Immunology / hemolymphatics Flashcards
Physiology (1 -> 60) - from Ninjanerd videos / notes and VECCademy power point - try to study in order
Classification of immune system
Components of immune system
Innate Immunity
Physical barriers and biologic processes that prevent infection
- Skin, MM, stomach acid, antimicrobial peptides
Specific cells
- PRRs on them bind compounds that are specific to microorganisms and trigger immune response - Pathogen-associated molecular patterns (PAMPs)
- Neutrophils
- Macrophages
- Natural killer cells
Acquired / Adaptive immunity
- Antigens activate T or B lymphocytes
- B lymphocytes differentiate into plasma cells and release antibodies that attack the antigen (humoral immunity)
- T lymphocytes produce receptors specific to the antigen and destroy it (cellular immunity)
- Small numbers of T and B cells persist as memory cells that “remember” that antigen -> quicker response if same antigen encountered again
The players of immune system
Granulocytes (aka polymorphonuclear leukocytes)
- Neutrophils
- Eosinophils
- Basophils/Mast cells
Lymphocytes
- B cells
- T cells
Monocytes/Macrophages
Granulocytes
Neutrophils
- First responders, phagocytosis of microbes
- Contain granules with proteases and defensins
- Cell membrane contains NADPH oxidase which produces ROS
- Release cytokines and other inflammatory mediators
- Can act as APCs in the spleen
Eosinophils
- Large numbers in GIT, respiratory and urinary mucosa
- Weak phagocytosis, hydrolytic enzymes, ROS, larvacidal polypeptide called major basic protein.
Basophils
- Granules contain histamine, heparin and other inflammatory mediators
- Involved in hypersensitivity reactions
Mast cells
- Granules contain proteoglycans, proteases, heparin and histamine
- Respond to allergens and parasites
Lymphocytes
B lymphocytes
- Become plasma cells and memory B cells
T lymphocytes
- Cytotoxic T cells
- Helper T cells - Type 1 and Type 2
- Suppressor T cells
- Memory T cells
Lymphocytes - B cells and immunoglobulins function
B cells differentiate into plasma cells that produce and release antibodies - immunoglobulins
Immunoglobulins functions:
Agglutination
Precipitation
Neutralization
Lysis
Lyse cell membranes
Opsonization - activation of complement system
Lymphocytes - immunoglobulins structure
- Made of light and heavy polypeptide chains
- Each chain has a variable portion that determines specificity of the antibody for antigens
- Each chain has a constant portion that determines things like diffusivity into tissues, adherence to structures within tissues, attachment to complement…
What is inflammation
Inflammation is some type of tissue damage/tissue infliction that initiates a set of vascular and cellular/molecular events that are designed to:
o Clean up any type of
▪ Cellular debris
▪ Infectious organisms/pathogens
o Initiate repair
Causes of inflammation
Physical trauma
Chemical trauma
Infectious microorganisms
o Bacteria
o Virus
o Fungi
o Parasites
Sunlight and burns
Characteristics of an antigen
Immunogenic
o Activate certain types of immune cells
▪ In response to that → immune cells start proliferating
Reactive
o Actual immune cells, specifically B cells can produce antibodies against the antigen
Incomplete antigens (hapten)
Example
o Poison ivy
o Poison oak
▪ Urushiol oil
Whenever these antigens get into the skin → bind with skin protein
o The antigen now becomes a complete antigen → Causes rash that we see with poison ivy
Examples of antigens
• Sugar molecules
• Protein molecules
• Glycoprotein
Inflammatory response - vascular events step 1
Whenever the endotoxins damage mast cells or activate the receptors on mast cells
o Mast cell can initiate a specific type of inflammatory response
▪ Sends signal to the nucleus
o Mast cell starts releasing tons of molecules
▪ Histamine - most important
▪ Leukotrienes
▪ Prostaglandins
There are plasma proteins from hemostasis
o Clotting protein → clotting factor XII
▪ Specific enzyme that converts prekallikrien →kallikrein
• Kallikrein gets into the tissue space
Inflammatory response - vascular events step 2
Another protein inside the tissue space → kininogen
o Kallikrein converts kininogen → bradykinin
▪ Kallikrein comes from thrombin
Kininogen can be circulating in bloodstream naturally and leak into tissue spaces due to
o High hydrostatic pressure
o Gaps present between the endothelial cells
Inflammatory response - vascular events step 3 - AA cascade
Whenever there’s any type of damage to cell membrane
o Cell membrane is phospholipid
o Phospholipids are being utilized
▪ There is an enzyme called phospholipase A2
▪ Phospholipase A2 starts breaking down phospholipids → arachidonic acid
Arachidonic acid (AA) will be converted by 2 enzymes
o Lipoxygenase → converts AA into leukotrienes
▪ Leukotriene C4
▪ Leukotriene D4
o Cyclooxygenase (COX1 or COX2) → converts AA into prostaglandin and thromboxane A2
▪ PGE2
▪ PGI2
▪ PGF2
Inflammatory response - vascular events step 4 - molecules
All these molecules (PG, leukotrienes, histamines and bradykinin) have things in common: they will work on endothelial cells and activate them:
1) P-selectin release
▪ Inside of the cell, there are preformed granules (Weibel-Palade bodies)
▪ These granules start migrating into the cell membrane surface -> they put the proteins up on the cell membrane → P-selectins
2) Causes the endothelial cells to contract
▪ When they contract → big gaps between the epithelial cells
▪ A lot of plasma can start leaking out through the intercellular clefts
▪ If plasma keeps leaking out - fluid starts accumulating in the interstitial space → swelling (edema)
Inflammatory response - vascular events step 5 - pain
Remember there are lots of pain receptors (nociceptors) in the tissues
o As the fluid in the space starts increasing:
→ Starts compressing the nociceptors
→ Activate the nociceptor and causes pain -> pain can also be caused by increased vascular permeability indirectly.
o Bradykinin also activates the nociceptors
Inflammatory response - vascular events - step 6 - heat & redness
Remember there are smooth muscles around the endothelial cells.
The histamines, leukotrienes, prostaglandins act on the smooth muscle cells:
→ Causes the smooth muscle relaxation → vasodilation in localized area -> Localized hyperemia → more blood flow into this area, therefore redness and heat.
Inflammatory response - vascular events - step 7 - selectines & cytokines - leukocyte migration - margination
White blood cells circulating in the blood plasma (most abundant → neutrophil & monocytes).
Naturally on their membrane, they have certain type of sugar molecules that interact with P-selectins -> can’t keep going → stuck -> we want the white blood cells to come and fight the infection.
Catches the white blood cell and prevents them from flowing pass the area -> these white blood cells will go from selectin to selectin, rolling on the endothelial cells - margination.
Inflammatory response - vascular events step 8- selectines & cytokines - leukocyte migration - diapedesis
There are molecules on the surface of endothelial cells called P-CAM-1
White blood cells try to squeeze through the gaps between endothelial cells interacting with P-CAM-1
Inflammatory response - vascular events - step 9 - selectines & cytokines - leukocyte migration - chemotaxis
We now have macrophages (derived from monocytes) and neutrophils in the tissues, and they have receptors on the membrane surface.
Histamines, leukotrienes, prostaglandins, and cytokines previously released from mast cells and from the AA cascade -> bind to the receptors of the white blood cells surface.
The bacteria are on one side and the white blood cell has to go that direction -> cytokines stimulating causing them to migrate where the infection is → positive chemotaxis
Inflammatory response - vascular events - step 10 - selectines & cytokines - leukocyte migration - E-selectin, I-CAM, V- CAM
Imagine there are gram negative bacteria and we have already recruited initially neutrophils and macrophages (from blood monocytes) to the damaged site.
Macrophages and neutrophils will fight these bacteria and they want to recruit more WBCs (this part comes a bit later in the inflammatory cascade, as IL1, IL8 and TNF-α have to be synthesized, whereas PG, leukotrienes… are already pre-synthesized):
Macrophages will release:
• IL-1 and TNF-α -> act on endothelial cell and stimulate production of E-selectins -> allow for monocytes and neutrophils to adhere with E-selectins.
