Here they are. This gives us all the info we're going to get. As I said before, pray hard and study hard, and it will turn out O.K. Merry Christmas, etc. if I do not see y'all Friday.
Sweatman-
This is graduate school. If you make a statement, prepare to back it up with an example. Even if you contradict something he said, if you can defend it, fine. He grades for structure and logic in your answer- beginning, middle,end, and don’t repeat yourself. Essay form, with flow. Illustrate does not mean draw pictures, it means give examples. Pictures do not substitute for text for him. He gave examples and papers to read- look over the papers for examples again before you try to answer his question.
Nutting- if he says describe, he means write out a description. A chart or diagram or flow chart with just a sentence will NOT do. The chart, graph or diagram is for illustrative purposes, NOT for answering the entire question. He wants text answers.
What are 3 main things he emphasized? hint about what he wants us to know about?
1. Sex determination and reproduction male and female
2. hypothalamus and pituitary and regulation (- and + feedback)
Be sure you are careful in words when talking about whether hormone secretion has changed or whether the tissue response has changed.
3. Calcium/phosphate/bones.
Handout- Look over those questions at the end. They exemplify his questions.
Which part of pituitary gland conmmunicates information directly via nerves? posterior pituitary.
Where is decision made- hypothalamus. Posterior pituitary hormones are synthesized in hypothalamus and get to posterior pituitary through center of axon, not surface as you might think from the diagram in the note packet. Stored at nerve terminal. Released as result of string of action potentials starting in hypothalamus. Hormone dumped into capillaries, to veins, to heart, to rest of body.
Anterior pituitary- how do signals get from hypothalamus to anterior pituitary? portal system via hypothalamic releasing hormones. Some stimulatory and some inhibitory. KNOW currently accepted RH names and hormones they control from table.
What are they?GnRH (stimulates FSH and LH-gonadotropins), CRH (stimulates ACTH or corticotropin, stimulates adrenal cortex), TRH(stimulates TSH, indirectly stimulates T3 and T4 from thyroid), PIH (inhibits prolactin secretion- is dopamine. A dopamine agonist decreases PRL secretion)
Hypothalamus is always suppressing PRL with dopamine. PRL stimulates milk synthesis. Takes a couple of hours. Suckling stimulates oxytocin to stimulate milk letdown. PRL is also stimulated by suckling.
Other RH: GHRH(stimulates GHsecretion- GH is also regulated in an inhibitory way by somatostatin. For a Burst of GH, the body releases GHRH and stops somatostatin. Most GH comes out in deep sleep. Most ACTH is secreted just before and when you get up. ACTH stimulates cortisol, which protects against stresses, like hypoglycemia from fasting- make more glucose in liver.
Couple others- ghrelin (like GHRH- not on test). Gonadotropins- there is not a known gonadotropin inhibiting hormone. There may be a gonadotropin surge attenuating factor in ovulating women.
Know FSH and LH forward and backward.
Be able for all the hormones to take the hormone and fill out the chart from fig 7. and draw in arrows for long loop and short loop inhibitory feedback, and any positive feedback that might occur.
Especially for male and female sex cells and hormones. Know what hormones feed back and what they affect. Understand negative and positive feedback.
Know general pattern of control of reproduction. Backward and forward. Estrogen briefly positively causes surge in LH resulting in ovulation in females.
Be able to draw and explain menstrual cycle graph. Be able to describe in the essay format Sweatman described: introduction,bulk, conclusion.
Likely to give you questions of a problem-solving nature like you had in the conference. Scenario- figure out what is going on- what would you measure and how would you get around problem. Might put in statement to refer to normal situation as appropriate. Do not draw a whole figure from the notes and say it is answer- write paragraph. Go for most likely causes of problems. Say to measure LH or FSH or hCG, for example. You do not have to give too many details about how to measure. hCG rescues corpus luteum from disintegrating. hCG is made by placenta (uterine lining) caused by implantation of blastocyst. Can also trigger hCG with grain of sand or other object in uterus. hCG peaks 1st trimester- goes away after placenta delivered. E and P increase until just before end. Beginning produced by corpus luteum (ovary). After a while- produced by placenta itself in ever increasing amounts. Another placental hormone- hPL- human placental lactogen which promotes expansion of milk glands- E and P block synthesis of milk during pregnancy. Placenta delivered- Estrogen,Progesterone, hPL plummet. Milk prod starts.
3rd area Ca/P
Suggestion: know what we take in and where it goes, normal Ca levels. In practice problems, a lot of normal values given.
What does PTH do? How is vitamin D activated? Where do you lose Ca? urine.
Ca not well absorbed. Net of oral absorption is about 10%. Know protective mechanism for hypocalcemia- enhance reabsorption in kidney. Ca regulated in distal tubule- most reabsorption proximal, little in loop, enhanced absorption in distal tubule.
Absorption from gut stimulated by 1,25 dihydroxy vitamin D. KNOW how it is activated. D2 (ergosterol) is from diet. cholecalciferol (D3) is from UV light on skin. Liver puts hydroxyl at 25. Kidney proximal tubule puts OH at 1. Stimulated by PTH. Liver damage- no 1st intermediate. Kidney problem- you get no 1,25. Give massive doses of 25 or calcitriol, which is really expensive. Vitamin D is like a steroid. Acts through nucleus and affecting gene transcription. PTH acts fast on kidney, moderately fast on bone. Vitamin D is like grease for a revolving door. Allows Ca into or out of bone to prevent hypocalcemia (fatal affect on heart and intercostal muscles, mostly heart)
End syllabus are study questions. Go through them.
Prob 2:
how do you answer it? follow the outline given.
Use figures like figure 1 in that section as well as description.
Problem 3:
phosphate- from meat and sodas. Excess is a problem- binds Ca. Excess is dumped in urine. PTH causes more bone breakdown- Ca and PO4 to blood- want to keep ionized Ca high, must eliminate phosphate in kidney. Decrease reabsorption PO4.
Prob 4: PTH secreting adenoma- plasma Ca up. Urine Ca- enhances reabsorption. Phosphate-inhibits reabsorption. Dump phosphate. Increase synththesis vitamin D. Get hypercalcemia. Plasma Ca chronically elevated, Ca excretion increased due to increased load. Figure out phosphate in plasma and urine.
Prob 5:
see paper.
Look over his questions.
Do not “vomit” on paper- organize your thoughts.
Tavalin
Ponder his self-study questions.
2. Think about stomach- sublingually the base would be right, but in the stomach the acid will be non-ionized. The base would be absorption in the small intestine, with better microenvironment.
3. Know volume of different compartments. 40 TBW, 4 blood. Unless stated, assume nothing. This drug retained in plasma compartment, bound to proteins in blood.
4. Descending phase is the parameter most affected by elimination. See beginning of his lectures.
5. cyt p450-functionalization or activation of compound to make it more polar, ionized, soluble.
More reactive for subsequent metabolism.
6. conjugation reactions- adding AAs, glucoronidation, sulfation, acetylation- add moiety to parent compound- make more polar generally.
Know metabolism of his favorite molecule, ethanol.
7. What are major routes of elimination- urine, feces, exhalation lungs, skin.
8. Make urine more basic to speed elimination. ion trapping- drug resides in a compartment due to its pH. Basic drugs tend to reside in stomach. Acidic will reside in intestines.
9. Be able to work it one way or other. Answer 24 hours.
With half lives, a constant fraction is removes per unit time.
10. You remove a constant amount every hour-zero order. HAlf lives are first order.
11. Assume act at same receptor with same response. Could give 100 mg every 2 hrs or 300 every 6.
12. Plateau principle- takes 5 half-lives to reach steady state. If administered every 6 hours-change dose interval, changes steady state level (final concentration) Interval dominated by elimination kinetics. Interval determines variability and plateau fluctuations (in perfect world). Longer interval, bigger fluctuations. Shorter interval, higher escalation drug concentration and less variability in drug concentration. When should you measure drug concentration to titrate? Measure at peak and trough (toxicity, and minimum effective dose).
13. Loading dose- relationship of dose and target concentration is similar to volume of distribution. Only difference is you have a bioavailability factor, which he said is 100% in this question. So it does not matter.
Volume of distribution is the theoretical volume in which administered dose appears to be contained.
Volume >TBW- it concentrates somewhere (lipids, etc). Dose is outside plasma compartment. concentration decreases in plasma.
14. 60%
His question has multiple parts- 6 or 7. With each part , be able to calculate or interpret like the thinking questions he gave us. For why- no diatribes, answer with a sentence or a small paragraph.
Wednesday, December 20, 2006
Saturday, December 16, 2006
Reviewing for exams
The posts here are all I have. There was no real review for the endocrinology and pharm sections in Systems Biology, though we do have thought questions from pharm and the presentation questions from those sections. I would go back and dig those up. Dr. Nutting says he likes thinking questions. Consider those at the end of the packet and the presentation question. I was able to come up with a few hints while reworking Dr. Cook's lecture notes for possible questions from him for CMB- see my notes, especially concerning the comparison of fatty acid oxidation and the second half of TCA. Pray hard and study hard, and you'll be fine. May God help us all. Merry Christmas/ Happy Hannukah/ Happy Kwanzaa/ Happy Break.
Mitzi
Mitzi
Review with Nelson
Look at space he alotted in notes for emphasis on exam. He won’t use the particular questions in the practice exam again. 6 lectures were 1.5 hrs each.
Starting with TCA Cycle: looking at big picture, know pyruvatedehydrogenase in detail and how subunits work. What is process with dihydrolipoylgroup? Look at similarities pyruvate dehydrogenase and other dehydrogenases and glycine cleavage system. TCA cycle is closed circle generating NaDHand FADH2, passed on to ETC to follow through redox centers,- to+ potential- energy captured in proton gradient.
Real insight was Peter Mitchel and chemiosmotic hypothesis. Use proton gradient, not high-energy intermediate.
Distinguish substrate level and oxidative phosphorylation. Mitchel proposed that inner mitochondrial membrane was imperm to protons to build up gradient.
TCA and other pathways- 1st 3 steps similar to other pathways. When pathways of AA mimic TCA, be aware.
TCA is generating NADH as produced. Cycle cannot cont w/o reoxidizing NADH- run out of substrate. Passed along to ETC complex1-> flavin NADH is obligatory 2electron donor, cannot reduce iron center. Flavin can take 2 and pass on one electron at a time.
Think about TCA depending on reoxidation NADH, depending on ETC.Some bacteria have other ways- some organisms miss parts of TCA, like Haemophilus. Influenza runs pieces of the cycle linearly in 2 directions. malate dehydrogenase to fumarase ran backwards to make succinyl coA to make heme. Consumes NADH from other side.
TCA is source of building blocks for other biosynthetic processes, alpha-ketoglutarate, oxaloacetate, etc. Asp in nucleic acid biosynthesis and urea cycle. Be familiar with anaplerotic reactions like pyruvate carboxylase to regenerate oxaloacetate.
Glyoxylate cycle- protists and plants and bacteria bypass decarboxylating steps in cycle. We cannot convert 2-C substrates into sugars because there is no net gain of C in TCA. Organisms that make sugars from Acetyl CoA do so by glyoxylate shunt. Isocitrate lyase yields glyoxylate and succinate. Glyoxylate +Acetyl CoA yield malate to continue the cycle. Target for drugs.
ETC lecture
FADH2 produced in succinate dehydrogenase (complex2)enzyme with dual role. FADH2 used instead of NADH because electrons do not have thermodynamic force to make NADH. Bypass complex 1- miss 4 protons- less ATP if you start with complex 2.
I large membrane sector with 7 subunits, arm in matrix has redox centers, flavin, iron sulfur centers, and ubiquinone. Keep movement of electrons through complexes in mind.
uQ binds to get reduced and oxidized in Q cycle.
bc1: Rieske FeS center and BL heme split path of electrons. one to cytochrome c1 and cytochrome c, one from Bl to BH, to UQ. Cycle repeats to reduce second ubiquinone at N center. Protons on uQ pumped out across membrane, driven by ability of structure to capture energy when electrons move.
complx4- electrons carried on cytochrome c. Found every organism, almost. Structure conserved. Heme in it to carry 1 electrons. Binds cytochrome C structure- to cytochrome c oxidase. Be fam w structures in 4, copper a and b binuuclear center. Oxygen binds betw Feand Cu. Protons to make water are chemical protns. Pumped ones are distinct. Certain number common inhibitors used- rotenone I, antamycin A , III, cyanide IV, be aware of where list of them in notes works.
ATP synthase: complex molecule. Be able to sketch bacterial structure with FO and F1, ball with alpha, beta, delta. b not crystallized.Through center- axle is gamma. Associated w epsilon. Stalk through ball toward membrane surface. C subunits form ring with 2 transmembrane segments each, with asp carboxyl important to the mechanism. Protons protonate the carboxyl group. know elevator model. Force of proton gradient turns 1/10 or 1/12 rotation. As proton goes around, finds exit channel to matrix. Be familiar with binding change mechanism. 3 conformations active site on beta subunit: Loose, tight, open. Conformations are interchangeable. Turning gamma distorts alpha and beta to change conformation. Change in conformation is responsible for releasing the ATP tightly bound at the tight site. Phosph bonds formed spontaneously and becomes tightly bound- force of H gradient needed to release ATP.
Remember reconstituted vesicle experiment with bacteriorhodopsin. Synthesize ATP with light to cause proton gradient in vesicle.Proved no high energy intermediate.
Evidence to prove ATP synthase turned- used Cys engineered in molecule between gamma and beta- oxidized-disulfide formed-could not turn. Reduce- restore function (wont ask about this). More elegant- beta with histidine tag on N terminal to anchor ATP synthase to nickel coating, held subunit down. Attached actin filament to gamma using biotin and avidin. Could see turning.
P/O ratio- consists of the number of ATP made for every electron. Look complex by complex. Complex 2- no protons. 14 p+per pair electrons. 1:4,3: 4,4: 2. ATP synthase produces 3 ATP per turn. 12 subunits- 12 protons required. 4 protons per ATP. 10= 3.3 protons per ATP. not much data.
Be aware of mitochondrial carrier proteins in membrane to translocate small negative molecules ATP, phosphate, FAs, malate, asp, alpha-ketoglutarate, etc. ATP must come out.Exchange ATP/ADP is high rate.
Similar amount phosphate through.
Metabolism AAs- 40 pathways. Do not memorize or draw structure.
Look at emphasis in notes- similar pathways to TCA, source of N from transamination, PLP involved in exchange, have glu dehydrogenase to remove amine from backbone- dehydrogenase removes and leaves as ammonia.
Basic principle- most compounds come from few starting compounds from glycolysis and TCA- break down to compounds in TCA. Keep intermediates in mind. Synthesis- break down into families based on starting elements- 5 families. See lecture for details.
Common features- activate carboxyl group by adding phosphate, AMP, succinyl coA.
Synthesis proline is different- cycle formed in an aldehyde reaction with amine at alpha carbon. alpha-ketoglutarate side chain carboxyl activated, Schiff base, forms ring. Synthesize arg, same method- acetylate alpha-amino group to protect- make ornithine, take acetyl group off.
Feature- metabolism of small molecule- easier to build uup, then break down. Gly up to Ser, then break down Ser. Where else does this happen? Carboxylate and split down middle.
Aromatic AAs- we do not have shikimate pathway. Be aware of herbicide Roundup and what it inhibits.
Metabolons predicted to exist, hard to isolate. Multiple enzymes exist in pathways, assembled into complex to channel substrate through. No release into media, evidence for channeling. Seen in trp pathway. Some genes fused, proteins coupled with disulfide bonds.
Reguation of pathways- complex branching- where does regulation take place? at branchpoints. end products feed back to inhibit at branch points. alternate ways to regulate in different organisms- aspartyl kinase in bacillus subtilis vs insects. chart of how differences in regulation look is in notes.
Urea cycle- 2 compartments- 2 steps in mitochondrion, 2 in cytosol in mammals. Dont worry about yeast. Synthesize carbamoyl phosphate in mitochondrial. transporters move ornithine in and citrulline out.Interesting step- Consumes ammonia- one N comes from Asp. Remember names of compounds. Split off fumarate- seen in purine and pyrimidine metabolism. Urea eliminates N in some mammals. Concentrated form of nitrogen. Live in air, cant diffuse out ammonia. For animals with weight concerns, take to uric acid to excrete as solid and not waste water (4Ns). CPS is distinct in cytosol (pyrimidine metabolism- uses gln for N) and mitochondrion (urea cycle).
Breakdown AAs- often see dioxygenases. 2 oxygen into product. Monoxygenase look like cytochrome p450- one oxygen in molecule, one to water. Used to break rings in degradation pathways.
Human diseases- have in mind as material for questions.Which enzymes causes which disease.
Branched chain- no questions.
Nucleic acid metabolism
De novo synthesis pathways in some detail- talk in meaningful way in terms of where inhibition act, defects in regulation, PRPS1 role in feedback regulation- can be overactive- too much purine- gout.
Causes of gout- PRPP activates pathway, oversynthesize purines with too much urate, precipitates in joints, causes gout. Pathway features- where Glu used, 2 sites of N10 formyl tethydrofolate as donor, affected by methatrexate. Talk about where impacts are on synthesis. Know GPAT, which affected by Gln and hydrofolate. Branch at IMP to AMP and GMP. See regulation of this. To make GMP requires ATP, ATP requires GTP. Allows uniform making of molecules.
Adenylosuccinate lyase- important to formation AMP, involved in splitting off fumarate in another rxn. as well. Disease associated is an autism disorder. Breakdown purines- costly to make, so salvage pathways used. HGPRT used. couples hypoxanthine and guanine w PRPP. Absence produces Lesch Nyhan syndrome.
Breakdown- adenosine deaminase in SCID. If compound builds, inhibits ribonucleic acid reductase which makes DNA nucleic acids. Block purine pathway, intersecting pathways affect each other.
Be aware of allopurinol. Analog hypoxanthine.Converted to inhibit xanthine oxidase, Treat gout.
Pyrimidine synthesis pathway- Gln nitrogen source. UMP has to be converted to TMP to make dTMP for DNA. Long and indirect path. Enzymes is thymidylate synthase. Look it up.Uses N5N10 tetrahydrofolate, converted to dihydrofolate, regenerate tetrahydrofolate by dihydrofolate reduction, which is inhibited by methotrexate. Treat infection and cancers by inhibiting DNA synthesis
Be aware of how bifluorouracil works.
see notes.
Azoserine and DON, too. AZT is analog of thymidine- interferes with reverse transcriptase. Incorporate- no 3’OH.Resistance develops to this compound, so there are inhibitors for proteases to process protein to make virus and other reverse transcriptase inhibitors. Look at notes. Will not pick obscure facts.
Starting with TCA Cycle: looking at big picture, know pyruvatedehydrogenase in detail and how subunits work. What is process with dihydrolipoylgroup? Look at similarities pyruvate dehydrogenase and other dehydrogenases and glycine cleavage system. TCA cycle is closed circle generating NaDHand FADH2, passed on to ETC to follow through redox centers,- to+ potential- energy captured in proton gradient.
Real insight was Peter Mitchel and chemiosmotic hypothesis. Use proton gradient, not high-energy intermediate.
Distinguish substrate level and oxidative phosphorylation. Mitchel proposed that inner mitochondrial membrane was imperm to protons to build up gradient.
TCA and other pathways- 1st 3 steps similar to other pathways. When pathways of AA mimic TCA, be aware.
TCA is generating NADH as produced. Cycle cannot cont w/o reoxidizing NADH- run out of substrate. Passed along to ETC complex1-> flavin NADH is obligatory 2electron donor, cannot reduce iron center. Flavin can take 2 and pass on one electron at a time.
Think about TCA depending on reoxidation NADH, depending on ETC.Some bacteria have other ways- some organisms miss parts of TCA, like Haemophilus. Influenza runs pieces of the cycle linearly in 2 directions. malate dehydrogenase to fumarase ran backwards to make succinyl coA to make heme. Consumes NADH from other side.
TCA is source of building blocks for other biosynthetic processes, alpha-ketoglutarate, oxaloacetate, etc. Asp in nucleic acid biosynthesis and urea cycle. Be familiar with anaplerotic reactions like pyruvate carboxylase to regenerate oxaloacetate.
Glyoxylate cycle- protists and plants and bacteria bypass decarboxylating steps in cycle. We cannot convert 2-C substrates into sugars because there is no net gain of C in TCA. Organisms that make sugars from Acetyl CoA do so by glyoxylate shunt. Isocitrate lyase yields glyoxylate and succinate. Glyoxylate +Acetyl CoA yield malate to continue the cycle. Target for drugs.
ETC lecture
FADH2 produced in succinate dehydrogenase (complex2)enzyme with dual role. FADH2 used instead of NADH because electrons do not have thermodynamic force to make NADH. Bypass complex 1- miss 4 protons- less ATP if you start with complex 2.
I large membrane sector with 7 subunits, arm in matrix has redox centers, flavin, iron sulfur centers, and ubiquinone. Keep movement of electrons through complexes in mind.
uQ binds to get reduced and oxidized in Q cycle.
bc1: Rieske FeS center and BL heme split path of electrons. one to cytochrome c1 and cytochrome c, one from Bl to BH, to UQ. Cycle repeats to reduce second ubiquinone at N center. Protons on uQ pumped out across membrane, driven by ability of structure to capture energy when electrons move.
complx4- electrons carried on cytochrome c. Found every organism, almost. Structure conserved. Heme in it to carry 1 electrons. Binds cytochrome C structure- to cytochrome c oxidase. Be fam w structures in 4, copper a and b binuuclear center. Oxygen binds betw Feand Cu. Protons to make water are chemical protns. Pumped ones are distinct. Certain number common inhibitors used- rotenone I, antamycin A , III, cyanide IV, be aware of where list of them in notes works.
