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237 Cards in this Set

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Where microbes live in your body
A. Microbes can live either extracellulary or intracellularly
Where microbes live in your body Extracellularly
i. the interstitial space, blood or lymph
ii. Epithelial cells, especially of the gut
what organisms live in the intestitial space blood or lymph
viruses, bacteria, protozoa, fungi, or worms
What immunity do we have against microbes that live in your body Extracellularly? How do they work against the microbes?
antibodies, complement, phagocytosis, and neutralization

Antibodies bind to the microbe and cause destruction by having white blood cells phagocytose the microbe and consequently destroy it.
Complement molecules can be activated by antibodies and cause lysis of the microbe. Opsimization by complement makes microbes more recognizable to be phagocytosed.
Some microbes excrete exotoxin. Antibodies can bind to exotoxin and neutralize it.
What organisms live in you gut?
1. such organisms are gonorrhea, worms, strep, cholera, ecoli, and heliobactor
How do you fight off organisms that live in you gut?
2. these organisms are delt with by the antibodies called IgA and antimicrobial peptides
a. IgA antibodies are produced by the GI track
b. Defencins also neutralize microbes on epithelial cells
Where can intracellular microbes live?
i. Can live in the cytoplasm
ii. Microbes intracellularly can also live vesicularlym ( in a compartement in the cell)
What are some intracellular microbes that live in the cytoplasm?
1. these microbes include viruses, chlamydia, rickets, listeria, and protozoa. They are part of the life cycle of the cell.
How do you destroy intracellular microbes that live in the cytoplasm?
2. To destroy microbes here you need cytotoxic T cells and natural killer (NK) cells
a. NK cells usually respond first and kill the infected cell before the cytotoxic T cells
What are some intracellular microbes that live vesiculary?
1. these include salmonella and yersinia pestis
How do you get rid of intracellular microbes that live vesiculary?
2. The immunity needed here are T cell and NK cell dependant macrophage activation. Specifically inflammatory T cells allow for the increased ability of the cell to kill the organism in the vesicle.
Ways bacteria can hurt
A. directly
a. exotoxin production. Exotoxins are harmful proteins
c. many viruses cause direct cytopathic effects (like cell lysis)
B. Indirect ways for tissue damage by pathogens
a. Immune complexes
b. Anti-host antibody
c. Cell mediated immunity
what are exotoxins? Give two examples
Exotoxins are harmful proteins that are produced by bacteria and can harm us directly.
i. in dipheria, not only does a pseudo membrane form in the through, but an exotoxin produced by the bacteria can cause heart failure
ii. in tetnus, the exotoxin interferes with neurons causing the patient to go into rigor. When lungs go into rigor the patient dies.
b. endotoxins are usually lipopolysacharides LPS that usually come from the walls of gram negative bacterium.
i. Gram negative means that they look pink when stained with the gram stain. These type of bacterium have more lipid in their cell wall. They cause a very powerful rxn called gram-negative sepsis that causes the circulatory system to collapse.
ii. Gram stain: 1st bacteria are stained with a purple stain that all bacteria take up. Then acetone is added. Acetone dissolves lipids and fat cell walls. Subsequently the purple stain is washed out from bacteria with a lot of lipid in their cell wall. Then a red stain is added making gram-negative bacteria red/pink.
Where are exotoxins from?
are usually lipopolysacharides LPS that usually come from the walls of gram negative bacterium.
What constitutes a gram negative vs gram positive bacteria?
i. Gram negative means that they look pink when stained with the gram stain. These type of bacterium have more lipid in their cell wall. They cause a very powerful rxn called gram-negative sepsis that causes the circulatory system to collapse.
ii. Gram stain: 1st bacteria are stained with a purple stain that all bacteria take up. Then acetone is added. Acetone dissolves lipids and fat cell walls. Subsequently the purple stain is washed out from bacteria with a lot of lipid in their cell wall. Then a red stain is added making gram-negative bacteria red/pink.
What are immune complexes?
in response to a pathogen a LOT of antibody-antigen complexes form and lead to complement activation. These large complexes can cause the glomeruli in the kidney to burst. (glomerulonephris)

an indirect way pathogens cause tissue damage
what is an anti-host antibody
an indirect way pathogens can cause tissue damage

an antibody can sometimes by chance also bind with a native host cell/molecule/body part and mark it for destruction.
i. An example is when strep leads to rheumatic fever in which the antibody reacts with the mitral valve of the heart and the patient gets a heart murmur.
How can cell mediated immunity hurt you?
an indirect way pathogens cause tissue damage

bystander cells are hurt by the immune response.
i. An example is tuberculosis when inflammation of infected lung cells hurt bystander cells and cause destruction of the lung
How organisms increase virulence
A. Pilus establishes infection by attaching the microbe to the tissue/cell and makes microbe hard to get rid of.
B. Capsule is a huge layer of sugars on a microbe that makes it hard to recognize by the immune system because sugars don’t cause a huge immune response.
C. Leukocidin (like in staph) kills cells that try to destroy the bacteria (phagocytic leukocytes)
D. Collagenase helps spread the bacterium from the initial infection site by breaking down collagen. (bone, skin and cartilage, in general ECM) causing gangrene
E. Fibrinolysin (streptokinase) helps spread the microbe from the initial infection site by dissolving fibrin clots.
a. Normally, an inflammation response produced clotting to stop the infection. Fibrinolysin breaks down the clot and allows the bacterium to spread.
b. In the 70s fibrinolysin was given to heart attack patients to break down clots. Now tissue plasminogen activator does the same thing.
what is a pilus?
A. Pilus establishes infection by attaching the microbe to the tissue/cell and makes microbe hard to get rid of.
what is a capsul?
B. Capsule is a huge layer of sugars on a microbe that makes it hard to recognize by the immune system because sugars don’t cause a huge immune response.
What is leukocidin
C. Leukocidin (like in staph) kills cells that try to destroy the bacteria (phagocytic leukocytes)
what is Collagenase
D. Collagenase helps spread the bacterium from the initial infection site by breaking down collagen. (bone, skin and cartilage, in general ECM) causing gangrene
what is fibrinolysin also called streptokinase
E. Fibrinolysin (streptokinase) helps spread the microbe from the initial infection site by dissolving fibrin clots.
a. Normally, an inflammation response produced clotting to stop the infection. Fibrinolysin breaks down the clot and allows the bacterium to spread.
b. In the 70s fibrinolysin was given to heart attack patients to break down clots. Now tissue plasminogen activator does the same thing.
What are the characteristics of innate immunity?
A. Is inherited. We share it with other organisms. Most components of innate imunity are present before the onset of infection and constitute a set of disease-resistant mechanisms that are not specific to a particular pathogen, but include cellular and molecular components that recognize classes of molecules particular to frequently encountered pathogens.
B. It is immediate, nonspecific and never learns from experience.
C. It deals with situations where white cells have PRRs (protein recognition receptors) that recognize PAMPs (Pathogen associated molecular/membrane patterns) which are on the microbe.
a. It recognizes PAMPs that are on many different organisms (are not unique) but not in our own body.
what are PRRs
protein recognition receptors

a part of innate immunity

they recognize PAMPs
what are PAMPS
Pathogen associated molecular/membrane patterns are on the microbe.

