1q interaction with C1r and C1s
o The activated MBL pathway then binds to C4, cleaves it so C4b covalently binds to bacterial surface and then C2 comes along and is cleaved and covalently binds C2a becoming C4b2a (aka C3 convertase)
• Alternative Pathway
o Activation is triggered by recognition of foreign surface structures (zymosan, endotoxin, etc.) or by complement itself
o Activators of the Alternative Pathway:
• Gm + and Gm – bacteria
• LPS from Gm – bacteria
• Teichoic acid from Gm + cell walls
• Fungal and yeast cell walls (zymosan)
• Some viruses and virus infected cells, some tumor cells, some parasites
• Human IgA, IgG and IgE in complexes
• Anionic polymers (dextran sulfate)
• Pure carbohydrates (agarose, inulin)
• Does NOT require specific antibody
o There are two different stages within the alternative pathway:
• Stage 1:
• C3 tickover = C3 + H2O reacts forming C3(H2O); this is always happening at slow steady state
• Factor B binds to this complex forming C3(H2O), B
• Factor D cleaves Factor B on the C3(H2O),B complex allowing Ba to float away and forming C3(H2O)Bb
• Stage 2:
• C3(H2O)Bb then encounters another molecule of C3 cleaving it into C3a (floats away) and C3b which covalently binds to the foreign surface
• Depending on the state of the surface it binds to, ‘activating’ or ‘non-activating,’ determines whether Factors I & H will not bind or bind C3b, respectively
o ‘Activating’ surface: C3b binds to Factor B, Factor D cleaves Factor B forming C3bBb and further amplifies the signal; C3bBb is stabilized by properdin (Factor P)
• Note, C3bBb and C4b2a = C3 convertase
• C3b is continuously deposited in random and non-specific ways
• When C3bBb associates with another C3b molecule to become C3bBbC3b, this molecule acts as a C5 convertase (C3bBbC3b)
o ‘Non-activating’ surface: if C3b binds to a non-activator surface, the C3b complex becomes C3bi (inactivated) by Factors I & H
• ‘Non-activating’ surfaces include those with sialic acid; human cells express sialic acid and thus are not usually targeted by the complement system
o Overall, amplification occurs in all three pathways and ultimately results in formation of:
• A single C3 convertase (Classic and MBL – C4b2a; Alt. – C3bBb) which can cleave many C3 molecules resulting in deposition of multiple C3b molecules on the surface
• These deposited C3b molecules associated with C3 convertases and form C5 convertases (Classic & MBL – C4b2aC3b; Alt. C3bBbC3b)
• A single C5 convertase which can cleave many C5 molecules, increasing the probability that a complete Membrane Attack Complex (MAC) will form
C5 and Terminal Lytic Pathway
• C5 is cleaved by C5 convertases from any of the activating pathways into two fragments
• C5a floats away and is involved in inflammation and T cell response
• C5b interacts with C6, C7, C8, & C9 according to the diagrams to the right and forms the Membrane Attack Complex (MAC) in the lipid membrane of the foreign surface (i.e. bacterial)
o Note, C5b-9 formation involves conformational changes, not enzymatic reactions
• MAC is a transmembrane channel that allows passage of ions and lysis of the cell
o If the interactions of C5b-9 occur not in close proximity to the membrane, this complex can float away and become sC5b-9 (soluble C5b-9) and is used as an indicator for excessive complement activation
o Alternatively, C5b-7 can bind to S protein in the fluid phase which prevents its membrane insertion and MAC formation
• Notes:
o Lysis can occur in the absence of C9, but it is slower
o Complement system activation can have important biological consequences even if C5b-9 does not deposit MAC and lead to lysis
o C3b and C4b are still covalently bound to the membrane and can result in other biological effects by interaction with complement receptors 1-4 (CR1-4)
Regulation of Complement System
• Activator vs. non-activator surfaces; sialic acid as a non-activating surface – found in human cells
• Lack of antibody to initiate classical pathway
• Short half-life of enzymes formed
• Inhibitors:
o Fluid phase control proteins: Factor H and Factor I
• If Factor H sees C3bBb is bound to non-activator surfaces it will bind to C3bBb and dissociates Bb inactivating this complex
o Membrane bound (found on human cells): C3b and C4b don’t attach
o Classical pathway:
• C1 inhibitor – blocks beginning of classical pathway
• C4 binding protein (C4BP) – binds at C4 site blocking progression of pathway
• S protein (vitronectin) – binds to C5b, C6, C7 complex and blocks its insertion
• CD59 (protectin); found on human membranes – controls membrane at MAC and won’t allow MAC to form
• Block C3 and C5 Activation: most effective inhibition of complement system since these steps are integral to all three complement pathways
• C1 Inhibitor Deficiency
aka Hereditary Angioedema
o Sx: recurrent episodes of localized edema in skin, GI tract, or larynx
o Functions of C1 inhibitor: inhibit C1 esterase, MASP, kallikrein, plasmin, Factor XIa and XIIa
o Uncontrolled complement activation leads to consumption of C4 and C2 (labs?)