ATP synthase: complex molecule. Be able to sketch bacterial structure with FO and F1, ball with alpha, beta, delta. b not crystallized.Through center- axle is gamma. Associated w epsilon. Stalk through ball toward membrane surface. C subunits form ring with 2 transmembrane segments each, with asp carboxyl important to the mechanism. Protons protonate the carboxyl group. know elevator model. Force of proton gradient turns 1/10 or 1/12 rotation. As proton goes around, finds exit channel to matrix. Be familiar with binding change mechanism. 3 conformations active site on beta subunit: Loose, tight, open. Conformations are interchangeable. Turning gamma distorts alpha and beta to change conformation. Change in conformation is responsible for releasing the ATP tightly bound at the tight site. Phosph bonds formed spontaneously and becomes tightly bound- force of H gradient needed to release ATP.
Remember reconstituted vesicle experiment with bacteriorhodopsin. Synthesize ATP with light to cause proton gradient in vesicle.Proved no high energy intermediate.
Evidence to prove ATP synthase turned- used Cys engineered in molecule between gamma and beta- oxidized-disulfide formed-could not turn. Reduce- restore function (wont ask about this). More elegant- beta with histidine tag on N terminal to anchor ATP synthase to nickel coating, held subunit down. Attached actin filament to gamma using biotin and avidin. Could see turning.
P/O ratio- consists of the number of ATP made for every electron. Look complex by complex. Complex 2- no protons. 14 p+per pair electrons. 1:4,3: 4,4: 2. ATP synthase produces 3 ATP per turn. 12 subunits- 12 protons required. 4 protons per ATP. 10= 3.3 protons per ATP. not much data.
Be aware of mitochondrial carrier proteins in membrane to translocate small negative molecules ATP, phosphate, FAs, malate, asp, alpha-ketoglutarate, etc. ATP must come out.Exchange ATP/ADP is high rate.
Similar amount phosphate through.
Metabolism AAs- 40 pathways. Do not memorize or draw structure.
Look at emphasis in notes- similar pathways to TCA, source of N from transamination, PLP involved in exchange, have glu dehydrogenase to remove amine from backbone- dehydrogenase removes and leaves as ammonia.
Basic principle- most compounds come from few starting compounds from glycolysis and TCA- break down to compounds in TCA. Keep intermediates in mind. Synthesis- break down into families based on starting elements- 5 families. See lecture for details.
Common features- activate carboxyl group by adding phosphate, AMP, succinyl coA.
Synthesis proline is different- cycle formed in an aldehyde reaction with amine at alpha carbon. alpha-ketoglutarate side chain carboxyl activated, Schiff base, forms ring. Synthesize arg, same method- acetylate alpha-amino group to protect- make ornithine, take acetyl group off.
Feature- metabolism of small molecule- easier to build uup, then break down. Gly up to Ser, then break down Ser. Where else does this happen? Carboxylate and split down middle.
Aromatic AAs- we do not have shikimate pathway. Be aware of herbicide Roundup and what it inhibits.
Metabolons predicted to exist, hard to isolate. Multiple enzymes exist in pathways, assembled into complex to channel substrate through. No release into media, evidence for channeling. Seen in trp pathway. Some genes fused, proteins coupled with disulfide bonds.
Reguation of pathways- complex branching- where does regulation take place? at branchpoints. end products feed back to inhibit at branch points. alternate ways to regulate in different organisms- aspartyl kinase in bacillus subtilis vs insects. chart of how differences in regulation look is in notes.
Urea cycle- 2 compartments- 2 steps in mitochondrion, 2 in cytosol in mammals. Dont worry about yeast. Synthesize carbamoyl phosphate in mitochondrial. transporters move ornithine in and citrulline out.Interesting step- Consumes ammonia- one N comes from Asp. Remember names of compounds. Split off fumarate- seen in purine and pyrimidine metabolism. Urea eliminates N in some mammals. Concentrated form of nitrogen. Live in air, cant diffuse out ammonia. For animals with weight concerns, take to uric acid to excrete as solid and not waste water (4Ns). CPS is distinct in cytosol (pyrimidine metabolism- uses gln for N) and mitochondrion (urea cycle).
Breakdown AAs- often see dioxygenases. 2 oxygen into product. Monoxygenase look like cytochrome p450- one oxygen in molecule, one to water. Used to break rings in degradation pathways.
Human diseases- have in mind as material for questions.Which enzymes causes which disease.
Branched chain- no questions.
Nucleic acid metabolism
De novo synthesis pathways in some detail- talk in meaningful way in terms of where inhibition act, defects in regulation, PRPS1 role in feedback regulation- can be overactive- too much purine- gout.
Causes of gout- PRPP activates pathway, oversynthesize purines with too much urate, precipitates in joints, causes gout. Pathway features- where Glu used, 2 sites of N10 formyl tethydrofolate as donor, affected by methatrexate. Talk about where impacts are on synthesis. Know GPAT, which affected by Gln and hydrofolate. Branch at IMP to AMP and GMP. See regulation of this. To make GMP requires ATP, ATP requires GTP. Allows uniform making of molecules.
Adenylosuccinate lyase- important to formation AMP, involved in splitting off fumarate in another rxn. as well. Disease associated is an autism disorder. Breakdown purines- costly to make, so salvage pathways used. HGPRT used. couples hypoxanthine and guanine w PRPP. Absence produces Lesch Nyhan syndrome.
Breakdown- adenosine deaminase in SCID. If compound builds, inhibits ribonucleic acid reductase which makes DNA nucleic acids. Block purine pathway, intersecting pathways affect each other.
Be aware of allopurinol. Analog hypoxanthine.Converted to inhibit xanthine oxidase, Treat gout.
Pyrimidine synthesis pathway- Gln nitrogen source. UMP has to be converted to TMP to make dTMP for DNA. Long and indirect path. Enzymes is thymidylate synthase. Look it up.Uses N5N10 tetrahydrofolate, converted to dihydrofolate, regenerate tetrahydrofolate by dihydrofolate reduction, which is inhibited by methotrexate. Treat infection and cancers by inhibiting DNA synthesis
Be aware of how bifluorouracil works.
see notes.
Azoserine and DON, too. AZT is analog of thymidine- interferes with reverse transcriptase. Incorporate- no 3’OH.Resistance develops to this compound, so there are inhibitors for proteases to process protein to make virus and other reverse transcriptase inhibitors. Look at notes. Will not pick obscure facts.
Cook Notes
Cook lectures- fatty acid metabolism
George Cook- pharmacology department
He covers fatty acid synthesis and metabolism. Following Stryer, which he says is a pretty horrible text for this..
2: Start with fat cell. Easy to isolate and use in solution. The cell in the photo is full of triacylglycerol. Very little cytoplasm.
3: This slide may be an exam question. 1 g glycogen stored- exists hydrated w 2 g water. Takes 6 g in cell to= 1 g fat. Fat is important energetically. Migratory birds could not fly without storing energy as fat.
Energy is from TCA –acetyl coA goes into TCA cycle.
Slide 4:Fatty acid oxidation- note similarity to TCA cycle:
1. the succinate dehydrogenase reaction to transform succinate to fumarate in TCA is like the oxidation(dehydrogenation)of the fatty acyl coA by acyl coA dehydrogenase(3 isozymes on slide 30) to form trans-delta2-Enoyl CoA. Both enzymes have a flavin, FAD, that forms FADH2 to donate electrons to the electron transport chain in the mitochondrion.
2. the conversion of fumarate to malate (hydration) in TCA is like the hydration of trans-delta2-Enoyl CoA to form L-3 - hydroxyacyl CoA by enoyl coA hydratase (see slide 31).
3. the conversion of malate to oxaloacetate (oxidation)is like the oxidation of L-3 hydroxyacyl CoA to form 3-ketoacyl coA (see slide 32) by beta-hydroxyacyl CoA dehydrogenase, which forms NADH. Then the ketoacyl-CoA is slplit to form an acylcoA shortened by 2 carbons and an acetyl-CoA that can go into the TCA cycle by thiolase (See slide 33). Why does it happen this way? When you find a way that works, you use it. See slides 29-33.
Look at the synthesis overview on the same slide(4) and refer to slides 63-71: Please note that all these reactions take place while the chain is attached to the Acyl Carrier Protein (ACP) in a massive complex of 7 proteins called Fatty Acid Synthase.
Activated acyl group +activated malonyl coA yields an acetoacyl group attached to the ACP. This condensation forms a diketoacyl ACP. Then there is reduction to form D-3 hydroxybutyryl-ACP, dehydration to form crotonyl ACP , and reduction using NADPH to form butyryl ACP, resulting in an activated acyl group to go through the cycle again until the cycle is finished with palmitate.
For synthesis and oxidation, the processes look like one is the reverse of the other, but the enzymes and locations are different. For these differences, see slide 62.
Slide 5: To do anything with fatty acids, you must get them into the cells first. Glycocholate is an example of a bile salt, which is important for the absorption of fat. Micelles form with the aid of bile salts in the intestinal lumen, which allow intestinal lipases access to digest the fats. Glucocholate is a Bile acid derived from cholesterol for lipase function.
Slide 6: Intestinal lipase clips triacylglycerol to hydrolyse off one fatty acid, to forms a diacylglycerol. Then further lipase action yields a monoacylglycerol in intestine, which is as far as metabolism goes there. No proteins were thought to transport fatty acids into the intestinal mucosal cells until recently. For short and medium-chain fatty acids, the monoacyl glycerol gets into membrane, flips, gets into cell that way. Long chain FAs appear to need help from fatty acid transporters (slide 7).. The fatty acids form triacylglycerols, which are packages in chylomicrons in the mucosal cells.
Chylomicrons go to lymph system. Why? Look at animal in fed state, see dilated blood vessels in intestine. The blood absorbs other nutrients, but chylomicrons to lymph. Do not want to deliver chylomicrons directly to liver, where they would overload the liver and be processed undesirably- want them to go to skeletal muscle and storage cells. Lymph empties in neck to general circulation. Lipids are not metabolized in liver first. Other Triacylglycerols get taken up and do go to liver.
In liver- some TAGs are put into VLDL. Good cholesterol- form HDL. Reverse cholesterol delivery- HDL brings cholesterol back to liver. Bad- LDL-leftovers when VLDLs get depleted a bit. LDLs form plaques.
FA transporters in fat tissue and muscle cells:
CD36 or Fatty Acid Transporter- most important. As originally described, it allows transport of FAs, docking to system, access so that lipases can hydrolyze triacylglycerol. 6 isoforms found in mouse and human cells. Rat different. Still questions about them . FA transport in yeast in bacteria-they form acyl coA. Free FAs have detergent function and are dangerous. FA transporters from mice can be explored in yeast. Isoforms 1 and 2 are transporters. 5 and 6 may be synthetases. 4 may be enzyme involved in cholesterol transport. Some transport not clear.
In adipose tissue cells resynthesize TAG. Stored in TAG form. To release- form free fatty acids. Lipases called hormone sensitive lipase and adipose tissue lipase are involved in forming free fatty acids for release into blood(slide 9). The glycerol liberated in adipose cells released by hormone sensitive lipase and adipose tissue lipase can be used for gluconeogenesis or glycolysis (slide 11). Hormone sensitive lipase clips first 2 fatty acids and monoacylglycerol lipase cleaves off the last fatty acid. Monoacylglycerol is active all the time.
Slide 10: Hormone sensitive lipase is highly regulated. Activated by epinephrine, norepinephrine, adrenocorticotropic hormone (ACTH). ACTH is secreted from the pituitary in response to corticotropin releasing hormone (CRH) from hypothalamus. ACTH acts on adrenal gland to produce norepinephrine and epinephrine from medulla. Hormone sensitive lipase has to be phosphorylated to be activated. Fatty acid binding protein is important in helping triacylglycerol lipase to associate with the lipid droplet after the enzyme has been phosphorylated.
11:Glycerol can be made into a primary substrate for gluconeogenesis.
12: Fatty acids released from adipose tissue are transported by albumin during fasting. Albumin has 3 binding sites. 6 disulfide bonds create hydrophobic lipophilic pockets.3 Fatty acids bound per molecule. Binds steroid hormones as well, but main function is fatty acid transport.
13: To oxidize fatty acids, must have acyl coA form. This form is necessary for oxidation or for esterification to form TAGs. ATP is hydrolyzed to inorganic pyrophosphate and AMP. The AMP is attached to the fatty acid, then HS-CoA joins to form acyl coA.
14: Steps to get to fatty acid oxidation (FRAGMENT- this comes BEFORE the steps in the summary in the beginning of the lecture, and allows the FAs to get into the mitochondrion.):
1. Acyl coA synthetase in mammals on external mitochondrial membrane catalyzes the reaction in slide 13, creating a high-energy coA bond.
2. carnitine palmitoyltransferase (CPT, his research chemical)- There isn’t an acyl-coA transporter to get through the impermeable inner mitochondrial membrane, so another form of the fatty acid has to be transported- mitochondrion has its own synthesis for coA, so we get it back.
15:Acyl carnitine is transported into mitochondrion, carnitine out. Carnitine can go in or out on transporter, but not acylcarnitine out.
Carnitine acyltransferase actually does the transfer across the membrane.CPT does the reaction to get the carnitine-acyl bond.
16:Malonyl coA inhibits fatty acid oxidation. It Is a product in synthesis used for making Fatty Acids. Oxidation and synthesis go on in same cell. Oxidation occurs in the mitochondrion, synthesis in cytosol. Malonyl coA is important to keep things in balance. Pathways are regulated in opposite directions.
There is more than one acetyl coA carboxylase. Acetyl-coA carboxylase I is free in cytosol for FA synthesis. Acetyl CoA carboxylase II has one transmembrane region and is bound to mitochondrion on outside to make malonyl coA. Heart and fatty tissue have both. There are 2 carnitine palmitoyl transferases,too. One on outside inhibited by malonyl coA and one on inside is not.
CPT activated by cardiolipin is found in inner membrane and contact sites. How FA crosses outer membrane has yet to be solved.Lipids will not go through porins- porin is too charged.
20: 3 isoforms of CPT-1. Outer membrane may contain CD36.
18: Peroxisomal fatty acid oxidation- very long chain polyunsaturated Fatty acids from fish oil are oxidized here. May have same transport- difficult to isolate. Yeast does not oxidize FA in mitochondrion- oxidize FA in peroxisome. CPT is in ER- found in microsomes. Transport systems may get FAs in to make VLDL and chylomicrons.
19:CPT on outer membrane- 2 transmembrane segments.
CPTII in matrix- has matrix targeting sequence. Cleaves and is active, but in vitro is active even without cleaving the targeting sequence.
Peroxisomal form has same specificity. COT metabolizes octanoic acid- same chain specificity as CPT.
carnitine acyl transferase (CAT)-short chain specificity. CPT I is only one with multiple isoforms.
20: alpha found in liver and kidney. Sensitive to inhibition by malonyl coA.
beta sensitive to malonyl coA.
Gamma- binds malonyl coA,important function, may be exclusive to brain. Not active with any tried substrate so far. Not known if it works on substrate. Alpha and beta are on outside of mitochondrion.
Malonly coA concentration does not vary a lot.
Mitochondrial CPT in fed animals- potently inhibited by malonyl coA. No inhibition in fasting.
Mostly level of malonyl CoA never gets over 5 micromolar.
22-23: Fasting and diabetes on hepatic CPT-Ialpha. Ki increases in diabetic animal. In liver- FA synthesized, do not want FA synthesis to be inhibited. Change prevents FA oxidation after synthesis in hepatocytes.
27:For CPT1-alpha, in liver levels change. Heart and brain- much less. Gamma really is not specific for brain.
26 and 28: More regulation- look at thyroid hormone T3 receptor. It has lots of accessory factors that bind and stimulate mRNA synthesis.PGC-1 added to cells stimulates transcription of CPT-1alpha.
29-32:Through CPT- fatty acid gets into mitochondrion- oxidized in matrix.Get acetyl coA.
First oxidation step uses FAD to get FADH2. Get 2 ATP out.
Long chain, med chain, short chain FA have their own dehydrogenases.
If you have unsaturated FAs, they come mostly from plants. All natural ones are cis to double bond. All same direction. Makes more flexibility in membrane.
See pathway on slide 38 for odd-chain fatty acid oxidation. Slides 35-45 show how propionyl coA is broken down. Most fatty acids have an even number of carbons, reflecting their synthesis. A few have odd numbers of carbons. Succinyl coA only found inside mitochondria.
Malonic acid inhibits the TCA cycle. No malonyl coA in mitochondrion.
Lecture 2:
email: gcook@utmem.edu
Will review as he goes today.
Yeast fatty acid oxidation is different from that of mammals.
Fat: The real high energy food slide could be exam question. Comparison slide of degradation and synthesis – list differences was question last year. Not on there this year, but good to know. May ask about comparison to TCA cycle.
Can also ask differences in peroxisomal and mitochondrial fatty acid oxidation See Slide 48 and Stryer 22.3.4, or if you have Lehninger 4th edition, a really nice diagram in figure 17-13..
Picture of peroxisome in slide 46- has single membrane packed with stuff. Crystalline structure may be urate oxidase, artifact of fixing cells. Shows how tightly packed they are.
Peroxisomal FA oxidation- important for long chain fatty acids. Phytol and phytanic acid degradation pathways are important. Toxic byproducts of degradation of chlorophyll. Can get a lttle energy from them. Literature of peroxisomal FA oxidation- he says FA are oxidized completely in peroxisome.
Zellweger syndrome- few peroxisomes. Retarded brain development.Another disease- deficiency acechoic (??wrong spelling, but that’s how he pronounced it) oxidase in peroxisomes- get same symptoms of retarded brain development and decreased myelin sheath and buildup of long chain FAs.
First step in peroxisome is acyl coA oxidase- oxidase, not dehydrogenase.
48: Early interest in peroxisome proliferators was in drugs for lipid lowering by increasing oxidation in peroxisomes. Stimulates synthesis carnitine palmitoyl transferase 1 amountt peroxisome formation in animals is more than in humans. Peroxisome proliferation in animals is associated with liver cancer - not necessarily in humans.
49:Looking at rat, mouse, and humans CPT1-alpha genes- why do FAs stimulate formation of CPT1 protein? All have long second intron. Lots of elements for thyroid hormone receptor binding there. Other regulatory properties there, including PPAR regulation.
50: Relation fatty acid oxidation and glucose oxidation- In heart and skeletal muscle- switch from FA to glucose oxidation. heart can live and pump in glucose solution happily for a few days. Add FAs- FAs oxidized, and glucose oxidation stops. End up w lactic acidosis-
Pyruvate dehydrogenase kinase is PDK.FA oxidation does not block glycolysis, so lactate builds up. Cant be transferred. To get lactate to pyruvate, must get rid of reducing equivalents. Heart has 4 isoforms of PDK, PDK4 most important. Pyruvate carboxylase is constitutively produced, not regulated.
51: Ketone body synthesis- fatty acids in liver and kidney are converted to ketone bodies.Muscle uses fatty acids for energy, converting them to CO2.
53: Ketone bodies are used by brain for energy during fasting.HMGcoA synthase is highly regulated, by cortisol (increased during fasting, increased gluconeogenesis thru PEP carboxykinase and stimulation of HMG coA synthase) and by glucocorticoids.
54: Lyase pushed to make more ketone bodies.
56: Measuring 3-hydroxybutyrate-acetoacetate ratio tells information about ketone body formation in liver.
57: Tissues that don’t make ketone bodies have enzymes to use them. Ketone body utilization happens all the time- Pathway not induced or capacity induced. Utilization is increased.
58: Ketone bodies go too high due to increased fatty acid oxidation- urine acidic.
59: Insulin regulates lipolysis and inhibits FA oxidation in liver.
60: Acetyl coA carboxylaseI in cytosol and fatty acid synthase reactions shown.Fatty acid synthase stops at palmitate. Acetyl coA carboxylase is Regulated by phosphorylation (see slide 61).
62 was a question last year.
63: ACP is a large carrier molecule with a phosphopantetheine group. These Enzymes are free in bacteria.
FA synthesis: see beginning of lecture and slides 64-71.
FA synthase genes are linked in mammals. Continuous AA sequence from one enzyme to another.
72: Some citrate from mitochondrion is transferred out by tricarboxylate carrier.Reacts using ATP-citrate lyase releases acetyl coA.
oxaloacetate to malate by malate dehydrogenase is another shuttle important to this pathway.
74: Peroxisomes- 2 fatty acid oxidation enzymes are combined.
75-6: Regulation of synthesis:
Acetyl CoA carboxylase- regulated by phosphorylation and by citrate. Citrate regulation slide he says is wrong. w 10mM citrate with pure enzyme, get same activation w phosphorylated and dephosphorylated form. No citrate- no activation in phosphorylated form, activation in dephosphorylated. Binding constant changes. Binds better or not as well.
77: Chicken ac coA carboxylase forms filaments in solution with citrate.
78: Enzyme important in obesity: data from gene chips-Stearoyl CoA desaturase induced in obesity. Stearoyl coA converts to monounsaturated oleoylcoA. Desaturated. Plants make unsaturated bonds by elongation of the chain and desaturation, alternating. Animals can insert an unsaturated bond after the chain is formed.
79: Linoleate and linolenate cannot be synthesized in animals.Essential for mammals.Omega-3 designation means 1st point of unsaturation (double bond) is 3 carbons from omega end.