They are recognized by PRRs of the innate immune system
What are the characteristics of adaptive immunity?
A. Is very specific. It recognizes unique markers on microbes
B. Takes longer than the innate IS to get activated
C. It has a memory. Each time you encounter a microbe it has a stronger and faster response.
Cells of the Immune System
A. neutrophils are cells of only the innate IS
a. They have PRRs that recognize PAMPs. They have reactive oxygenizing proteins that disable microbes. They are phagocytotic.
B. macrophages are part of both the innate and adaptive IS
a. they are also phagocytotic, help case inflammation response, help upregulate the adaptive immune response, also have oxidizing agents and complement proteins.
C. Dendritic cells are part of both the innate and adaptive IS
a. They are even better at activating the adaptive IR. They are called langerihan cells when they are younger and are phagocitic at this point. When they mature and are able to induce the AIS they are no longer phagocytic. They are also antigen presenting cells and produce interferon and cytokinines.
D. Natural Killer Cells (NK) cells are mostly part of the innate immune system
a. They use a complicated recognition system and are responsible for lysis of viral-infected cells
b. They produce interferon (gamma interferon) which activates macrophages
neurophils
are cells of only the innate IS
a. They have PRRs that recognize PAMPs. They have reactive oxygenizing proteins that disable microbes. They are phagocytotic.
B. macrophages
are part of both the innate and adaptive IS
a. they are also phagocytotic, help case inflammation response, help upregulate the adaptive immune response, also have oxidizing agents and complement proteins.
C. Dendritic cells
are part of both the innate and adaptive IS
a. They are even better at activating the adaptive IR. They are called langerihan cells when they are younger and are phagocitic at this point. When they mature and are able to induce the AIS they are no longer phagocytic. They are also antigen presenting cells and produce interferon and cytokinines.
D. Natural Killer Cells (NK) cells
are mostly part of the innate immune system
a. They use a complicated recognition system and are responsible for lysis of viral-infected cells
b. They produce interferon (gamma interferon) which activates macrophages
What are the features of inflammation
Inflammation is characterized by redness, swelling, pain and heat. The innate IR causes dilation of blood vessles that become leaky, then swollen with fluid hot and red and stimulates pain fibers.
Once phogocytosis of a microbe occurs how does the cell kill th e microbe?
oxygenation
I. Innate immune response molecules
a. Defensin
b. Collectins
a. Defensin
an innate immune response molecule

a. Defensin has repeating cysteine units. It is made by macrophages and Paneth cells. They are produced in respose to a phagocytic episode. They make holes in bacteria that have phagocytized or can be released by phagocytic cells extracellularly to act on bacteria that have not yet been phagocytized.
i. Paneth cells are like neutraphils. They are found in the intestinal track
what are panneth cells?
aneth cells are like neutraphils. They are found in the intestinal track. they make defensin.
b. Collectins
b. Collectins are protein molecules that have a collagen configuration and have a moity (lectins) that can bind sugars.
i. Mannose binding lectins (MBL) recognize mannose on microbes and make those microbes more easy to be phagosatized. It is also a soluble PRR (MBL)that recognizes PAMPs (mannose in this case).
What is MBL? How does it generally work?
i. Mannose binding lectins (MBL) recognize mannose on microbes and make those microbes more easy to be phagosatized. It is also a soluble PRR (MBL)that recognizes PAMPs (mannose in this case).
ii. MBL can be produced by the liver. When microphages eatup bacteria they produce Interleuken I Beta. IL I travels to the liver and induces it to produce a lot of soluble acute phase proteins (APPs). MBL is a type of APP. MBL in turn recognizes mannose, which makes the microbe easily recognizable for other phagocytes.
1. Interleukin is a molecule that is produced by white blood cells and has an effect on other white cells.
what is an interleukin?
1. Interleukin is a molecule that is produced by white blood cells and has an effect on other white cells.
What is an APP? what are some APPs?
acute phase proteins
Mannose n=binding lectin is an app.
i. C reactive protein (CRP) – has 5 repeating units and is therefore a member of the pentraxin family of molecules. CRP binds to choline on prokaryotes. It is a primitive antibody. It produces phagocytosis.
what is CRP?
an app

