Exam 1 - Week 1 Flashcards
1
Q
Primary pathogens cause disease in - host while opportunistics cause disease in - host?
**Define a pathogen
A
- Primary pathogens cause disease in ANY HOST (whether healthy or sick)
- Opportunistics only cause disease in ONLY HOST WITH IMPAIRED/DAMAGED/WEAK IMMUNE/DEFENSE mechanism/system.
**Pathogen is any disease causing microorganism.
2
Q
1-4. What is the difference between infection, virulence and avirulence? How does it differ from pathogen?
- What is the state where you get infected by a pathogen for a long period of time but you have no clinical symptoms? Give 1 example
A
- Infection - microbe enter host tissue - multiplies - damage tissue - cause host to respond - immune response (ability of microbe to infect you, not necessarily cause disease).
- Virulence; ability of organism to cause disease (degree to which microbe caused disease)
- Avirulence; organism can’t or do not cause disease
- Pathogen; disease causing organism
- Asymptomatic carrier e.g Typhoid Mary.
3
Q
There are 3 general sources in which humans are exposed to pathogens (disease causing agents).
1. Give 3 ways that EXOGENOUS MICROBES can be transmitted human to human, diseases caused (and how these can be controlled?)
A
- Aerosols (respiratory or salivary spread - coughing, sneezing, yelling, cursing); TB, mycoplasma, legionella, cold viruses, influenza. Not readily controllable
- Fecal-oral spread. Controlled by public health measure (hepatitis?)
- Venereal spread; difficult to control cause of social factors (HIV from sex?)
4
Q
There are 3 general sources in which humans are exposed to pathogens (disease causing agents).
- What is this called when exogenous microbes are transmitted from animals to humans ? (Zoonistic infections) or human to insect/vertebrate to human.
Identify the 3 types
- Ticks spread Rocky Mountain spotted fever called rickettsial and mosquitoes spread plasmodia, Zika and dengue virus (ant). 3 other diseases?
- dogs and bats spread rabies virus (animal).
- bubonic plaque spread from rat to fleas to man? (Ant - animal - man)
A
- Vector (biting arthropod - ant to man); malaria, sandfly fever, typhus (louse borne) (MST)
- Vertebrate reservoir (from animal) ; brucellosis, rabies, Q fever, lassa fever, salmonellosis (BRQLS)
- Vector-vertebrate reservoir; bubonic plague, trypanosomiasis, yellow fever. (PTY)
5
Q
- Yellow fever can be gotten from what route of transmission? (Same as bubonic plaque)
- Sandfly fever?
- Q and Lassa fever?
- Flu? (What is best way of getting flu?)
- Typhoid? (Same for malaria)
- Salmonellosis?
- Shigella or salmonella?
- Mycoplasma, legionella? (*Hint same as TB)
A
- Vector-vertebrate reservoir (ant to animal to man)
- Vector - Biting arthropod (ant to man)
- Vertebrate reservoir (animal to man)
- Aerosols (human to human; most exposure from SNEEZING more than coughing.
- Vector (biting arthropod)
- Vertebrate reservoir (animal to man)
- Venereal spread (human to human genitals)
- Aerosols (human to human)
6
Q
There are 3 general sources in which humans are exposed to pathogens (disease causing agents).
- What is the third and how can it cause disease?
A
ENDOGENOUS AGENTS part of NORMAL FLORA
- don’t normally cause disease except if damage result in gut contents leaking into adjacent tissues (SEVERE TRAUMA, SURGERY).
7
Q
There are 9 specific MECHANISMS of transmission. Identify all
A
- Formites; inanimate objects e.g toys, toothbrush, towels, bedding
- Food and water; salmonellosis from picnic lunch, contaminated wells
- Insect vectors; ticks, mosquitoes
- Direct contact; hand shake spread enteric virus (GI tract infection)
- Sexual transmission; HIV, herpes, chlamydia, syphilis
- Social ills; shared needles (HIV, hep B)
- World travel; (spread infectious disease country to country)
- Compromised living conditions ; crowded, dirty, poor places
- Nosocomial infections; crowded dirty hospital - become antibiotic resistant. E.g klebsiella outbreak.
8
Q
The most common source of human infections is when microbes moves from their normal habitat to sterile areas of the body. What are the 3 sterile sites that normal flora (microbes) should NOT be found?
A
- Blood
- Alveoli
- Muscle (deep tissues)
**BAM (we are in trouble if microbes enter BAM)
9
Q
Habitats are laden with microbes which have evolved to establish a symbiotic relation with host.
- Name 4 external surfaces that you find normal flora
- 11 general places you find normal flora
- Which part of body can you find normal flora that is pathogenic but do not cause disease
- Total cell density of normal flora range from 10^3 to the 10^10 per gram fecal material. Which part of GI has highest vs lowest density.
- Is there any group of normal flora present in high freq? (Most common)
A
- A) Skin (3). B) GI tract (1). C) Urogenital surfaces (2). D) Upper respiratory (5)
- General; scalp, teeth, throat, skin, nose, mouth, lung, intestine, urethra and vagina, groin and perineum, feet
- NASOPHARYNX (throat) e.g strep pyrogens and strep pneumonia
- Upper GI has lowest density of 10^3 per gram. Lower GI has highest density (make sense - rectum has poop so lots of bacterial).
- Yes. Especially in large bowel/poop 100% bactericides spp, E. coli etc.
10
Q
Name 4 ways in which normal flora are beneficial to host (Important roles of normal flora **High yield)
A
- Source of NUTRIENTS (e,g Vit K)
- Occupy a NICHE; Block access to pathogens and consume nutrients so pathogens don’t grow
- Elaborate/EXPAND BACTERIAL TOXINS that kill potential pathogens. E.g bacteriocin like colicin can kill E.coli, normal flora have high pH that kill pathogen
- Stimulate host IMMUNE response; normal flora make colony that build up immune system.
11
Q
Identify 3 mechanisms of opportunistic infection (host factors that lead to opportunistic infection).