80: Prostaglandin synthesis-
comes from FAs.Arachidonates come from phospholipids. Phospholipase A2 cuts off middle FA of TAG. to make arachidonic acid, then prostaglandins using cyclooxygenase. NSAIDS inhibit synthesis by inhibiting cyclooxygenase. COX 2 is present in inflammation- inducible enzyme. aspirin prevents inflammation by blocking COX in many tissues. Some prostaglandins protect stomach. Others protect heart and kidneys, so inhibiting them is bad. Glucocorticoids are also antinflammatory. They bind to nuclear receptors and regulate phospholipase A2 expression- knock it down.
For test:
First question usually about comparing one thing and another. What are possibliities for differences...? He has 2 questions on the test.
George Cook- pharmacology department
He covers fatty acid synthesis and metabolism. Following Stryer, which he says is a pretty horrible text for this..
2: Start with fat cell. Easy to isolate and use in solution. The cell in the photo is full of triacylglycerol. Very little cytoplasm.
3: This slide may be an exam question. 1 g glycogen stored- exists hydrated w 2 g water. Takes 6 g in cell to= 1 g fat. Fat is important energetically. Migratory birds could not fly without storing energy as fat.
Energy is from TCA –acetyl coA goes into TCA cycle.
Slide 4:Fatty acid oxidation- note similarity to TCA cycle:
1. the succinate dehydrogenase reaction to transform succinate to fumarate in TCA is like the oxidation(dehydrogenation)of the fatty acyl coA by acyl coA dehydrogenase(3 isozymes on slide 30) to form trans-delta2-Enoyl CoA. Both enzymes have a flavin, FAD, that forms FADH2 to donate electrons to the electron transport chain in the mitochondrion.
2. the conversion of fumarate to malate (hydration) in TCA is like the hydration of trans-delta2-Enoyl CoA to form L-3 - hydroxyacyl CoA by enoyl coA hydratase (see slide 31).
3. the conversion of malate to oxaloacetate (oxidation)is like the oxidation of L-3 hydroxyacyl CoA to form 3-ketoacyl coA (see slide 32) by beta-hydroxyacyl CoA dehydrogenase, which forms NADH. Then the ketoacyl-CoA is slplit to form an acylcoA shortened by 2 carbons and an acetyl-CoA that can go into the TCA cycle by thiolase (See slide 33). Why does it happen this way? When you find a way that works, you use it. See slides 29-33.
Look at the synthesis overview on the same slide(4) and refer to slides 63-71: Please note that all these reactions take place while the chain is attached to the Acyl Carrier Protein (ACP) in a massive complex of 7 proteins called Fatty Acid Synthase.
Activated acyl group +activated malonyl coA yields an acetoacyl group attached to the ACP. This condensation forms a diketoacyl ACP. Then there is reduction to form D-3 hydroxybutyryl-ACP, dehydration to form crotonyl ACP , and reduction using NADPH to form butyryl ACP, resulting in an activated acyl group to go through the cycle again until the cycle is finished with palmitate.
For synthesis and oxidation, the processes look like one is the reverse of the other, but the enzymes and locations are different. For these differences, see slide 62.
Slide 5: To do anything with fatty acids, you must get them into the cells first. Glycocholate is an example of a bile salt, which is important for the absorption of fat. Micelles form with the aid of bile salts in the intestinal lumen, which allow intestinal lipases access to digest the fats. Glucocholate is a Bile acid derived from cholesterol for lipase function.
Slide 6: Intestinal lipase clips triacylglycerol to hydrolyse off one fatty acid, to forms a diacylglycerol. Then further lipase action yields a monoacylglycerol in intestine, which is as far as metabolism goes there. No proteins were thought to transport fatty acids into the intestinal mucosal cells until recently. For short and medium-chain fatty acids, the monoacyl glycerol gets into membrane, flips, gets into cell that way. Long chain FAs appear to need help from fatty acid transporters (slide 7).. The fatty acids form triacylglycerols, which are packages in chylomicrons in the mucosal cells.
Chylomicrons go to lymph system. Why? Look at animal in fed state, see dilated blood vessels in intestine. The blood absorbs other nutrients, but chylomicrons to lymph. Do not want to deliver chylomicrons directly to liver, where they would overload the liver and be processed undesirably- want them to go to skeletal muscle and storage cells. Lymph empties in neck to general circulation. Lipids are not metabolized in liver first. Other Triacylglycerols get taken up and do go to liver.
In liver- some TAGs are put into VLDL. Good cholesterol- form HDL. Reverse cholesterol delivery- HDL brings cholesterol back to liver. Bad- LDL-leftovers when VLDLs get depleted a bit. LDLs form plaques.
FA transporters in fat tissue and muscle cells:
CD36 or Fatty Acid Transporter- most important. As originally described, it allows transport of FAs, docking to system, access so that lipases can hydrolyze triacylglycerol. 6 isoforms found in mouse and human cells. Rat different. Still questions about them . FA transport in yeast in bacteria-they form acyl coA. Free FAs have detergent function and are dangerous. FA transporters from mice can be explored in yeast. Isoforms 1 and 2 are transporters. 5 and 6 may be synthetases. 4 may be enzyme involved in cholesterol transport. Some transport not clear.
In adipose tissue cells resynthesize TAG. Stored in TAG form. To release- form free fatty acids. Lipases called hormone sensitive lipase and adipose tissue lipase are involved in forming free fatty acids for release into blood(slide 9). The glycerol liberated in adipose cells released by hormone sensitive lipase and adipose tissue lipase can be used for gluconeogenesis or glycolysis (slide 11). Hormone sensitive lipase clips first 2 fatty acids and monoacylglycerol lipase cleaves off the last fatty acid. Monoacylglycerol is active all the time.
Slide 10: Hormone sensitive lipase is highly regulated. Activated by epinephrine, norepinephrine, adrenocorticotropic hormone (ACTH). ACTH is secreted from the pituitary in response to corticotropin releasing hormone (CRH) from hypothalamus. ACTH acts on adrenal gland to produce norepinephrine and epinephrine from medulla. Hormone sensitive lipase has to be phosphorylated to be activated. Fatty acid binding protein is important in helping triacylglycerol lipase to associate with the lipid droplet after the enzyme has been phosphorylated.
11:Glycerol can be made into a primary substrate for gluconeogenesis.
12: Fatty acids released from adipose tissue are transported by albumin during fasting. Albumin has 3 binding sites. 6 disulfide bonds create hydrophobic lipophilic pockets.3 Fatty acids bound per molecule. Binds steroid hormones as well, but main function is fatty acid transport.
13: To oxidize fatty acids, must have acyl coA form. This form is necessary for oxidation or for esterification to form TAGs. ATP is hydrolyzed to inorganic pyrophosphate and AMP. The AMP is attached to the fatty acid, then HS-CoA joins to form acyl coA.
14: Steps to get to fatty acid oxidation (FRAGMENT- this comes BEFORE the steps in the summary in the beginning of the lecture, and allows the FAs to get into the mitochondrion.):
1. Acyl coA synthetase in mammals on external mitochondrial membrane catalyzes the reaction in slide 13, creating a high-energy coA bond.
2. carnitine palmitoyltransferase (CPT, his research chemical)- There isn’t an acyl-coA transporter to get through the impermeable inner mitochondrial membrane, so another form of the fatty acid has to be transported- mitochondrion has its own synthesis for coA, so we get it back.
15:Acyl carnitine is transported into mitochondrion, carnitine out. Carnitine can go in or out on transporter, but not acylcarnitine out.
Carnitine acyltransferase actually does the transfer across the membrane.CPT does the reaction to get the carnitine-acyl bond.
16:Malonyl coA inhibits fatty acid oxidation. It Is a product in synthesis used for making Fatty Acids. Oxidation and synthesis go on in same cell. Oxidation occurs in the mitochondrion, synthesis in cytosol. Malonyl coA is important to keep things in balance. Pathways are regulated in opposite directions.
There is more than one acetyl coA carboxylase. Acetyl-coA carboxylase I is free in cytosol for FA synthesis. Acetyl CoA carboxylase II has one transmembrane region and is bound to mitochondrion on outside to make malonyl coA. Heart and fatty tissue have both. There are 2 carnitine palmitoyl transferases,too. One on outside inhibited by malonyl coA and one on inside is not.
CPT activated by cardiolipin is found in inner membrane and contact sites. How FA crosses outer membrane has yet to be solved.Lipids will not go through porins- porin is too charged.
20: 3 isoforms of CPT-1. Outer membrane may contain CD36.
18: Peroxisomal fatty acid oxidation- very long chain polyunsaturated Fatty acids from fish oil are oxidized here. May have same transport- difficult to isolate. Yeast does not oxidize FA in mitochondrion- oxidize FA in peroxisome. CPT is in ER- found in microsomes. Transport systems may get FAs in to make VLDL and chylomicrons.
19:CPT on outer membrane- 2 transmembrane segments.
CPTII in matrix- has matrix targeting sequence. Cleaves and is active, but in vitro is active even without cleaving the targeting sequence.
Peroxisomal form has same specificity. COT metabolizes octanoic acid- same chain specificity as CPT.
carnitine acyl transferase (CAT)-short chain specificity. CPT I is only one with multiple isoforms.
20: alpha found in liver and kidney. Sensitive to inhibition by malonyl coA.
beta sensitive to malonyl coA.
Gamma- binds malonyl coA,important function, may be exclusive to brain. Not active with any tried substrate so far. Not known if it works on substrate. Alpha and beta are on outside of mitochondrion.
Malonly coA concentration does not vary a lot.
Mitochondrial CPT in fed animals- potently inhibited by malonyl coA. No inhibition in fasting.
Mostly level of malonyl CoA never gets over 5 micromolar.
22-23: Fasting and diabetes on hepatic CPT-Ialpha. Ki increases in diabetic animal. In liver- FA synthesized, do not want FA synthesis to be inhibited. Change prevents FA oxidation after synthesis in hepatocytes.
27:For CPT1-alpha, in liver levels change. Heart and brain- much less. Gamma really is not specific for brain.
26 and 28: More regulation- look at thyroid hormone T3 receptor. It has lots of accessory factors that bind and stimulate mRNA synthesis.PGC-1 added to cells stimulates transcription of CPT-1alpha.
29-32:Through CPT- fatty acid gets into mitochondrion- oxidized in matrix.Get acetyl coA.
First oxidation step uses FAD to get FADH2. Get 2 ATP out.
Long chain, med chain, short chain FA have their own dehydrogenases.
If you have unsaturated FAs, they come mostly from plants. All natural ones are cis to double bond. All same direction. Makes more flexibility in membrane.
See pathway on slide 38 for odd-chain fatty acid oxidation. Slides 35-45 show how propionyl coA is broken down. Most fatty acids have an even number of carbons, reflecting their synthesis. A few have odd numbers of carbons. Succinyl coA only found inside mitochondria.
Malonic acid inhibits the TCA cycle. No malonyl coA in mitochondrion.
Lecture 2:
email: gcook@utmem.edu
Will review as he goes today.
Yeast fatty acid oxidation is different from that of mammals.
Fat: The real high energy food slide could be exam question. Comparison slide of degradation and synthesis – list differences was question last year. Not on there this year, but good to know. May ask about comparison to TCA cycle.
Can also ask differences in peroxisomal and mitochondrial fatty acid oxidation See Slide 48 and Stryer 22.3.4, or if you have Lehninger 4th edition, a really nice diagram in figure 17-13..
Picture of peroxisome in slide 46- has single membrane packed with stuff. Crystalline structure may be urate oxidase, artifact of fixing cells. Shows how tightly packed they are.
Peroxisomal FA oxidation- important for long chain fatty acids. Phytol and phytanic acid degradation pathways are important. Toxic byproducts of degradation of chlorophyll. Can get a lttle energy from them. Literature of peroxisomal FA oxidation- he says FA are oxidized completely in peroxisome.
Zellweger syndrome- few peroxisomes. Retarded brain development.Another disease- deficiency acechoic (??wrong spelling, but that’s how he pronounced it) oxidase in peroxisomes- get same symptoms of retarded brain development and decreased myelin sheath and buildup of long chain FAs.
First step in peroxisome is acyl coA oxidase- oxidase, not dehydrogenase.
48: Early interest in peroxisome proliferators was in drugs for lipid lowering by increasing oxidation in peroxisomes. Stimulates synthesis carnitine palmitoyl transferase 1 amountt peroxisome formation in animals is more than in humans. Peroxisome proliferation in animals is associated with liver cancer - not necessarily in humans.
49:Looking at rat, mouse, and humans CPT1-alpha genes- why do FAs stimulate formation of CPT1 protein? All have long second intron. Lots of elements for thyroid hormone receptor binding there. Other regulatory properties there, including PPAR regulation.
50: Relation fatty acid oxidation and glucose oxidation- In heart and skeletal muscle- switch from FA to glucose oxidation. heart can live and pump in glucose solution happily for a few days. Add FAs- FAs oxidized, and glucose oxidation stops. End up w lactic acidosis-
Pyruvate dehydrogenase kinase is PDK.FA oxidation does not block glycolysis, so lactate builds up. Cant be transferred. To get lactate to pyruvate, must get rid of reducing equivalents. Heart has 4 isoforms of PDK, PDK4 most important. Pyruvate carboxylase is constitutively produced, not regulated.
51: Ketone body synthesis- fatty acids in liver and kidney are converted to ketone bodies.Muscle uses fatty acids for energy, converting them to CO2.
53: Ketone bodies are used by brain for energy during fasting.HMGcoA synthase is highly regulated, by cortisol (increased during fasting, increased gluconeogenesis thru PEP carboxykinase and stimulation of HMG coA synthase) and by glucocorticoids.
54: Lyase pushed to make more ketone bodies.
56: Measuring 3-hydroxybutyrate-acetoacetate ratio tells information about ketone body formation in liver.
57: Tissues that don’t make ketone bodies have enzymes to use them. Ketone body utilization happens all the time- Pathway not induced or capacity induced. Utilization is increased.
58: Ketone bodies go too high due to increased fatty acid oxidation- urine acidic.
59: Insulin regulates lipolysis and inhibits FA oxidation in liver.
60: Acetyl coA carboxylaseI in cytosol and fatty acid synthase reactions shown.Fatty acid synthase stops at palmitate. Acetyl coA carboxylase is Regulated by phosphorylation (see slide 61).
62 was a question last year.
63: ACP is a large carrier molecule with a phosphopantetheine group. These Enzymes are free in bacteria.
FA synthesis: see beginning of lecture and slides 64-71.
FA synthase genes are linked in mammals. Continuous AA sequence from one enzyme to another.
72: Some citrate from mitochondrion is transferred out by tricarboxylate carrier.Reacts using ATP-citrate lyase releases acetyl coA.
oxaloacetate to malate by malate dehydrogenase is another shuttle important to this pathway.
74: Peroxisomes- 2 fatty acid oxidation enzymes are combined.
75-6: Regulation of synthesis:
Acetyl CoA carboxylase- regulated by phosphorylation and by citrate. Citrate regulation slide he says is wrong. w 10mM citrate with pure enzyme, get same activation w phosphorylated and dephosphorylated form. No citrate- no activation in phosphorylated form, activation in dephosphorylated. Binding constant changes. Binds better or not as well.
77: Chicken ac coA carboxylase forms filaments in solution with citrate.
78: Enzyme important in obesity: data from gene chips-Stearoyl CoA desaturase induced in obesity. Stearoyl coA converts to monounsaturated oleoylcoA. Desaturated. Plants make unsaturated bonds by elongation of the chain and desaturation, alternating. Animals can insert an unsaturated bond after the chain is formed.
79: Linoleate and linolenate cannot be synthesized in animals.Essential for mammals.Omega-3 designation means 1st point of unsaturation (double bond) is 3 carbons from omega end.
80: Prostaglandin synthesis-
comes from FAs.Arachidonates come from phospholipids. Phospholipase A2 cuts off middle FA of TAG. to make arachidonic acid, then prostaglandins using cyclooxygenase. NSAIDS inhibit synthesis by inhibiting cyclooxygenase. COX 2 is present in inflammation- inducible enzyme. aspirin prevents inflammation by blocking COX in many tissues. Some prostaglandins protect stomach. Others protect heart and kidneys, so inhibiting them is bad. Glucocorticoids are also antinflammatory. They bind to nuclear receptors and regulate phospholipase A2 expression- knock it down.
For test:
First question usually about comparing one thing and another. What are possibliities for differences...? He has 2 questions on the test.
Review with Dr. Marion
First thing to consider when pathogen enters:
Within minutes- innate immunity responds- receptors include TLR. When pathogen or agent engages receptors, (do not use term bind- they may not bind to things they "sense"-how is not clear). There has been a paradigm shift in self-nonself recognition.
Question to think about: If you think about self/nonself from Janwway perspective, self/nonself discrimination by innate response is absolute, while for adaptive it is relative. Recpetors are designed to only recognize things on pathogens in innate response. In new paradigm that Janeway uses, he views self/nonself as job of APCs that sense whether something is a pathogen or not.
To have good immune response and make an antibody: Immunize mice. Add adjuvant with mycobacterium. Difficult to induce immune response without activating APC.
Why is self-nonself recognition by lymphocytes relative?
Within any individual, individual inherits set of molecules to respond to microbes, but TCR and BCR repertoire are broader due to somatic recombination.
When you think about function MHCI and II- what does it signal- intra or extracellular infection. Lymphocytes can’t see what it is (virus or bacterium or toxin). The cell knows by class I(intracellular) or II(outside brought in) APC presentation.
We inherit complete repertoire for TCR, derived somatically. Review thymic selection process – how thymus trains TCR to recognize MHC. Thymus is T cell school. 2exams-+selection. No binding to self-gone. Negative- do they recognize self too well-fail. Think about it like this:
Recognition generates +survival signal at beginning. Small number TCR expressed at the time. Want to be sure it recognizes MHC well. Peptide is not so relevant- low signal.
2nd half- lots of TCR expressed.
Since we have that generated potential, select only useful ones.
Pathogen in, innate recognition, produce cytokines. Cytokines induce inflammation.
Inflammation’s good side: immune response dependent on it for
1. activation of APC
2. transportation
One cytokine released quickly- TNF alpha- causes vascular dilation, increases expression of adhesion molecules (PCAM, integrins)on surface of endothelium (see Fitzpatrick lecture). Chemokine gradient is established so cells will follow it.
Another thing inflammation does- intiates clotting cascade. Look at endothelium- microclots form at either end of region of insult. Prevents pathogen from leaving. Forces accumulation of fluid in tissue to go to lymph system. Inflammation provides means to accumulate cells to pick up pathogens, block pathogen dispersal, and deliver APCs where immune response can occur.
Delivered to lymph node- best cells for delivering and initiating immune response?
dendritic cells. Why? What do they do in periphery? Roslaniec showed they endocytose stuff efficiently in periphery. Get schlepped into lymph and go to lymph node. Character of the cells changes- no longer endocytosing. Expression class I and II- go from eating to presenting. Change occurs when cell gets into lymph nodes.
Do not want response in periphery to minimize tissue damage and maximize presentation of antigen to T cells to find correct one. Lots of MHC and B7.1 and B7.2. Lots of activation of T cells.
If you monitor efferent lymph at lymph node, massive number of cells through daily, until you get an infection. Then almost no cells through. Why? Dendritic cells and T cells express integrins to help them stick together. Integrins expressed early to allow cells to extravasate, then dendritic cells go to lymph- Character and kinds of adhesins expressed changes. Integrins special so T cells can stick efficiently. Stop and sit. Interrogate surface. T cell specific, activated. 1st 24 hrs- T cells are expanding and expressing molecules causing them to stick to dendritic cells. They stay around long enough to get a lot of them.
Activated- express molecule to get out ,but bind to activated endothelium. Full circle.Bind to spot where it needs to be to deal w pathogen.
Big picture-innate cells induce inflammation, get pathogens to lymph nodes, activation of lymphocytes, clonal expansion, released to promote ability to go to tissue and do their jobs.
Other things important:
complement-
what does it do to bacteria? promotes phagocytosis.
Soluble proteins-part of innate immune function. Adaptive focuses response. Complement can amplify its effector function. It can recognize pathogens because it binds to structures on their surfaces. Once activates, nonspecific in function. What distinguishes pathogen and our cells- we have inhibitors. Inhibitors inactivate C3 convertase. Bacteria don’t have these inhibitors. Complement on- more gets deposited. Forms handles for phagocytes to grab bacteria. Also generate anaphylotoxins to generate inflammation. Provides mechanism for antibodies to direct phagocytes where they need to be.
Want big picture- clonal selection hypothesis. Everything about immune response follows those rules. Have to have clonal expansion, self/nonself, control immune response. Need system regulation to block activation of cells. Whole system is messy. No absolutes.Things generally go in one direction, but based on probability.
Impairment of either system- mess up response.
Few examples of kinds of questions- he asks based on our presentations in class- see those questions.
Going through a few examples:
Dendritic cells have scavenger receptors that pick up cellular debris. Have Fc recpetors for IgG. How they bind some things for endocytosis is not entirely known.
For viruses, some direct their own fate by getting in by infecting dendritic cell. they trigger innate response inside cell that cause cell to present peptides from the virus. Recognition from RNA from the virus. Dr. English’s lecture.