• IL-8 - binds to endothelial cells receptors -> it activates the cell and stimulate the synthesis
of ICAM and VCAM
• ICAM = intracellular cell adhesion molecule
• VCAM = vascular cell adhesion molecule
The actual neutrophils and monocytes have specific inactive proteins on cell membrane → integrins.
Then IL-8 activates neutrophil -> activates the integrin -> neutrophil interacts with V-CAM and I-CAM to be “fixed” to the endothelium and then start diapedesis interacting with P-CAMs.
▪ White blood cells squish through the capillary with amoeboid motion → diapedesis
▪ Follow the chemoattractant molecule → positive chemotaxis.
Inflammatory response - vascular events - step 11 - fever
- IL-1 and TNF-𝜶 cause the fever
- They will act on brain, liver and bone marrow
Brain: act on hypothalamus
- IL-1 and TNF-𝛼 stimulates the secretion of PGE2 -> PGE2 resets the body temperature (raise the setpoint) and initiates fever.
Fever significance
o Denatures some of the proteins, DNA of the bacteria - makes it harder to survive.
o Speeds up the metabolism → initiate quicker healing process.
Liver: in response to IL-1 and TNF-𝛼, it will produce acute phase reactant proteins
o C-reactive peptide (CRP)
o Ceruloplasmin
o Haptoglobin
o Fibrinogen
o Ferritin
Good for determining active inflammation → C-reactive peptide (CRP)
IL-6 also can act on liver and cause the release of acute phase reactant proteins (produced by macrophages and lymphocytes).
Bone marrow:
TNF-𝛼, IL-1, IL-3, IL-5 act on the stem cells → Stimulate the bone marrow to produce more white blood cells - causes leukocytosis.
Inflammatory response - recap of VASCULAR events on inflammation
Inflammatory response - cellular events - step 1 - phagocytosis (neutrophils and macrophages)
1) Pseudopods
Macrophages and neutrophils will make pseudopods
o Cytoskeleton → actin filaments and myosin filaments
▪ Able to change shape toward to literally come around the bacteria
▪ Latch onto it and pull it inside the cytosol inside specific endosome/vesicle (phagosome)
2) Phagosomes + lysosome → phagolysosome
Inside the macrophage and neutrophil, there are vesicles with the G- bacteria (phagosome) - the bacteria still have their antigens (proteins, sugar molecules or glycoproteins) on their cell walls.
Inside of the cell, macrophage and neutrophil have another specific vesicles → lysosome (has hydrolytic enzymes that helps to break down bacteria - cell walls, peptidoglycan, internal structures).
These two vesicles will fuse together → phagolysosome. After the fusion, hydrolytic enzymes are released -> starts breaking down the cell wall and internal structures.
3) Overall result - destruction of the bacterial cell wall, fungi cell wall, viral, fungal, parasitic. Only leave antigens at the end and all the bacterial and viral substances are gone.
Inflammatory response - cellular events - step 2 - neutrophils and NETs
Neutrophil is not antigen-presenting cell
The phagolysosome fuses with cell membrane → exocytosis -> spits out the antigens into the interstitial space and they go to LN - adaptive immunity.
Sometimes -> bacteria that are hard to break down with necessary lysosomes -> it requires the sacrifice of the neutrophil itself.
Oxidative / respiratory burst - Neutrophils have capability of taking O2 and convert it into free radicals:
O2 →O2- →H2O2 →OH- or HOCl
o O2- (superoxide anion)
o H2O2 (hydrogen peroxide)
o OH- (hydroxide free radicals)
o HOCl (hypochlorite acid)
▪These free radicals come in and destroy the bacteria’s protein, cell wall, DNA.
▪ Free radicals aren’t specific -> it can also destroy the neutrophil’s protein, membrane.
If this neutrophil is about to die because of the free radical reactions -> whenever it undergoes of fragmentation, it can start releasing its own DNA/chromatin (chromatin is made of DNA + histone proteins) -> Neutrophil Extracellular Traps (NETs).
NETs -> the chromatin histone proteins can bind on the foreign bacteria antigens -> now it is possible to produce antibodies against this complex. It also can activate another enzyme → cathepsin G. Now the pathogen can be destroyed by cathepsin G or opsonization by white blood cells.
Inflammatory response - cellular events - step 3 - macrophages and MHC II
Macrophage is an antigen-presenting cell (they can present antigens on their cell membrane)
They have specific gene sequence on chromosome 6 responsible for producing specific proteins -> there’s recombination where the DNA within that area can actually shuffle around and produce different types of mRNA → different types of proteins.
Example:
There are 3 types of antigen (circle, square, triangle) within the phagolysosoma of the macrophage. Macrophages be able to produce different proteins specific to fit that antigen, called MHC, Major histocompatibility complex, specifically type II.
MHC II will bind to different types of antigens, and then those molecules will be exposed on the cell membrane bound with antigens.
Additional information - there are MHC I - found in ALL nucleated cells in the body.
Therefore, antigen-presenting cells also have MHC I molecules because they’re nucleated.
Inflammatory response - Complement proteins - step 1 - Introduction
Complement is a part of the innate immune system (nonspecific).
- Proteins made by liver in inactive form.
- Normally complement proteins are circulating in the plasma.
- Inflammatory response causes ↑ vascular permeability -> complement proteins are activated and leave blood and enter tissue to attack the pathogens (together with phagocytosis cascade).
- This complement system includes 3 pathways.
Inflammatory response - Complement proteins - step 2 - Classical pathway
Starts at C1 and is antibody mediated:
- There are antibodies constantly circulating plasma and lymphatic fluid.
- In inflammatory reactions, specific memory antibodies can leak out into tissues (increased vascular permeability).
- These are antibodies that maybe have previously been exposed to the bacteria before (e.g. IgM, IgG, and antibodies) and can bind to the bacterial antigens via the Fc portion -> the Fc portion is very attractive to the complement proteins.
- As soon as the antibody binds to the antigen, this attracts the first complement protein called C1 to attach to the Fc portion.
- Then another protein called C4 binds to the C1, and the chain is as follows:
C1 → C4 → C2 → C3 - There is an enzyme called C3 convertase cleaves the C3 protein into C3a and C3b.
- C3a moves away and C3b remains in the pathway and continue the chain as follows: C1→C4→C2→C3b→C5b→C6→C7→C8→C9
- The C5 protein undergoes the same cleaving process as the C3, this means an enzyme called C5 convertase cleaves C5 into C5a and C5b
- C5a moves away and C5b remains in the pathway.
- Mast cells released various molecules like histamines, leukotrienes, prostaglandins… (at the beginning of the process, vascular events).
- During this process, mast cells release proteases that act on C3a and C5a (that where cleaved off C3 and C5) to activate them.
- Active C3a and C5a become great chemotactic agents and ENHANCE the inflammatory response.
Inflammatory response - Complement proteins - step 3 - Classical pathway - MAC
- C5b→C6→C7→C8→C9 can break off from the main pathway and form a pentameric protein channel - Membrane Attack Complex, MAC.
- This Membrane Attack Complex (MAC) forms a membrane pore that can bind into the bacterial cell membrane.
The purpose of MAC: there is an open movement between the inside and outside the pathogenic cells -> water, Na+, can move in and cause CELL LYSIS.
Inflammatory response - Complement proteins - step 4 - Classical pathway - opsonization & phagocytosis
Once the pentameric protein channel is formed (MAC), what is left of the complement still attached to the bacteria is:
C1→ C4→ C2→ C3b
- C3b is now exposed -> C3 it is an opsonin, making the pathogen more susceptible (more yummy!) to phagocytosis.
- C3b binds to a receptor called C3b receptor on macrophages / neutrophils and triggers phagocytosis and engulfs all the complement proteins and the pathogenic cells.