A
- Compromised host; lower the immune response - age (newborn, child, old), cancer, nutrition status, genetic (inherited immune deficiency), immunosuppressants (to prevent transplant rejection), HIV
- Breach of normal barriers; cuts, surgery, burns, medical devices
- Use of antibiotics ; wipe out normal flora and cause C-diff.
12
Q
** What is the reasoning behind Koch’s postulates (function?)
**What are the 4 Koch’s postulates
A
** To establish causality - conclusively show that a bacterium is responsible for a particular disease.
- *
1. Bacterium should be found in people and parts of body affected by the DISEASE. (Not in healthy people)
2. Bacterium should be ISOLATED from lesions of affected person and able to be maintained in pure culture
3. Should cause disease when inoculated into healthy organism (pure culture reproduce the disease symptoms)
4. Should be RE-ISOLATED in pure culture from intentionally infected organism.
13
Q
Identify 5 limitations of Koch’s postulates
A
- Ignores the role of host (GENETICS); Susceptibility to disease is inherited e.g AID and TB. So if you are RESISTANT you will be asymptomatic. (Reduce correlation of postulate 1 that bacterium cause disease)
- Some organisms are FASTIDIOUS/FUSSY. Require lots of nutrients to grow. Some virus take long time to culture while some can’t be cultured at all. (Iimit postulate 2 cause you can’t isolate)
- Avirulent - VARIABILITY IN VIRULENCE of single bacterial species. Can acquire new virulence traits by genetic exchange e.g lysogenic conversion.
- Can’t do in humans (ETHICS) - postulate 4 (show disease in intentionally infected organisms)
- POLYMICROBIAL DISEASE - caused by various pathogens.
14
Q
Identify 4 classes of organisms
A
- Fungi
- Parasite
- Virus
- Bacteria
15
Q
Identify these group of cells
- include; archaebacteria and eubacteria
- Replicate by binary fission
- translation begins with N-formymethionine
- few introns
**identify 9 total xters; respiration? Differentiation? Repeated DNA? Ribosome type? Genetic material?
A
PROKARYOTES
- No nuclear membrane; lack membrane bound nucleus
- Replicate by binary fission
- Genetic material is DNA
- Limited amount of repeated DNA (1-5%)
- Very few introns (splicing can occur)
- 70S ribosomes (30S + 50S)
- Translation begins with N-formymethionine
- Respiration occurs in plasma membrane
- Limited capacity to differentiate.
16
Q
Identify group of cells that have defined nucleus
**9 xters
A
EUKARYOTES
- Well defined nucleus (membrane bound)
- True mitotic apparatus
- Genetic material is DNA
- Lots of repeated genome (10-40%)
- Most genes have introns
- 80S ribosomes (40S + 60S)
- Methionine
- Respiration occur in mitochondria
- Many cell types in multicellular species.
17
Q
Identify the class of organisms
- UNICELLULAR prokaryotic organisms
- lack nuclear envelope and membrane-bound organelles
- *Does this Class all have cell wall?
- *Another name fo this class? (9 Xters?)
A
BACTERIA
- most have cell wall (not all)
- bacteria and PROKARYOTE used interchangeably
18
Q
Yeast and molds are under what class of organisms? Difference btw yeast and mold
- what group of cells?
- 9 xters?
**What is unique to this class of organism?
A
FUNGI; include yeast (unicellular), molds (multicellular with filament), and dimorphism fungi (switch between yeast to mold). E.g of dimorphism fungi is Candida albicans.
**EUKARYOTES; fungi are eukaryotes that have well defined nucleus, membrane bound cytoplasmic organelles and A CELL WALL.
***Remember; Prokaryote with a cell wall is bacteria. Eukaryotes with a cell wall is Fungi.
19
Q
Identify organism
- prokaryote, eukaryotes or virus that require a living host for at least part of their cycle and cause disease in the host
3e. g - unicellular or multicellular EUKARYOTES that require a living host for at least part of their life and cause disease in host.
A
PARASITES (broad definition); obligate intracellular bacteria, viruses, parasitic worms.
PARASITES (narrow definition) ; parasitic amoebae, plasmodia, worms
20
Q
- Give examples of multicellular parasites you can see with naked eye
- Give examples of single cell protozoan and bacteria you need microscope to see
A
- Tape worm and other worms like larval worm. **Anything below 10^-3 need microscope
- Protozoa, bacteria, virus (biggest to smallest)
21
Q
Identify organism
- intracellular parasites that lack cell structure
- consist of nucleic acid genome surrounded by a protein coat
- require a cellular host for replication
**Describe xters of the genome (genetic material, size, structure - what protects genome form degradation? What surround outer coat and what is it made of (polar/nonpolar?)
A
VIRUSES
**included in broad definition of parasite
VIRAL GENOME
- either DNA or RNA (bot NOT BOTH)
- genome range in size form 5-200kb
- structure; CAPSID is a primary outer structure that protects genome from degradation and mediates attachment to host cell proteins called HOST CELL RECEPTORS. Some virus have additional outer coat that surround capsid called ENVELOPE
- ENVELOPE is a LIPID bilaterally embedded with glycoproteins (spikes).
22
Q
There are 2 types of viral infection
Identify this
- few viruses infect a cell, replicate within and produce 1000s of viruses which can be released by lysing the host cell
- animal cells are not lysed but rather harbor viral genome or allow the replication of low number of viruses (what is a similar phenomenon?)
A
- Lytic cycle
- Persistent/latent infections; similar phenomenon called LYSOGENY - occurs for bacteriophage which can lie dormant within the host bacterium.
23
Q
Simpler than virus
- RNA only (mostly in plants) - can get hepatitis delta from this
- protein only (no DNA or RNA). Still capable of causing disease - mad cow, scrapie (consume your brain), Creutzfeldt-Jakob disease.
A
- Viroids
2. Prions
24
Q
Identify 3 physicochemical properties of drugs and how they are determined?
A
- Molecular size
- size is determined by number and type of atoms present in drug molecule and how they are arranged in space (straight or branched chains, stereochemistry).