Compare and contrast the innate immune responses that control bacterial replication from those that control viral replication, with a focus on the following:
1. recognition of infected cells
2. effector cells and mechanisms responsible for limiting replication of the pathogen
Global question asking for overview. From Miller’s lecture. Innate have different means to recognize infection with bacterium or virus. How does it work? Which more imprtant for virus- class I, peptides gen intracellulary. class II binds to peptides in phagolysosome. effector cell for viral infection eliminate primary infection.day three thymectomy of neonatal mice produces profound autoimmunity including gastritis, thyroiditis, and type I diabetes as the mice develop. Would you expect adoptive transfer of bone marrow hematopoietic cells from an adult mouse to prevent the autoimmunity? Why, or why not?NO. Assume mice are same strain. Tregs wont develop- no thymus.Took adult peripheral T cells, could fix it.
Discuss role of cytokines (TNFalpha in inflammation)/chemokines in the process of leukocyte migration.Include in your answer:
1. Identify the principle cytokine mediating acute inflammatory response.
2. (went too fast to type)
What characteristics distinguish immune responses from adaptive immune response?
receptors germ line or somatic? clonal or non?
Affinity maturation is caused by somatic mutation and clonal selection. B cells alter their receptors- get better, chosen. CD40 is signal 2 for B cells. Hyper IgM syndrome is disease of this. Solution to this disease included people at St Jude- people had mutation in CD40. Got no IgG. What part of lymph node underdeveloped- follicles. Did not generate secondary activation of B cells and memory.
He likes those kinds of questions.
How would 2nd signal be generated for IgM without CD40- from innate immune ligands on pathogens. Enough antigen stimulates B cells, and T cells (to make a lot of cytokines)- enough antigen to crosslink receptors and enough cytokines- will get some B cell activation. Activation depends on getting enough stuff at a place to initiate phosphorylation cascade and get cell signalling.
He will post sample questions on blackboard.
Within minutes- innate immunity responds- receptors include TLR. When pathogen or agent engages receptors, (do not use term bind- they may not bind to things they "sense"-how is not clear). There has been a paradigm shift in self-nonself recognition.
Question to think about: If you think about self/nonself from Janwway perspective, self/nonself discrimination by innate response is absolute, while for adaptive it is relative. Recpetors are designed to only recognize things on pathogens in innate response. In new paradigm that Janeway uses, he views self/nonself as job of APCs that sense whether something is a pathogen or not.
To have good immune response and make an antibody: Immunize mice. Add adjuvant with mycobacterium. Difficult to induce immune response without activating APC.
Why is self-nonself recognition by lymphocytes relative?
Within any individual, individual inherits set of molecules to respond to microbes, but TCR and BCR repertoire are broader due to somatic recombination.
When you think about function MHCI and II- what does it signal- intra or extracellular infection. Lymphocytes can’t see what it is (virus or bacterium or toxin). The cell knows by class I(intracellular) or II(outside brought in) APC presentation.
We inherit complete repertoire for TCR, derived somatically. Review thymic selection process – how thymus trains TCR to recognize MHC. Thymus is T cell school. 2exams-+selection. No binding to self-gone. Negative- do they recognize self too well-fail. Think about it like this:
Recognition generates +survival signal at beginning. Small number TCR expressed at the time. Want to be sure it recognizes MHC well. Peptide is not so relevant- low signal.
2nd half- lots of TCR expressed.
Since we have that generated potential, select only useful ones.
Pathogen in, innate recognition, produce cytokines. Cytokines induce inflammation.
Inflammation’s good side: immune response dependent on it for
1. activation of APC
2. transportation
One cytokine released quickly- TNF alpha- causes vascular dilation, increases expression of adhesion molecules (PCAM, integrins)on surface of endothelium (see Fitzpatrick lecture). Chemokine gradient is established so cells will follow it.
Another thing inflammation does- intiates clotting cascade. Look at endothelium- microclots form at either end of region of insult. Prevents pathogen from leaving. Forces accumulation of fluid in tissue to go to lymph system. Inflammation provides means to accumulate cells to pick up pathogens, block pathogen dispersal, and deliver APCs where immune response can occur.
Delivered to lymph node- best cells for delivering and initiating immune response?
dendritic cells. Why? What do they do in periphery? Roslaniec showed they endocytose stuff efficiently in periphery. Get schlepped into lymph and go to lymph node. Character of the cells changes- no longer endocytosing. Expression class I and II- go from eating to presenting. Change occurs when cell gets into lymph nodes.
Do not want response in periphery to minimize tissue damage and maximize presentation of antigen to T cells to find correct one. Lots of MHC and B7.1 and B7.2. Lots of activation of T cells.
If you monitor efferent lymph at lymph node, massive number of cells through daily, until you get an infection. Then almost no cells through. Why? Dendritic cells and T cells express integrins to help them stick together. Integrins expressed early to allow cells to extravasate, then dendritic cells go to lymph- Character and kinds of adhesins expressed changes. Integrins special so T cells can stick efficiently. Stop and sit. Interrogate surface. T cell specific, activated. 1st 24 hrs- T cells are expanding and expressing molecules causing them to stick to dendritic cells. They stay around long enough to get a lot of them.
Activated- express molecule to get out ,but bind to activated endothelium. Full circle.Bind to spot where it needs to be to deal w pathogen.
Big picture-innate cells induce inflammation, get pathogens to lymph nodes, activation of lymphocytes, clonal expansion, released to promote ability to go to tissue and do their jobs.
Other things important:
complement-
what does it do to bacteria? promotes phagocytosis.
Soluble proteins-part of innate immune function. Adaptive focuses response. Complement can amplify its effector function. It can recognize pathogens because it binds to structures on their surfaces. Once activates, nonspecific in function. What distinguishes pathogen and our cells- we have inhibitors. Inhibitors inactivate C3 convertase. Bacteria don’t have these inhibitors. Complement on- more gets deposited. Forms handles for phagocytes to grab bacteria. Also generate anaphylotoxins to generate inflammation. Provides mechanism for antibodies to direct phagocytes where they need to be.
Want big picture- clonal selection hypothesis. Everything about immune response follows those rules. Have to have clonal expansion, self/nonself, control immune response. Need system regulation to block activation of cells. Whole system is messy. No absolutes.Things generally go in one direction, but based on probability.
Impairment of either system- mess up response.
Few examples of kinds of questions- he asks based on our presentations in class- see those questions.
Going through a few examples:
Dendritic cells have scavenger receptors that pick up cellular debris. Have Fc recpetors for IgG. How they bind some things for endocytosis is not entirely known.
For viruses, some direct their own fate by getting in by infecting dendritic cell. they trigger innate response inside cell that cause cell to present peptides from the virus. Recognition from RNA from the virus. Dr. English’s lecture.
Compare and contrast the innate immune responses that control bacterial replication from those that control viral replication, with a focus on the following:
1. recognition of infected cells
2. effector cells and mechanisms responsible for limiting replication of the pathogen
Global question asking for overview. From Miller’s lecture. Innate have different means to recognize infection with bacterium or virus. How does it work? Which more imprtant for virus- class I, peptides gen intracellulary. class II binds to peptides in phagolysosome. effector cell for viral infection eliminate primary infection.day three thymectomy of neonatal mice produces profound autoimmunity including gastritis, thyroiditis, and type I diabetes as the mice develop. Would you expect adoptive transfer of bone marrow hematopoietic cells from an adult mouse to prevent the autoimmunity? Why, or why not?NO. Assume mice are same strain. Tregs wont develop- no thymus.Took adult peripheral T cells, could fix it.
Discuss role of cytokines (TNFalpha in inflammation)/chemokines in the process of leukocyte migration.Include in your answer:
1. Identify the principle cytokine mediating acute inflammatory response.
2. (went too fast to type)
What characteristics distinguish immune responses from adaptive immune response?
receptors germ line or somatic? clonal or non?
Affinity maturation is caused by somatic mutation and clonal selection. B cells alter their receptors- get better, chosen. CD40 is signal 2 for B cells. Hyper IgM syndrome is disease of this. Solution to this disease included people at St Jude- people had mutation in CD40. Got no IgG. What part of lymph node underdeveloped- follicles. Did not generate secondary activation of B cells and memory.
He likes those kinds of questions.
How would 2nd signal be generated for IgM without CD40- from innate immune ligands on pathogens. Enough antigen stimulates B cells, and T cells (to make a lot of cytokines)- enough antigen to crosslink receptors and enough cytokines- will get some B cell activation. Activation depends on getting enough stuff at a place to initiate phosphorylation cascade and get cell signalling.
He will post sample questions on blackboard.
Friday, December 15, 2006
Miller Notes
This was a really bad one to miss. He put things together in a wonderful way that really helped us understand. For those looking for Cook's notes- it'll probably be tomorrow before they are up. It will take a lot of rewriting.
Contact information on handouts.
After today- understand how immune system response to bacteria or viral infection.
2 parts: innate and acquired
Chart of phases of immune response shows all we will talk about today.
3: Innate immune response is critical. number organisms vs time- normal- rapid increase, then plateau, then decline. Without T or B cells, but intact innate, initial infection same, then slow increase due to innate immune response
WITH T or B but without neutrophils, mouse dies fast.
4: variety of microorganisms.
All bacteria have common components.
Rapid response: recognize by preformed nonspecific effector cells- macrophages. Not diverse or specific, but for general features of bacteria.
1st defense is mechanical, chemical, microbiological . Most important mechanical. Lots of cells produce antibacterial peptides. Normal flora compete with potential pathogens. Most potential infections never occur.
3 ways to activate the complement cascade:
classical, lectin, alternative. Alternativen was the 1st in evolution to develop- complement is constantly in circulation. C3b deposition is most important event in cascade- is primary opsonin recognize by macrophage with complement receptors. Makes bacterium more palatable to macrophage and neutrophil. See cascade. Understand role in innate immune response. C3 always cleaved- if deposits on cell surface, then initiates complement activation.
Bacteria destroyed by macrophage- pattern recognition receptors recognize LPS, glycans, etc. When macrophage recognizes through engaging of receptor, uptakes material, activation signal makes macrophage more bacteriocidal, secretes cytokines. Amplifies response. Recruits more phagocytic cells. No long-term protective immunity from this.
Early induced response- macrophage initiates inflammatory response- involves changes in local vascular permeability and vasodilation. Heat- slows pathogen replication and increases activation of immune cells. Swelling- fluid drain to lymph nodes to carry antigen and APCs to initiate acquired immune response. Pain alerts you to problem. Increased number of phagocytes can clear infection.
Receptor-
LPS from gram negative bacteria.
mannose- most bacteria
glycans-bacteria
Engage- signal through TLR for cytokine production. Phagocyte promotes inflammation though cytokines and becomes more bacteriocidal.
TLR- LPS signal is thru TLR-4. Review these.
Macrophage coordinates innate response thru cytokines.
IL-1 activate vascular endothelium, lymphocytes, local tissue destruction- prevents spread of infection. Walls off organisms in inflamed area.
IL-1,6, tnf alpha induce fever
IL-8 attract innate cells, increase access of effector cells by altering adhesion marker profile. Activates integrins. Also activate PMNs.
TNF- see slide. Key cytokine in producing fever. If it produces response systemically- can result in shock.
IL-6- activate lymphocytes, antibody produced,
IL-12 critical for activation NK cells in viral infection. promotes acquisition of response for Th1 development.
Acute phase response- triad of cytokines act on a variety of tissues. See slide. Bone marrow has huge stores of neutrophils. Released on command- kamikazes with a short half life that eat bacteria and die. .million enter oral cavity every day.
hypothalamus- increases body temp. Systemic heat induction.
fat and muscle- protein and energy mobilization
Acute phase response-
acute phase protein- mannose binding lectin and C reactive protein from liver into blood. Complement system.
fibrinogen- part of clotting cascade- helps block off infection site
Creactive protein acts as opsonin or 1st step of complement cascade.
Systemic production cytokines can be deadly-remember shock.
B-1 B cells are CD5+. Produce IgM to bacterial surface. Recognize capsular polysaccharides and cell wall components. Crosslink receptors- get IgM- opsonin. No IgM receptors on phagocytes, but initiate complement cascade. Deposit C3b, then the bacteria get taken up. No memory. No clonal expansion and no memory.
Controlling viral infection:
Viruses induce cells to produce type 1 interferon, Alpha and beta. Activate NK cells, and make adj uninfected cells less susceptible to infection. Cells upregulate MHC class I expression on surface.
Time vs virus titer- after few days up, then down due to acquired response. Rate of viral replication low at first- held down by NK cells. IL-12 is from dendritic cells infected with virus.
NK cells kill cells that don’t express MHC class 1. Most cells express it. Neural cells do not. Why NK cells dont attack neurons-he does not know. Viruses take over protein production machinery of cell and turn off host cell protein production to provide viral protein. After few hours, MHC class 1 molecules are not replaced. Once the cell has lost MHC class 1, it becomes a target for NK killing.
NK cells perforin and granzymes(initiate apoptotic pathway)- release granules to kill in antibody cell-mediated killing (acquired response) or cells without MHC class 1- have activation and inhibition receptors. MHC rec by inh receptor. Inh killng cell.
Other adhesion molecules initiate reaction- sampling of surface occurs.No MHC-dump granules and kill cell. If you experimentally Bind MHC1 with an antibody to mask its presence- NK cell can kill healthy cell.
Without MHC, how do CD8 cells recognize sick cells? They don’t. They recognize cells early in infection. MHC class 1 will be full of viral protein at the time. Late- class 1 gone and NK cells can kill. T cells recognize early in replication cycle, and destroy. NK cells can’t completely control a viral infection because they kill late in replication cycle and viruses are released. T cells better limit virus replication than NK cells.
Acquired immune response slides
1- summary
2- typical course infection. Immune system is designed to respond to intermediate amounts of antigen, above a certain threshold.
After 4-5 days- transport of antigen to secondary lymphoid tissue promotes recognition, clonal expansion and differentiation- removal infectious agent.
Different mechanisms as we go.
Immature dendritic cells are Langerhans cells- can’t present antigen because they do not express costimulatory B-7 molecule. Why does presentation have to happen in lymphatics? APC contacts large number T cells in lymph node- better probability of running into cell with right specificity. Take up antigen in periphery, mugrate to T cell zones of lymphoid tissue, Constitutively produce B-7, progress.
REALLY understand MHC class I and II. 2 pathways allow recognition of intracellular pathogen or extracellular pathogens.
Class II- extracellular pathogen. Most bacteria. Bacterium is phagocytosed, goes to endocytic vesicle, to lysosome, dismantled, class II-peptide complex takes peptide anigen to surface, present to naive T cells. Some class II antigen may get into class I or antigen from class I pathway may get into class II, but mostly works. CD4 cells recognize these.
Class 1- intracellular bacteria, or all viruses intracellular. Protein production in cytoplasm. Fed into proteasome. Presented to CD8 T cells.
T cell activation- 2 signals. 1st is TcR on naive T- recognize cognate peptide Only activate with B7-CD28 signal.B7 is costimulatory molecule on APC, CD28 on T cell. Only get 1st signal- anergic and die. No B-7 – means it is response to self antigen. b-7 upregulated in infection. This is peripheral tolerance (most tolerance is thymic).
Once T cells fully differentiate, armed and proliferate. Most new cells become effector T cells. No further costimulation is necessary. Recognize peptide- perform function. Express different adhesion molecules.
CD8 cell does NOT activate in secondary lymphoid tissue due to different adhesion molecules.
Il-2 is autocrine growth factor. They also produce high affinity IL-2 growth factor so the cell stimulates itself.
Cytotoxic T cells only kill recognized cell. Can kill many cells due to regeneration of lytic granules. CTLs recognize MHC class 1 molecules.
Adhesion molecules initiate reaction.
3 types effector T cells to know:
CD8 are CTL. killer CD8s.
CD4:
differentiate into Th1 ot Th2. depends on cytokines.
Th1- most of CD4 cells. These direct response to intracellular pathogens. Activate macrophages. MHCII mostly on APC (outside thymus). Th1 recognize class II- activate more bacterocide, more inflammatory cytokines. Many initracellular bacteria live inside macrophage, which can kill them if it is activated.
Th2- extracellular-promote response to extracellular pathogens. Activate B cells.
Different T helper cells:
All CD4+ naive cells are Th0- not precommitted.
Activate, proliferate, then differentiate to Th1 or Th2 depending on cytokine signals. If effector T differentiate toward Th1, activate macrophage, B cells for opsonization of antigen. Th2- b cells for neutralizing antigen. Th1 and th2 can supply different cytokines for 2nd signal to B cells. Th1 activate class switching for intracellular pathogens. Th2 for extracellular.
What drives differentiation Th0 cells?
cytokine environment. is primary driver. CD4- IL-12+IFNgamma-Th1
CD4- IL-4 (from NK 1.1)- Th2 phenotype. Produces IL-4 and 5- promote class switch to antibody for extracellular pathogens.
Antigen also influences pathway. Antigen low affinity or lowconcentration Th2
High affinity or high concentration- Th1. Get more concentration from intracellular infection.
After Th cell is produced- Th2 inhibits Th1,and vice versa. response highly polarized.
What do Th1 do? see slide.p7.
diapedesis- aloow macrophages into infection site. Chemotactic factor draws macrophages in.
B cell activation and differentiation:
1. Engage membrane bound antibody molecule- endocytose complex, degrade, load class II molecule, to surface, present to naive T cells.T cell signals back- B cell becomes plasma cell. Not-B cell anergic.
B cell migrate into T cell zone, interacts with effector T cells, recognizes peptide MHCII complex, T helper activate B cell, proliferates, pairs of B cell-T cell move into follicle and replicate to form germinal center. All cells with same antigen specificity- affinity maturation happens here. High rate mutation in complementarity determining regions of heavy and light chain produce antibodies with different affinity for antigen than initial B cell receptor had.some lower, some higher. Higher affinity is selected to continue development. Follicular dendritic cells are different. Supply survival signals and hold antigen for long period of time. Lower affinity B cells die.Affinity of group increases significantly over time. Surrounded by helper Ts proliferating- make cytokines to promote class switching to different types antibody.
What happens in germinal center- 2 important things:
affinity maturation
isotype swtching
1st isotype always IgM- always constant, and affinity does not increase over time. Over time- switch to IgG eventually, and affinity increases after 3rd exposure to vaccine. concentration antibody also increases over time.
Should know Antibody functions w 3 +++. IgA- mucosal. IgE-mast cell sensitization.
Mechanisms antibodies to clear infection:
opsonization- Fc receptors bind antibody only when it is bound to antigen. complement is further opsonin.
Other key- neutralize antigen. Bacterial toxin usually binds to receptor on cell to produce damage. Antibody binds to the toxin to prevent toxin access to cell and render it palatable for macrophage destruction.
Another role antibody- NK-Mediated ADCC
NK have Fc receptors.
Mast cells- deal with parasites, extracellular, like GI pathogens. Granules promote powerful inflammatory response. Sytemic- explosive diarrhea, vomiting, can result in anaphylactic shock. Have no antigen specific receptors- have IgE receptors. Serum IgE low- taken up through FcE reseptors on mast cells. Used as antigen receptors with different specificities- crosslink with antigen- mast cell degranulates. Hives- local degranulation.
Know protective immune response is mediated by T cell and/or B cell responses. Innate not amplified or changed.
B cell differentiation results in plasma cell or memory B cell. May be some T cell memory, or not . Not well understood.
Initiate response- control infection. Infection few wks later- handled quickly.
Be able to understand what happens if infection with particular organism- virus or bacterium.
Contact information on handouts.
After today- understand how immune system response to bacteria or viral infection.
2 parts: innate and acquired
Chart of phases of immune response shows all we will talk about today.
3: Innate immune response is critical. number organisms vs time- normal- rapid increase, then plateau, then decline. Without T or B cells, but intact innate, initial infection same, then slow increase due to innate immune response
WITH T or B but without neutrophils, mouse dies fast.
4: variety of microorganisms.
All bacteria have common components.
Rapid response: recognize by preformed nonspecific effector cells- macrophages. Not diverse or specific, but for general features of bacteria.
1st defense is mechanical, chemical, microbiological . Most important mechanical. Lots of cells produce antibacterial peptides. Normal flora compete with potential pathogens. Most potential infections never occur.
3 ways to activate the complement cascade:
classical, lectin, alternative. Alternativen was the 1st in evolution to develop- complement is constantly in circulation. C3b deposition is most important event in cascade- is primary opsonin recognize by macrophage with complement receptors. Makes bacterium more palatable to macrophage and neutrophil. See cascade. Understand role in innate immune response. C3 always cleaved- if deposits on cell surface, then initiates complement activation.
Bacteria destroyed by macrophage- pattern recognition receptors recognize LPS, glycans, etc. When macrophage recognizes through engaging of receptor, uptakes material, activation signal makes macrophage more bacteriocidal, secretes cytokines. Amplifies response. Recruits more phagocytic cells. No long-term protective immunity from this.
Early induced response- macrophage initiates inflammatory response- involves changes in local vascular permeability and vasodilation. Heat- slows pathogen replication and increases activation of immune cells. Swelling- fluid drain to lymph nodes to carry antigen and APCs to initiate acquired immune response. Pain alerts you to problem. Increased number of phagocytes can clear infection.
Receptor-
LPS from gram negative bacteria.
mannose- most bacteria
glycans-bacteria
Engage- signal through TLR for cytokine production. Phagocyte promotes inflammation though cytokines and becomes more bacteriocidal.
TLR- LPS signal is thru TLR-4. Review these.
Macrophage coordinates innate response thru cytokines.
IL-1 activate vascular endothelium, lymphocytes, local tissue destruction- prevents spread of infection. Walls off organisms in inflamed area.
IL-1,6, tnf alpha induce fever
IL-8 attract innate cells, increase access of effector cells by altering adhesion marker profile. Activates integrins. Also activate PMNs.