- When it breaks the pathogen cells down, antigen then gets exposed with the MHC II molecule (in macrophages) -> recall: neutrophil undergoes the same process but releases the product via exocytosis and into the LN in the end.
Overall result of Complement Classical pathway:
- MAC formation - PRODUCES CELL LYSIS
- Opsonization - ENHANCES PHAGOCYTOSIS
- Release of C3a and C5a -> ENHANCES CHEMOTAXIS
Inflammatory response - Complement proteins - step 5 - Alternative pathway
The difference in this pathway is that there is NO antibody mediated effect (no antibody attached to the surface of the pathogen):
- In some cases, C3b protein can bind directly to the bacterial antigen, and the main chain is as follows: C3b → C5b → C6 → C7 → C8 → C9
Note: In between C3b and C5b, there is a molecule called Factor B
C3b → Factor B →C5b → C6 → C7 → C8→ C9
C3a and C5a are released and interact with the proteases released by the histamines - this enhances the inflammatory response (via chemotaxis) (same as in Classical pathway).
The same process of the Classical Pathway occurs:
- C5b→C6→C7→C8→C9 break off and form the MAC -> this gets pushed into the bacterial cell membrane -> MAC initiates lysis of the pathogenic cell.
- Again, when these molecules break off, C3b is exposed again.
- The macrophages/neutrophils/dendritic cells bind via the C3b receptor and opsonization occurs (enhancement of phagocytosis process).
Inflammatory response - Complement proteins - step 6 - Lectin pathway
- Remember antigens on pathogenic cell can be sugar molecules, proteins or glycoproteins…
- Some pathogen cells have specific type of antigen called Mannose.
- Mannose binding lectin circulating the blood stream can bind to the mannose antigen on the pathogen cell.
- This forms a mannose binding lectin complex.
- C4 binds to this complex and the chain is as follows: C4→C2→C3b→C5b→C6→C7→C8→C9
- And the same process occurs again: C5b→C6→C7→C8→C9 break off and form the MAC -> triggers lysis of the cell.
- C3a and C5a are released and interact with the proteases released by the histamines which enhances the inflammatory response.
- What remains is: C4→C2→C3b
- And again, the exposed C3b binds to macrophages/neutrophils/dendritic cells and opsonization and phagocytosis occurs.
Inflammatory response - Complement - summary
Inflammatory response - interferon and toll-like receptors - step 1 - Introduction
1) Toll-like receptors
• Proteins that are present in the cell membrane or the vesicles inside of the cell that respond to pathogens and elicit via specific responses.
• Part of the pattern recognition receptors (PRRs) that bind PAMPs and DAMPs
• Expressed on dendritic cells, macrophages, B cells, cytotoxic T cells as well as endothelial and epithelial cells
• Activation leads to up regulation of inflammatory pathways NFkB and mitogen activated protein kinase (MAPK) -> acute phase proteins, coagulation factors, TNF-alpha, IL-1, IL-6, IL-8, IL-12, interferon
2) Interferons
• Signal nearby host cells to let them know the presence of pathogens so that they can start making anti-viral peptides.
• Signal macrophages and natural killer cells to proliferate
Inflammatory response - interferon and toll-like receptors - step 2 - IRF signaling
IRF – Interferon Regulatory Factors
- When a virus infects a generalized tissue cell or a macrophage, usually, it results to death of the cell (lytic cycle).
- Whenever a cell is damaged by virus, IRF TRANSCRIPTION FACTOR will activate IRF gene -> will produce an mRNA that will be translated to proteins forming interferons – alpha, beta, gamma.
- Those interferons will be secreted out of the cell.
Inflammatory response - interferon and toll-like receptors - step 3 - Interferon subtypes
1) Alpha and Beta
Both can be secreted by generalized tissue cells
Beta-interferons can also be secreted by platelets
Effects
Activate Protein Kinase R cascade of a nearby unaffected cell - Protein kinase R (antiviral peptide) -> if the virus tries to penetrate the cell with Protein kinase R, it will be destroyed.
Activate nearby natural killer cells -> some infected cells might down regulate the expression of MHC I molecules -> NK cells will notice cells that do not have MHC class I and induce apoptosis.
2) Gamma
Secreted by immune system cells like lymphocytes
Effects
Binds to macrophage receptors activating macrophage proliferation.
It will also make the macrophage bigger, hungrier, and increase its expression of MHC I and MHC II receptors.
Inflammatory response - interferon and toll-like receptors - step 4 - what happen to cells when they get infected - generalized tissue cells
- When generalized tissue cells are infected, virus can activate the cell’s IRF signaling mechanisms.
- Interferons alpha and beta are secreted and in turn activate a healthy cell.
- Healthy cell produces protein kinase R and other antiviral peptides
- Protein Kinase R act like scissors, chopping the virus into pieces once it enters the healthy cell -> inhibiting the virus from infecting the healthy cell.
- NK cells are also activated causing apoptosis of MHC-I deficient cells.
Inflammatory response - interferon and toll-like receptors - step 5 - what happen to cells when they get infected - macrophages
- When a macrophage gets infected, virus can also activate its IRF signaling mechanisms.
- Interferons alpha and beta are secreted and in turn activate a healthy cell.
- Healthy cell produces protein kinase R and other antiviral peptides.
- Protein Kinase R acts like scissors, destroying the virus once it enters the healthy cell -> inhibiting the virus from infecting the healthy cell.
- NK cells are also activated causing apoptosis of MHC -I deficient cell.
- Interferon gamma is also secreted causing macrophage proliferation and activation, resulting to a stronger immune response.
Inflammatory response - interferon and toll-like receptors - step 6 - toll-like receptors
TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages as well as non-immune cells such as fibroblast cells and epithelial cells.
Subtypes:
Technically, there are 11 subtypes.
However, TLR-10, its function is unknown.
TLRs dimerize when activated - combine with another TL receptor nearby to form a complex
TLR1, TLR-2, TLR-3,…., TLR-9, TLR-11
There can be TLR in the membrane, or in the inside of the cell on membrane of vesicles (endosomes).
Inflammatory response - interferon and toll-like receptors - step 7 - toll-like receptors
Cascade of events:
TLR in the cell membrane, when activated, stimulate the vesicular TLRs.
Membrane dimers:
- TLR 1 + TLR 2 -> respond to GPI - anchoring proteins - parasites
- TLR 2 + TLR 6 -> respond to zymogens (fungi) and LTA (gram +)
- TLR 4 + TLR 4 -> respond to LPS (gram -)
- TLR 5 + TLR 5 -> respond to flagellin (E. coli)
- TLR 11 + TLR 11 -> respond to bacteria that damage the urogenital system
Vesicular TLRs, in turn, will activate certain transcription factors
- TLR 3 + TLR 3 -> responds to double stranded RNA virus
- TLR 9 + TLR 9 -> responds to CpG DNA within bacteria
- TLR 7 + TLR 7 -> responds to single stranded RNA
- TLR 8 + TLR 8 -> responds to double stranded RNA
Transcription factors cause activation of GENES that will produce mRNAs encoding proteins - AP1, IRF, NFKB
Inflammatory response - interferon and toll-like receptors - step 8 - toll-like receptors - genes activated by vesicular TLR
- AP1 Transcription Factor - activates protein signaling molecules
- Interferon Regulating Factors - stimulate the production of interferons
- Nuclear Factor Kappa Beta - stimulate the production of cytokines such as
o Tumor Necrosis Factor Alpha (TNF-α)
o Interleukin 1-Beta (IL-1β)
o Interleukin 18 (IL-18) -can also activate NK cells
IL-β and IL-18 still need to be acted upon by CASPASES for them to be activated.