i. C reactive protein (CRP) – has 5 repeating units and is therefore a member of the pentraxin family of molecules. CRP binds to choline on prokaryotes. It is a primitive antibody. It produces phagocytosis.
what part of the IS produces tumor necrosis factor alpha and Interferon?
d. Macrophages
what is TNFα and what does it do>
tumor necrosis factor alpha produces a profound inflammatory response. Also called cachectin.
1. Cacgectin or TNFα causes cachexia- or muscle wasting (especially in cancer patients) Why?
a. TNFα is a powerful inducer of APPs. APPs are made of amino acids. Muscles are a huge store of aa. TNFα can shunt aa from muscles to liver to make the needed APPs. In chronic inflammatory response causes problems but works well in acute inflammatory response.
what is interferon alpha? what produces it? what does it do?
ii. Macrophages also produce interferon α
1. Interferon α interferes with the replication of a virus so it doesn’t spread and induces NKC which kill virus infected cells.
What are TLRs?
e. Toll like Receptors TLRs are a kind of PRR
i. They are similar to toll receptors in fruit flies
1. in flies they allow normal embryonic development esp of the dorsal ventral area.
2. They also are involver with preventing fungal infection
ii. In vertebrates TLRs are involved in the innate host defense (located on macrophages)
iv. There are many TLRs. Each one recognizes same PAMP.
1. TLR4 that binds to LPS (endotoxins from gram negative bacteria) and leads to the activation of the cell.
How do TLRs work?
ii. In vertebrates TLRs are involved in the innate host defense (located on macrophages)
1. They are on the surface of the cell and bind to an infectious agent. A transmembrane protein.
2. Through a signal transduction pathway they activate:
a. Nuclear Factor kappa B (NFκB), a transcription factor, that travels to the nucleus and causes transcription of new genes. (like IL-I)
b. MAP kinases (Mitogen Activated Protein kinases) that phosphorylate transcription factors which enter the nucleus and also cause transcription of genes such as IK-I
What do TLRs look like?
iii. Look like a hook
1. exterior domain has leucine rich repeats
2. The interior domain or TIR domain has boxes that bind to different transcription factors and activate them upon binding with a loigand
f. soluble molecules of the innate immune system
i. lysozyme
ii. Interferon
iii. Complement
i. lysozyme
a hydrolytic enzyme found in tears and mucousal secretions that is able to cleave the peptioglycan layer of bacterial cell walls.
ii. Interferon
ii. Interferon is a group of proteins produced by virus-infected cells that has the ability to bind to nearby cells and induce a generalized anti-viral state.
iii. Complement
a group of serum proteins that circulate in an inactive state. Specific and nonspecific immuno mechanisms can convert the inactive form into an active state the has the ability to damage membranes of pathogenic organisms, either by destroying the pathogen or facilitating their clearance. (straddle the adaptive and inate immune system)
II. Adaptive Immune Response cells
a. Lymphocytes are found mostly in the AIR
what are the two type of lymphocytes?
i. B lymphocytes are produced in the bone marrow and produce antibodies
ii. T lymphocytes develop in the thymus gland
1. Tc (cytotoxic T cells) kill virally infected cells
2. T helper I cells help Tc cells become cytotixic and can become inflammatory T cells that produce many molecules (such as gamma interferon)
3. T helper II cells help T cells produce antibodies
1. Tc (cytotoxic T cells)
kill virally infected cells
2. T helper I
help Tc cells become cytotixic and can become inflammatory T cells that produce many molecules (such as gamma interferon)
3. T helper II cells
help B cells produce antibodies
Titer
amount of antibody
b. Adaptive immune response kinetics
i. Response gets stronger and faster upon repeated exposure to antigen.
1. Antibody response is called a humeral immune response
1. Antibody response is called
a humeral immune response
rejection of foreign tissue is mediated by?
ii. Cellular immune response
What happens when tissue is rejected?
1. Experimental system is grafting skin from one strain of inbred mouse to another.
a. At first skin gets vascularized but after 1-2 weeks the skin dies off and gets rejected.
b. Rejection response also gets stronger and faster. (Skin gets rejected earlier) when same graft is reintroduces
2. Rejection is caused by cytotixic cells and inflammatory T cells (not by antibodies). Why?
a. Major Histocompatibility Complex (MHC) are molecules on the surface of out cells and everyone has different ones and they lead to the major rejection response.
What is MHC?
i. MHC I are heterodimers of a large alpha protein (each person has 6 alleles for alpha) and a small molecule, β2 microglobulin that is highly variant. MHC I is expressed in every nucleated cell. (So not in red blood cells)
2. When a cell is infected by a virus it displays viral proteins on its surface in connection with MHC class I. Tc cells have an adhesion molecule callsed cluster of differentiation (CD) which recognizes class I and viral protein. Tc cells cause infected cell to commit suicide.
ii. MHC II are heterodimer or a large α and a large β subunit and both are variant. MHC II are mostly expressed by antigen presenting cells (such as macrophages and dendritic cells)
1. When a microphage recognizes (via PAMP) a bacterium and eats it, it breaks it down and expresses the bacterial proteins (processed peptide) on the surface of its membrane in a binding grove between the α and β subunits.
2. Then a T helper one cell has on its surface a receptor called a T cell receptor (TCR). The TCR has a heterodimer α and β chain that recognizes two things: (must recognize both at same time) the class II molecule and processed peptide. Then the T helper cell is activated and causes signal transduction to produce gamma interferon
What cells recognize MHC type I receptors?
Tc cells have an adhesion molecule callsed cluster of differentiation (CD) which recognizes class I and viral protein. Tc cells cause infected cell to commit suicide.
What cells recognize MHC type II receptors?
helper t cells
What is the process of a helper T cell to act on a antigen?
1. When a microphage recognizes (via PAMP) a bacterium and eats it, it breaks it down and expresses the bacterial proteins (processed peptide) on the surface of its membrane in a binding grove between the α and β subunits of the MHC class II molecule.
2. Then a T helper one cell has on its surface a receptor called a T cell receptor (TCR). The TCR has a heterodimer α and β chain that recognizes two things: (must recognize both at same time) the class II molecule and processed peptide. Then the T helper cell is activated and causes signal transduction to produce gamma interferon
a. Gamma interferon (from adaptive IR) causes macrophage (innate IR) to produce more class II (becoming a better AP) , and more Interleukin I upregulating it. (IL I also helps helper T cells become active) So there is a positive feedback system between the AIS and the IIS.
what is gamma interferon
a. Gamma interferon (from adaptive IR) causes macrophage (innate IR) to produce more class II (becoming a better AP) , and more Interleukin I upregulating it. (IL I also helps helper T cells become active) So there is a positive feedback system between the AIS and the IIS.
Tc cells are called
class I restricted T cells or CD8 cells (b/c) they produce CD8
ThI are called
class II restricted T cells or CD 4 cells
Normal ratio of CD 4 to CD 8 cells is
2:1
III. Stem Cells are
are cells that give rise to 1 daughter cell and one stem cell
a. Totapotent stem cells are zygotes. They can turn into anyting
b. Pleurapotent stem cells are embryonic stem cells. They can turn into anything but embryonic fetal membranes
c. Mulitpotent stem cells are found in the bone marrow and can turn into lots of things.
i. They are found in the bone marrow of pelvic bones, sternum, skull flat bones, and vertabrea
d. unipotent stem cells can turn into one type of daughter cell (ie skin)
a. Totapotent stem cells
are zygotes. They can turn into anyting
b. Pleurapotent stem cells
are embryonic stem cells. They can turn into anything but embryonic fetal membranes
c. Mulitpotent stem cells
are found in the bone marrow and can turn into lots of things.
i. They are found in the bone marrow of pelvic bones, sternum, skull flat bones, and vertabrea
d. unipotent stem cells
can turn into one type of daughter cell (ie skin)
a. Mulitpotent stem cells in marrow give rise to
myeloid progenitors and lymphoid progenitors. (Which are not stem cells themselves)
i. Lymphoid progenitors
produce NK cells, T cell progenitors, B cell progenitors, and dendritic cells.
ii. Myeloid progenitors give rise to
dendritic cells, monocytes, granulocytes, megakaryocytes, and erythrocyte progenitors.
1. monocytes
are attracted to infected areas where they get more PRRs and oxydaze and become phagocytic macrophages
2. basophil
is a type of granulacyte. It takes up the basic stain and looks purple to blue. It is closely related to mast cells and may be involved in allergic response.
3. eosidophil
are also granulacytes and take up an acid stain. Looks pink. Involved in allergic response and getting rid of infectious worms.
4. neutrophil
are the most common of the granulacytes and takes up both acid and basic stains. It is a quintessential phagocyte. It has a lot of PRRs. It is first on the scene of an infection. It is not antigen presenting cells so don’t make a good bridge btw adaptive and innate IR.
How can you select step cells that will be appropriate for a bone marrow transplant?
by selecting for cells that show on their surface CD34. If a cells has this CD, it is undifferentiated. This allows for fewer cells to be transfused and the cells are less likely to be rejected.
What leads to various differentiated cells?
Complicated but there are many cytokines that if present at the right time in the right amount during bone marrow cell development lead to the differentiation of different cells types in the bone marrow.
Important Cytokines for differentiation of stem cells:
I. Mutli CSF (colony stimulating factor) is the same thing as Interleuken 3 (IL-3) is involved in many cell types. To get a specific cell you need different cytokined in correct molar ratio at the correct time in development.
II. GM-CSF (granulocyte/ monocyte solony stimulation factor)
a. Used commercially to ppl who undergo chemo to increase bone marrow cells
III. EPO – erythropoyatine is made by the kidney and helps with red blood cell production.
I. Mutli CSF
a cytokine important for differentiation of stem cells

I. Mutli CSF (colony stimulating factor) is the same thing as Interleuken 3 (IL-3) is involved in the development many cell types. To get a specific cell you need different cytokined in correct molar ratio at the correct time in development.
II. GM-CSF
a cytokine important for differentiation of stem cells