TNF- see slide. Key cytokine in producing fever. If it produces response systemically- can result in shock.
IL-6- activate lymphocytes, antibody produced,
IL-12 critical for activation NK cells in viral infection. promotes acquisition of response for Th1 development.
Acute phase response- triad of cytokines act on a variety of tissues. See slide. Bone marrow has huge stores of neutrophils. Released on command- kamikazes with a short half life that eat bacteria and die. .million enter oral cavity every day.
hypothalamus- increases body temp. Systemic heat induction.
fat and muscle- protein and energy mobilization
Acute phase response-
acute phase protein- mannose binding lectin and C reactive protein from liver into blood. Complement system.
fibrinogen- part of clotting cascade- helps block off infection site
Creactive protein acts as opsonin or 1st step of complement cascade.
Systemic production cytokines can be deadly-remember shock.
B-1 B cells are CD5+. Produce IgM to bacterial surface. Recognize capsular polysaccharides and cell wall components. Crosslink receptors- get IgM- opsonin. No IgM receptors on phagocytes, but initiate complement cascade. Deposit C3b, then the bacteria get taken up. No memory. No clonal expansion and no memory.
Controlling viral infection:
Viruses induce cells to produce type 1 interferon, Alpha and beta. Activate NK cells, and make adj uninfected cells less susceptible to infection. Cells upregulate MHC class I expression on surface.
Time vs virus titer- after few days up, then down due to acquired response. Rate of viral replication low at first- held down by NK cells. IL-12 is from dendritic cells infected with virus.
NK cells kill cells that don’t express MHC class 1. Most cells express it. Neural cells do not. Why NK cells dont attack neurons-he does not know. Viruses take over protein production machinery of cell and turn off host cell protein production to provide viral protein. After few hours, MHC class 1 molecules are not replaced. Once the cell has lost MHC class 1, it becomes a target for NK killing.
NK cells perforin and granzymes(initiate apoptotic pathway)- release granules to kill in antibody cell-mediated killing (acquired response) or cells without MHC class 1- have activation and inhibition receptors. MHC rec by inh receptor. Inh killng cell.
Other adhesion molecules initiate reaction- sampling of surface occurs.No MHC-dump granules and kill cell. If you experimentally Bind MHC1 with an antibody to mask its presence- NK cell can kill healthy cell.
Without MHC, how do CD8 cells recognize sick cells? They don’t. They recognize cells early in infection. MHC class 1 will be full of viral protein at the time. Late- class 1 gone and NK cells can kill. T cells recognize early in replication cycle, and destroy. NK cells can’t completely control a viral infection because they kill late in replication cycle and viruses are released. T cells better limit virus replication than NK cells.
Acquired immune response slides
1- summary
2- typical course infection. Immune system is designed to respond to intermediate amounts of antigen, above a certain threshold.
After 4-5 days- transport of antigen to secondary lymphoid tissue promotes recognition, clonal expansion and differentiation- removal infectious agent.
Different mechanisms as we go.
Immature dendritic cells are Langerhans cells- can’t present antigen because they do not express costimulatory B-7 molecule. Why does presentation have to happen in lymphatics? APC contacts large number T cells in lymph node- better probability of running into cell with right specificity. Take up antigen in periphery, mugrate to T cell zones of lymphoid tissue, Constitutively produce B-7, progress.
REALLY understand MHC class I and II. 2 pathways allow recognition of intracellular pathogen or extracellular pathogens.
Class II- extracellular pathogen. Most bacteria. Bacterium is phagocytosed, goes to endocytic vesicle, to lysosome, dismantled, class II-peptide complex takes peptide anigen to surface, present to naive T cells. Some class II antigen may get into class I or antigen from class I pathway may get into class II, but mostly works. CD4 cells recognize these.
Class 1- intracellular bacteria, or all viruses intracellular. Protein production in cytoplasm. Fed into proteasome. Presented to CD8 T cells.
T cell activation- 2 signals. 1st is TcR on naive T- recognize cognate peptide Only activate with B7-CD28 signal.B7 is costimulatory molecule on APC, CD28 on T cell. Only get 1st signal- anergic and die. No B-7 – means it is response to self antigen. b-7 upregulated in infection. This is peripheral tolerance (most tolerance is thymic).
Once T cells fully differentiate, armed and proliferate. Most new cells become effector T cells. No further costimulation is necessary. Recognize peptide- perform function. Express different adhesion molecules.
CD8 cell does NOT activate in secondary lymphoid tissue due to different adhesion molecules.
Il-2 is autocrine growth factor. They also produce high affinity IL-2 growth factor so the cell stimulates itself.
Cytotoxic T cells only kill recognized cell. Can kill many cells due to regeneration of lytic granules. CTLs recognize MHC class 1 molecules.
Adhesion molecules initiate reaction.
3 types effector T cells to know:
CD8 are CTL. killer CD8s.
CD4:
differentiate into Th1 ot Th2. depends on cytokines.
Th1- most of CD4 cells. These direct response to intracellular pathogens. Activate macrophages. MHCII mostly on APC (outside thymus). Th1 recognize class II- activate more bacterocide, more inflammatory cytokines. Many initracellular bacteria live inside macrophage, which can kill them if it is activated.
Th2- extracellular-promote response to extracellular pathogens. Activate B cells.
Different T helper cells:
All CD4+ naive cells are Th0- not precommitted.
Activate, proliferate, then differentiate to Th1 or Th2 depending on cytokine signals. If effector T differentiate toward Th1, activate macrophage, B cells for opsonization of antigen. Th2- b cells for neutralizing antigen. Th1 and th2 can supply different cytokines for 2nd signal to B cells. Th1 activate class switching for intracellular pathogens. Th2 for extracellular.
What drives differentiation Th0 cells?
cytokine environment. is primary driver. CD4- IL-12+IFNgamma-Th1
CD4- IL-4 (from NK 1.1)- Th2 phenotype. Produces IL-4 and 5- promote class switch to antibody for extracellular pathogens.
Antigen also influences pathway. Antigen low affinity or lowconcentration Th2
High affinity or high concentration- Th1. Get more concentration from intracellular infection.
After Th cell is produced- Th2 inhibits Th1,and vice versa. response highly polarized.
What do Th1 do? see slide.p7.
diapedesis- aloow macrophages into infection site. Chemotactic factor draws macrophages in.
B cell activation and differentiation:
1. Engage membrane bound antibody molecule- endocytose complex, degrade, load class II molecule, to surface, present to naive T cells.T cell signals back- B cell becomes plasma cell. Not-B cell anergic.
B cell migrate into T cell zone, interacts with effector T cells, recognizes peptide MHCII complex, T helper activate B cell, proliferates, pairs of B cell-T cell move into follicle and replicate to form germinal center. All cells with same antigen specificity- affinity maturation happens here. High rate mutation in complementarity determining regions of heavy and light chain produce antibodies with different affinity for antigen than initial B cell receptor had.some lower, some higher. Higher affinity is selected to continue development. Follicular dendritic cells are different. Supply survival signals and hold antigen for long period of time. Lower affinity B cells die.Affinity of group increases significantly over time. Surrounded by helper Ts proliferating- make cytokines to promote class switching to different types antibody.
What happens in germinal center- 2 important things:
affinity maturation
isotype swtching
1st isotype always IgM- always constant, and affinity does not increase over time. Over time- switch to IgG eventually, and affinity increases after 3rd exposure to vaccine. concentration antibody also increases over time.
Should know Antibody functions w 3 +++. IgA- mucosal. IgE-mast cell sensitization.
Mechanisms antibodies to clear infection:
opsonization- Fc receptors bind antibody only when it is bound to antigen. complement is further opsonin.
Other key- neutralize antigen. Bacterial toxin usually binds to receptor on cell to produce damage. Antibody binds to the toxin to prevent toxin access to cell and render it palatable for macrophage destruction.
Another role antibody- NK-Mediated ADCC
NK have Fc receptors.
Mast cells- deal with parasites, extracellular, like GI pathogens. Granules promote powerful inflammatory response. Sytemic- explosive diarrhea, vomiting, can result in anaphylactic shock. Have no antigen specific receptors- have IgE receptors. Serum IgE low- taken up through FcE reseptors on mast cells. Used as antigen receptors with different specificities- crosslink with antigen- mast cell degranulates. Hives- local degranulation.
Know protective immune response is mediated by T cell and/or B cell responses. Innate not amplified or changed.
B cell differentiation results in plasma cell or memory B cell. May be some T cell memory, or not . Not well understood.
Initiate response- control infection. Infection few wks later- handled quickly.
Be able to understand what happens if infection with particular organism- virus or bacterium.
Wednesday, December 13, 2006
Brand notes
Dear everyone,
Do look at the review article associated with this one. It helps. Also, my hands are starting to hurt more, so my notes will be getting a little more terse, if that is possible. And if the professor hands out complete works in class, I won't be typing anything in excess. That's why Nelson's lectures are not posted. Here are my notes on Brand:
Regulation and control of Immune Function: Tolerance
He works with regulatory Tcells.
Big questions on slide 2.
3: checkpoints are there to minimize generation of self –reactive lymphocytes. A few escape.
4: layers of self-tolerance
peripheral anergy- cells get weak signal without costimulus.
clonal exhaustion- signalled for death in secondary lymphoid tissue- similar to selection process, but later
cytokine deviation- instead of becoming a TH1, Th0 directed to become a more suppressive TH2.
we will focus on regulatory cells.
8: induced tolerance- we use processes that take place naturally to our advantage
intravenous tolerance or mucosal.
induced RA-adjuvant and collagen produces a model of arthritis in mouse. feed soluble collagen, or give IV before injecting the collagen+adjuvant- no disease.
12: specific mechanisms in gut help us tolerate food proteins. Lack of costimulation, cells, etc.
14: It is a normal process for self-reactive lymphocytes to make it to periphery. If regulatory T cells are deleted from normal blood, and you move the blood into an athymic system, get autoimmunity.
CD4CD25 double + cells keep autoimmunity in check.
15: + selection for Treg is higher affinity than T effector cells. State of heightened activation CD25 is elevated early in periphery on them (normally in proliferating cells). regulatory T cells suppress effector T cells. Problems when these do not work are listed on the slide. Some tumors may be allowed by too much regulation.
16: Remove thymus around day 3, systemic autoimmunity ensues. If give adult spleen cells- no disease because of regulatory T cells.18: How to identify
Naive animal- double positive is Tregulatory. In vitro- CD25 goes up in other T cells, too.
19: Fox p3 is a nuclear transcription factor. It is in the nucleus, so not good for functional studies.Can’t fish out Tregs very easily using foxp3, because you have to fix and permeabilize the cell. IS specific to Tregs.
Problem: no unique cell surface molecule to discriminate. We trade purity of preparation for getting most of them or vice versa.
22: Mutating foxp3 results in lethal autoimmune problems
25: Tnfrs18 encodes GITR. (glucocorticoid induced TNF-receptor family-related gene/protein), which is a potential marker for Tregs, though not exclusive.
CTLA-4 is uniformly upregulated on Treg cells- negative regulator.
Flow cytometer distinguishes cells based on size and granularity. With antibodies, can look at specific proteins on surface.
34: Tube of cells placed on cytometer. Data is collected on relative fluorescnece while cells go through.
Cells have been fixed and permeabilized so that antibodies can get in.
37: CD25 is found in a spectrum in T cells in the spleen
Lymph node- most regulatory Ts express higher CD25.
39: Transgenic mouse with TcR specific for collagen. Administer collagen- Tregs become 42% of peripheral T cells in blood!
40: AIR gene- autoimmune regulator gene in thymus- produces 2-3,000 peripheral proteins in thymus for negative selection of high-affinity cells.
43: In synovial fluid in autoimmunity, lots of antiself T cells are there- and lots of regulatory Ts. Looks like where autoimmunity is happening, the regulatory T cells are there.
46: look at foxp3 vs GITR- good correlation. May be differences between cells with foxp3 that are bright or dark for CD25. Mice given GITR low cells died earlier.
Mice with CD25 had a different spectrum of diseases. Take away GITR high- may be taking away more Tregs.
GITR and CTLA-4 are used to delimeate Tregs.
Cannot use a single marker yet. combinations work better, but you miss some cells. These markers are higher on Tregs.
Read the review paper in his file on blackboard.
Tregs depend on IL-2 for survival from effector cells (drives proliferation), but they downreg IL-2 transcription in those cells. without it, get autoimmune disease. High doses IL-2 upregulate CD25 expression.
55: Take home- balance between autoreactive T cells and regulatory T cells. All this is being worked out now.
CTLA-4 delivers a suppressive signal. Anti CTLA4 antibodies increase autoimmune responses. BUT CTLA-4 deficient mice can have Treg-like cells . Exact mechanism of suppression is not worked out.
Do look at the review article associated with this one. It helps. Also, my hands are starting to hurt more, so my notes will be getting a little more terse, if that is possible. And if the professor hands out complete works in class, I won't be typing anything in excess. That's why Nelson's lectures are not posted. Here are my notes on Brand:
Regulation and control of Immune Function: Tolerance
He works with regulatory Tcells.
Big questions on slide 2.
3: checkpoints are there to minimize generation of self –reactive lymphocytes. A few escape.
4: layers of self-tolerance
peripheral anergy- cells get weak signal without costimulus.
clonal exhaustion- signalled for death in secondary lymphoid tissue- similar to selection process, but later
cytokine deviation- instead of becoming a TH1, Th0 directed to become a more suppressive TH2.
we will focus on regulatory cells.
8: induced tolerance- we use processes that take place naturally to our advantage
intravenous tolerance or mucosal.
induced RA-adjuvant and collagen produces a model of arthritis in mouse. feed soluble collagen, or give IV before injecting the collagen+adjuvant- no disease.
12: specific mechanisms in gut help us tolerate food proteins. Lack of costimulation, cells, etc.
14: It is a normal process for self-reactive lymphocytes to make it to periphery. If regulatory T cells are deleted from normal blood, and you move the blood into an athymic system, get autoimmunity.
CD4CD25 double + cells keep autoimmunity in check.
15: + selection for Treg is higher affinity than T effector cells. State of heightened activation CD25 is elevated early in periphery on them (normally in proliferating cells). regulatory T cells suppress effector T cells. Problems when these do not work are listed on the slide. Some tumors may be allowed by too much regulation.
16: Remove thymus around day 3, systemic autoimmunity ensues. If give adult spleen cells- no disease because of regulatory T cells.18: How to identify
Naive animal- double positive is Tregulatory. In vitro- CD25 goes up in other T cells, too.
19: Fox p3 is a nuclear transcription factor. It is in the nucleus, so not good for functional studies.Can’t fish out Tregs very easily using foxp3, because you have to fix and permeabilize the cell. IS specific to Tregs.
Problem: no unique cell surface molecule to discriminate. We trade purity of preparation for getting most of them or vice versa.
22: Mutating foxp3 results in lethal autoimmune problems
25: Tnfrs18 encodes GITR. (glucocorticoid induced TNF-receptor family-related gene/protein), which is a potential marker for Tregs, though not exclusive.
CTLA-4 is uniformly upregulated on Treg cells- negative regulator.
Flow cytometer distinguishes cells based on size and granularity. With antibodies, can look at specific proteins on surface.
34: Tube of cells placed on cytometer. Data is collected on relative fluorescnece while cells go through.
Cells have been fixed and permeabilized so that antibodies can get in.
37: CD25 is found in a spectrum in T cells in the spleen
Lymph node- most regulatory Ts express higher CD25.
39: Transgenic mouse with TcR specific for collagen. Administer collagen- Tregs become 42% of peripheral T cells in blood!
40: AIR gene- autoimmune regulator gene in thymus- produces 2-3,000 peripheral proteins in thymus for negative selection of high-affinity cells.
43: In synovial fluid in autoimmunity, lots of antiself T cells are there- and lots of regulatory Ts. Looks like where autoimmunity is happening, the regulatory T cells are there.
46: look at foxp3 vs GITR- good correlation. May be differences between cells with foxp3 that are bright or dark for CD25. Mice given GITR low cells died earlier.
Mice with CD25 had a different spectrum of diseases. Take away GITR high- may be taking away more Tregs.
GITR and CTLA-4 are used to delimeate Tregs.
Cannot use a single marker yet. combinations work better, but you miss some cells. These markers are higher on Tregs.
Read the review paper in his file on blackboard.
Tregs depend on IL-2 for survival from effector cells (drives proliferation), but they downreg IL-2 transcription in those cells. without it, get autoimmune disease. High doses IL-2 upregulate CD25 expression.
55: Take home- balance between autoreactive T cells and regulatory T cells. All this is being worked out now.
CTLA-4 delivers a suppressive signal. Anti CTLA4 antibodies increase autoimmune responses. BUT CTLA-4 deficient mice can have Treg-like cells . Exact mechanism of suppression is not worked out.
Tuesday, December 12, 2006
Fitzpatrick notes (Monday)
Take home some general properties of chemokines and cytokines and leucocyte migration. Do not memorize all of them.
3:Chemokines- small proteins tell leucocytess where to go. These molecules are often referred to as cytokines. Term from old literature. About 30 homologous polypeptides that tell naive T or B cells where to go. N terminal C residue defines their classification- divide into 4 families.
4:
1. CC1,2,3- chemokine. CCL-ligand. CCR-receptor.
2. CXC- ELR+ means ELR before the C.
6: Chart from immunobiology book. Ex of CXC class. Numbered based on when they were identified.
A lot of these have been renamed over time. Chemokines produced by a lot of cell types and can bind to more than one kind of receptor.
7: Chart continued. Do not memorize.
8: General Features
Endothelial- line blood vessels.
2 times produced:
inflammatory response-chemokines to tell where infection is. Upregulated during infection, then disappear.
normal homeostasis- tell leucocytes to go to lymphoid organs. Constitutively produced.
9: receptors named for ligands. T cells have a number of different chemokine receptors to tell them to do things at different times.
10: General scheme of receptor- 7 transmembrane helices. Bind to G proteins. Activate a number of pathways in cell.
11:
Chemokines are important for migration as well as other roles.
12:
Immune response- want innate system to respond to site of infection. Chemokines needed to tell cells where to go, and adhesion molecules need to be regulated. Immune cells need to adhere to extracellular matrix of collagen fibers and move through tissue. Binding of adhesion molecules allows cells to move.
13:3 families
selectins induce rolling along the endothelium (blood vessel lining). Slows neutrophil down- bind and release.
integrins- tight adhesion- stops and binds. Then can move into tissue
Ig superfamily- T cell binding to APC. Maintains contact.
14: What happens during inflammation? Endothelial cells normally do not express P selectin. as a result of infection, TNF and IL-1 cause P selectin to be expressed in an area of endothelium near the infection. Cells roll along.
15: In tissue- IL-8 produced and binds to receptor on endothelial cell to produce high-affinity integrin to bind to ligand. Contacts, binds to endothelial cell, and causes shape change in cell so it can squeeze between endothelial cells and into tissue. Inside- more chemokines cause crawling along structural framework to where infection is. Downregulate epxression- cell stops to do its job. Different chemokines attract different cells for the most efficient response.
16: Normal immune cell trafficking:
HOw do cells know to go to lymph node? Stromal cells produce CCL21. Dendritic cells have receptor CCR7. They migrate , bind, migrate into lymph node. T cells have same receptor. Lymph node has a lot of consttitutive production of CCL21,19, etc.
17:Cytokines:
regulate inflammatory response. Autocrine or paracrine.
18: General properties
1. Gene transcribed- there are bursts of cytokine production, and mRNA is degraded fast. All regulated by different means: mRNA degradation, cleavage for activation, post-translational modification, etc. Only neutrophils may store them in granules. All other cells secrete them.
19:
pleiotropic- act on different cell types.
20:
Cytokine cascades exist.
21:
Most produced locally and act locally. Sometimes large infections cause huge amount TNF and IL1.
22:
Bind specific receptors- high affinity. SMall amount produced, but physiologically important.
23: Stimulate T or B cell- upregulates cytokine expression.
24:
Cells respond by differentiating (T cells), activating function (macrophages and radical production), and cell proliferation (Tcells and IL-2- allows to enter S phase)
25: Receptors have similar features.
26: rest of talk. Same cytokines can have a lot of diff functions.
28: What happens when you have infection:
What immune cells normally live in tissues? macrophages and dendritic cells can bind microbes through pattern recognition receptors. Bind to cell wall and generate features. Pull in bug, chew it up, stimulate production cytokines. IL-1, 6,TNf act on vascular endothelium to tell them to upregulate adhesion molecules and produce more chemokines. Also increase vasccular permeability. Loosen tight junction. Gets inmmune cells and products to site.
Neutrophils are great killers .Live up to 24 hours and tend to move in first. IL-8 attracts them to chew up bacteria.
29: TNF
Block TNF, inhibit inflammatory response.
30: TNF- tumor necrosis factor- can cause apoptosis in some tumors.
31: Septic shock- systemic production of TNF, IL-1, IL-6. When in circulation, can affect organs. Fever, liver, appetite suppression, smooth muscle and heart problems.
32: whole body effects from cytokines.
36: local effects are favorable to clear infection. Systemic- blood volume decreases because of leaky vessels. Cells move into un- needed tissues. neutropenia signals influx from bone marrow. multiple organ failure results in death. Principle mediator- TNF. In human patients- blocking TNF does not work to prevent shock.
39:To stop inflammatory response, anti-inflammatory cytokines reduce response.