Inflammatory response - interferon and toll-like receptors - step 9 - overall effect
To enhance the inflammatory response via different mechanisms
- Chemotaxis - via secretion of protein signaling molecules
- Protect healthy cells from the viruses - via secretion of interferons
- Initiate leukocytosis, activation of pyrogenic effect, activation of bone marrow, activation of liver to produce acute phase reactive proteins - via secretion of TNF-α, IL-1β, and IL-18
Inflammatory response - adaptive immunity - step 1 - recap of cellular events
Before entering the lymph nodes, there are cellular events happening in order to bring the antigens inside the lymph node:
1) Macrophages - the antigens that were pulled away from bacterial microbe may be expressed on MHC II molecules on cell surface.
- These macrophages are referred as Antigen Presenting Cells (APCs)
- The APCs interact then with T-helper cells
Antigen Presenting cells (APCs)
- Macrophages
- Dendritic Cells
- B-cells
2) Neutrophils - the antigens are exocytosed into interstitial fluid and then carried to nearby lymph nodes - this is the free antigen.
Inflammatory response - adaptive immunity - step 2 - B-lymphocytes
- Lymph nodes contain germinal centers which contains large amount of B-cells (AKA B-lymphocytes)
- B-lymphocytes contains specific types of receptor on their membrane which is called the B-cell receptors - they are formed through recombination -> each of these B-cell receptors have different binding domains -> hence, each receptor can bind different types of antigen.
- B-cell receptors - they are IgD antibodies.
Inflammatory response - adaptive immunity - step 3 - Naïve / activated B-lymphocytes
Before the binding, the B-lymphocyte is naïve since it hasn’t gone any immunogenicity response.
-> Activation happens when the free antigen circulating inside the LN, by random chance binds onto the B-cell receptor that was designed to fit that antigen.
-> Activates B-cells through signaling mechanisms to the nucleus.
-> the B-lymphocytes, now more mature, undergo receptor mediated endocytosis of antigen-antibody complex into B cell.
-> Chromosome number 6 of the B cell produce MCH II molecules which fit perfectly to the antigen that has been phagocytosed.
-> MCH II with antigen fuses on to the cell membrane - activated B-lymphocyte (Antigen Presenting Cell).
- > Activated B-lymphocyte -> have B-cell receptors, including the MHC II molecules with the foreign antigen on its membrane → important to make more specific types of antibodies specific to the antigens. It still cannot undergo proliferation.
Inflammatory response - adaptive immunity - step 4 - Interactions of APCs with T-cells
Before interacting with the antigens, the T helper cell is a naïve T-helper cell → have receptors which can respond to the molecule but still not activated and unspecific
Remember -on the membrane of the macrophages, there are:
o MHC I molecules with some type of self-antigen - all nucleated cells must have it.
o MHC II molecules which expresses the antigen.
Inflammatory response - adaptive immunity - step 5 - Interactions of APCs with T-cells - activation
T naïve cells can become either T helper 1 or T helper 2, depending on the factors that activate them. IL2 and IL4 will produce T helper 2 cells.
Primary signal
MHC II molecules on cell membrane of macrophages -> will interact with CD4 (CD = cluster of differentiation) molecule on T helper cells
Antigen which is expressed on the MHC II molecule -> will interact with T-cell receptor (TCR) specific to that antigen on T-helper cells (like the B-cell, due to recombination, every T-cell have different types of receptors specific to the type of antigens). This interaction sends primary signals to the nucleus through CD3 molecules.
Secondary signal
B7 molecules on macrophage surface -> will interact with CD28 on naïve T helper cells → send secondary signal / co-stimulation to the nucleus together with CD3.
Third signal
The macrophages secrete IL1 molecule → this sends the third signal to the nucleus.
Activation of the T-cell leads to the production and secretion of IL-2:
- IL2 can bind on the T-cell (autocrine - binding to the same cell that produces it)
- IL2 and IL4 will stimulate proliferation of the now called T helper 2 cells.
- T helper 2 cells will produce IL4, IL5 and IL6
Inflammatory response - adaptive immunity - step 6 - B-cell proliferation - cytokines released
Now with the production of IL4 and IL5 by T helper 2 cells, B-lymphocytes can proliferate.
The B-cell and T-helper cell interaction as was listed, triggers T-helper cells to release cytokines: IL-4 and IL-5 → converts naïve T cell into TH2 cells
IL-4
- Activates B-cells to start proliferating (clonal expansion) → become immunocompetent.
- This B-cell have specific B-cell receptor to the specific foreign antigen and MHC II molecules with the foreign antigen, expresses on its membrane -> can recognize any types of antigens due to the specific B-cell receptor.
IL-5
- Promotes differentiation of proliferated B cells into memory B cells and plasma cells
IL-5/IL-6
- Stimulate plasma cells to produce antibodies against specific antigens on pathogens:
o Neutralization - the antibodies bind to all the surface antigens on the pathogen -> block the antigen from attaching to healthy host cells which can cause damage.
o Precipitation reaction
- Antibody bind to the freely circulating antigen (causes precipitation and enhances opsonization)
- Free antigen-antibody complexes may deposit into tissues causing type 3 hypersensitivity
o Lysis
- Antibodies binds to the same antigens on the pathogen - stimulates the complement system -> produces membrane attack complex - cell lysis.
o Agglutination
- When there’s a mismatch/incompatible blood - will have antigens on the RBCs surface -> antibodies bind to the antigen on the RBCs - agglutination.
o Opsonization
- When the pathogen is marked for destruction by the antibody → the pathogen will be destroyed by the macrophages either through the complement cascade as already mentioned (lysis) or directly via antibody- dependent cellular phagocytosis.
Humoral immunity: activation of B lymphocytes, activation of T helper cells into T 2 helper and leading to B cell proliferation, maturation into plasma cells and leads to the production of antibodies.
Inflammatory response - adaptive immunity - step 7 - cytokines released
- Hormone like molecules called interleukins that act to regulate immune responses
- Over 100 identified -> IL1, IL2, IL4, IL5, IL6, IL8, IL10, IL11, IL12, TNF alpha, TNF beta, TGF beta, INF alpha, INF beta, INF gamma
IL1 – activates T cells and macrophages, induces synthesis of adhesion molecules on endothelium, up regulates iNOS and COX2, increases corticosteroid release, causes fever
IL2 – activates macrophages, NK cells, and lymphocytes
IL4 – activates lymphocytes and monocytes, stimulates IgE production
IL5 – causes differentiation of eosinophils
IL6 – activates lymphocytes and causes differentiation of B cells, stimulates production of acute phase proteins, causes fever
IL8 – causes chemotaxis of neutrophils, basophils and T cells
IL10 – anti-inflammatory
IL11 – stimulates production of acute phase proteins
IL12 – stimulates INFgamma production from TH1 cells and NK cells, induces TH1 cells
TNF alpha and beta – general pro-inflammation
TGF beta – causes immunosuppresion
INF alpha and beta – induces cells to resist viral infection
INF gamma – activates macrophages and inhibits TH2 cells
Inflammatory response - adaptive immunity - step 8 - cell mediated immunity
Triggered by endogenous antigen - the antigen is INSIDE the cell causing damage to the cell
Mediated through the cytotoxic T-cells - recognize MHC - I in infected / damaged cells by CD8.
T cytotoxic cells have TCR receptors - normally, they do not recognize the self antigen attached to the MCH - I (would lead to autoimmune disease), but when the cell is infected, the self antigen is modified and recognized by the TCR on the T cytotoxic cell.
Inflammatory response - adaptive immunity - step 9 - cell mediated immunity - T cytotoxic cells
Kills virus infected cells and neoplastic (cancerous) cells
When cells are infected by virus or are cancerous this happens:
- The virus can get integrated into the DNA → creates viral proteins that can get integrated into the self-peptide → leads to expression of viral antigen or cancer antigens on MHC-I complex
- MHC-1 complex of infected or cancerous cells interact with CD8 molecule on cytotoxic T cell
- The cancerous or viral antigen on infected or cancerous cells (on MHC - I) interact with TCR on cytotoxic T-cell - gets activated -> they release perforins and granzymes
Perforins -> creates pores in infected or cancerous cells
Granzymes - moves through the pores and activates pro-apoptotic genes, leading to cell apoptosis.