o Prevalence: 2-10 / 100,000
o Treatment:
• Anabolic steroids (increase synthesis of C1 inhibitor)
• Purified C1 Inhibitor
• Kallikrein inhibitors and B2 receptor inhibitors; excess kinin formation causes edema
• DAF (CD55) and CD59 Deficiencies
o Leads to:
• Increased susceptibility of erythrocytes to MAC-mediated lysis
• Complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria
o Etiology: defect in post-translational modification of peptide anchors that bind these proteins to cell membrane
o Treatment: Eculizumab (antibody to C5) – reduces hemolysis
• Various deficiencies of complement components often present as infections:
o Pyogenic infections and infections with encapsulated bacteria (classical and alternative)
• Opsonization and phagocytosis are a primary host defense
o Neisseria infections (C3, alternative pathway and terminal lytic pathway)
o Serious pyogenic infections with MBL deficiency
o Immune complex or autoimmune disease (Classical pathway or C3 deficiencies)
Biological Effects of Complement System
• Mediates Inflammation through C3a and C5a (anaphylatoxins)
o C3a and C5a can mimic the symptoms of inflammation and anaphylaxis
o Chemotaxis, smooth muscle contraction, increased vascular permeability, degranulation of mast cells, etc.
o Have distinct receptors on many cell types and are implicated in pathology of many inflammatory diseases
• Opsonization: C3b, C4b, and their degraded products are opsonins
o C3b and C4b:
• Participate in continued pathway activation leading to MAC formation and lysis
• Interact with CR1 → opsonization clearance of the immune complex
• Degrade into fragments and interact with CR2 and CR3 → opsonization, clearance of IC, augment humoral immunity
• Solubilization and clearance of immune complexes
o CR1 receptor (CD35)
• Major ligands: C3b, C4b
• Found on monocytes, macrophages, PMNs, eosinophils, RBCs, B and T cells
• Functions: transports ICs by RBC, promote immune adherence (binding of opsonin), promotes phagocytosis (cooperates with Fc receptors), blocks formation of C3 convertase
• There is a high incidence of immune complex disease in individuals whom are deficient in C1, C2, C3, or C4; complement plays a huge role in tissue damage in IC diseases
o Solubilization of antigen-antibody complexes by binding of C3b allows these complexes to remain soluble and be cleared from the body preventing aggregation
o Transport of these ICs from the body is a crucial function of complement:
• CR1 receptors on RBCs transport ICs to the reticuloendothelial system (macrophages in spleen, etc.) for clearance
• The IC is coated with C3b and transferred from the RBC CR1 to the macrophage CR1 for internalization and degradation
• Augmentation of Humoral Immunity
o CR2 receptor (CD21)
• Major Ligands: C3d, C3dg, iC3b; Note C3 degradation= C3 → C3b → C3bi → C3dg →C3d
• Found on B cells, activated T cells, and epithelial cells
• Functions: increased humoral immune response through CR2/CD19/CD81 complex, has affinity for EBV envelope allowing the virus to enter the B cell
• Note, when co-stimulated by C3d and C3dg, the B cell makes more antibody
o CR3 (CD11b/CD18) & CR4 (CD11c/CD18)
• Major Ligand: C3bi
• Found on monocytes, macrophages, PMNs, NK cells, T cells
• Functions: phagocytosis, cell adhesion
• Contraindications to Vaccination
o Valid: anaphylactic reaction to vaccine, prior high fever after vaccination, immunodeficiency, pregnancy, significant acute illness
o Invalid: minor illness, mild/moderate local reaction or fever following a prior dose, antimicrobial therapy, disease exposure or convalescence, pregnancy or immunosuppression in household, premature birth, breastfeeding, allergies to products not in vaccine, family Hx (unrelated to immunosuppression)
Whole Blood
Plasma + RBC + platelets + WBC
Massive Hemorrhage
Red Cells
RBC + few WBC + few platelets + little plasma
Low hemoglobin
Leukocyte-reduced Red Cells
RBC + no WBC + rare platelets + little plasma
Reduced alloimmunization and allergic reactions
Frozen Red Cells
RBC + few WBC
Used for storage of rare blood types
Granulocytes
Neutrophils
Sepsis in neutropenic patients that do not respond to antibiotics
Fresh Frozen Plasma
Plasma
Bleeding due to multiple factor deficiencies
Cryoprecipitate
Low fibrinogen, von Willebrand disease, hemophilia A or XIII def
• Describe the method and principles of the type (forward and reverse), screen, and crossmatch tests.