II. GM-CSF (granulocyte/ monocyte solony stimulation factor)
a. Used commercially to ppl who undergo chemo to increase bone marrow cells
III. EPO
a cytokine important for differentiation of stem cells
erythropoyatine is made by the kidney and helps with red blood cell production.
a. It is not surprising that kidney produced EPO because it gets 20% of Cardiac output of blood and is very sensitive to change in Oxygen levels. If O2 levels fall à additional EPO produced.
b. Used in ppl with chemo to increase levels of RBC
c. Also a doping drug
d. Use too much EPO can lead to heart problems because the viscosity of the blood is increased; the heart works harder, and can lead to clotting.
To test what cytokines are needed to develop a particular cell:
1. Put stem cells in salt Ager plate with bone marrow stroma (proteins that are analogous to the ones in bone marrow)
2. Add cytokines at specific concentrations
3. Colonies grow
4. Examine colonies to see what cell types are growing there
5. Based in what you put in the plate and at what amounts infer what is happening in vivo
Types of Immune Organs
I. Primary Lymphoid Organs – bone marrow and thymus gland
II. Secondary Lymphoid Organs- usually involved in activation of mature
III. Tertiary lymphoid structure/site
a. an infected area
I. Primary Lymphoid Organs
bone marrow and thymus gland
a. Thymus gland located
in the throat it gets smaller after puberty.
anatomy of thymus gland
1. Outer part called Cortex
a. Where positive selection takes place.
2. Inner part called Medula
what cells are in the thymus
b. Cells in thymus called thymocytes. They are immature T cells.
what happens in the thymus?
cells undergo two processes in thymus gland. Positive and Negative selection.
what is positive selection? where does it take place?
i. Positive selection takes place in the Cortex of the thymus and is carried out primarily by cortical epithelial cells. CEC are NOT bone marrow derived. They have a rich array of class I and class II molecules on surface.
1. Thymocytes have, at this point a randomly created T cell receptor. The receptor is called αβ receptor. If and only if the T cell receptor can bind to one of the class I or class II molecules then and only then will it survive. Otherwise will die.
2. These thymocytes are being educated how to interact with antigen presenting cells (its own class I or class II receptors)
3. Otherwise cell dies by apoptosis (organized cell death, no damage to other cells)
what cells carry out positive selection?
primarily by cortical epithelial cells. CEC are NOT bone marrow derived. They have a rich array of class I and class II molecules on surface.
what is apoptosis? How does it occur?
(organized cell death, no damage to other cells)
a. Capsases turn on apoptotic processes.
i. Cell destroys its DNA so it cant be released
ii. Cell blebbing- when cell disintegrated it is covered by a membrane and can’t release its cytoplasmic content to hurt other cells.
a. Capsases
turn on apoptotic processes.
ii. Cell blebbing
- when cell disintegrated it is covered by a membrane and can’t release its cytoplasmic content to hurt other cells.
what is negative selection? where does it take place? what cells participate?
ii. In medulla of the thymus there are dendritic cells (idd cells) having rich arrays of class I and class II molecules. They ARE bone marrow derived. They perform negative selection in thymocytes.
1. If a T cell had a receptor that recognizes own molecules that are not MHC related, or a class I molecule too well, or a class II molecule too well, it may be auto reactive.
2. To prevent an auto immune response, these t-cells that are too active it also undergoes apoptosis.
what cells participate in negative selection?
dendritic cells (idd cells) having rich arrays of class I and class II molecules. They ARE bone marrow derived. They perform negative selection in thymocytes.
II. Secondary Lymphoid Organs
usually involved in activation of mature lymphocytes (not development, not specificity of lymphocytes)