Inflammatory response - adaptive immunity - step 10 - Natural Killer cells
- They are NOT a part of the adaptive/acquired immunity, but the mechanism of action is very similar to cytotoxic T- cells
- NK cells are large agranular lymphocytes
- The activated natural killer cells release perforins and granzymes which trigger apoptosis of viral infected cells
They can kill by 3 ways:
- Absent MHC-I expressed on surface
- Different MHC-I expressed on surface (MICA)
- Through IgG mechanism
Inflammatory response - adaptive immunity - step 11 - Natural Killer cells
1) Absent MHC I molecule expressed on the surface
- All nucleated cells must have MHC I molecule expressed on the surface
- If a viral pathogen infects tissue cells -> virus induces abnormal MHC I complex or inhibits MHC I formation.
- This foreign MHC I or Absent MHC I due to viral infection → activates natural killer cells
2) Different type of MHC I expressed on the surface (MICA)
- MICA doesn’t contain beta 2 molecule (normal structure of MHC I molecule contains the alpha 1, alpha 2, alpha 3 and beta 2 subunits).
- This foreign MHC I like molecule activates natural killer cells
3) Through IgG mechanism
- If IgG antibodies made by plasma cells bind viral antigens expressed on MHC-I complex this allows natural killer cells to bind to Fc portion of IgG antibody via their CD-16 protein this activates the natural killer cells -> then they release perforins and granzymes and trigger apoptosis.
Hypersensitivity reactions
Mediated by immunoglobulins binding to antigens and then effector cells binding to that immune complex.
- Type I -> Immediate reaction within 15-30 minutes usually, but can be up to 12 hours post-exposure to antigen. IgE mediated
- Type II -> Reaction within minutes to hours. IgG or IgM mediated
- Type III -> Reaction up to 10 hours post-exposure. IgG or IgM mediated with complement involved
- Type IV -> Delayed reaction typically 2-3 days post-exposure, but can be longer. T cell mediated
Autoimmune disease
Loss of immune tolerance resulting in destruction of body’s own tissues
- Alloimmune reactions -> antibody from member of same species reacts with antigens on affected individual’s cells: transfusion reaction, transplant reaction
- Primary reaction: antibody directed against a self antigen
- Secondary reaction: antibody directed against absorbed antigen on cell surface
Drugs that affect the immune system (more in pharmacology section, this is only an outline)
- Glucocorticoids -> decrease circulating T lymphocytes, inhibit neutrophil and monocyte chemotaxis and phagocytosis, inhibit cytokines, decrease mast cell numbers and histamine synthesis
- Cyclosporine -> inhibits T lymphocyte growth (primarily T helper cells), inhibits cytokines (IL2)
- Azothiaprine -> antagonizes purine metabolism inhibiting RNA and DNA synthesis and mitosis, inhibits coenzyme formation
- Cyclophosphamide -> alkylating agent inhibits RNA and DNA synthesis, phosphorylating activity causes cytotoxicity, exact immunosuppressive mechanism of action not known
- Mycophenolate -> non-competitive reversible inhibition of inosine monophosphate dehydrogenase (required for purine synthesis in T and B cells)
Future directions of immunology
1) Microparticles
- Membrane derived exocytic vesicles formed when there is disruption of phospholipid asymmetry
* Phosphatidylserine (PS) and phosphatidylethanolamine (PE)
* Phosphatidylcholine and sphingomyelin
- Produced by -> platelets, endothelial cells, erythrocytes, neutrophils, monocytes and lymphocytes
- Contain membrane, cytosolic and nuclear components of their cell of origin
- Associated with pro-inflammatory and procoagulant effects -> targeting these effects can be a novel therapeutic option for many disease states
- Serve as biomarkers of vascular injury and inflammation
2) Targeted immunosuppressives -> research into medications that suppress specific cytokines and proteins within the inflammatory cascade are ongoing
3) Inflammation in Sepsis -> research is more fully elucidating the complex pathways and roles of inflammatory mediators in sepsis
- Regulatory T cells have recently been found to play a larger role in the immune dysfunction seen with sepsis
- Loss of balance between the inflammatory response in sepsis and the compensatory anti-inflammatory response is being investigated
Four mechanisms of neutrophilia
- Increased production (appropriate)
- Increased production (inappropriate)
- Demargination
- Decrease egress from circulation
Where are the storage pool of mature neutrophils in dogs and cats?
Bone marrow and marginated neutrophils along endothelial walls - both serve as buffer for fluctuations in peripheral demand.
T/F - early stages of inflammation in dogs are most commonly associated with neutrophilia that is independent of bone marrow production
TRUE - dogs have up to a 5 days supply of neutrophils in the bone marrow storage pool (based on normal rates of utilization). If the inflammatory nidus is acute and fulminant with rapid depletion of bone marrow neutrophil stores prior to any regeneration, neutropenia may ensue.
What occurs if the mature stored neutrophils are not enough to control the inflammatory stimulus?
De novo generation of neutrophils is required - occurs via myeloid progenitor cell proliferation in the bone marrow - complex response known as emergency granulopoiesis.
Major granulopoetic cytokines and growth factors involved in emergency granulopoiesis
G-CSF (granulocyte - colony stimulating factor)
GM-CSF (granulocyte monocyte - colony stimulating factor)
IL3
IL6
FLT3 ligand
What is a left shift
To produce new neutrophils takes time
To tide over the lag period between the need of neutrophils and releasing new ones -> previously produced band neutrophils or even earlier granulocytic precursors are released into circulation -> left shift.
How can we monitor progression of the neutrophil response?
With serial monitoring every 24-48h due to their short half-life (6-8h) and the dynamic interplay between production and consumption.
What is a degenerative left shift (DLS)
When granulocytic precursors outnumber mature neutrophils in circulation.
It has been found to increase the risk of death or euthanasia in hospitalized dogs and cats.
In cats, not only the presence but the severity of the DLS (ratio of mature to immature neutrophils, N/I index), predicts a higher risk of mortality.
What is a leukemia response
Marked leukocytosis due to a strong inflammatory stimulus that can mimic a neoplastic response.
Dogs and cats with extreme neutrophilia secondary to neoplasia were more likely to die than other etiologies.
Neutrophils - toxic changes
- Signs of dysplasia, associated with increased granulopoiesis and inflammation
- Changes are mostly in cytoplasm -> presence of Dohle bodies, increased cytoplasmic basophilia, vacuolation of the cytoplasm and rarely, the presence of primary granules.
- Giantism of neutrophils and neutrophilic precursors is also a sign of toxicity.
- Can be helpful in differentiating cases of inflammation (toxic changes often present) from steroids/stress and epinephrine response (toxic changes not present).
- Presence of toxic changes is associated with increased hospitalization time in both dogs and cats, and degree of toxicity is associated with risk of mortality in dogs.
Neutrophils - inappropriate increased production
- Rarely seen in dogs and cats
- Due to neoplasia:
- Primary in the bone marrow (granulocytic leukemia - very rare)
- Paraneoplastic response secondary to release of growth factors from a wide array of different neoplasms: HSA, mast cell tumors, thymoma, ovarian carcinoma, pulmonary adenocarcinoma.
Neutrophils - demargination
- In cats, approx. 70% of total blood neutrophils are in the marginated pool vs 50% in dogs.
- As they are marginated, not circulating, normally not part of the measured neutrophil concentration - demmargination can dramatically increase the neutrophil concentration -> this is seen in response to corticosteroids and epinephrine.
Neutrophils - demargination - steroids response
- Stress leukogram
- Both endogenous and exogenous steroids can cause demargination of neutrophils and decreased transit into tissues.
- Magnitude of neutrophilia - 15K - 25K cells / uL in dogs and cats.
- Maximal response within 8h
- Count goes back to normal in 24h if no more steroids are administered
- Steroids - hallmark is lymphopenia - peripheral lymphocyte count is low due to redistribution.