o Forward type: add antibody (A & B) to patient’s blood + AHG and observe for agglutination
• Note, AHG = anti-human globulin
o Reverse type: add reagent red cells (both type A & B) to patient’s plasma (have patient antibodies) + AHG and observe for agglutination
o Cross-match: take patient’s serum + donor RBC + AHG and observe for agglutination
o Antibody screen: applies to the other systems (Duffy, Lutheran, etc.) and test for agglutination
• Acute hemolytic transfusion reactions
patient has ABO antibodies against donor red cells
Dx: Hgb in serum and urine; ↓ haptoglobin, ↑ bilirubin, DAT positive; type and cross-match shows ABO mismatch
• Delayed Hemolytic transfusion reactions
hemolysis occurs days after transfusion caused by antibodies to non-ABO antigens
Dx: falling Hgb after transfusion, DAT positive. Antibody screen IDs the Ab
• Febrile Transfusion Reactions
recipient antibodies against donor WBC which causes cytokine release → fever, headache, nausea, chest pain
Dx: rule out everything else
Tx: Tylenol. Leukocyte-reduced components
• Allergic Transfusion Reactions
host reaction to donor plasma proteins
Tx: antihistamines
o Circulatory overload
occurs when blood is given too quickly
• Symptoms: hypertension, congestive heart failure
• Tx: stop transfusion, give diuretics
o Iron overload
too much iron can damage the heart and liver; patients with chronic anemias are at biggest risk
Complication Risk
Allergic Reaction 1 in 100 (severe: 1 in 20,000)
Febrile Reaction 1 in 200
Circulatory Overload 1 in 3,000
Delayed hemolysis 1 in 4,000 (fatal: 1 in 4 million)
Acute Hemolysis 1 in 20,000 (fatal: 1 in 600,000)
GVHD Unknown
• Tx: iron-chelating agents
o Graft-vs-host disease
donor lymphocytes attack host
• Symptoms: Fever, rash, hepatitis, marrow failure; usually fatal
• Populations at biggest risk: immunocompromised pts or pts w/ blood-relative donors
• Prevent by irradiating products
Hemolytic Disease of the Newborn
• Diagnosis and Prevention of Rh HDN
o Diagnosis: DAT (baby), IAT (mom)
o Rhogam (anti-D Ab) administration at 28 weeks and within 72 hours of delivery
o Must quantify amount of fetomaternal hemorrhage to dose Rhogam; use Kleihauer-Betke test or flow cytometry
• Kleihaur-Betke Test:
• Make smear of mom’s blood
• Expose smear to acid bath (removes HbA)
• Stain smear
• Baby’s cells appear pink; mom’s appear ghostly
• Flow Cytometry Test for Fetal Cells:
• Use mom’s blood
• Apply anti-HbF antibody
• Run flow
• Baby cells, if present, are intensely positive
• IDA Pearls:
o History must include:
• Bleeding risks: menorrhagia, frequent epistaxis
• GI bleeding risks: NSAIDs or anticoagulation, previous EGD or colonoscopy, prior surgeries (Roux-en-Y)
o Iron supplements?
o IDA leads to low iron and high TIBC & transferrin
o ACD leads to low iron and low/normal TIBC & transferrin
o All labs must be interpreted in the clinical context
o The gold-standard test is bone marrow iron stain
• Pernicious Anemia Pearls
o Associated with vitiligo & auto-immune disorders.
o By definition is slow in onset.
o More than just anemia. Neurological symptoms too.
o When in doubt, send for MMA (methylmelonic acid) test
• AIHA Pearls
o Always get a thorough history that focuses on other autoimmune conditions (RA, SLE, hyper/hypothyroid, scleroderma), drugs (especially new ones) and recent transfusions of any blood product.
o AIHA is associated with lymphoid malignancies (esp. CLL) and may precede the lymphoma diagnosis.
o Prolonged high dose steroids will lead to Cushing syndrome and comes with increased risk of PJP.
o Always give folate supplements with hemolytic anemias.
o These patients present problems with transfusions
• Nucleated RBC Pearls
o They should not be overlooked
o Present in any ‘overcrowding’ disorder of the bone marrow
o Usually present when the reticulocyte count is high