Ex: IL-10 shuts off recruitment of cells. Downregulate MHC class II and costimulatory molecules.
p13:
viral infections:
IFN alpha and beta secreted to act on other cell types. Bind to alphabeta receptor. Other cells stimulated to produce proteins to inhibit oligodenylate synthetase, to inhibit transcription of RNA and upregulate Class I MHC for presentation to cytotoxic T cells. ALso promotes development of Th1 cells and inhibits proliferation of cells.
14:
Macrophages and innate response activated. Cytokines like IL12 tell adaptive response what to do.
Th2- important for extracellular bacteria. Il-4 present directs this difference.
IL-12-Th1 response- cell-med response. Activate macrophages, etc.
IL-12- mediates innate and links innate and adaptive. Tells adaptive that infection is intracellular
Il-2: growth factor for T cells. Produced by activated T cells- autocrine growth factor.
Interferon gamma- no antiviral properties- principal activator macrophages.
19: during inflammation, cytokines can tell bone marrow to produce more cells.
Colony stimulating factors- stimulate differentiation progenitor cells.
back of handout- cartoon of cytokines and cell lineages.
3:Chemokines- small proteins tell leucocytess where to go. These molecules are often referred to as cytokines. Term from old literature. About 30 homologous polypeptides that tell naive T or B cells where to go. N terminal C residue defines their classification- divide into 4 families.
4:
1. CC1,2,3- chemokine. CCL-ligand. CCR-receptor.
2. CXC- ELR+ means ELR before the C.
6: Chart from immunobiology book. Ex of CXC class. Numbered based on when they were identified.
A lot of these have been renamed over time. Chemokines produced by a lot of cell types and can bind to more than one kind of receptor.
7: Chart continued. Do not memorize.
8: General Features
Endothelial- line blood vessels.
2 times produced:
inflammatory response-chemokines to tell where infection is. Upregulated during infection, then disappear.
normal homeostasis- tell leucocytes to go to lymphoid organs. Constitutively produced.
9: receptors named for ligands. T cells have a number of different chemokine receptors to tell them to do things at different times.
10: General scheme of receptor- 7 transmembrane helices. Bind to G proteins. Activate a number of pathways in cell.
11:
Chemokines are important for migration as well as other roles.
12:
Immune response- want innate system to respond to site of infection. Chemokines needed to tell cells where to go, and adhesion molecules need to be regulated. Immune cells need to adhere to extracellular matrix of collagen fibers and move through tissue. Binding of adhesion molecules allows cells to move.
13:3 families
selectins induce rolling along the endothelium (blood vessel lining). Slows neutrophil down- bind and release.
integrins- tight adhesion- stops and binds. Then can move into tissue
Ig superfamily- T cell binding to APC. Maintains contact.
14: What happens during inflammation? Endothelial cells normally do not express P selectin. as a result of infection, TNF and IL-1 cause P selectin to be expressed in an area of endothelium near the infection. Cells roll along.
15: In tissue- IL-8 produced and binds to receptor on endothelial cell to produce high-affinity integrin to bind to ligand. Contacts, binds to endothelial cell, and causes shape change in cell so it can squeeze between endothelial cells and into tissue. Inside- more chemokines cause crawling along structural framework to where infection is. Downregulate epxression- cell stops to do its job. Different chemokines attract different cells for the most efficient response.
16: Normal immune cell trafficking:
HOw do cells know to go to lymph node? Stromal cells produce CCL21. Dendritic cells have receptor CCR7. They migrate , bind, migrate into lymph node. T cells have same receptor. Lymph node has a lot of consttitutive production of CCL21,19, etc.
17:Cytokines:
regulate inflammatory response. Autocrine or paracrine.
18: General properties
1. Gene transcribed- there are bursts of cytokine production, and mRNA is degraded fast. All regulated by different means: mRNA degradation, cleavage for activation, post-translational modification, etc. Only neutrophils may store them in granules. All other cells secrete them.
19:
pleiotropic- act on different cell types.
20:
Cytokine cascades exist.
21:
Most produced locally and act locally. Sometimes large infections cause huge amount TNF and IL1.
22:
Bind specific receptors- high affinity. SMall amount produced, but physiologically important.
23: Stimulate T or B cell- upregulates cytokine expression.
24:
Cells respond by differentiating (T cells), activating function (macrophages and radical production), and cell proliferation (Tcells and IL-2- allows to enter S phase)
25: Receptors have similar features.
26: rest of talk. Same cytokines can have a lot of diff functions.
28: What happens when you have infection:
What immune cells normally live in tissues? macrophages and dendritic cells can bind microbes through pattern recognition receptors. Bind to cell wall and generate features. Pull in bug, chew it up, stimulate production cytokines. IL-1, 6,TNf act on vascular endothelium to tell them to upregulate adhesion molecules and produce more chemokines. Also increase vasccular permeability. Loosen tight junction. Gets inmmune cells and products to site.
Neutrophils are great killers .Live up to 24 hours and tend to move in first. IL-8 attracts them to chew up bacteria.
29: TNF
Block TNF, inhibit inflammatory response.
30: TNF- tumor necrosis factor- can cause apoptosis in some tumors.
31: Septic shock- systemic production of TNF, IL-1, IL-6. When in circulation, can affect organs. Fever, liver, appetite suppression, smooth muscle and heart problems.
32: whole body effects from cytokines.
36: local effects are favorable to clear infection. Systemic- blood volume decreases because of leaky vessels. Cells move into un- needed tissues. neutropenia signals influx from bone marrow. multiple organ failure results in death. Principle mediator- TNF. In human patients- blocking TNF does not work to prevent shock.
39:To stop inflammatory response, anti-inflammatory cytokines reduce response.
Ex: IL-10 shuts off recruitment of cells. Downregulate MHC class II and costimulatory molecules.
p13:
viral infections:
IFN alpha and beta secreted to act on other cell types. Bind to alphabeta receptor. Other cells stimulated to produce proteins to inhibit oligodenylate synthetase, to inhibit transcription of RNA and upregulate Class I MHC for presentation to cytotoxic T cells. ALso promotes development of Th1 cells and inhibits proliferation of cells.
14:
Macrophages and innate response activated. Cytokines like IL12 tell adaptive response what to do.
Th2- important for extracellular bacteria. Il-4 present directs this difference.
IL-12-Th1 response- cell-med response. Activate macrophages, etc.
IL-12- mediates innate and links innate and adaptive. Tells adaptive that infection is intracellular
Il-2: growth factor for T cells. Produced by activated T cells- autocrine growth factor.
Interferon gamma- no antiviral properties- principal activator macrophages.
19: during inflammation, cytokines can tell bone marrow to produce more cells.
Colony stimulating factors- stimulate differentiation progenitor cells.
back of handout- cartoon of cytokines and cell lineages.
Marion 9 (Friday)
Humoral Immunity
2 types: innate and adaptive.
innate- soluble factors- talking about complement. It has been bane of immunology teachers and students because it involves lots of components. How proteins are activated can be complicated if you focus on details. He has provided notes that go through details. Think about the concept, what it does, why it is important.
Proteins constitute about 12 proteins involved in activation and about 6 or more involved in regulation and control.
Slide 1: 3 ways complement system engaged:
1. classical pathway
discovered by von Behring early 1900s. Erlich discovered antibodies, but von Behring and Kitasato realized antibodies were not enough to eliminate bacteria. Had to be something else. They called it complement, because it complements antibodies.
Cl path activated by immune complexes. Antibodies can neutralize viruses and toxins, and provide handles for phagocytes, but they need help.
2. lectin pathway-
most recent to be discovered. Involves protein in circulation that recognize mannans. Almost the only place to find exposed mannose is on pathogens, mostly bacteria. Not absolutely true. Humans- mannoses get glycosylated, mostly with sialic acid. Our cells only rarely show exposed mannose. There are a few nonsialated exceptions. All the mannoses on cell wall and capsule of bacteria are targets for protein to activate lectin pathway- mannan binding proteins (MBP), and have associated mannan binding protein associated serine proteases. This mechanism activates potent innate immune response dependent on series of complement proteins to recognize mannans.
2. alternative pathway
discovered in 1950s- classical was only one known at the time. Immune complexes were not only way to get activation.
works like lectin pathway, with differences. Depends on natural turnover of complement components. C3 hydrolysed to active C3b at low rate all the time. If it happens near a bacteria, can bind to surface. Then other proteins can use it as focus to activate more complement. Pathway becomes more important- once classical or lectin activated, alternative pathway allows for process of amplification for much more complement activation than in its absence. Components of this pathway are heat labile- why you heat inactivate fetal bovine serum before incubating cells in it.
Slide 2: Most important point about complement activation- generate something called C3 convertase (serine protease). It converts C3 to C3b +C3a and C3b binds to cell surface (mostly bacteria). C3a is lost to fluid phase, but it is an anaphylatoxin- induces inflammation
**important**1. cell- bound components are opsonins- "to prepare to eat". There are receptors for these components on phagocytic cells (PMNS, macrophages, phagocytic cells). Erythrocytes have them, too. Relevant for classical pathway- red cells are primary means for removal of immune complexes from blood to transfer to liver Kupfer cells- strip off complexes and get rid of them without damaging red cells.
2. soluble components induce inflammation.
What else can complement do?
Once you generate C3 convertase (cell bound), it is formed on cell surface. Converts C3 to C3b, binds more cells, etc.
Slide 3: C3 convertase, when you generate C3b, creates C5 convertase. Serine protease with different substrate, C5. Converts C5 into C5b, cell bound, and soluble C5a (anaphylatoxin- the most potent inflammation inducer from complement activation because of receptors on vascular endothelium to induce vascular dilation. Mast cells- histamine release. Smooth muscle- bronchial restriction).
4: C5b is on cell surface. C6(1),7(1),8(1),9(9-10) now polymerize around C5b to generate the MAC (membrane attack complex). Form pore to induce cell lysis. Looks like perforin complexes.
Complements cannot distinguish self from nonself surface. Complement activation is tightly regulated.
Soluble inhibition - keeps enzyme cascade in check. Once C3b is generated, get amplification because of alternative pathway.
cell surface inhibition- on our cells, not bacterial. Inhibits C3 convertase, inhibits binding of Clq to immune complexes.As burden of pathogen reduced, control mechanismss rein in system. Also inhibits MAC formation to protect our cells
See slides for review with diagrams from text.
Complement provides 3 means of effector function. For all 3 pathways, goal is to generate C3 convertase. C3b is ligand for handle for phagocytic cells**), Membrane complex lyses cell. Photomicrograph shows holes. Same structure as perforin. Sequence comparison: Structure C9 is similar to perforin.
Humoral Adaptive Immunity
Antibody (Ab)- selected during development of B cells to be good at binding pathogens. Generated by B cells. B cell has to be activated from naive inexperienced B cell to activated B cell, or plasmablast.
Plasmablast can go one of 3 directions:
1. memory B cell for later use
2. plasma cell- cell specialized for Ab secretion
3. die
2 types B cells:
Slide 6: B-1
Slide 5 and 7: B-2 are B cells that migrate to lymph node and spleen depending on expression of molecule on surface called CD62L. This molecule helps new B cell get into lymph node through HEV. HEV has receptors on lumenal side for this ligand. B cell goes to paracortex (T cell zone). Want to migrate to follicle. As B cell goes through, it interacts w T cells, + antigen brought in in lymph as well as dendritic and phagocytic cells. If the B cell interacts with a T cell and antigen to become activated, it forms a specialized structure called a germinal center (slides 10, 11). No contact, just goes into follicle and sits about 3 wks, recirculates. Lasts about 3 wks in follicle. Not in follicle the B cell lasts 3 days and dies. Samples all antigens from afferent lymph and with T cells interacts with APCs. In paracortex, or T cell zone (PALS) of spleen If B cell there and activated helper T cell, B cell found antigen it can bind and take up because receptors are crosslinked by antigen, binding to antigen delivers to B cell signal 1 through Ig receptor (aggregation of Ig receptor on surface is signal 1)
If antigen can be processed and peptides generated from it, recognized by T cell receptor (activated by dendritic cell, which had MHC+peptide and IL-2), cognate interaction happens.
If T cell did not have to see antigen on B cell, B with self and B with nonself would be equal.
Only way B cell helped by T cellis if B cell expresses same MHC peptide that activated T cell in the first place. Signal 2: For B cell to get help from T cell, needs signal 2. Signal 2 from T cell is in form of CD40 ligand or CD 154. Activated T cell expresses CD40 ligand. B cell has CD40 on surface. Molecule engages ligand on T cell for signal 2. B cell differentiate into memory or plasma cell in response to cytokines from Tcell, but signal 2 is essential for formation of germinal center.
If T cell does not see antigen but does see CD40, may see FAS and trigger apoptosis. signal 2 delivered by CD40 on activated T cell. When B cell gets signals in paracortex, migrates into follicle to generate germinal center, where clonal expansion (10^3-10^4 fold), affinity maturation (driven by T cells and dependent on CD40 ligand and cytokines including IL-4), and isotype switching. Also differentiation to plasma cell or memory cell. Signals for differentiation are unknown. Memory cells can sit and make antibodies for months to years.
Slide 12: Affinity maturation is due to somatic mutation plus selection. Selection more important. Ability of limited antigen to select B cells with best receptors to form memory cells is important. Follicular dendritic cells are not lymphoid derived. Have long dendrites all through follicle forming reticular network. They have on surface FCgamma and C3b receptors and derivatives. They bind immune complexes. They can hold immune complexes for long time- reservoir for B cell sampling. Antigen released from follicular cell to B cell, B cell expresses peptide in MHC, gets activated. May be one way long term memory is maintained. Read Gray paper.
Germinal centers – where above processes occur. B cell undergoes somatic mutation. In process generates dysfunctional receptor- dies by apoptosis. Self reactivity- dies by apoptosis. Why? because B cells have in germinal center as they proliferate in dark zone (where somatic mutation occurs)- move into light zone- proliferation slows down, but undergo test. Must be able to generate specific antigen in order to generate signal. Need receptor engagement on FDC and T cell help again – if not- dies by apoptosis. Failsafe mechanism maintains tolerance. B cell not relevant, it drops out. Passes- proliferates more, become plasma cell or sits in follicle as memory B cell.
Other points:
What about B-1 B cells?
These cells live in marginal zone of spleen and peritoneal cavity. They are selected for these areas because of specificity for bacterial carbohydrates. Activation is different. Signal 1 is same. Ig receptor. Signal 2 derives from antigen. Like innate immune B cell. Still activated in clonotypic fashion, but gets signal through TLR or other innate receptors. When cells are activated, limited proliferation (not much)occurs, very limited isotype switching, no somatic mutation, no affinity maturation, no memory.
Best Ab for bacteria are carbohydrates, but it is impossible to develop a strictly carbohydrate vaccine. Immune response is IgM and relatively low affinity antibody and no memory. Solution for haemophilus influenza- hook onto carrier to get T cell epitopes. B cell receptor complex other molecules important CD21,81,19
19 important because cytoplasmic tail binds cytoplasmic tyrosine kinases
21 complement receptor- binds immune complexes.
81 binds whole thing together.
Ig receptor comes together with CD19 to get more effective B cell activation than with B cell receptor alone.
In children, last immune development is response to carbohydrates- the lack increases susceptibility to infection.
See slides again for review.
IgA prevents colonization of surfaces.
IgE sits on surface of mast cells waiting for amtigen.
Secretory IgA- B cells in Peyer’s patch . Binds to poly Ig receptor, transported, released on lumenal side by cleavage of receptor, leaves secretory piece.
IgE found on mast cells lining epithelium. IgG throughout. IgM in circulation.
Look in his information he sent us.
2 types: innate and adaptive.
innate- soluble factors- talking about complement. It has been bane of immunology teachers and students because it involves lots of components. How proteins are activated can be complicated if you focus on details. He has provided notes that go through details. Think about the concept, what it does, why it is important.
Proteins constitute about 12 proteins involved in activation and about 6 or more involved in regulation and control.
Slide 1: 3 ways complement system engaged:
1. classical pathway
discovered by von Behring early 1900s. Erlich discovered antibodies, but von Behring and Kitasato realized antibodies were not enough to eliminate bacteria. Had to be something else. They called it complement, because it complements antibodies.
Cl path activated by immune complexes. Antibodies can neutralize viruses and toxins, and provide handles for phagocytes, but they need help.
2. lectin pathway-
most recent to be discovered. Involves protein in circulation that recognize mannans. Almost the only place to find exposed mannose is on pathogens, mostly bacteria. Not absolutely true. Humans- mannoses get glycosylated, mostly with sialic acid. Our cells only rarely show exposed mannose. There are a few nonsialated exceptions. All the mannoses on cell wall and capsule of bacteria are targets for protein to activate lectin pathway- mannan binding proteins (MBP), and have associated mannan binding protein associated serine proteases. This mechanism activates potent innate immune response dependent on series of complement proteins to recognize mannans.
2. alternative pathway
discovered in 1950s- classical was only one known at the time. Immune complexes were not only way to get activation.
works like lectin pathway, with differences. Depends on natural turnover of complement components. C3 hydrolysed to active C3b at low rate all the time. If it happens near a bacteria, can bind to surface. Then other proteins can use it as focus to activate more complement. Pathway becomes more important- once classical or lectin activated, alternative pathway allows for process of amplification for much more complement activation than in its absence. Components of this pathway are heat labile- why you heat inactivate fetal bovine serum before incubating cells in it.
Slide 2: Most important point about complement activation- generate something called C3 convertase (serine protease). It converts C3 to C3b +C3a and C3b binds to cell surface (mostly bacteria). C3a is lost to fluid phase, but it is an anaphylatoxin- induces inflammation
**important**1. cell- bound components are opsonins- "to prepare to eat". There are receptors for these components on phagocytic cells (PMNS, macrophages, phagocytic cells). Erythrocytes have them, too. Relevant for classical pathway- red cells are primary means for removal of immune complexes from blood to transfer to liver Kupfer cells- strip off complexes and get rid of them without damaging red cells.
2. soluble components induce inflammation.
What else can complement do?
Once you generate C3 convertase (cell bound), it is formed on cell surface. Converts C3 to C3b, binds more cells, etc.
Slide 3: C3 convertase, when you generate C3b, creates C5 convertase. Serine protease with different substrate, C5. Converts C5 into C5b, cell bound, and soluble C5a (anaphylatoxin- the most potent inflammation inducer from complement activation because of receptors on vascular endothelium to induce vascular dilation. Mast cells- histamine release. Smooth muscle- bronchial restriction).
4: C5b is on cell surface. C6(1),7(1),8(1),9(9-10) now polymerize around C5b to generate the MAC (membrane attack complex). Form pore to induce cell lysis. Looks like perforin complexes.
Complements cannot distinguish self from nonself surface. Complement activation is tightly regulated.
Soluble inhibition - keeps enzyme cascade in check. Once C3b is generated, get amplification because of alternative pathway.
cell surface inhibition- on our cells, not bacterial. Inhibits C3 convertase, inhibits binding of Clq to immune complexes.As burden of pathogen reduced, control mechanismss rein in system. Also inhibits MAC formation to protect our cells
See slides for review with diagrams from text.
Complement provides 3 means of effector function. For all 3 pathways, goal is to generate C3 convertase. C3b is ligand for handle for phagocytic cells**), Membrane complex lyses cell. Photomicrograph shows holes. Same structure as perforin. Sequence comparison: Structure C9 is similar to perforin.
Humoral Adaptive Immunity
Antibody (Ab)- selected during development of B cells to be good at binding pathogens. Generated by B cells. B cell has to be activated from naive inexperienced B cell to activated B cell, or plasmablast.
Plasmablast can go one of 3 directions:
1. memory B cell for later use
2. plasma cell- cell specialized for Ab secretion
3. die
2 types B cells:
Slide 6: B-1
Slide 5 and 7: B-2 are B cells that migrate to lymph node and spleen depending on expression of molecule on surface called CD62L. This molecule helps new B cell get into lymph node through HEV. HEV has receptors on lumenal side for this ligand. B cell goes to paracortex (T cell zone). Want to migrate to follicle. As B cell goes through, it interacts w T cells, + antigen brought in in lymph as well as dendritic and phagocytic cells. If the B cell interacts with a T cell and antigen to become activated, it forms a specialized structure called a germinal center (slides 10, 11). No contact, just goes into follicle and sits about 3 wks, recirculates. Lasts about 3 wks in follicle. Not in follicle the B cell lasts 3 days and dies. Samples all antigens from afferent lymph and with T cells interacts with APCs. In paracortex, or T cell zone (PALS) of spleen If B cell there and activated helper T cell, B cell found antigen it can bind and take up because receptors are crosslinked by antigen, binding to antigen delivers to B cell signal 1 through Ig receptor (aggregation of Ig receptor on surface is signal 1)
If antigen can be processed and peptides generated from it, recognized by T cell receptor (activated by dendritic cell, which had MHC+peptide and IL-2), cognate interaction happens.
If T cell did not have to see antigen on B cell, B with self and B with nonself would be equal.
Only way B cell helped by T cellis if B cell expresses same MHC peptide that activated T cell in the first place. Signal 2: For B cell to get help from T cell, needs signal 2. Signal 2 from T cell is in form of CD40 ligand or CD 154. Activated T cell expresses CD40 ligand. B cell has CD40 on surface. Molecule engages ligand on T cell for signal 2. B cell differentiate into memory or plasma cell in response to cytokines from Tcell, but signal 2 is essential for formation of germinal center.