- Lymphopenia faster than neutrophilia - seen within in 4h, and takes longer to resolve.
Neutrophils - demargination - epinephrine
- Release of epinephrine with excitement causes marginated neutrophils to be released into circulation.
- Effect is seen immediately and resolves within 30min.
- Epinephrine normally results in a lymphocytosis, secondary to mobilization from the thoracic duct and blockage of entry into lymphoid organs.
Neutrophils - decreased transit into tissues
- Most important cause is corticosteroids
- Irish setters - genetic disorder, leukocyte adhesion deficiency (LAD) - neutrophils lack surface receptors (CD18) to be able to bind to endothelial cells therefore they cannot enter the tissues and they accumulate in blood.
- Extremely high neutrophil concentrations occur (50,000 - 100,000+ cells /uL) and many hypersegmented neutrophils may be seen.
- Animals are susceptible to infections and die young.
Neutrophils - neoplasia - chronic lymphocytic leukemia (CCL)
- Circulating neoplastic cells can occasionally cause leukocytosis.
- Middle-aged to older dogs (less common in cats)
- Marked leukocytosis - 15K to 1600K cells / uL
- Neoplastic cells: small, mature lymphocytes
- Indolent and slowly progressive
Neutrophils - neoplasia - acute leukemias
- Associated with large, immature circulating cells.
- Aggressive, high-grade malignancies.
- Grave prognosis.
- Associated with concurrent cytopenias.
- Machines do not identify reliably neoplastic cells - blood smear
Increases in other than neutrophil cell lines
Can frequently give clues to underlying disease processes.
Treatment of neutrophilia
- Address underlying condition.
- Magnitude of neutrophilia often correlates with the severity of the disease process.
- Monoclonal therapy against neutrophils - tested in lab animals
- Induces a leukocyte adhesion deficiency syndrome that may impose a risk for exacerbating sepsis.
- Argument against this therapies - alterations in neutrophil morphology, mechanics and motility - worse outcomes noted in septic patients with larger and less granular neutrophils.
Mechanism of neutropenia
- Decreased production by bone marrow
- Increased utilization
- Increased destruction
Neutrophils - decreased production by the bone marrow
- Non-bacterial infectious diseases the most common etiology in dogs and cats
- These agents most likely target bone marrow precursors
- Dogs -> parvovirus accounted for 53% cases of neutropenia - increasing severity of leukopenia associated with increased mortality vs a WBC > 4.5x10^3/uL at 24h post admission - positive predictive value for survival of 100%.
- Cats -> FIV / FeLV accounted for 10% and 24% of neutropenic patients. Feline panleukopenia - also a potent cause of neutropenia, similar to parvo, increased severity of neutropenia associated with increased risk of mortality.
- Other diseases: neoplasia, necrosis, myelofibrosis, RT and drug toxicity.
- When is due to drugs / toxins - many are accompanied by anemia, thrombocytopenia or both.
Neutrophils - drugs, hormones and toxins associated with neutropenia in dogs and cats
Neutropenia - increased utilization
Neutropenia secondary to increased demand from marked inflammation, bacterial sepsis or endotoxemia accounted for 17% of feline cases and 10% of canine cases.
Early stages of sepsis can be associated with neutropenia:
* Experimental model of endotoxin mediated sepsis in dogs - severe leukopenia observed 3h after induction of sepsis. * Leukopenia is due to neutropenia - lymphocytes and monocytes stay constant * By 24h - rebound leukocytosis secondary to neutrophilia seen. * Neutropenia suspected due to transient margination of neutrophils secondary to endotoxin-mediated up regulation of adhesion molecules on neutrophils.
Neutropenia - destruction
Immune-mediated destruction of neutrophils is rarely seen in dogs, even more rare in cats.
Dogs with corticosteroid-responsive idiopathic neutropenia - young (<4y) and lower neutrophils compared to patients with known causes of neutropenia.
Lymphopenia
- Can be severe enough to cause mild leukopenia
- Most cases is due to endogenous or exogenous steroids - hallmark of stress leukogram
- Other causes -> lymph losing diseases, viral dz (including parvo) and other infectious dz.
Treatment of neutropenia
- Treat underlying condition
- Most vets - do not initiate treatment until neutrophils are < 2500 cells / uL
- Human guidelines - propensity to develop an infection is significant when neutrophils
< 1000 cells / uL. - Goal of management - prevent infection.
- Febrile neutropenia secondary to chemotherapy -> patients expected to have a short nadir in neutrophil concentration may be easily managed as outpatients.
- Therapy with atb: based on degree of fever, magnitude of leukopenia / neutropenia and patient characteristics. Important information:
- Has the patient been previously on antibiotics?
- Results of a culture?
- What pathogen is most likely?
- What are the regional susceptibility patterns?
- Broad spectrum atb should be initiated in all febrile neutropenic patients - potentiated penicillin or fluoroquinolone.
- If patient has signs of SIRS or sepsis -> combination of amino glycoside with a B-lactam antimicrobial is preferred - change based on culture and susceptibility when available.
- Hospitalization vs outpatient - weight risks.
- G-CSF has been recommended to aid in the earlier increase of neutrophils from bone marrow - potential to produce more leukocytes - study in parvo ineffective in increasing neutrophils and improving survival.
- Canine recombinant G-CSF - shown to improve neutrophil concentrations in neutropenic patients - option when available?
Classification of anemia
- Regenerative - blood loss or hemolysis. Acute development of anemia will appear non-regenerative due to insufficient time elapsed to see a regenerative response.
- non-regenerative - ineffective erythropoiesis (primary bone marrow pathology or systemic disease causing bone marrow suppression).
Erythropoiesis
- Pluripotent hematopoietic step cells in the bone marrow give rise to all adult stages of erythrocytes, leukocytes and platelets.
- Red cell line undergoes several mitoses -> each successive cell division is smaller than the previous one, with increasing hemoglobin content.
- Once achieving a critical hemoglobin concentration, reticulocytes are released into the peripheral circulation via diapedesis.
- Within 48h any residual basophilic material in the reticulocyte is lost, forming mature erythrocytes.
Erythropoietin
- Glycoprotein produced by kidneys in response to hypoxia.
- Primary regulator of erythropoiesis.
- Other substances like thyroxine or cortisol can augment erythropoiesis.
- Sufficient iron stores are essential for Hb function.
- Life-spam: erythrocytes persist for approximately 104 days in the periphery of the dog and 73 in the cat.
- Aged erythrocytes - cleared by erythrophagocytic system.
Pathophysiological response to non-regenerative anemia
- Symptoms are non-specific and milder than with regenerative anemia.
- Chronic anemia has some degree of physiological compensation.
- Increased CO via NO mediated vasodilation
- Rheological changes promoting lower systemic vascular resistance.
- Down side of this relative hypovolemia - increased sympathetic discharge and RAAS activity, risking hypervolemia and cardiac remodeling over time.
- Volume overload is considered a risk in chronic anemia, especially in cats.
- Compensatory increases in synthesis of 2,3-DPG with a right shift of the oxygen dissociation curve can also occur in dogs.
What is erythron
All mature and immature erythrocyte stages.
PCV with estimation of TS is extremely valuable.
Hematocrit can be calculated from the RBC count and mean cell volume (MCV) from automated machines - higher error than with manual PCV.
Why are EDTA anti coagulated blood samples preferred to run hematology
Due to superior leukocyte staining.
Anemia cut off value
Depends on institution, but normally dogs are considered anemic when PCV <35% and cats, <30%.
Expected characteristics of regenerative and non-regenerative anemia
Characteristics of a blood smear (Romanowsky, AKA Diff-Quick stain) with signs of regeneration
- Polychromasia
- Anysocytosis
- Nucleated red cells
- Polychromatophil -> recitulocytes that are basophilic due to the staining of their nuclear material.
Gold standard stain to assess regeneration
Methylene blue.
How many varieties of reticulocytes cats have?
Two: aggregate and punctate.
Aggregate - used to assess regeneration - shorter lived than punctate.