- small size is important for passing through pores (e.g channels) and spaces between cells. - Fat and water solubility
- determined by overall polarity (function of the chemical groups present in the molecule eg -OH, -Cl, -COOH)
- measured by determining PC - Charge (ionic character)
- determined by acidic, basic and neutral character of drugs.
25
If solubility of drug is determined by polarity, how is polarity then determined? (What is formula)
** What value is polar vs non-polar?
PARTITION COEFFICIENT
PC = [Drug]fat/[Drug]water
**Use water and 1-octagon as fat/lipid to measure.
PC>1 is lipophilic/hydrophobic
PC<1 is hydrophilic
26
There are 3 major chemical classification of drugs
Identify with 5 examples
- donates protons to proton acceptor - dissolving media
**What is major component that make up this chemical group?
**What is the acid and base form and polarity?
Weakly ACIDIC drugs
E.g; carboxylic acid (diuretics), sulfonamines, sulfonylureas, imides and ureides.
**COOH is major component
**HA is acid form (RCOOH) and is lipophilic
A- is conjugate base (RCOO-) and is hydrophilic cause of charge
HA + H20 <> H30+ + A-
27
There are 3 major chemical classification of drugs
Identify with 2 examples
- accepts a proton (H+) from an acid in dissolving media
**What is acid and base form?
Weakly BASIC Drugs
E.g Amides (antihistamines) and guanidines
**Base form is B:
Conjugate acid form is BH+
B: + H30+ <> H20 + BH+
28
There are 3 major chemical classification of drugs
Identify with examples
- neither donate nor accept protons at physiological pHs
- can be charged or uncharged (give e.g)
NEUTRAL DRUGS
E.g
* Neutral charged; quaternary ammonium salts of R4N+
* Neutral uncharged; alcohols (C2H5OH), aldehydes, amides, esters, ethers, ketones, halides and nitro, nitriles, lactam, acetylene
29
Equilibrium with weak acids involve - and depend on -?
What concept is this?
**What is pH and pK equivalent to?
**Formula?
Involve an equilibrium constant (Keq) and are pH dependent.
Henderson-Hasselback equation
**
pH = -log [H+]
PK = -log Keq
* *pH = pK + log [base or A-]/[acid or HA]
* *-log [H+] = -log Keq + log [A-]/[HA]
30
Identify 4 ways that drugs cross biological membranes?
1. Filtration through pores
2. Passive diffusion - across lipid like membrane
- simple passive diffusion (no energy, no carrier)
- carrier facilitated diffusion (no energy but need carrier)
3. Active transport - across membranes (require energy)
4. Endocytosis (minor pathway - not many drugs use this).
31
Give example of lipophilic vs hydrophilic drug and why it is important?
1. Lipophilic - Cocaine (weak base). Go across brain barrier. Could be neutral uncharged or acid form of weak acid or base form of weak base.
2. Hydrophilic - furosemide. Do not enter blood streams, tissues. Only used for kidney. Could be neutral charged or conjugate base of weak acid or conjugate acid of weak base.
32
Identify the mode of drug transportation
- depend on size. Need physical force (force of filtration) to push it through. Can’t be bound. Must be FREE.
- this rate of transport depends on what 2 factors?
FILTRATION THROUGH PORES
- Purely physical process in which the driving force for movement is a PRESSURE GRADIENT
- rate of filtration depends on PRESSURE GRADIENT and SIZE OF DRUG in relation to size of pore.
33
Identify the mode of drug transportation
- IONIC FORMS (ionized) Can transport against gradient.. anything that affects energy can affect transport e.g CO, cyanide (prevent ATP production) so transport cant happen cause no ATP
- they used to extract penicillin from urine. So they give additional drug that competes with elimination of penicillin so was more effective for patient.
* *rate of transport depends on?
* *What must drug posses to be transported?
* *Can there be competition in transport?
ACTIVE TRANSPORT
- Rate of transport depends on; BINDING CAPACITY of drug with its carrier and LIMITED AVAILABILITY of the carrier.
- must posses full negative or full positive charge to be actively transported.
- Yes if you transport 2 drugs that require active transport, they will compete.
34
Identify the mode of drug transportation
- non-ionic diffusion - (unionized forms) ; must be LIPOPHILIC. Move from high to low concentration by dissolving.
- rate affected by
PASSIVE DIFFUSION
- must possess ability to dissolve in aqueous phases bathing the membranes and also dissolve in lipid-like network of membrane itself.
- drug transport occur in direction of CONCENTRATION GRADIENT and in proportion to PHYSICAL FORCE provided by the gradient.
35
How can neutral drugs (uncharged and charged) go through a membrane ?
1. Neutral uncharged (aldehydes, lactams etc)
- may pass through pores (FILTRATION)
- no active transport
- PASSIVE DIFFUSION (if PC is favorable/high)
2. Neutral charged (quaternary ammonium salts)
- may pass through pores (FILTRATION)
- may do ACTIVE TRANSPORT (because they are permanent cations)
- little or no passive diffusion (very low PC)
36
How do acid and base forms of weak base and acid go through membrane?
**similarities? Differences?
1. Weak acid (HA; acid form)
- may pass through pores
- no active transport
- may do passive diffusion (if PC is favorable)
* *Similar to neutral uncharged drugs
2. Weak acid (A-; conjugate base form)
- may pass through pores
- may do ACTIVE TRANSPORT (due to an anionic character)
- little or no passive diffusion (becuase of low PC - it is polar/hydophilic)
* *similar to neutral charged drugs
3. Weak base (N: base form)
- May pass through pores
- no active transport
- may passively diffuse
* *similar to neutral uncharged and HA acid form of weak acid
4. Weak base (NH+ acid form)
- may pass through pores
- may be actively transported (cationic)
- little or no diffusion (low PC)
* *similar to neutral charged and A- conjugate form of weak acid.
37
Identify the following;
1. Drug go into blood stream. When in blood stream, it can then be stored, biontransformed, distributed. **End product of biotransformation is ?