If T cell does not see antigen but does see CD40, may see FAS and trigger apoptosis. signal 2 delivered by CD40 on activated T cell. When B cell gets signals in paracortex, migrates into follicle to generate germinal center, where clonal expansion (10^3-10^4 fold), affinity maturation (driven by T cells and dependent on CD40 ligand and cytokines including IL-4), and isotype switching. Also differentiation to plasma cell or memory cell. Signals for differentiation are unknown. Memory cells can sit and make antibodies for months to years.
Slide 12: Affinity maturation is due to somatic mutation plus selection. Selection more important. Ability of limited antigen to select B cells with best receptors to form memory cells is important. Follicular dendritic cells are not lymphoid derived. Have long dendrites all through follicle forming reticular network. They have on surface FCgamma and C3b receptors and derivatives. They bind immune complexes. They can hold immune complexes for long time- reservoir for B cell sampling. Antigen released from follicular cell to B cell, B cell expresses peptide in MHC, gets activated. May be one way long term memory is maintained. Read Gray paper.
Germinal centers – where above processes occur. B cell undergoes somatic mutation. In process generates dysfunctional receptor- dies by apoptosis. Self reactivity- dies by apoptosis. Why? because B cells have in germinal center as they proliferate in dark zone (where somatic mutation occurs)- move into light zone- proliferation slows down, but undergo test. Must be able to generate specific antigen in order to generate signal. Need receptor engagement on FDC and T cell help again – if not- dies by apoptosis. Failsafe mechanism maintains tolerance. B cell not relevant, it drops out. Passes- proliferates more, become plasma cell or sits in follicle as memory B cell.
Other points:
What about B-1 B cells?
These cells live in marginal zone of spleen and peritoneal cavity. They are selected for these areas because of specificity for bacterial carbohydrates. Activation is different. Signal 1 is same. Ig receptor. Signal 2 derives from antigen. Like innate immune B cell. Still activated in clonotypic fashion, but gets signal through TLR or other innate receptors. When cells are activated, limited proliferation (not much)occurs, very limited isotype switching, no somatic mutation, no affinity maturation, no memory.
Best Ab for bacteria are carbohydrates, but it is impossible to develop a strictly carbohydrate vaccine. Immune response is IgM and relatively low affinity antibody and no memory. Solution for haemophilus influenza- hook onto carrier to get T cell epitopes. B cell receptor complex other molecules important CD21,81,19
19 important because cytoplasmic tail binds cytoplasmic tyrosine kinases
21 complement receptor- binds immune complexes.
81 binds whole thing together.
Ig receptor comes together with CD19 to get more effective B cell activation than with B cell receptor alone.
In children, last immune development is response to carbohydrates- the lack increases susceptibility to infection.
See slides again for review.
IgA prevents colonization of surfaces.
IgE sits on surface of mast cells waiting for amtigen.
Secretory IgA- B cells in Peyer’s patch . Binds to poly Ig receptor, transported, released on lumenal side by cleavage of receptor, leaves secretory piece.
IgE found on mast cells lining epithelium. IgG throughout. IgM in circulation.
Look in his information he sent us.
Sunday, December 10, 2006
Roslaniec Notes
Structure and Function of MHC and Antigen Presentation
Wednesday, 12/6/06, 8:30:00-9:50 AM
Ed Rosloniec- immunologist
in VA medical center. Contact information in UT directory.
3 lectures in 24 hours on immune response and steps to link innate and adaptive. 3 subjects listed in the Topics of MHC Structure and Function 1 slide are all 3 lectures.
T cell ontogeny is development and maturation of T cells.
2:Big picture (abridged)
We will focus on left. Need to understand how pathogenic antigens enter immune system, how presence is communicated to adaptive immune system, and how T cells are generated. Particulate and souble antigens are handled differently. We will focus on soluble protein antigen. We will discuss APCs, capable of grabbing pieces of pathogens and preparation for recognition by adaptive immune system. Today about T cell expression and path they follow.
Today looking at ability of APC to collect piece of pathogen and get it in context of MHC. T cell can survey, identify, mount response.
3: MHC most polymorphic locus in mammals. It has large numbers of allelic variants. Not hypervariable- not result of recombination. Human MHCs are called HLA. Mouse is H-2. Large loci with 3 classes, I,II,III. Not discussing III today. I and II have similar function despite different organization. DP, DQ, DR are class II. Class I are A, B , C.
Mouse: see slide.
within MHC- lots of other genes. They are chaperones and other genes with different function. For the most part they are involved in antigen presentation in one way or another.
4: Class I and II are heterodimeric glycoproteins. I has a single transmembrane region and a single small cytoplasmic chain.
II two of each (two transmembrane domains and two small cytoplasmic chains).
Short domains: 28 amino acids. Role in signalling limited. Primary function is to bind antigen peptide and display on the surface of the cell as a surveillance mechanism. Complex is ligand for T cell receptor.
5 and 6: Where are molecules expressed?
Class I is expressed in almost every cell.
II is expressed on Tcells, B cells, macrophages and APCs. It is also expressed on the epithelium of the thymus.
No class I or II on red blood cells.
Malaria affects red blood cells, but they cannot tell the immune system they are infected with no class I MHCs to present antigens.
7: Function of MHC molecules:
MHC is the link between innate and adaptive immunity. Regulates ability to produce a specific immune response. Ligand for T cell receptor.
8-10: Class I structure:
3 extracellular domains, 4th is beta2 microglobulin with few variants. It stabilizes molecule.
Beta-2 molecule noncovalently associates with alpha-domains. alpha- helical regions form binding cleft for peptides. All class I and II proteins look like the structure shown.
11-14: Class II:
gene organization is different, but crystal structure is almost identical to class I. No function in either chain alone. Must be paired to get to surface. DR is most represented of loci.
Ie and Dr are similar.
Class II crystal structure shows covalent linkage.
How to generate effective immune response?
specificity is interesting. Biochemical interactions tend to be highly specific. Well defined. MHC has variety, but there are rules. Each MHC can bind a wide variety of peptides. If you take one cell- isolate MHCs and kick off peptides- find at least 1000 different peptides. A single cell has associated with the same MHC molecule a large number of peptides. But there is specificity.
15: Different Class I and Class II function:
1. length peptide bound: Class I 8-10 AAs.
2. Specificity and mechanism of peptide binding: II has a different binding motif or pocket
3. where they pick up peptides
4. presentation to and stimulation of T cells
Peptide binding:
CLass I: short peptide, 8-10 AAs II: longer, extended peptide longer than 12-13 AAs, 30-
100AAs in vitro
closed ends, peptide buried into corners open ends on binding cleft
bind between int at N and C terminus of peptide much tighter definition of what it binds. Most 1,4,6,9 binding pocket motif with some variation.
to bind largermolecules- kink out molecule in Not all binding
middle pockets are always used.
too big- disrupts binding. Binding motifs- can see 1,4,6,9.
19-21:Class II:There are contacts with backbone as well as side chains of peptides. p1 has a conserved deep pocket and serves as the primary anchor position.
Binding motifs: p4-positive AAs.
Green are conserved residues. Rest have little conservation.
Peptides go into cleft in alpha helical or extended helical form.
2 crystal structures of peptides: both Y and F in p1. T cell specificity comes from the surface of the molecule from side chains and orientation presented to T cell.
23-24: Biosynthesis-
Class I is synthesized as one chain with beta-2 microglobulin associated for stability. Synthesized in ER, exit through Golgi to surface. Stabilized by calnexin, then beta-2 microglobulin. Class I also requires peptide in cleft for stability. Peptide enters ER through TAP molecules. They pump peptides into ER. Not MHC specific. Peptides come from large subunit called proteosome. Cleaves peptides into small molecules, picked up by TAP and pumped into ER, then bind if compatible to MHC.
25: Schematic diagram of TAP:
pumping is an energy dependent process . TAP has an ABC binding domain.
27: Class II totally different. It gets antigens through the endocytic process. Particulates in vesicles are brought in, the vesicles are acidified, proteases degrade, fuse with late endosomal vesicles with class II MHCs.
28-32: Details:
Class II is synthesized in ER, but stable in absence of peptide. Can grow in lab and purify. Has built in second domain. Biosynthesis- no peptides are there until late stages of biosynthesis and movement to surface of cell. Green- invariant chain Occupies peptide binding site during biosynthesis. Clip peptide in red binds all class II molecules. Shown in trimeric association. Invariant is called Type II molecule topogenically. Antiparallel interaction gives ability to bind.
Class 2 goes through stages- clip peptide is left at end. Clip binds tightly. How do we get rid of it? It gets displaced by second class II molecule with filled binding cleft. HLA-DM in mouse and human. Acts as chaperone to stabilize the molecule so clip can be released. Then molecule acquires antigenic peptides that have entered cell.
32:
Mechanisms of acquisition.
Can work for ingested bacteria in cell.
Also works for bound antigen on antibody from B cell. Extremely good antigen presenters.
33-34: Peptide binding facts:
1st point important. MHCs do not discriminate between self and pathogenic peptides. Lots of self bound.
2. Peptide with the right amino acid sequence will bind to MHC regardless of source.
3. Single MHC allele presents thousands of peptides at the same time.
Problem autoimmunity. Why don’t we generate response to all the peptides on the MHCs? Later.
MHC locus most polymorphic- multiple class I and II genes.
DR, DP, DQ: polymorphisms are significant. They are highly polymorphic, with polymorphic meaning 8-9 amino acids difference from one allele to another.
36-39: Polymorphisms have effect. Almost all surround peptide binding cleft and tend to be clustered.
Class II: polymorphisms grouped more tightly. Frequently referred to as 1,2,3,4 regions.
If you are completely heterogeneous at all alleles, express 6 different alleles for I and 6 for II. Expression of 12 different alleles is rare. Which alleles you express tends to depend on your ancestry and ethnicity.
Developing vaccines for therapeutic purposes- if alleles differ, different people will not mount a response to the same determinant. Can find peptide with broad binding specificity.
T cell receptor Restriction-
MHC complex is ligand for T cells. T cell receptor interaction with complex is highly specific and based on ability to recognize antigen peptide as well as to recognize the MHC presenting the antigen.
T cell receptor is restricted to only recognize specific MHC molecules. Develops in thymus.
DR1 with peptide:
significant portion of peptide is buried. Points of contact with T cell receptor are limited. >50% buried- usually 60-70% buried in class II. Specificity is high and limited in terms of contacts.
T cell receptor is specific for peptide only in context of MHC.
T cell receptor has to have both interactions. Interacts at an angle with the peptide and MHC.
45: Alloreactivity- unintended recognition of MHC by T cell receptor. Historically MHCs were first discovered in experiments with skin grafts in mice. Rejection led to observations of MHC. Rejection is not an intended function of MHC. Immune system has ability for T cell receptor to recognize nonself MHC class II and generate a response. Tcell from someone else- get response based on T cell receptor interacting with wrong MHC- high affinity with peptide or strong reaction with MHC withoout regard to peptide. Nonself MHC class II.
T cell receptor genes from mouse A can interact with mouse B’s MHC molecules. Plastic set of gene segments allow these molecules to interact even though they have never "seen" each other.
47: Last thing: superantigens.
Molecules with ability to put MHC and T cell receptor together and act as molecular clamp. Stimulate T cells for huge release of cytokines and chemokines. They are from bacteria and viruses. Discovered because of their role in toxic shock. The superantigen acts as a molecular vise to crosslink MHC and T cell receptor.
T Cell Development (Wed PM)
specialized process. MHC plays a pivotal role. None of this is generation of immune response- all preparing to respond to antigen.
Mostly occurs in thymus.
2: Big Picture- ability of cells to migrate and develop into T cells.
3:Class II interacts with CD4+ T cells.
Class I interacts with CD8- extra stability to increase affinity. Deciding and signalling to cell to be helpers or cytotoxic.
4-5: CD4 interacts with a small area of the beta subunit.
CD8 interacts with the alpha chain.
6-7:Where cells come from: T cells from bone marrow. Lineage diverges-see slide 6. Cells out of bone marrow- not distinct. Enter thymus and maturation starts from top. They mature as they move down pathway. Screening in thymic selection goes on as well. Identifies cells with receptors capable of interacting with CD4 and CD8 and eliminates cells that interact with self. T cell receptor has no concept of MHC. Somehow through recombination the T cell receptors become able to respond to MHC.
First screen: interact with MHC?- yes, then stimulated to differentiate and multiply. No- try again or apoptose.
Second screen: React too well? Recognize self ?yes-apoptose.no-mature into fully mature T cell.
Small numbers are released compared to what you start with.
8: Micrograph of cells in thymic stroma.
9: Australian immunology:
Importance of thymus:
scid mouse has a poor immune system, but a functioning thymus.
nude mouse- no thymus and no peripheral T cells as a result- has pre Ts, but they do not mature.
Combine- put nude mouse bone marrow into scid mouse- gets normal T cells –defect in bone marrow, not thymus.
Reverse- thymus from scid in nude mouse- T cells released. Transplanting thymus- can put it anywhere.
Adult thymus involutes, which means it shrinks over time. You have your maximum number of T cells in your teens. We produce lots of memory T cells, so it works out.
Properties- see slide 10.
After puberty- can live without the thymus if it is removed.
11: T cell life cycle in the thymus:
proliferate 7 days, develop lots of new cells, 98% die. Reasons they die: Nonfunctional receptor, untranslatable transcript, protein that does not recognize MHC, self recognition. You Produce 2 million cells per day.
Cells can be eliminated by apoptosis.
13: Stages and receptors:
14-15:: Pre-Ts emerge from bone marrow CD3,4,8 negative.
Into thymus, gammadelta and alphabeta pre-receptors form. Selection drives the cells into one developmental pathway or the other.
Cells that are double positive are CD4 and CD8 positive. Selection then occurs for that direction. Then commitment to lineage occurs and export to periphery.
Other changes at cell surface: CD25 is IL2 receptor for proliferation. Expressed just before serious proliferative phase in thymus.
17-23: delta locus is between valpha and jalpha locus.
1st you get CD25 expression and DJ rearrangement. This is on slides in series.
What receptor you get is a matter of statistical probability.
24:Important markers: RAG genes, TdT, preTalpha, signal transducers.
Allelic exclusion- once beta chain rearranges on one chromosome, shuts down other locus.
If T cell receptor doesn’t work, can go through other chromosome.
T cell can get 2 alpha chains expressed, but one will be nonproductive.
27: Gammadelta cells- less variability in T cell receptor cells, and found in interesting places like mucosal surfaces and lining reproductive tract. Not same variability. Ligands are different as well. Seem to be first line defense.
28: Irradiation-bone marrow chimeras-
F1 mouse with axb genotype- irradiate to wipe out bone marrow. Thymic epithelium is radiation resistant and expresses MHC I and II.
Irradiate type A, and give bone marrow from axb, repopulate APCs- T cell responses- only A because thymic cells select for A and are radiation resistant.
What role do the the resident epithelial cells of thymus have? Series of experiments addressed this with donors and acceptors. Periphery and thymus must match. Is this + or – selection? Giving mouse APCs of a type allows for selection of that type. Positive selection is mediated by thymic epithelial cells.
T cell receptor transgenic mouse- all T cells recognize MHCI CD8+. Receptor downregulates CD4 +T cells.
Negative selection examines response to self peptides. If self peptide is recognized, generates apoptotic signal.
Negative selection avoids producing self-reactive cells.
36:Thymus slide- orange spots are apoptotic cells. Peptide injected into thymus resulted in increased apoptosis of cells. Transgenic mouse- normal development is driven by endogenous T cell receptors.Added peptide was ligand for transgenic receptor expressed by almost every cell. Peptide is not recognized as self because of compartmentalization of +/- selection. In medulla- cells present foreign and self peptide with the injection. Part of thymus teaches what is self. Peptide is available to bone marrow derived cells- presented as self peptide and that is why apoptosis happens. Thymus epithelial cells work at top.
Bone marrow APC needed for negative selection- see slide.
skin graft not rejected- negative selection removed b responsive T cells.
38:Why are cells released at all?
39:Avidity hypothesis- avidity means movement of receptors together to act as a unit. It is the net of affinity of the individual receptors.
see slide. Weak signal is in part of thymus where the cell has few receptors anyway.
Cells go through differentiation after + selection, resulting in production of lots of receptor, so negative selection involves a much stronger signal.
Qualitative hypothesis- signalling events are different. Evidence hard to come by.
Other possibilities- thymic epithelial cells may process antigens differently.
Are all proteins from body expressed in thymus-no.
41-42: Overview slide is at end. Zap-70 and Lck are important for signalling.
Lecture 3:
T-Cell Activation and Cellular Immunity
Thursday, 12/7/06, 8:30:00-9:50 AM
2:Effector T cells are generated during immune response. Cells that have passed through selection but not been exposed to presented antigens outside the thymus are considered naive. They are released from the thymus and reside in spleen, lymph nodes, Peyer’s patches in the intestines, tonsils, etc. There they wait for signs of infection or required immune response. These T cells recirculate in the lymphatic system in a constant mechanism to be available for response.
T cells are activated by APCs: dendritic cells, macrophages, B cells.
3: Example of cell-mediated immunity:
Different types of T cells response to different pathogens. Viruses are intrracellular, so their proteins get into class I MHCs. Antigens encountered through extracellular mechanisms, including extracellular bacteria or bacteria in vesicles, generate TH1 T cell response mediated by class II complex.
Humoral- CD4 and class II activate B cell to make antibody.
4-5:Dendritic cells: most powerful and effective APC. Sit in surveillance mode. Become activated to be APC. Immature dendritic cells can be stained (green (class II)and red (lysosomal proteins)make yellow). Lysosomal proteins and classII MHC are colocalized. Sit in periphery (not lymphoid organs) until they encounter the antigen and change– separate green fluorescent signal from class II and red from lysosomal proteins are observed when the dendritic cells are activated. Surface of the dendritic cell becomes ruffled to allow increased surface area for more cells to interact so as many as possible will be activated. Dendritic cells take up by phagocytosis as much as possible. Once activated through innate receptor, they stop phagocytosis and start migration to lymphoid organs, and express class II on the cell surface. They migrate to lymph nodes- reside there- T cells scan.
6:Dendritic cells become antigen presenting machines.
7: Within lymphoid structure, where cells are found is shown on slide. Dendritic cells are in the cortex of lymph node, macrophages throughout, B cells in follicles. Antigen presenting cells may have different functions based on location, but whether this is true and to what extent is unknown.
8:T cells sample surface dendritic cell in lymph node. If there is no specific stimulation, they recirculate.
Recognize antigen- congregate around dendritic cell. A large number of T cells is necessary for proper response, but they number will be small compared to the total cohort of T cells in the body. Successful CD4 expression may be 1% of total repertoire. Small % in long run. Expansion and proliferation are important.
9: Adhesion molecules in dendritic sampling by T cells: no T cell receptor or MHC yet. T cell needs slowing down. LFAs, ICAMs, VCAMs get system started. ICAM3 and CD209 are specific to dendritic cells.
10: Once slowed down, T cell receptor samples MHC, CD4 comes in to enhance contact, interface determines whether T cell activates.
12-13: T cell activation requires 2 signals. This is a 2-signal process. Requires 2nd signalling event . One through MHC-T cell receptor, the other is co-stimulation through CD28:CD80/CD86 (B7.1,B7.2). APCs express B7.1 and B7.2.
14:Crosslink of CD28 gives costimulatory signal for proliferation and induces expression of CTLA-4 (or CD152) eventually. It binds B7 and sends negative signal to T cell to limit proliferative response. B7 only expressed by upregulation by stimulation of APC. Another layer of control.
Do not want uncontrolled proliferation of T cells. Superantigens do this, and it makes people really sick.
This is why costimulation is necessary.
Cell becomes cytotoxic- they kill other cells. No costimulation for effector function. Costimulation is only necessary for primary function.
16: Why is costimulation important?
Lack of B7 keeps cells from being injured by naive T cells. Cell encounters ligand without costimulatory signal. The cell is stimulated through T cell receptor- without signal through CD28- cell goes into nonresponsive state,and will not respond even to APC.
Get costimulation alone- no effect on T cell.
B7 must be induced on dendritic cells. Phenotype changes.
19: Macrophages- operate differently. Still have costimulatory system. Stimulated by bacterial infection. Toll-like receptor recognizes parts of bacteria, presents antigen, activates T cells.
Potential situation:
macrophage can encounter bacteria and get stimulated, but have some self peptides. Need only 10 MHCs on a cell with same peptide to stimulate in vitro. Say 20% occupied by bacterial proteins- others by self. It is possible to encounter a T cell which recognizes self peptides. Despite this. the link between bacterial infection and autoimmune response is weak. Some bacterial proteins are like ours, too- molecular mimicry could also cause autoimmune response.
20:B cells can endocytose antigen, act as affinity columns to sequester antigens, and bring them into cells through the Ig receptor. Large % MHC contain peptides. Efficient APC. Costimulatory molecule also required for induction on these cells.
21: Summary and comparison slide
Why don’t we develop autoimmunity with every bacterial infection?Epithelial cells are important to make self-reactive T cells anergic all the time. No phagocytic capacity or B7. Macrophages are within epithelium, but they have separate activity.
Lyme disease is a rickettsial disease that is poorly diagnosed. About 10% of cases progress to autoimmune arthritis due to a lack of treatment. Chronic infection yields autoimmune response.
22:T cell not activated- low affinity IL-2 receptor. Stimulate, upregulate alpha subunit, get a high affinity IL-2 subunit.
Add IL-2- get proliferation.
Class one and Cytotoxic T cell is major pathway for CD8 function.
25: CD4- Class II pathway- produces TH1 and TH2 cells. Primary means of identifying TH1 and TH2 cells is cytokines they produce.