lymph nodes, spleen, MALT and GALT
location of Lymph nodes
a. Lymph nodes are located in throat (anterior cervical lymph nodes), back of neck (posterior cervical), under armpit (axillary), clavical (supraclavical), thorasix lymph nodes, lots in the abdomen, behind knee (poplidial), elbows (epitroclial)
i. Draining lymph node
is nearest lymph node to infected site. Swells up when actively fighting an infection.
what is the purpose of a lymph node?
ii. Circulation through a lymph node allows lymphocytes maximum opportunity to recognize its antigen.
What is the "plumbing" of a lymph node
two types of cirulation:
1. Blood circulatory system
a. Artery drains into node. Is broken down into capillaries, and leaves in vein.
b. Special site – post capillary venule has endothelial cells that are plumped up and are called HEV (high endothelial venule cells). At HEV and primarily there, lymphocytes can leave the blood and enter the lymph node. MOST lymphocytes enter through here.
c. when a lymphocyte enters the lymph nose and recognized/ encounters the right antigen it stays in the lymph node and becomes activated. (Replicate, etc)
d. If lymphocyte does not encounter antigen it leaves through the efferent lymphatic vessel. Will die after a month or so if it doesn’t find antigen in another lymph vessel.
2. Lymphatic circulatory system
a. Lymph vessels drain interstitial fluid. These are the afferent lymphatic vessels that bring in the fluid to the node.
b. Fluid peculates down and leaves through efferent lymphatic vessel.
c. Many efferent lymphatic vessels join together to form larger vessels and most of the liquid ends up in the thoracic duct (in chest).
d. The thoracic duct connects to the left subclavian vain (drains left arm of blood) and re-enters blood stream.
describe the blood circulatory system through a lymph node
a. Artery drains into node. Is broken down into capillaries, and leaves in vein.
b. Special site – post capillary venule has endothelial cells that are plumped up and are called HEV (high endothelial venule cells). At HEV and primarily there, lymphocytes can leave the blood and enter the lymph node. MOST lymphocytes enter through here.
c. when a lymphocyte enters the lymph nose and recognized/ encounters the right antigen it stays in the lymph node and becomes activated. (Replicate, etc)
d. If lymphocyte does not encounter antigen it leaves through the efferent lymphatic vessel. Will die after a month or so if it doesn’t find antigen in another lymph vessel.
post capillary venule
b. Special site in the lymph node– post capillary venule has endothelial cells that are plumped up and are called HEV (high endothelial venule cells). At HEV and primarily there, lymphocytes can leave the blood and enter the lymph node. MOST lymphocytes enter through here.
what happens when a lymphocyte enters the lymph nose and recognized/ encounters the right antigen
it stays in the lymph node and becomes activated. (Replicate, etc)
what happens when a lymphocyte does not encounter antigen in the lymph node
it leaves through the efferent lymphatic vessel. Will die after a month or so if it doesn’t find antigen in another lymph vessel.
describe the lymphatic circulatory system?
a. Lymph vessels drain interstitial fluid. These are the afferent lymphatic vessels that bring in the fluid to the node.
b. Fluid peculates down and leaves through efferent lymphatic vessel.
c. Many efferent lymphatic vessels join together to form larger vessels and most of the liquid ends up in the thoracic duct (in chest).
d. The thoracic duct connects to the left subclavian vain (drains left arm of blood) and re-enters blood stream.
afferent lymphatic vessels
a. Lymph vessels drain interstitial fluid. These are the afferent lymphatic vessels that bring in the fluid to the node.
efferent lymphatic vessel
how lymph leaves lymph node.
c. Many efferent lymphatic vessels join together to form larger vessels and most of the liquid ends up in the thoracic duct (in chest).
thoracic duct
(in chest)
where most liquid that left the lymph node in efferent lymphatic vessels ends up
d. The thoracic duct connects to the left subclavian vain (drains left arm of blood) and re-enters blood stream.
iii. Anatomy of lymph node
1. Cortex (B cell area)
a. Contains more B cells than any other lymphocyte but has B, T and antigen presenting cells
b. A unique cell called the Follicular Dendritic cell (FDC) is found in the cortex.
c. Primary lymphoid follicle – if lymph node is not undergoing activation against and antigen, the cortical area is modest in size but there are little clusters of cells that contain FDC. Not very active.
d. If node is activated, cortex swells and primary lymphoid follicle starts producing cells and is now called the germinal center. In germinal center there is rapid proliferation of lymphocytes.
2. Paracortex (T cell area)
a. Contains more T cells than any other lymphocyte but has B, T and antigen presenting cells.
3. Medulla
cortex of a lymph node
a. Contains more B cells than any other lymphocyte but has B, T and antigen presenting cells
b. A unique cell called the Follicular Dendritic cell (FDC) is found in the cortex.
c. Primary lymphoid follicle – if lymph node is not undergoing activation against and antigen, the cortical area is modest in size but there are little clusters of cells that contain FDC. Not very active.
d. If node is activated, cortex swells and primary lymphoid follicle starts producing cells and is now called the germinal center. In germinal center there is rapid proliferation of lymphocytes.
FDC
Follicular Dendritic cell (FDC) is found in the cortex of a lymph node
c. Primary lymphoid follicle
if lymph node is not undergoing activation against and antigen, the cortical area is modest in size but there are little clusters of cells that contain FDC. Not very active. cortex of lymph node.
germinal center
d. If node is activated, cortex swells and primary lymphoid follicle starts producing cells and is now called the germinal center. In germinal center there is rapid proliferation of lymphocytes.
2. Paracortex of lymph node
(T cell area)
a. Contains more T cells than any other lymphocyte but has B, T and antigen presenting cells.
b. Spleen
is located on the left side of the abdomen. It is in essence a large lymph node that is good at detecting blood born antigens. (vs in interstitial space as with most lymph nodes)
anatomy of the spleen
i. Red pulp
1. for recycling red blood cells
2. most of the spleed
ii. White pulp has various areas
1. PALS (periarterial lymphatic sheath) analogous to paracortex and therefore most enriched with T cells.
2. Follicular area is analogous to cortex and enriched with B cells.
3. Marginal zones contain dendridic cells.
1. PALS
periarterial lymphatic sheath)of the spleen is analogous to paracortex and therefore most enriched with T cells.
2. Follicular area
2. Follicular area of spleen is analogous to cortex and enriched with B cells.
3. Marginal zones
3. Marginal zones of spleen contain dendridic cells
c. MALT
mucosal associated lymphoid tissue is not as organized and more diffuse as lymph node and is found in many places
d. GALT
gastrointestinal associated lymphoid tissue) is MALT in GI tract. On a daily basis largest Ab produced against microbes in intestinal tract (IgA).
i. Intestinal tact anatomy
1. Innermost part of GI tract is mucosal epithelium
a. Interspersed are M cells that translocate antigen from lumen into lamina propria.
i. Antigen moves into M cell and into the “pocket” where we have the antigen presenting cell and lymphocyte
2. Under mucosal epithelium is lamina propria
a. GALT is located in the lamina propria
i. Large amounts of IgA are made against the antigen and is translocated into the leumen
M cells
translocate antigen from lumen into lamina propria.
i. Antigen moves into M cell and into the “pocket” where we have the antigen presenting cell and lymphocyte
lamina propria
2. Under mucosal epithelium is lamina propria
a. GALT is located in the lamina propria
i. Large amounts of IgA are made against the antigen and is translocated into the leumen
Antigens
to be a good antigen a molecule must be foreign. The more distantly related the better. Size is also important, the bigger the better (over 10,000 daltons). Heterogeneous molecules are better antigens than homogeneous. (all alanine not as good as a combo of aa)
Immunogenicity
- a molecule that leads to an immune reaction (large proteins, large sugars, large polynucleotides
Antigenicity