2. out of blood delivered to all over the body
3. Drug is no longer pharmacologically active and can either be in the body or out of the body
4. Drug is out of body and not pharmacologically active
1. Absorption
- End product of biotransformation is metabolites
2. Distribution
3. Elimination
4. Excretion ; it is a form of elimination.
38
Identify 3 routes of drug administration and different modes
1. Enteral; place drug in some part of GI TRACT to be absorbed (passive diffusion).
* *Modes - oral, sublingual, rectal
2. Parenteral; place drug in some part of body OTHER THAN GI TRACT
* *Modes; subcutaneous, IM, IV, IA, inhalation, intrathecal
3. Topical; place drug onto surface of tissue (local or systemic effect)
* *Modes; patch, derm and ophthalmic, intravaginal
39
Nitro is mostly administered via what route and mode?
**What mode can’t it survive?
**How is it transported
ROUTE - Enteral (can also use topical - nitro patch)
Mode - Sublingual
can’t survive ORAL
**Under tongue - general circulation - liver (absorption is via passive diffusion).
40
1. What are the 2 main absorption site for oral drugs? 2. What is the main site and why (2)?
3. How absorped?
4. Rate of absorption depends on? (4)
1. Stomach and small intestine
2. Small intestine is main site;
- has longer transit time in SI
- SI has larger surface area
3. Absorped by passive diffusion (has to be in solution), there are few exceptions though.
- carried from GI tract to liver (by portal system)
4. ROA depend on what various factors?
A) dissolution rate - how quick it dissolves.
B) PC (partition coefficient) of drug - want it to be lipophilic to dissolve faster,
C) chemical nature of drug - acid, base, neutral
D) others; anticholinergic drugs delay gastric emptying, food can speed up (alcohol with no food) or delay (wine with food) absorption. You cant give some drugs orally
41
Identify mode of drug transportation
- absorbed by passive diffusion
- Go into bloodstream before liver.
- Can use for unconscious, vomiting, stubborn patients, kids.
RECTAL (e,g suppository)
**all enteral route (oral, sublingual, rectal) is absorbed by passive diffusion.
42
1. What is the elimination effect of oral administration CALLED?
2. How are drugs carried from liver to GI tract in oral administration?
1. FIRST PASS ELIMINATION EFFECT
- extensive hepatic clearance via biotransfromation (hepatic clearance of metabolites) before it go back to bloodstream?
2. PORTAL SYSTEM - go to liver first where biotransformation take place
43
Identify how the following is absorbed
1. Neutral uncharged particles ; major site of absorption? What if you have ulcer? E.g?
2. HA (weak acid); fast or slow? Why? Polarity? E.g?
3. N: (weak base); fast or slow? Why? E.g?
1. The major area of absorption for X (neutral uncharged) is the SMALL INTESTINE (oral). If you have ulcer in GI tract, you end up going into tissues and blood vessel to be absorbed which can affect things e.g person they gave a drug (curare?) that has a ulcer and ended up with hearing loss. ACETAMINOPHEN is neutral.
2. HA (weakly acidic) absorb right away from stomach because it is LIPOPHILIC (SI still major absorption site). Take some away from stomach to liver? E.g ASPIRIN?
3. N (weakly basic). Different from HA because it get protonated **Doesnt not do passive diffusion. Have to wait to get into small intestine first - bicarb secreted then cause pH to rise before absorbed?. E,g ANTIHISTAMINE
44
Identify the 6 parental administration of drugs
1.
- Passive diffusion through capilllary membranes - lipid soluble drugs
- small molecules enter through pores, larger water soluble molecules water blood via pores but slowly
- ROA depend on blood flow to injection site
2. Passive diffusion, depot preparation
3. No absorption - placed in blood. Good for emergency or hospital patients
4. No absorption. High Conc of drug into e.g liver (one body site). CANCER
5. Passive diffusion, go through pulmonary before blood
6. Injection into spinal arachnoid space. CNS CHEMOTHERAPy
1. Subcutaneous (S.c) e,g insulin.
2. Intramuscular (IM). Advantages - can use larger volumes and more irritating during. E,g penicillin (labile). DEPOT PREP (release drug over long periods of time) e.g contraceptives or if going to 3rd world countries that dont have the drug.
3. Intravenous (IV) - NO ABSORPTION. Advantage - rapid onset, known amount. Disadvntage - once it is in you are stuck, if you give wrong drug (OYO lo wa)
4. Intraarterial (IA) - NO ABSORPTION. You want a high conc of the drug is a specific area and no where else inn body cause it can be toxic e,g cancer drugs.
5. Inhalation; gas anesthetic and analgesics, illegal drugs (cocaine), must first go into pulmonary fluid before bloodstream (all passive diffusion)
6. Intrathecal; spinal anesthesia, cancer or antimicrobial drug, CNS chemotherapy. Get effect immediately.
45
IDENTIFY Form of the class of drugs and what determines rate of diffusion.
- cardiac glycoside (digoxin)
- skeletal muscle relaxants
- general anesthetic (e.g acetaminophen?)
- Chloramphenol
- many steroid
NEUTRAL UNCHARGED DRUGS
- dont carry charge at physiological pH so ROA/ROD depend soles on PC
- if PC <1; hydrophilic and will have low to no passive diffusion
- if PC >1; lipophilic and have high passive diffusion.
46
IDENTIFY Form of the class of drugs and what determines rate of diffusion.
- quaternary ammonium salts
- ganglionic blockers
- some autonomic (anticholinergic)
- anthemintics
NEUTRAL CHARGED
- permanent charge so very low PC
- little to no GI absorption
- anticholinergic delay gastric emptying
47
IDENTIFY Form of the class of drugs and what determines rate of diffusion.
- penicillins
- cephalosporins
- antibacterial sulfa
- nitrodurantoin
- barbiturates
- ANTICOAGULANT
- DIURETICS
- LAXATIVES
- salicylate
- oral hypoglycemia
- Glucoronic conjugates
- prostaglandins e.g ASPIRIN
WEAK ACID
HA more likely to diffuse (only if it has high PC)
48
IDENTIFY Form of the class of drugs and what determines rate of diffusion.