What else other than signs of regeneration can we assess in a blood smear?
Presence of parasites
Schisctocytosis
Spherocytosis
Erythrophagocytosis
What other information in the hematology can we use to characterize anemias?
MCV - average erythrocyte size
MCHC - hemoglobin content.
MCV/MCHC in anemias
- Non-regenerative: normocytic normochromic (normal MCV and MCHC)
- Regenerative - classically considered macrocytic (high MCV) and hypochromic (low MCHC).
- Iron deficiency - microcytic (low MCV) and hypochromic (low MCHC). - most common reason for iron deficiency is chronic blood loss, typically from GI, that becomes non-regenerative over time.
- Diagnostic accuracy of MCV and MCHC to identify regeneration - 70% and 66% respectively.
- Other diseases can result in macrocytosis (FeLV) and microcytosis (PSS), which limits the sensitivity of MCV as the sole indicator of regeneration.
RDW and anemia
- Indicates variation in erythrocyte size -> regenerative anemia associated with high RDW, non regenerative with a low RDW.
- Helps identify regeneration in dogs - reported diagnostic accuracy of 69%.
- Combination of RDW and polychromatic improved diagnostic performance to 79%.
Anemia and other cell lines
- Other cell lines should also be assessed.
- Pancytopenia - suggests primary BM pathology.
- Preservation of other cells can be bone marrow or extramarrow disease.
Why cytology and biopsy of bone marrow should always be submitted concurrently?
- To characterize the pathology as accurate as possible.
- Cytology could say low-cellularity -> could be a non-diagnostic sample or a truly hypoplastic process.
- CBC should also be submitted along with the bone marrow sample to guide interpretation.
Bone marrow sampling
- Animals should be deeply sedated or anesthetized, with analgesia.
- Anatomical locations - proximal humerus, iliac crest and trochanteric fossa of the femur are some possible locations.
- Sternal marrow aspiration using hypodermic needles has been described in beagles -> provided good-quality diagnostic samples and associated with lower pain scores.
- bone marrow needle depends on patient size -> generally Illinois sternal needles are preferred for smaller patients, and Jamshidi for larger.
How much volume is needed for a bone marrow aspirate?
- Small, <0.5mL
- Larger samples -> risk of hemodilution
- Transfer it promptly to glass slides -> should contain visible spicules of bone marrow.
- Same procedure repeated to take a core biopsy but removing the stylet.
T/F - Anemia of primary bone marrow disorders produce a less severe anemia than extra marrow suppression
FALSE - produces a MORE severe anemia.
What should we suspicious off when there is bicytopenia or pancytopenia?
- Of aplastic anemia, where hematopoietic marrow space is replaced with adipose tissue.
- Neutrophils have the shortest half-life, neutropenia might be evident within 5 days, followed by thrombocytopenia after 8-10 days, and then anemia weeks later.
Causes of pancytopenia (some examples)
- Idiopathic
- Infections (E. canis, parvovirus)
- Toxins / drugs (azathioprine, procarbazine, estrogens, phenylbutazone)
- Immune-mediated diseases.
Cats:
- Idiopathic
- FeLV
- Renal disease
- Drug reactions
Guarded prognosis.
Is bone marrow transplant performed in vetmed?
Not routinely. It could offer a cure.
Examples of 2 variants of immune-mediated erythrocyte precursor destruction
Pure red cell aplasia (PRCA) and Non regenerative IMHA (NRIMHA)
- WBCs and PLT are unaffected.
- Absence of all red cell precursors on bone marrow = PRCA
- NRIMHA = destruction of later stages of erythrocyte development.
- NRIMHA vs peripheral IMHA -> spherocytosis is uncommon, icterus not observed and Cooms positivity is rare with NRIMHA.
- Both managed with long-term immunosuppressive agents.
- Response to therapy between 61% to 77% in PRCA, 88% of cats alive 60 days after dx.
- Time to recovery is slow in both dogs and cats
- Potential for need of >1 blood transfusion.
- In contrast to IMHA - thrombosis is rare and thromboprophylaxis not routinely used.
- Secondary PRCA and NRIMHA - described and associated with FeLV in cats, parvovirus in dogs, adverse effects of human recombinant erythropoietin therapy and estrogen toxicity.
Non regenerative anemia and neoplasia
- Should always be considered.
- Leukemia involves the neoplastic proliferation of one hematopoietic cell line in the marrow, with secondary suppression of the other cell lines - anemia develops later than neutropenia and thrombocytopenia.
- Disseminated lymphoma, malignant histiocytoma (MH) - both can cause nr anemia.
- MH can cause regenerative anemia too due to erythrophagocytosis by malignant histiocytes.
- Myelodysplasic syndromes - dysplasic rather than neoplastic hematopoietic precursor development.
Secondary extramarrow disorders
- Anemia or renal disease is common - likely multifactorial.
- EPO deficiency considered the major cause.
- Increased concentrations of cytokines in uremia may also inhibit erythropoiesis.
- Functional iron deficiency has been reported in cats with renal disease.
- Uremic gastropathy causing ulceration - uncommon cause of anemia in dogs and cats.
- Thyroxine augments the effects of EPO - mild NR anemia may be seen in hypothyroid dogs.
- Some dogs with Addison’s - anemia -> supports role of cortisol in erythropoiesis - inconsistent finding. Concurrent GI bleed could contribute to the anemia.
- Anemia of inflammatory disease (AKA anemia of chronic disease) - usually mild, normocytic and normochromic.
- IL1, IL6 and TNF alpha - may result in up regulation of acute phase protein hepcidin -> promotes a state of relative iron deficiency, blunting erythropoiesis and promoting a low-grade anemia.
Therapies for NR anemia
- Blood products and hemoglobin oxygen-carrying solutions may be required in animals with hemodynamic compromise due to their anemia.
- Disease-specific considerations -> immunosuppression, exogenous EPO…
- Anabolic steroids (nandrolone) may be considered in chronic NR anemia, especially of renal disease - proven efficacy lacking in small animals.
- Lithium - psychiatric medication -> could have applications for aplastic pancytopenia - no data available in vetmed.
What is an hemolytic anemia?
- Anemia that results from increased erythrocyte destruction and shortened red blood cell lifespan.
- Usually extravascular, with macrophages erythrocyte destruction in spleen, liver and bone marrow. Splenomegaly frequently present.
- Intravascular less common, leads to hemoglobinemia and hemoglobinuria.
- With either, the rate of erythrocyte destruction can exceed hepatic clearance, resulting in hyperbilirrubinemia and icterus.
Clinical signs, history details relevant to hemolytic anemias and PE findings
- Lethargy, weakens, exercise intolerance and inappetence. Some might have pigmenturia.
- Hx: details on medications, diet, travel and past illnesses. Dietary indiscretion (onions, zinc) may result in hemolysis. Travels to certain locations - possibility of tick-borne disease or erythroparasites.
- PE - tachycardia, tachypnea, cardiac murmur, gallop sounds, snappy pulses, pallor, jaundice, splenomegaly and pigmentaruria.
Hemolytic anemia - CBC
- Anemia - confirmed via decreased PCV / Ht / Hb.
- Reticulocyte count is essential - hemolytic anemia are almost always regenerative - absolute reticulocyte count >60K/uL in the dog, or aggregate reticulocyte count > 50k/uL in the cat.
- Can be NR anemia if: early in the disease process prior to BM response or if it targets the RBCs precursors.
- Always evaluate a blood smear and SAT - we can detect spherocytes, Heinz bodies, eccentrocytes, erythroparasites and schistocytes that might help our ddx.
DDX for hemolytic anemia (broad categories)
- Immune-mediated hemolytic anemia (IMHA)
- Alloimmune hemolytic anemia
- Oxidative hemolyisis
- Zinc & Copper toxicity
- Erythroparasites
- Inherited erythrocyte defects
- Microangiopathic hemolysis
- Hypophosphatemia
IMHA - causes
- Most common cause of hemolysis in dogs.