a molecule that can bind to an antibody but may be too small to activate the immune response
Keyhole limpet hemocynin (KLH)
is a commonly used good antigen
folding of the antigen is important for...
not important for...
becuase...
For a secondary ANTIBODY immune response to occur the folding of the antigen is important. A denatured protein will not produce a secondary immune response
A secondary cell mediated TDTH cell relies on PROCESSED proteins and will occur even if a protein is denatured.
To quantify primary vs secondary DTH response:
1. inject mouse with antigen. Skin reddens and hardens. This is area of DTH.
2. second exposure are or redness increases and is harder and this happens faster.
Serum
what is left over after blood clots. (Plasma minus clotting factor)
Plasma
when blood is not clotted
what do B cells need to recognize antigen?
B cells recognize soluble antien (hydrophilic and unprocessed) without any help (can bind). B cell antigen receptor is the antibody that B cells will secrete stuck in the membrane. So any hydrophobic parts of an antigen are not recognized by B cells.
epitope.
Can be sequential (aa in a row)
Can be conformational (near each other b/c of folding of protein, aa not actually near each other in sequence)
Denaturing proteins gets rid of the conformational epitopes.
what do T cells need to recognize antigen
T cells always recognized processed antigen displayed on MHC molecules of antigen presenting cells. Most T cell epitopes are antapathic.
Primary T cell receptor type is
Primary T cell receptor type is αβ.
haptens
- molecules too small to lead to the production of antibodies. Example: dinitralphenol
If you inject DNP into mouse, you will not get an immune response,
If you link the hapten to a large backbone protein such as BSA (cover the BSA in hapten),
you will get antibody production to the hapten that is the immunodominant epitope. The protein backbone helps T cells help B cells make antibodies. Even though this production of antibodies against DNP will lead to an antibody response against it, there will be no DTH (t cell) response against the DNP or a cytotoxic T cell response.
I. Antibody Structure
a. Ab is a tetromer of two identical light chains and two identical heavy chains.
b. Three major components in cell membrane antibody
i. Extracellular component
ii. Tranmembrane domain
iii. And a small cytoplasmic tail
c. Near antibodies there are Ig-α and Ig-β molecules that have small extracellular domains but big cytoplasmic tail and are involved in cell signaling pathways
d. Disulfide bonds hold the Ab together
i. Interchain disulfide bonds stabilize interaction between heavy and light chain
ii. Intrachain disulfide bonds stabilize interaction between two heavy chains
1. These also give a balloon like configuration that is called an immunoglobulin domain, associated with immune cells (Ab, T cell receptors)
e. amino end of heavy and light chains together binds epitope and is called the antigen binding fragment. (FAB)
f. Ab molecule has a valence of two (can bind to two epitopes)
g. Carboxyl end of the Ab can crystallize out in solution and is called the Fc fragment. It gives the specificity of what TYPE of Ab it is.
b. Three major components in cell membrane antibody
i. Extracellular component
ii. Tranmembrane domain
iii. And a small cytoplasmic tail
Ig-α and Ig-β molecules
c. Near antibodies there are Ig-α and Ig-β molecules that have small extracellular domains but big cytoplasmic tail and are involved in cell signaling pathways
i. Interchain disulfide bonds
stabilize interaction between heavy and light chain
ii. Intrachain disulfide bonds
stabilize interaction between two heavy chains
immunoglobulin domain
disulfide bonds give a balloon like configuration that is called an immunoglobulin domain, associated with immune cells (Ab, T cell receptors)
e. amino end of heavy and light chains together
binds epitope and is called the antigen binding fragment. (FAB)
(FAB
e. amino end of heavy and light chains together binds epitope and is called the antigen binding fragment. (FAB)
f. Ab molecule has a valence of
of two (can bind to two epitopes)
Fc fragment
g. Carboxyl end of the Ab can crystallize out in solution and is called the Fc fragment. It gives the specificity of what TYPE of Ab it is.
experiment that showed where Ab migrates?
i. Immunize rabbit against OVA
ii. Run serum through electrical field
iii. Various peaks form from immune serum that refers to various proteins. One peak is gamma globulin.
iv. Take same serum and add OVA to it. Gamma globulin peak disappears.
v. Therefore Ab migrates in serum in the gamma globulin peak and Ab is called gamma globulin.
experiment that proves Ab is a tetromer of 2 light and 2 heavy chains
i. Mercaptoethenol – is a disulfide reducing agent. Breaks apart disulfide bonds.
1. Papain- protease that cleaves antibody at place where FAB connects to Fc region.
ii. Add mercoptoethenol to Ab, the heavy and light chains disassociate with each other.
iii. Put purified antibody over column with beads that have micropores. Small molecules get in beads and take a long time to get through column.
1. Calibrate with known proteins of known molecular weight.
iv. can determine molecular weight of unknown molecule
v. intact Ab have mw of 150kd
vi. first (heavy) thing to come off of column has mw of 50 kd
vii. second (light) thing comes off colums at 25 kd
viii. heavy to light thing come off at equal molar ratio
ix. Therefore Ab has two heavy chains and two light chains
i. Mercaptoethenol
is a disulfide reducing agent. Breaks apart disulfide bonds.
1. Papain
protease that cleaves antibody at place where FAB connects to Fc region.
c. Oucherlony Assay
i. Take ager plate and drill out holes on opposite sides of plate. In first hole put antigen. In second put antibody. As antigen and AB diffuse, when they meet in right molar ratio with precipitate out and you get a white line.
ii. Ab not against the antigen à no reaction à no line
oucherlony assay to show that the Fc domain is made up of H chain
Make three holes. Put Heavy chain in one hole, Light chain in another, and an antibody against the Fc part in the third
a. The antibody can be a goat-anti mouse Ab for example
2. Line forms only between HC and Ab implying that the HC make up FC domain.
oucherlony assay to show that FAB fragment is made of H and L cahins
1. Make three holes one with HC, one with LC, and one with Ab against FAB fragment
2. Two lines form implying the the FAB fragment is made form both HC and LC
d. Fingerprinting Assay
i. Add a protease to separate the antigentic peptide
ii. Use electrophoresis to separate by charge
iii. Use chromatography to separate by molecular weight
iv. Do it for OVA, BSA, KLH, Has etc…
v. Some blobs line up exactly regardless of antigen and are constant regions
vi. Some blobs do not and are variable regions of the Ab
plasma cytoma.
a cancer where the individual only produce a single type of antibody (monoclonal). Since they also have kidney problems, the H and L chains get into the urine and are called Bence Jones proteins. At 50 C they precipitate out and can be harvested.
Bence Jones proteins
the H and L chains get into the urine
At 50 C they precipitate out and can be harvested.
b. We can induce plasma cytoma in a mouse by
injecting mineral oil into the abdominal cavity of the mouse. These cytomas are called MOPC’s. We can then crystallize out the Ab.
MOPC’s
b. We can induce plasma cytoma in a mouse by injecting mineral oil into the abdominal cavity of the mouse
variable domains
b. There are three very variable domains in Ab. They are the epitope binding regions or complimentary determining regions. CDRs. There are 3 CDRs on each chain. You need 6 CDRs to bind to an epitope. There are 12 CDRs on each antibody.
complimentary determining regions
b. There are three very variable domains in Ab. They are the epitope binding regions or complimentary determining regions. CDRs. There are 3 CDRs on each chain. You need 6 CDRs to bind to an epitope. There are 12 CDRs on each antibody.
c. Variable regions are determined to be so using
the variability index:
i. Number of aa at position/ frequency of aa at all positions
d. There are two main epitope-Ab interactions
i. small epitopes are completely enveloped by CDR’s
ii. large epitopes induce a planar configuration of binding,
e. There are 3 divisions to an antibody
i. The isotype of an Ab gives it its phenotype of where it can go (IgA, IgG etc…)
ii. allotype
iii. idiotype
isotype
i. The isotype of an Ab gives it its phenotype of where it can go (IgA, IgG etc…)