```
Amines
- Narcotic analgesics
- phenothiazines
- local anesthetic
- most autonomic
- antihistamines
Amniglucoside antibiotics
-all plant alkaloids
```
WEAK BASIC
- uncharged N more likely to diffuse (only if it has high PC)
49
Identify the 3 topical administration of drugs and if it is systemic/local effect
Give examples
1. Patches - getting more common. Whole pioint is SYSTEMIC NOT local absorption. Systemic effect is spread out not just site of application. E.g nitro, contraceptives, nicotine, clonidine etc
2. Dermatological and ophthalmic - LOCAL absorption (at site of application) .
3. Intravaginal; antimicrobials for a localized effect. Can get systemic effect if it is absorped in blood stream.
50
Identify 5 factors which influence drug distribution
1. Physicochemical properties (size, charge, solubility); lipophilic or hydrophilic.
2. Biological membranes encountered
3. Protein binding/storage
4. Blood perfusing a given tissue
5. Disease states ; CHF affect how blood perfuse tissues
**PBPBD
51
1. What is the FIRST BARRIER to drug distribution? (I.e last place before it is taken from blood to destination - interstitium - target cells/intracellular)
* *What are the other 2 barriers?
2. Anything less than what protein can go through pores?
3. How do drugs leave blood (2)?
4. Special capillary beds exist in what 2 sites? (Which one lacks a complete endothelial cell lining?)
1. Capillary Endothelial Membrane (between plasma and interstitium)
* *
- Cellular membrane is between interstitium and intracellular
- Epithelium barrier is before plasma
2. Albumin
3. Drugs leave blood same way they enter blood. 2 processes;
- Passive diffusion (concentration gradient)
- Movement through pores (filtration - pressure gradient)
4.
- Kidney; large pores in glomerulus
- Liver; lacks a complete endothelial cell lining.
52
Of the 3 barriers a drug must cross to get to target cell? (List all 3 first). Which Barriers can a lipophilic drug vs a hydrophilic drug go through.
Lipophilic
- epithelial barrier to plasma - capillary endothelium membrane to interstitium - cellular membrane to intracellular (have biological effect)
Hydrophilic
- epithelial barrier to plasma - capillary endothelium membrane to interstitium.
**Hydrophilic Do not go through cellular membrane so no biological effect
53
1. What is BBB and how do drugs cross it?
| 2. What is BCB and how do drugs cross it?
1. Blood brain barrier - tight junctions between brain capillary endothelial cells. (Astrocyte stealth)
* *Drugs cross BBB by PASSIVE DIFFUSION (must be lipophilic and small size?)
2. Blood-CSF barrier
* *Drugs enter CSF via PASSIVE DIFFUSION at ventricles or via ACTIVE TRANSPORT (choroid plexus). Cells that make up choroid plexus can take up cation and anion by active transport.
* *Movement mainly out of CSF and into blood
54
What is the fetal barrier called?
**What is the limitation of this barrier?
PLACENTA - sometimes a barrier to the fetus
- Placenta is a large capillary bed that allows for exchange of fluid and nutrients with mother.
- If mother smokes, CO can build up in blood and can spread to the fetus.
55
Identify the fluid
1. The pH is 7.4
2.
- The pH is 6.4
- ion trapping can occur in the gland (explain how)
- blood supply is low relative to other tissues
**Where else can ion trapping occur and why? (Hint - ion trapping is the build up of ionized base)
1. Plasma fluid
2. Prostatic fluid
- Ion trapping occur only in basic drugs because they enter by passive diffusion and become protonated/ionized/BH+ (acid is HA and has no charge)
- the ionic form of basic drug can’t diffuse out of prostate so it build up (remember that prostatic fluid has lower pH than plasma fluid so it is more acidic and with attract base) - the build up of ionized base is called ION TRAPPING.
**Ion trapping occur where there is acid so STOMACH is another place it can occur
56
1. What 2 places do protein binding occur?
2. Can protein binding affect phamacological activity? How?
3. How many binding sites do proteins normally have? What is the exception to this?
- which binding site of albumin is important to know and why
4. what are other binding sites of albumin
1. Protein binding occurs in PLASMA and in TISSUES
2. YES, as long as drug is bound to the protein, it will not be pharmacologically active
3. Proteins normally have 1 binding site. However ALBUMIN has 5 binding sites
- One of albumin binding sites is WARFARIN. Important to know because there is a thin line between active vs toxic level
- for example; if you have drug A 99% bound to albumin warfarin site with 1% unbound and active. Then you have drug B 97% bound to albumin warfarin site with 3% unbound and active. You have tripled the amount of warfarin left unbound/active which can cause patient to bleed to death (warfarin is anticoagulant)
4. Other binding sites of albumin; digitoxin, azapropazone warfarin, bilirubin, indole and benzodiazepine, fatty acid.
57
1. What types of bonds exist between protein and drug? (7)
| - which is strongest and may be irreversible
1. COVALENT - strongest and may be irreversible
2. ionic
3. Hydrogen bond
4. Ion-dipole
5. Dipole-dipole
6. Van deer Waals
7. Hydrophobic bonding
**Bonds between drugs and proteins are the same as between any two molecules
58
What is the process called when a substance, another drug, non-drug xenobiotic, endogenous substance competes with a drug for binding to a protein?
WHat is the consequence?
Give example
1. Protein binding interactions
2. The free concentration of the drug could be increased with or without significant consequences
**Important for drugs with low therapeutic ratios.
for example; if you have drug A 99% bound to albumin warfarin site with 1% unbound and active. Then you have drug B 97% bound to albumin warfarin site with 3% unbound and active. You have tripled the amount of warfarin left unbound/active which can cause patient to bleed to death (warfarin is anticoagulant)
59
Identify drugs and chemicals that can be stored in fat?