- Immune targeting of and damage to the erythrocyte membrane.
- Hemolysis is most commonly extravascular, but can be intravascular.
- Can be secondary to an inciting cause, or part of a systemic immune disease.
- If cause not discovered -> autoimmune hemolytic anemia (AIHA).
IMHA - overrepresented breeds
- American Cocker Spaniel
- Miniature Schnauzers
- English springer spaniels
- Old English sheepdogs
- Poodles
- Spayed females.
Less common in cats.
IMHA - diagnosis
- Demonstration of immune-mediated RBC destruction by one or more of the following:
- Presence of spherocytes on a blood smear
IMHA - diagnosis
- Demonstration of immune-mediated RBC destruction by one or more of the following:
- Presence of spherocytes on a blood smear
- Positive SAT
- Positive direct Coombs test
IMHA - what is a spherocyte
- Result from phagocytosis of a portion of the RBC membrane.
- Microcytic, spherical erythrocytes w/o central pallor
- Hard to distinguish from normal erythrocytes in cats - not reliable for IMHA in this species.
IMHA - saline agglutination test
- Assesses autoagglutination
- Drop of blood mixed with an equal or greater volume of 0.9% NaCl on a slide
- Macroagglutination can be grossly visualized
- Microagglutination should be assessed by the presence of rouleaux.
- True autoagglutination will persist after saline addition.
IMHA - direct Coombs
- Detects antibodies or complement on the RBCs surface.
- Sensitivity of 60% to 70 %.
IMHA - complementary diagnostics
- Depends on clinical findings and geographical location.
- CXRAY, AUS, UA, UC, serology / PCR for tick-borne pathogens.
IMHA and coagulation
- IMHA is thombophilic - incidence of thromboembolism is significant and a frequent cause of mortality.
- Assessment of hypercoagulability should be done via TEG.
- D-dimers - occur in dogs w/ IMHA, uncertain if reliable as indicator of hypercoagulability.
IMHA - treatment
- Immunosuppression to decrease the rate of erythrocyte destruction.
- Treat underlying disease if any.
- Prophylactic antithrombotics.
- pRBCs transfusion if necessary.
IMHA - treatment: immunosuppressive therapy.
- Corticosteroids the mainstay of immunosuppressive therapy.
- Prednisone or prednisolone at 1-2mg/kg q12h. Large dogs do not exceed 40-60mg/day.
- Cats: prednisolone preferred - results in higher plasma concentrations.
- If oral therapy cannot be tolerated - dexamethasone at 0.15 - 0.3 mg/kg IV / Sq q12h.
- Response seen within 2-10 days (stabilization or increase in RBCs. In NRIMHA it might take weeks to months.
- Continued at initial dose for 3-4 weeks - then slowly taper based on response to treatment. Faster tapering is possible if underlying condition is removed.
- Adjunctive immunosuppressive therapy might be necessary - azathioprine, mycophenolate, cyclosporin, human IVIg, cyclophosphamide, danazol and leflunomide. Cyclophosphamide no longer recommended - no benefit or increased mortality in different studies.
IMHA - antithrombotic therapy
- Optimal therapy not established yet.
- A study in dogs with IMHA suggested improved survival when ultra-low-dose aspirin (0.5-1mg/kg q24h) was included.
- One study showed improved survival with individually adjusted dose of UF heparin based on anti-FXa activity vs fixed low-dose UF heparin administration.
IMHA - other treatments
- Splenectomy has been described as treatment option for patients that are non-responsive to drug therapy.
- Plasmapheresis has been described in one patient, but has to be further evaluated.
IMHA and prognosis
- Reported mortality - 40% to 70%
- Numerous negative prognostic indicators have been proposed - thrombocytopenia, tbil > 5mg/dL, increased BUN, presence of band neutrophils, prolonged PT/PTT, evidence of hypocoagulable state via TEG, hypoalbuminemia, increased ALT, increased ALP. Little agreement between studies.
Alloimmune hemolytic anemia
Occur with hemolytic transfusion reactions and neonatal isoerythrolysis
Neonatal isoerythrolysis
- Acute hemolysis that occurs in a neonate due to incompatibility between maternal and neonatal blood types.
- Usually seen in cats when a type A or type AB kitten is born to a type B queen.
- Does not occur in utero because cats have endotheliochorial placentation.
- Queens type B secrete anti-type A antibodies in their colostrum and milk - can be absorbed by neonatal kittens for the first 16 hours of life.
- Kittens are born healthy but develop hemolysis at a few days of age, and rarely live more than 1 week.
- Can be avoided by typing queens and toms to ensure compatible coupling.
- if incompatible coupling - kittens born of a type B queen and type A tom, can be placed with a type A queen for the first 24h, or fed commercial milk for 24h and 1-3mL of serum from a type A cat can be administered PO/SQ to transfer passive immunity.
What happens to the RBCs with oxidative injury?
- Denaturation of hemoglobin resulting in Heinz body formation
- RBC membrane damage
- Oxidation of ferrous iron to the ferric form, resulting in methemoglobinemia.
Which species is more susceptible to oxidative injury?
Feline erythrocytes due to a greater number of reactive sulfhydryl groups an reduced ability to metabolize oxidative agents.
Oxidative hemolysis - Heinz bodies
They cause erythrocyte rigidity and resultant shortened lifespan.
Causes of HB formation:
- Allium species (onion, garlic)
- Methylene blue
- DL-methionine
- Phenacetin
- Compounds which also cause methemoglobinemia (acetaminophen, benzocaine and phenazopyridine)
- DKA, hyperthyroidism and lymphoma in cats can lead to HB formation.
- Commercial foods and drug preparations with propylene glycol are associated with HB formation, but do not generally result in anemia.
Oxidative hemolysis - Heinz body anemia - how can we best visualize the HBs?
Best identified in a blood smear with new methylene blue staining
Oxidative hemolysis - HB anemia - how can we diagnose it?
Dx of HB anemia requires a history of exposure and visualization of HBs in many erythrocytes, together with evidence of hemolysis.
Oxidative hemolysis - HB anemia - treatment?
- Removal of oxidant agent
- Antioxidant therapy (NAC or SAMe)
- Supportive care
Hemolysis - zinc toxicity
- From ingestion of zinc-containing objects: US pennies minted after 1982, bolts screws or topical skin protectants.
- Severe intravascular hemolysis, GI signs and occasionally, pancreatitis.
- Should be suspected in any patient with acute hemolytic anemia in the absence of autoagglutiation - AXRAY!
Give an example of a erythroenzymopathy, describe briefly the pathophysiology
Pyruvate kinase (PK) deficiency PK –> typically has a chronic course, with mild clinical signs. Dogs, but not cats, with PK deficiency frequently develop myelofibrosis and osteosclerosis, and will usually die from hemosiderosis-induced hepatic failure or bone marrow failure
Breeds: basenjis, beagles, dachshunds, toy Eskimos, West Highland white terriers, and cairn terriers and in cats
Phosphofructokinase (PFK) deficiency –> characterized by chronic, well-compensated hemolysis with acute bouts of hemolytic anemia associ- ated with exertion. The hyperventilation and alkalemia that accompany exertion or stress result in accelerated hemolysis of the alkaline-fragile erythrocytes.
Breeds: cocker spaniels and English springer spaniels
Which approach/es would you take before trying to start a third immunosuppresive drug or peform an splenectomy on a IMHA patient?
- Evaluate for trigger factors
- Perform Therapeutic drug monitoring (TDM)
- IVIG
Always consider:
*Diagnostic confidence
* Drug dose
* Client compliance
*Poor GI absorption
*Administration with food
List the paraneoplastic syndromes
- Hypercalcemia of malignancy
- hypoglycemia
- Polycytemia
- anemia
- thrombocytopenia
- coagulopathy/DIC
- Hypertrophic osteopathy
- Fever
List exogenous pyrogens
List medications that you could consider for treatment for anaphylaxis with their respective dose