1. located on the Fc region
2. there are 5 different isotypes (with subdivions)
3. κ (kappa) and λ(lamda) are the isotype son light chains. They don’t have much effect on function. An Ab has either two kappa or two lamda.
4. all members of a species carry the same collection of constant-region genes.
5. Within a species each individual will express all iso types in the serum.
a. So Ab of different species are recognized as such
b. Anti-isotype antibody is used in research.
ii. allotype
1. individual variability on isotypes
2. inherited in a mendelian fashion
iii. idiotype
1. the amino end of the H and L chains are highly variable
2. this is what binds antigen
how many domains do heavy chains of Ab have
4 or 5
i. In the Fc part there is 1 variable and 3 constat Hc domains
ii. The CH2 domain sometimes but not always activates compliment. (Top of I)
iii. The CH1 domain (bottom of V) stabilizes interaction
iv. The CH3 domain (bottom of I) can bind to FC receptor and cause phagocytosis
CH2 domain
sometimes but not always activates compliment. (Top of I)
CH1 domain
bottom of V) stabilizes interaction
CH3 domain
(bottom of I) can bind to FC receptor and cause phagocytosis
g. IgG
i. Has 3 constant domains
ii. Can activate complement
iii. Can cross placental membrane and is responsible for mother à infant immunity
h. IgM
i. Pentamer (made up of 5 Ab linked together by J chains)
1. Valence of 10
ii. Stays in the plasma and it is hard to get it into the interstitial space
iii. BEST at compliment cascade
iv. Produced first against a new microbe
1. Amount of IgM in blood is used to determine how long a pathogen has been in system
v. 4 constant domains (3 domains in H chain vs the 2 on IgG)
vi. when bound to antigen has a clawlike configuration and only then can activate complement.
i. IgA
i. Most common Ab
ii. Dimer linked with J chain
1. valence is 4
iii. Found in mothers milk à transferred to children
1. Main component of mother à child Ab transfer
iv. Urine and respiratory tract
v. Can pull bacteria out of solution, bacteria gets excreted
vi. In GI tract
vii. Can’t activate complement
why is IgA not broken down in GI tract?
1. not easily broken down by proteases because it is attached to a secretory component that is covalently linked to the Ab.
a. After IgA is produced and secreted by plasma cells it is transcellularly translocated through an epithelial cell.
i. There is a receptor on the submucossal side (poly Ig) that is specific to IgA
ii. After binding with IgA, both the Ab and receptor are endocytosed
iii. In the process of translocation, enzymatic cleavage of the receptor from the vesicle membrane. (it is still attached to the IgA)
iv. IgA is secreted with the receptor, which becomes the secratory component that protects sites of protease activity.
j. IgE
i. 4 constant domains
ii. involved in allergic response
iii. can’t activate complement
iv. smallest concentration in serum
v. purpose to get rid of matazoen parasites
k. IgD
i. 3 constant domains
ii. B cell development marker
1. if B cell expresses IgM and IgD it is a mature B cell and is ready to bind antigen
I. Antigen-Antibody interactions
a. H bonds
b. Ionic bonds
c. Hydrophobic interactions
d. Van der Walls interactions
e. NO COVALENT INTERACTIONS EVER
II. Affinity of Antibody
a. Described by Ka (association constant) and Kd (disassociation constant)
i. High Ka à high affinity
ii. Low Kd à high affinity
Avidity
b. When one epitope binds to antibody it can change tertiary structure and can effect (positively) the binding to the second epitope à Avidity
c. Kinetics of affinity
K1
i. As + Ab D As-Ab
K-1
ii. Ka = [As-Ab] / [As] [Ab] = K1/K-1 (L/mol)
iii. Kd = 1/ Ka
iv. Units
1. K1 = L/mol sec
2. K-1 = 1/sec
d. How to determine affiny
i. Surface plasma resonance
ii. Equilibrium dialysis
ii. Ka
= [As-Ab] / [As] [Ab] = K1/K-1 (L/mol)
iii. Kd
iii. Kd = 1/ Ka
iv. Units
1. K1
L/mol sec
units k-1
1/sec
i. Surface plasma resonance
1. determines affinity based on intensity of light
ii. Equilibrium dialysis
1. solution separated by semipermiable memrane allows small antigens to pass through, but not Abs
2. In control with no Ab present put radiolabled antigen on both sides and allow to equillibriate.
a. Measure concentration (based on lable) over time.
b. Side B and A concentration reaches same concentration over time.
3. Experiment. Put Ab on one side (A), antigen on other side (B)
a. Antigen moves to side A
b. Some antigen binds with Ab and is essentially taken out of solution.
c. More antigen moves to side A
d. The higher the affinity of antibody to antigen, at the state of equilibrium, there will be more antigen on side A than side B
e. Different of concentration à affinity
i. Immunoprecipitation
1. In serum there is a particular antigen (T3 lets say). We want to take it out of solution.
2. add antibody against T3
a. binding precipitates out or centrifuges out (sometimes
3. precipitation doesn’t always work so add protein A to Fc region of antibody.
a. Pour serum on plate with protein A and only the Ab will stick to plate
4. take Ab and add bead to it to make it heavier and can easily centrifuge out
5. Add iron filing to Ab and remove bound Ab-antigen with magnet
6. Change pH to disassociate Ab to antigen
ii. ELISA
Enzyme Linked Immuno abSorbance Assay) can be used to detect amount of antibody or antigen from a sample.
1. Used clinically as an HIV test
how doe elisa for an Ab work?
2. Antigen is bound to the bottom of plastic wells
3. As a control know amount of increasing concentration of antibody are added to the wells.
a. Binding occurs à wash off unbound Ab
b. secondary Ab (anti isotypic) is added with enzyme peroxidase
c. Hydrogen peroxide is added + substrate that produces a color when peroxidated by 2ndary antibody.
d. More binding à darker color (optical density)
4. A standard curve is made (y is optical density, x is antibody concentration)
5. An unknown is put in well and intensity of color is used to determine concentration
iii. Immunoflourescence
If in a mass of cells if you want to find a particular cell (lets say one with CD8)]
1. Uses biotin- a molecule that binds avadin with a high affinity.
a. Looks like a 4 leaf colver
b. Each cleft can bind 1 avadin (so can bind 4 avaidins)
2. Make a primary antibody
a. It has no marker
b. They are hard to make so you don’t want to mess with it
3. Make secondary (anti-isotypic) antibody against the primary antibody that is biotinylated
4. Add avadin that is with a fluorescent dye
a. flourescin- yellow label
b. rhodomine – red label
c. phycoeretheryn- red
5. Can see fluorescence under microscope
a. Use of secondary antibody and biotin to amplify fluorescence signal
biotin-
a molecule that binds avadin with a high affinity.
a. Looks like a 4 leaf colver
b. Each cleft can bind 1 avadin (so can bind 4 avaidins)
isotypic Ab
secondary Ab against an isotype (goat anti mouse)
a. flourescin
yellow label
b. rhodomine
red label
c. phycoeretheryn
red
iv. ELISPOT
is used to detect what cytokine or Ab a cell produces or to Id a cell with a type of cytokine or Ab
ELISPOT procedure
1. Have a dish of cells that produce cytokines that adhere to the plastic surface
2. Wash off cells top get just the cytokines
3. Add antibody that will stick to cytokines
4. Add horseradich peroxidase in presence of Ab and substrate will produce a spot
5. Variation: Well growing with B cells that are producing Ab from a single clone (monoclonal) (want to know what Ab these cells produce)
a. Wash off cells, Ab left behind
b. Add antigen, wash
c. Add secondary antibody to antigen
v. FACS
fluorescence activated cell sorter is used to count and separate different cells that are labled with different fluorescent labels.
1. Make Ab with fluorescence against antigen
a. One color against one type of cell another against another cell
2. machine makes droplets that contain only one cell
3. as droplets pass through a laser detects what kind of fluorescence is associated with the cell
4. can separate and count cells
5. Readout:
a. Log of color 1 (red) on y axis and log of color 2 (green) on x axis
b. 4 quadrent readout
i. Quad 3 (bottom left) is dbl negatives (cells w/o any color)
ii. Quad 4 (top left) are red labeled cells
iii. Quad 1 (top right) are dbl positives (both colors)
iv. Quad 2 (bottom right) are green labeled cells
c. Contour mad readout
FACS readout
a. Log of color 1 (red) on y axis and log of color 2 (green) on x axis
b. 4 quadrent readout
i. Quad 3 (bottom left) is dbl negatives (cells w/o any color)