**What can accumulate slowly in fat?
**What other place can drugs be stored apart from in fat?
1. Lipid soluble Drugs (e.g thiopental)
2. Environmental chemicals (polychlorinated biphenyls, dioxins etc). They are lipophilic and stored in adipose tissue (pesticides)
**Poor Blood supply - accumulate slowly and efflux slowly.
PASSIVE DIFFUSION is the mechanism of uptake and efflux
Drugs can also be stored in BONE and KIDNEY
60
What drug can complex calcium or replace calcium in the bone?
What drug get stored in the kidney instead of going to target tissues?
What drug stay in fat instead of going to target site?
TETRACYCLINES - complex calcium in bone (bind to teeth and cause grey color of teeth)
LEAD- Replace calcium in bone (stay for long time)
AMINOGLYCOSIDES, CEPHALOSPORINS - stay in kidney
ANESTHETICS - stay in fat
61
***High yield
What action can terminate the phamacological action of many drugs especially lipophilic drugs.
*Depends on what factors?
REDISTRIBUTION
**Initial distribution depends on blood flow to a body region But Redistribution depend on other factors
62
What parts of body do blood; rapidly perfuse (4), less rapidly perfuse (2) and poorly perfuse (2). How does this affect drug concentration in these body parts?
Rapidly perfused
- Kidneys
- Heart (musculature)
- Brain
- Liver (hepatic artery flow)
* *Concentration of drug has rapid build up and then it falls
Less rapidly perfused
- Skin
- Muscle (resting)
* *Muscle has phospholipid so average buildup of anesthetic then drops off
Poorly perfused
- Fat tissue (adipose)
- Connective tissue
* *least build up of drug concentration and then falls.
63
A change in chemical structure caused by a living system is called? Aka? Product called?
**What body systems can this occur ? (6) One primary place
Biotransformation
Drug metabolism
Metabolite
- Liver (primary)
- kidney
- lung
- nervous tissue
- plasma or GI tract (epithelial cells, gastric pH, digestive enzymes, gut flora)
64
What are the results of biotransformation? (Basically what can you biotransform from what to what?) - (4)
**Whats the advantage and disadvantage of a metabolite?
1. Activate an inactive drug (prodrug)
2. Inactivate an active drug (MAIN function)
3. Convert active drug to active metabolite. E.g antidepressant discovered as an active metabolite of another drug
4. Convert active drug or metabolite to a toxic metabolite (disadvantage)
**Metabolites are normally more polar, more water soluble and excreted faster than a parent drug.
65
Biotransformation reactions are classified into 2 phases. Identify the reactions in each phase
**what speeds up this reactions?
**How do you induce enzyme activity? Give example of enzyme?
Phase 1: adding oxygen or hydrogen or water
• Oxidation;
• Reduction;
• Hydrolysis
Phase 2: adding other things than oxygen and hydrogen. Most catalyzed by NON-MICROSOMAL enzymes?
• conjugation or synthesis
Catalyzed by enzymes
• microsomal enzymes; substrates have to be lipophilic to get into cell and SER (where microsomal enzymes are located). You can induce the enzyme activity and synthesis by (drugs, chemicals, cig, alcohol)
◦ Cytochrome P450 enzymes is not a single enzyme - it is superfamily of enzymes. 20-30 active in humans. Very important
66
Microsomal enzymes help catalyze biotransformation reactions and are located in SER (smooth endoplasmic reticulum. Identify the following types of microsomal enzymes (2)?
**What induce the activity of microsomal enzymes that catalyze biotransformation?
1. Oxidative enzymes - MFO (mixed function oxidase)
2. Cytochrome P450 (CYP) enzymes (terminal oxidase)
**Drugs, environmental chemicals, cigarettes smoke and ethanol.
67
1. How are cytochrome P450 family members grouped?
Identify the following groups ? What are other inducers and drugs being induced (i.e what induces what drugs to be metabolized -inactivated)
2. induced by cig smoke (pyrene) - affects acetaminophen, warfarin, tricyclics antidepressant. You may have to give more warfarin to the patient. Other drugs like anticonvulsant that treat epilepsy can also induce these enzymes
3. responsible for the biotransformation of most drugs. If its a prodrug, it is good becuase it metabolize more than the active form?
4. responsible for only metabolizing 5% of drugs. One is acetaminophen. Ethanol can induce the activity and make it toxic - cause liver injury.
1. According to similarity
2. 1A2
Inducers; tobacco smoke and anticonvulsants (carbamazepine)
Drug metabolism induced: Haldol, acetaminophen, WARFARIN, tricyclics antidepressants
3. 3A4 and 3A5
Inducers; barbiturates, steroids, anticonvulsants
drug metabolism induced; most drugs
4. 2E1
Inducers; ethanol, isoniazid
drug metabolism induced; acetaminophen, ethanol (minor), halothane
68
Identify the biotransformation reactions catalyzed by microsomal enzymes?
Which reactions do microsomal enzyme NOT catalyze?
Phase 1
1. Oxidation reactions
- side-chain or alkyl hydroxylation
- aromatic ring hydroxylation
- N-,O- and S- demethylation
- oxidative deamination
- sulfoxide formation
- N-oxidation or hydroxylation
- dehalogenation
2. Reduction reactions (not common)
- nitro group
- Azo group
3. Hydrolysis reactions
NONE
4. Conjugation reactions (conjugation makes the metabolites more polar)
- glucuronide formation
69
1. Where in cell are nonmicrosomal enzymes found? (3)
| 2. can they be induced?
1. Located in;
- Cytosol
- Mitochondria in cells
- Blood (esterases); important for breaking down drugs.
2. Activity is generally NOT INDUCIBLE
70
Identify the phenomenon
- a lot of changes that occur in DNA that cause more or less activity than normal. Either become rapid metabolites or slow/no metabolites
Genetic polymorphism
71
Identify the non-microsomal enzymes and reaction type it catalyze
1. Asians and Europeans have reduced amount - if they drink alcohol - enzyme dont convert the acid aldehyde so it build up- get nauseous and sick.
* What countered this enzyme to help alcoholics stop drinking?