Th2- IL-4,10, other cytokines. We cannot differentiate these cells phenotypically. No decent markers. we use what they produce.
24: Categories usually used in textbooks for TH1 and TH2 are not quite true. This slide is pretty good. Th1 can produce gamma interferon. Promotes IgG2A production (antibody) Th2 promote more IgG-1. Real differences- types of infection they combat.
Th1 predominates in autoimmunity and bact infection with intracellular aspects.
Th2 in allergy and parasitic infection. Both have uses and bad sides.
26-27: There may be crosstalk between 2 systems. CD4 T cells may help CD8 get stimulated. If you get class I and II on same cell, upregulates costimulatory molecules to encourage participation of both responses.APCs express all class I and II. Macrophages respond to gamma interferon to become bacteriocidal
Th17- appears to be separate lineage from T0 cells. TH17 cells produce IL-17. Seem to come out of Th0 lineage and play role in autoimmunity.
29: CTL function: Immunological synapse: Cytotoxic T cell has toxic granules- released to cell to kill.
30: Green is microtubules and red is cytotoxic granules.
31: SMAC-supermolecular adhesion complex. Includes T cell receptor, CD4, CD28, MHC:peptide, other proteins on slide. CTLs kill targets one at a time.
35-37: How they kill- perforin punches hole, granzymes and granulysin induce apoptosis.
In movie, dark area between peripheral and central adhesion zones is secretory zone where cytotoxic granules are delivered.
Gamma interferon causes macrophage to upregulate bacteriocidal effect.
Wednesday, 12/6/06, 8:30:00-9:50 AM
Ed Rosloniec- immunologist
in VA medical center. Contact information in UT directory.
3 lectures in 24 hours on immune response and steps to link innate and adaptive. 3 subjects listed in the Topics of MHC Structure and Function 1 slide are all 3 lectures.
T cell ontogeny is development and maturation of T cells.
2:Big picture (abridged)
We will focus on left. Need to understand how pathogenic antigens enter immune system, how presence is communicated to adaptive immune system, and how T cells are generated. Particulate and souble antigens are handled differently. We will focus on soluble protein antigen. We will discuss APCs, capable of grabbing pieces of pathogens and preparation for recognition by adaptive immune system. Today about T cell expression and path they follow.
Today looking at ability of APC to collect piece of pathogen and get it in context of MHC. T cell can survey, identify, mount response.
3: MHC most polymorphic locus in mammals. It has large numbers of allelic variants. Not hypervariable- not result of recombination. Human MHCs are called HLA. Mouse is H-2. Large loci with 3 classes, I,II,III. Not discussing III today. I and II have similar function despite different organization. DP, DQ, DR are class II. Class I are A, B , C.
Mouse: see slide.
within MHC- lots of other genes. They are chaperones and other genes with different function. For the most part they are involved in antigen presentation in one way or another.
4: Class I and II are heterodimeric glycoproteins. I has a single transmembrane region and a single small cytoplasmic chain.
II two of each (two transmembrane domains and two small cytoplasmic chains).
Short domains: 28 amino acids. Role in signalling limited. Primary function is to bind antigen peptide and display on the surface of the cell as a surveillance mechanism. Complex is ligand for T cell receptor.
5 and 6: Where are molecules expressed?
Class I is expressed in almost every cell.
II is expressed on Tcells, B cells, macrophages and APCs. It is also expressed on the epithelium of the thymus.
No class I or II on red blood cells.
Malaria affects red blood cells, but they cannot tell the immune system they are infected with no class I MHCs to present antigens.
7: Function of MHC molecules:
MHC is the link between innate and adaptive immunity. Regulates ability to produce a specific immune response. Ligand for T cell receptor.
8-10: Class I structure:
3 extracellular domains, 4th is beta2 microglobulin with few variants. It stabilizes molecule.
Beta-2 molecule noncovalently associates with alpha-domains. alpha- helical regions form binding cleft for peptides. All class I and II proteins look like the structure shown.
11-14: Class II:
gene organization is different, but crystal structure is almost identical to class I. No function in either chain alone. Must be paired to get to surface. DR is most represented of loci.
Ie and Dr are similar.
Class II crystal structure shows covalent linkage.
How to generate effective immune response?
specificity is interesting. Biochemical interactions tend to be highly specific. Well defined. MHC has variety, but there are rules. Each MHC can bind a wide variety of peptides. If you take one cell- isolate MHCs and kick off peptides- find at least 1000 different peptides. A single cell has associated with the same MHC molecule a large number of peptides. But there is specificity.
15: Different Class I and Class II function:
1. length peptide bound: Class I 8-10 AAs.
2. Specificity and mechanism of peptide binding: II has a different binding motif or pocket
3. where they pick up peptides
4. presentation to and stimulation of T cells
Peptide binding:
CLass I: short peptide, 8-10 AAs II: longer, extended peptide longer than 12-13 AAs, 30-
100AAs in vitro
closed ends, peptide buried into corners open ends on binding cleft
bind between int at N and C terminus of peptide much tighter definition of what it binds. Most 1,4,6,9 binding pocket motif with some variation.
to bind largermolecules- kink out molecule in Not all binding
middle pockets are always used.
too big- disrupts binding. Binding motifs- can see 1,4,6,9.
19-21:Class II:There are contacts with backbone as well as side chains of peptides. p1 has a conserved deep pocket and serves as the primary anchor position.
Binding motifs: p4-positive AAs.
Green are conserved residues. Rest have little conservation.
Peptides go into cleft in alpha helical or extended helical form.
2 crystal structures of peptides: both Y and F in p1. T cell specificity comes from the surface of the molecule from side chains and orientation presented to T cell.
23-24: Biosynthesis-
Class I is synthesized as one chain with beta-2 microglobulin associated for stability. Synthesized in ER, exit through Golgi to surface. Stabilized by calnexin, then beta-2 microglobulin. Class I also requires peptide in cleft for stability. Peptide enters ER through TAP molecules. They pump peptides into ER. Not MHC specific. Peptides come from large subunit called proteosome. Cleaves peptides into small molecules, picked up by TAP and pumped into ER, then bind if compatible to MHC.
25: Schematic diagram of TAP:
pumping is an energy dependent process . TAP has an ABC binding domain.
27: Class II totally different. It gets antigens through the endocytic process. Particulates in vesicles are brought in, the vesicles are acidified, proteases degrade, fuse with late endosomal vesicles with class II MHCs.
28-32: Details:
Class II is synthesized in ER, but stable in absence of peptide. Can grow in lab and purify. Has built in second domain. Biosynthesis- no peptides are there until late stages of biosynthesis and movement to surface of cell. Green- invariant chain Occupies peptide binding site during biosynthesis. Clip peptide in red binds all class II molecules. Shown in trimeric association. Invariant is called Type II molecule topogenically. Antiparallel interaction gives ability to bind.
Class 2 goes through stages- clip peptide is left at end. Clip binds tightly. How do we get rid of it? It gets displaced by second class II molecule with filled binding cleft. HLA-DM in mouse and human. Acts as chaperone to stabilize the molecule so clip can be released. Then molecule acquires antigenic peptides that have entered cell.
32:
Mechanisms of acquisition.
Can work for ingested bacteria in cell.
Also works for bound antigen on antibody from B cell. Extremely good antigen presenters.
33-34: Peptide binding facts:
1st point important. MHCs do not discriminate between self and pathogenic peptides. Lots of self bound.
2. Peptide with the right amino acid sequence will bind to MHC regardless of source.
3. Single MHC allele presents thousands of peptides at the same time.
Problem autoimmunity. Why don’t we generate response to all the peptides on the MHCs? Later.
MHC locus most polymorphic- multiple class I and II genes.
DR, DP, DQ: polymorphisms are significant. They are highly polymorphic, with polymorphic meaning 8-9 amino acids difference from one allele to another.
36-39: Polymorphisms have effect. Almost all surround peptide binding cleft and tend to be clustered.
Class II: polymorphisms grouped more tightly. Frequently referred to as 1,2,3,4 regions.
If you are completely heterogeneous at all alleles, express 6 different alleles for I and 6 for II. Expression of 12 different alleles is rare. Which alleles you express tends to depend on your ancestry and ethnicity.
Developing vaccines for therapeutic purposes- if alleles differ, different people will not mount a response to the same determinant. Can find peptide with broad binding specificity.
T cell receptor Restriction-
MHC complex is ligand for T cells. T cell receptor interaction with complex is highly specific and based on ability to recognize antigen peptide as well as to recognize the MHC presenting the antigen.
T cell receptor is restricted to only recognize specific MHC molecules. Develops in thymus.
DR1 with peptide:
significant portion of peptide is buried. Points of contact with T cell receptor are limited. >50% buried- usually 60-70% buried in class II. Specificity is high and limited in terms of contacts.
T cell receptor is specific for peptide only in context of MHC.
T cell receptor has to have both interactions. Interacts at an angle with the peptide and MHC.
45: Alloreactivity- unintended recognition of MHC by T cell receptor. Historically MHCs were first discovered in experiments with skin grafts in mice. Rejection led to observations of MHC. Rejection is not an intended function of MHC. Immune system has ability for T cell receptor to recognize nonself MHC class II and generate a response. Tcell from someone else- get response based on T cell receptor interacting with wrong MHC- high affinity with peptide or strong reaction with MHC withoout regard to peptide. Nonself MHC class II.
T cell receptor genes from mouse A can interact with mouse B’s MHC molecules. Plastic set of gene segments allow these molecules to interact even though they have never "seen" each other.
47: Last thing: superantigens.
Molecules with ability to put MHC and T cell receptor together and act as molecular clamp. Stimulate T cells for huge release of cytokines and chemokines. They are from bacteria and viruses. Discovered because of their role in toxic shock. The superantigen acts as a molecular vise to crosslink MHC and T cell receptor.
T Cell Development (Wed PM)
specialized process. MHC plays a pivotal role. None of this is generation of immune response- all preparing to respond to antigen.
Mostly occurs in thymus.
2: Big Picture- ability of cells to migrate and develop into T cells.
3:Class II interacts with CD4+ T cells.
Class I interacts with CD8- extra stability to increase affinity. Deciding and signalling to cell to be helpers or cytotoxic.
4-5: CD4 interacts with a small area of the beta subunit.
CD8 interacts with the alpha chain.
6-7:Where cells come from: T cells from bone marrow. Lineage diverges-see slide 6. Cells out of bone marrow- not distinct. Enter thymus and maturation starts from top. They mature as they move down pathway. Screening in thymic selection goes on as well. Identifies cells with receptors capable of interacting with CD4 and CD8 and eliminates cells that interact with self. T cell receptor has no concept of MHC. Somehow through recombination the T cell receptors become able to respond to MHC.
First screen: interact with MHC?- yes, then stimulated to differentiate and multiply. No- try again or apoptose.
Second screen: React too well? Recognize self ?yes-apoptose.no-mature into fully mature T cell.
Small numbers are released compared to what you start with.
8: Micrograph of cells in thymic stroma.
9: Australian immunology:
Importance of thymus:
scid mouse has a poor immune system, but a functioning thymus.
nude mouse- no thymus and no peripheral T cells as a result- has pre Ts, but they do not mature.
Combine- put nude mouse bone marrow into scid mouse- gets normal T cells –defect in bone marrow, not thymus.
Reverse- thymus from scid in nude mouse- T cells released. Transplanting thymus- can put it anywhere.
Adult thymus involutes, which means it shrinks over time. You have your maximum number of T cells in your teens. We produce lots of memory T cells, so it works out.
Properties- see slide 10.
After puberty- can live without the thymus if it is removed.
11: T cell life cycle in the thymus:
proliferate 7 days, develop lots of new cells, 98% die. Reasons they die: Nonfunctional receptor, untranslatable transcript, protein that does not recognize MHC, self recognition. You Produce 2 million cells per day.
Cells can be eliminated by apoptosis.
13: Stages and receptors:
14-15:: Pre-Ts emerge from bone marrow CD3,4,8 negative.
Into thymus, gammadelta and alphabeta pre-receptors form. Selection drives the cells into one developmental pathway or the other.
Cells that are double positive are CD4 and CD8 positive. Selection then occurs for that direction. Then commitment to lineage occurs and export to periphery.
Other changes at cell surface: CD25 is IL2 receptor for proliferation. Expressed just before serious proliferative phase in thymus.
17-23: delta locus is between valpha and jalpha locus.
1st you get CD25 expression and DJ rearrangement. This is on slides in series.
What receptor you get is a matter of statistical probability.
24:Important markers: RAG genes, TdT, preTalpha, signal transducers.
Allelic exclusion- once beta chain rearranges on one chromosome, shuts down other locus.
If T cell receptor doesn’t work, can go through other chromosome.
T cell can get 2 alpha chains expressed, but one will be nonproductive.
27: Gammadelta cells- less variability in T cell receptor cells, and found in interesting places like mucosal surfaces and lining reproductive tract. Not same variability. Ligands are different as well. Seem to be first line defense.
28: Irradiation-bone marrow chimeras-
F1 mouse with axb genotype- irradiate to wipe out bone marrow. Thymic epithelium is radiation resistant and expresses MHC I and II.
Irradiate type A, and give bone marrow from axb, repopulate APCs- T cell responses- only A because thymic cells select for A and are radiation resistant.
What role do the the resident epithelial cells of thymus have? Series of experiments addressed this with donors and acceptors. Periphery and thymus must match. Is this + or – selection? Giving mouse APCs of a type allows for selection of that type. Positive selection is mediated by thymic epithelial cells.
T cell receptor transgenic mouse- all T cells recognize MHCI CD8+. Receptor downregulates CD4 +T cells.
Negative selection examines response to self peptides. If self peptide is recognized, generates apoptotic signal.
Negative selection avoids producing self-reactive cells.
36:Thymus slide- orange spots are apoptotic cells. Peptide injected into thymus resulted in increased apoptosis of cells. Transgenic mouse- normal development is driven by endogenous T cell receptors.Added peptide was ligand for transgenic receptor expressed by almost every cell. Peptide is not recognized as self because of compartmentalization of +/- selection. In medulla- cells present foreign and self peptide with the injection. Part of thymus teaches what is self. Peptide is available to bone marrow derived cells- presented as self peptide and that is why apoptosis happens. Thymus epithelial cells work at top.
Bone marrow APC needed for negative selection- see slide.
skin graft not rejected- negative selection removed b responsive T cells.
38:Why are cells released at all?
39:Avidity hypothesis- avidity means movement of receptors together to act as a unit. It is the net of affinity of the individual receptors.
see slide. Weak signal is in part of thymus where the cell has few receptors anyway.
Cells go through differentiation after + selection, resulting in production of lots of receptor, so negative selection involves a much stronger signal.
Qualitative hypothesis- signalling events are different. Evidence hard to come by.
Other possibilities- thymic epithelial cells may process antigens differently.
Are all proteins from body expressed in thymus-no.
41-42: Overview slide is at end. Zap-70 and Lck are important for signalling.
Lecture 3:
T-Cell Activation and Cellular Immunity
Thursday, 12/7/06, 8:30:00-9:50 AM
2:Effector T cells are generated during immune response. Cells that have passed through selection but not been exposed to presented antigens outside the thymus are considered naive. They are released from the thymus and reside in spleen, lymph nodes, Peyer’s patches in the intestines, tonsils, etc. There they wait for signs of infection or required immune response. These T cells recirculate in the lymphatic system in a constant mechanism to be available for response.
T cells are activated by APCs: dendritic cells, macrophages, B cells.
3: Example of cell-mediated immunity:
Different types of T cells response to different pathogens. Viruses are intrracellular, so their proteins get into class I MHCs. Antigens encountered through extracellular mechanisms, including extracellular bacteria or bacteria in vesicles, generate TH1 T cell response mediated by class II complex.
Humoral- CD4 and class II activate B cell to make antibody.
4-5:Dendritic cells: most powerful and effective APC. Sit in surveillance mode. Become activated to be APC. Immature dendritic cells can be stained (green (class II)and red (lysosomal proteins)make yellow). Lysosomal proteins and classII MHC are colocalized. Sit in periphery (not lymphoid organs) until they encounter the antigen and change– separate green fluorescent signal from class II and red from lysosomal proteins are observed when the dendritic cells are activated. Surface of the dendritic cell becomes ruffled to allow increased surface area for more cells to interact so as many as possible will be activated. Dendritic cells take up by phagocytosis as much as possible. Once activated through innate receptor, they stop phagocytosis and start migration to lymphoid organs, and express class II on the cell surface. They migrate to lymph nodes- reside there- T cells scan.
6:Dendritic cells become antigen presenting machines.
7: Within lymphoid structure, where cells are found is shown on slide. Dendritic cells are in the cortex of lymph node, macrophages throughout, B cells in follicles. Antigen presenting cells may have different functions based on location, but whether this is true and to what extent is unknown.
8:T cells sample surface dendritic cell in lymph node. If there is no specific stimulation, they recirculate.
Recognize antigen- congregate around dendritic cell. A large number of T cells is necessary for proper response, but they number will be small compared to the total cohort of T cells in the body. Successful CD4 expression may be 1% of total repertoire. Small % in long run. Expansion and proliferation are important.
9: Adhesion molecules in dendritic sampling by T cells: no T cell receptor or MHC yet. T cell needs slowing down. LFAs, ICAMs, VCAMs get system started. ICAM3 and CD209 are specific to dendritic cells.
10: Once slowed down, T cell receptor samples MHC, CD4 comes in to enhance contact, interface determines whether T cell activates.
12-13: T cell activation requires 2 signals. This is a 2-signal process. Requires 2nd signalling event . One through MHC-T cell receptor, the other is co-stimulation through CD28:CD80/CD86 (B7.1,B7.2). APCs express B7.1 and B7.2.
14:Crosslink of CD28 gives costimulatory signal for proliferation and induces expression of CTLA-4 (or CD152) eventually. It binds B7 and sends negative signal to T cell to limit proliferative response. B7 only expressed by upregulation by stimulation of APC. Another layer of control.
Do not want uncontrolled proliferation of T cells. Superantigens do this, and it makes people really sick.
This is why costimulation is necessary.
Cell becomes cytotoxic- they kill other cells. No costimulation for effector function. Costimulation is only necessary for primary function.
16: Why is costimulation important?
Lack of B7 keeps cells from being injured by naive T cells. Cell encounters ligand without costimulatory signal. The cell is stimulated through T cell receptor- without signal through CD28- cell goes into nonresponsive state,and will not respond even to APC.
Get costimulation alone- no effect on T cell.
B7 must be induced on dendritic cells. Phenotype changes.
19: Macrophages- operate differently. Still have costimulatory system. Stimulated by bacterial infection. Toll-like receptor recognizes parts of bacteria, presents antigen, activates T cells.
Potential situation:
macrophage can encounter bacteria and get stimulated, but have some self peptides. Need only 10 MHCs on a cell with same peptide to stimulate in vitro. Say 20% occupied by bacterial proteins- others by self. It is possible to encounter a T cell which recognizes self peptides. Despite this. the link between bacterial infection and autoimmune response is weak. Some bacterial proteins are like ours, too- molecular mimicry could also cause autoimmune response.
20:B cells can endocytose antigen, act as affinity columns to sequester antigens, and bring them into cells through the Ig receptor. Large % MHC contain peptides. Efficient APC. Costimulatory molecule also required for induction on these cells.
21: Summary and comparison slide
Why don’t we develop autoimmunity with every bacterial infection?Epithelial cells are important to make self-reactive T cells anergic all the time. No phagocytic capacity or B7. Macrophages are within epithelium, but they have separate activity.
Lyme disease is a rickettsial disease that is poorly diagnosed. About 10% of cases progress to autoimmune arthritis due to a lack of treatment. Chronic infection yields autoimmune response.
22:T cell not activated- low affinity IL-2 receptor. Stimulate, upregulate alpha subunit, get a high affinity IL-2 subunit.
Add IL-2- get proliferation.
Class one and Cytotoxic T cell is major pathway for CD8 function.
25: CD4- Class II pathway- produces TH1 and TH2 cells. Primary means of identifying TH1 and TH2 cells is cytokines they produce.
Th2- IL-4,10, other cytokines. We cannot differentiate these cells phenotypically. No decent markers. we use what they produce.
24: Categories usually used in textbooks for TH1 and TH2 are not quite true. This slide is pretty good. Th1 can produce gamma interferon. Promotes IgG2A production (antibody) Th2 promote more IgG-1. Real differences- types of infection they combat.
Th1 predominates in autoimmunity and bact infection with intracellular aspects.
Th2 in allergy and parasitic infection. Both have uses and bad sides.
26-27: There may be crosstalk between 2 systems. CD4 T cells may help CD8 get stimulated. If you get class I and II on same cell, upregulates costimulatory molecules to encourage participation of both responses.APCs express all class I and II. Macrophages respond to gamma interferon to become bacteriocidal
Th17- appears to be separate lineage from T0 cells. TH17 cells produce IL-17. Seem to come out of Th0 lineage and play role in autoimmunity.
29: CTL function: Immunological synapse: Cytotoxic T cell has toxic granules- released to cell to kill.
30: Green is microtubules and red is cytotoxic granules.
31: SMAC-supermolecular adhesion complex. Includes T cell receptor, CD4, CD28, MHC:peptide, other proteins on slide. CTLs kill targets one at a time.
35-37: How they kill- perforin punches hole, granzymes and granulysin induce apoptosis.
In movie, dark area between peripheral and central adhesion zones is secretory zone where cytotoxic granules are delivered.
Gamma interferon causes macrophage to upregulate bacteriocidal effect.
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