ii. Quad 4 (top left) are red labeled cells
iii. Quad 1 (top right) are dbl positives (both colors)
iv. Quad 2 (bottom right) are green labeled cells
III. Monoclonal antibodies
arise from a single B call against only one epitope
a. Natural antibodies are all polyclonal
b. Monoclonal Ab are useful for experiments because they ensure replication
c. They can also be used to activate or inactivate certain CDs on cells (CD3)
d. To make monoclonal Ab
three techniques
i. Fuse a normal activated B cell with a myeloma cell (a cancerous plasma cell) to produce a hybrid called a hybridoma cell. This cell possesses immortal-growth properties and can secrete antibodies
ii. For industry get mouse embryo stem cells
1. KO the heavy and light chain genes.
2. add human artificial chromosome with human H and L chains
3. breed chimeric mouse
4. selctivly interbreed to produce mouse that produces human Ab
5. infect with antigen
6. get human Ab production from a mouse
iii. Take a B cell and isolate the variable genes that code for the H and L chains
1. recombine the genes randomly to make all possible combos of Ab
2. package into phage
3. grow in ecoli
4. make a library of antibodies
5. incubate Ab on a plate with immobilized antigen
6. purify bound phage(one against the antigen)
7. enrich
8. purify Ab
making monoclonal antibodies using myeloma cells
i. Fuse a normal activated B cell with a myeloma cell (a cancerous plasma cell) to produce a hybrid called a hybridoma cell. This cell possesses immortal-growth properties and can secrete antibodies
making monoclonal Ab using stem cells
ii. For industry get mouse embryo stem cells
1. KO the heavy and light chain genes.
2. add human artificial chromosome with human H and L chains
3. breed chimeric mouse
4. selctivly interbreed to produce mouse that produces human Ab
5. infect with antigen
6. get human Ab production from a mouse
making monoclonal Ab using phage
iii. Take a B cell and isolate the variable genes that code for the H and L chains
1. recombine the genes randomly to make all possible combos of Ab
2. package into phage
3. grow in ecoli
4. make a library of antibodies
5. incubate Ab on a plate with immobilized antigen
6. purify bound phage(one against the antigen)
7. enrich
8. purify Ab
I. Complement
a. Can lyse foreign cells by causing a membrane boring pore called a membrane altering complex
b. Can opsonize which promotes phagocytosis of antigens
c. Activate inflammatory response
i. Can make proinfalmatory anaphylotoxins that increase leakiness of blood vessles, increase basal dilation and are chemoatracted to neutrophils and cause degranulation
d. can bind antigen-antibody complexes and allow them to be cleared
II. To activate complement there are three pathways
a. Classical Antigen-Antibody mediated
b. Alternative pathway mediated by the innate IS
i. Most prokaryotes have low amounts of sialic acid while euk have lots
c. Leptin
i. Manose binding leptin (part of innate immunity)
III. Compliment activation cascade (classical pathway)
a. The IgG antibody has a CH2 domain that is a complement activating domain. It binds to an antigen and a conformational change in the Fc region exposes a binding site for C1.
b. C1qr2s2 is a complement molecule.
i. It has 6 C1q heads
ii. Each C1qr2s2 must bind its C1q heads to at least two Fc sites
1. that means that there must be two IgG Abs bound to activate complement but only one IgM. (b/c IgM is a pentamer and IgG is not)
c. binding of the C1q domain of C1qr2s2 causes a conformational change in the C1r domain. The molecule now becomes an active serine protease that cleaves C1s.
d. After cleavage C1s becomes a protease that can cleave C4 and C2
e. C4 is cleaved into C4A which diffuses away and C4B that sticks to the cell surface.
f. C4B now has a binding site for C2.
g. C1s cleaves C2 into C2B which diffuses away and C2A which sticks to the bacteria along with C4B
i. We now have stuck on the bacteria a C4b2a
h. The C4b2a complex is now an enzyme called C3 convertase. It cleaves C3 into C3a which diffuses away and C3b which also sticks to the bacteria
i. We now have stuck to the antigen C4b2a3b
i. C4b2a3b is an enzyme (c5 convertase) that converts C5 into C5a (diffuses away) and C5b which binds to the surface of the cell.
j. C5b acts as a crystallization site for C6 and C7. It first binds C6 and cleaves it to become C5b6 and then binds C7. This complex exposes hydrophobic regions, which serve as binding sites for membrane phospholipids, allowing the complex to be inserted into the bacterial phospholipid bilayer.
k. This attracts C8 which bind to the complex. The C5b678 complex creats a small pore.
l. C8 complex polymerizes many C9s to bind and makes the pore bigger allowing ions to leave the bacteria, ruining osmotic balance, and killing the bacteria.
complement activating domain
CH2 domain It binds to an antigen and a conformational change in the Fc region exposes a binding site for C1.
b. C1qr2s2
is a complement molecule.
i. It has 6 C1q heads
ii. Each C1qr2s2 must bind its C1q heads to at least two Fc sites
1. that means that there must be two IgG Abs bound to activate complement but only one IgM. (b/c IgM is a pentamer and IgG is not)
c. binding of the C1q domain of C1qr2s2 causes
a conformational change in the C1r domain. The molecule now becomes an active serine protease that cleaves C1s.
d. After cleavage C1s becomes
a protease that can cleave C4 and C2
e. C4 is cleaved by/into
cleaved by C2
cleaved into C4A which diffuses away and C4B that sticks to the cell surface.
f. C4B now has a binding site for C2.
C4B binds
C2.
g. C1s cleaves C2 into
C2B which diffuses away and C2A which sticks to the bacteria along with C4B
i. We now have stuck on the bacteria a C4b2a
h. The C4b2a complex is now
an enzyme called C3 convertase. It cleaves C3 into C3a which diffuses away and C3b which also sticks to the bacteria
i. We now have stuck to the antigen C4b2a3b
i. C4b2a3b is
an enzyme (c5 convertase) that converts C5 into C5a (diffuses away) and C5b which binds to the surface of the cell.
j. C5b acts as
a crystallization site for C6 and C7. It first binds C6 and cleaves it to become C5b6 and then binds C7. This complex exposes hydrophobic regions, which serve as binding sites for membrane phospholipids, allowing the complex to be inserted into the bacterial phospholipid bilayer.
the C5b67 complex
attracts C8 which bind to the complex. The C5b678 complex creats a small pore.
l. C8 complex
polymerizes many C9s to bind and makes the pore bigger allowing ions to leave the bacteria, ruining osmotic balance, and killing the bacteria.
IV. Alternative Complement Pathway
a. This pathway is not as efficient as the classical one because it relies on the spontaneous breakdown of C3
b. C3, other than playing a role in the normal pathway, also spontaneously breaks down into C3a and C3b.
c. C3b can bind to cells. It is inactivated by sialic acid (found on eukaryotic cells and NOT on prokaryotic cells). If not inactivated, C3b can bind factor B to form a complex
d. The complex can now activate factor D
e. Factor D cleaves factor B so that the bond complex is now C3bBb
f. C3bBb is now an active protease (C3 convertase) and is analogous to C4bC2a. It converts more C3 into an active form and now C3bBb3b becomes C5 convertase and is analogous to C4bC2aC3b

pathway is then same as classical...

g. C3bBb3b converts C5 into C5a (diffuses away) and C5b which binds to the surface of the cell.
h. C5b acts as a crystallization site for C6 and C7. It first binds C6 and cleaves it to become C5b6 and then binds C7. This complex exposes hydrophobic regions, which serve as binding sites for membrane phospholipids, allowing the complex to be inserted into the bacterial phospholipid bilayer.
i. This attracts C8 which bind to the complex. The C5b678 complex creats a small pore.
c. C3b can bind to cells. It is inactivated by
by sialic acid (found on eukaryotic cells and NOT on prokaryotic cells). If not inactivated, C3b can bind factor B to form a complex
C3bB complex can
activate factor D
e. Factor D cleaves
factor B so that the complex is now C3bBb
f. C3bBb is
now an active protease (C3 convertase) and is analogous to C4bC2a. It converts more C3 into an active form
C3bBb3b
becomes C5 convertase and is analogous to C4bC2aC3b
g. C3bBb3b converts C5 into C5a (diffuses away) and C5b which binds to the surface of the cell.
V. Mannose Binding Leptin
a. MBL looks like C1qrs
b. After MBL binds to 2 mannose sugars it begins activation of the same pathway