2. hydrolyze esters and amides to carboxylic acid
3. Group of reactions
• -; body has low level to carry it out for long period
• -; fast and slow acetylators.
• - is a 3 amino acid peptide - protect us from reactive chemical species (neutralizes them)
1. aldehyde dehydrogenase - Reduction reaction and oxidative reaction
* Antabuse drug worked to counter the aldehyde dehydrogenase ( to help you stop drinking).
2. Esterases and amidases - hydrolysis reactions
3. Conjugation reactions
- Sulfation
- acetylation
- glutathione
72
What factors affect the rate of biotransformation? (7)
1. Enzyme activity inducers (drugs, smoking)
2. Enzyme activity inhibitors (competing drugs); competition that inhibit metabolism (TABLE) e.g grapefruit and SSRI inhibit warfarin metabolism (bleed out form increase warfarin)
3. Age; the very young and very old are susceptible
4. nutrition; for co-factors
5. genetic polymorphism
6. gender; enzymes in control of testosterone vs estrogen
7. Liver function/disease
73
What are the 2 major routes of excretion
**Other routes?
1. Biliary excretion (in bile)
2. Renal excretion (in urine)
**Others; sweat, saliva, breast milk and exhalation
74
There are 2 types of excretion
1. Explain biliary excretion
- 2 ways blood enter lobule
- where to from lobule?
- what 2 fates can the drug have?
- what 2 fates if drug is in bile?
- Advantage of drug in bile that has >PC? Example of drug? (Phenomenon called enterohepatic cycling)
* * Lobule - sinusoids - central vein - general circulation (LSCC)
* *Lobule - sinusoids - hepatocytes - bile cannaliculus - bile ductule - small intestine (LSHBBS)
- BILE; blood comes into lobule from 2 ways - PORTAL VEIN (oral drug to liver), HEPATIC ARTERY (general circulation to liver) - all end up going to sinusoids (come in contact with hepatocytes) - either go to central vein to general circulation (re-distributed) or transport to hepatocytes and products go to bile cannaliculus to bile ductule to small intestine (called biliary excretion). Just because a drug does biliary excretion does not mean it will go to feces..
• Once in bile several things can happen - if it has >PC - go back up to liver - general circulation or bile and process go on and on. This PROLONGS the TIME that a drug can be in the body (enterohepatic cycling).
• Some drugs like morphine make conjugate - dump to intestine. MORPHINE has longer half life and activity becuase it has a favorable PC so easily re-absorbed.
75
There are 2 types of excretion
Explain renal excretion
- Must drug be free or bound? Why?
- which part of kidney transport conjugate base of acid and conjugate acid of base? Secreted where? Passed out where?
- urine is acidified in what part of kidney? What chemical group is absorbed more and why? Why acidify urine?
* has to be free so it can be filtered at the glomerulus
* There are transport systems at the proximal TB that transport conjugate forms of acid and base and be secreted to luminal fluid and passed out in urine?
* Urine acidified in distal tubule. If you have weakly acidic drug - can have more absorption in DT (unionized form). Weakly basic drug not reabsorped because it is in ionized form. ACIDIFY URINE TO PRMOTE EXCRETION OF THE DRUG.
76
1. What is the study of body’s defense against infection?
2. What is an intricate system of molecular and cellular components that have evolved to defend the host against pathogens (virus, bacteria, fungi and parasites)?
1. IMMUNOLOGY
- how body defend itself
- how body develop long term immunity
- from word immunitas which means exemption from service or duty.
2. Immune system
77
Identify phenomenon
1. Components of the immune system that can over-react
2. Components of the immune system that can sometimes fail to react
3. Components of the immune system that can turn on the host
3b. Abnormal growth of cells, recognized as self by the immune system
4. Immune system is a complex interplay of what 2 cells
* *How are they all connected? (2 systems)
5. How do the cells communication?
6. What organs are immune cells produced and where do they function respectively?
1. Hypersensitivity; overactive immune system. Sometimes a simple ordinary response gets out of hand. ALLERGIES are the result of hypersensitivity
2. Immuno-deficiency; ineffective immune response. Some are hereditary and manifest sometime after birth with others can be acquired later in life (AIDS)
3. Autoimmunity - inappropriate reactions to self antigens
3b. CANCER - suppress the immune system too
4. Interplay of immune cells and connective tissue cells
- interactions occur throughout the body in various tissues and organs - all connected by a vascular network (LYMPHATICS AND VASCULAR SYSTEM)
5. Communicate through secretion of protein hormones (cytokines and chemokines) - work over short distances (micro environment of disease)
6. Immune cells produced in PRIMARY LYMPHOID ORGANS (bone marrow, thymus) and perform effector functions in SECONDARY LYMPHOID ORGANS - tissues (spleen, lymph nodes).
**Lymphatic system is the communication as fluid leave circulatory system and go back to vascular system.
78
How are immune cells capable of causing a deleterious response to invading pathogens, tumor cells, transplanted tissue or normal cells (3)?
1. Direct cell-cell contact (kiss of death)
2. Release of soluble factors/mediators (cytokines)
3. Recruitment of other cells to site of injury (cytokines and chemokines)
79
Explain the drive to survive phenomenon
• There is an intricate system that allows me to be me - called HLA system of proteins. One of the ways we pick our mate in life - influences smell so you smelll someone different from you cause we want heterogeneity - different offspring (DRIVE TO SURVIVE). Organisms change because they want to survive
80
Identify if it is self or non-self
1. Me
2. Neighbor
3. Pathogen
4. Transplant
5. Tumor
**What recognized foreign (non-self0 if in your body?
1. Self
2. Non-self
3. Non-self (virus, bacteria, fungi is not you - it is foreign)
4. Non-self (transplant form someone is foreign)
5. Has component of self but also non-self
*HLA proteins - Histo compatibility complex (influence how people smell - drive to survive)
81
Size of pathogen predicts what you do to defend yourself.
1. Identify pathogens form biggest to smallest?
2. How big is an immune cell and what helps you recognize if the cell is infected or normal?
1. Worms (3-5m) > Protozoa (10-100um) > yeast (4-12 um) > extracellular bacteria (1-5um) > intracellular virus (<100nm)
2. Immune cell the size of yeast
- erythrocytes (7um)
- leukocyte (7-18 um))
3. HLA proteins helps you recognize what size are normal and which are infected. (Kiss of death to infected cells)
82
Identify which is intracellular vs extracellular pathogens?
1.
- many BACTERIA, fungi and most large parasite. These are sometimes larger than host cells and must be dealt with appropriately
2.
- VIRUSES and some bacteria typically
- integrate innate and acquired mechanism to deal with
1. Extracellular pathogen
- mostly bacteria though some reside in phagocytes
2. Intracellular pathogen
83
1. Any substance than can be recognized by the immune system that stimulates an immune response is called ?
2. (The word originated from what notion)?
3. Give examples
4. (what can be complexed with this examples to counter the effect)
1. ANTIGEN
2. notion that they can stimulate antibody generation
3. examples; PROTEINS OR polysaccharides
4. lipid and nucleic acids when complexed with proteins or polysaccharides can be antigenic.
84
1. What is the portion of antigen molecule called - that interacts with the immune effector molecule or receptor such as antibodies, BCR or TCR respectively?
2. The portion on the immune effector molecule or receptor is called?
3. The interaction between 1 and 2 define/determine what?
4. What forms/shape can antigens take? (2)
1. Epitope
2. Paratope
3. Antigen specificity (epitopes of antigen bind to paratopes on antibody BCR or TCR to define antigen specificity)
4.
- Linear (variety of epitopes on surface)
- 3-D (folded up)
85
Identify the 2 characteristics of antigens
1. The capacity to stimulate the development of innate or acquired immunity
2. The ability to bind to immune effector molecules
* *Give 3 types of immune effector molecules
3. What is the main aim of the immune system
4. Immune system declines with?
1. Immunogenicity
2. Antigenicity
* * (antibodies, BCR or TCR)
3. HOMEOSTASIS
- If you get out of balance - problem. If you dont clear infection - lead to chronic inflammation - lead to Gastric, prostate cancer, celiac disease. Becuase you are beyond the homeostatic environment.
4. Age has a role to play on immune system
• I am at my prime of immunity - T cells, B cells all developed
• 40s,50s - downhill. Immune system declines with age. Cancer increases with age. Occurs mostly in young kids and greatly in old people.
• Why do i get the flu a lot and my grand father doesn’t? I am just being introduced to it while my grandpa has lots of memories to protect against the flu.
86
Immune response - 4 effector mechanisms
Identify
1. Innate immunity
- bind antigens with B CELL secreted antibodies. Prevents the antigens from binding to the target cell where they could cause damage
2. Innate immunity
- facilitates inflammation and can cause direct lysis of a pathogen
3. Innate immunity
- cells eating cells. Bind to foreign cell, particulate matter or Ag/Ab complex; ingest it, kill it and digest it.
4. Adaptive immunity
- death signal or “kiss of death” is delivered to a cell harboring foreign antigens which initiate a self programmed death event called APOPTOSIS. This is followed by digestion of the killed cell by enzymes
How Immune system works;
1. Neutralization; try to neutralize what is foreign (bind antibody to toxin and neutralize them before they bind to target cell).
2. Complement cascade activation; one of first things that becomes activated. If boo left a board with a nail and you step on it, you activate this system to destroy any bacteria you got form the nail. This complement system is shared with lower vertebrates
* *complement can also coat the toxin with opsinin to make it more edible - called opsinization.
3. Phagocytosis; cells eat other cells. E.g Neutrophils, macrophage? (Lysosomes)
4. Cytotoxic reaction; e.g T cells bind to target cell and induce death “kiss of death”.
* First 3 - innate immunity
* 4 works more specifically in acquired immune response.
• Immunology is not black and while (its gray). Different functions for different things so that they all work together.
87
Cytotoxic reaction is example of adaptive immunity and they make use of T cells. How do these worK?
Types of T cells
1. Direct cell contact (kiss of death)
2. Direct contact with virus infected cell and kill it
3. Release cytokines, bind to macrophage and activate th macrophage that release cytokines
4. Release cytokines and bind to B cell - becomes plasma cell and release antibody
1. cytolytic T cell
2. CD8 T cell
3. CD4 T cell (specific helpers Th0-Th1 -cell mediated immunity)
4. CD4 T cell (Th0 - Th2 - humoral inmmunity)
***Immune system communicate through CYTOKINES - they are released with synapses of cell-cell contact
88
Identify 2 types of immunity
Innate versus Adaptive
1. Response time
2. Number of specificity
3. Constant or changing
4. Types
1. Innate immunity (2 types - immediate and induced response)
- primitive, immediate, fast (minutes to 4hours)
- limited number of specificity
- Fixed mechanisms
- Constant during response
- e.g neutralization (antibodies), complement cascade, phagocytosis
2. Adaptive immunity
- slow response (days to week)
- Numerous highly selective specificities
- Variable mechanism
- improve during response
- e.g cytotoxic reactions (T cells)
**Both work in harmony in the battle against pathogens
89
1. In acquired immunity, how many cells and peptides are recognized?
2. Can memory occur?
**How can antibodies improve specificity?
1. 1 cell, 1 peptide recognized
- very specific protective mechanism
- there is a vast universe of distinct antigenic epitopes and a vast capacity for the recognition of these antigens through acquired immunity
2. Yes, acquired immunity results in immunologic memory (B cells and T cells ) (vaccines utilize acquired immunity)
* *There is tolerance of self antigens in acquired immunity. Adaptation during life of the host can also ocxur.
* *Antibodies can improve their specificity through MUTATION
90
Identify the hormone
- made by immune effector cells
- can effect cells of origin (autocrine) or neighboring cells (paracrine)
- ACT OVER SHORT DISTANCES
- often release into cell-cell synapse and HAVE A SHORT HALF LIVE
**What are the actions of the hormone? (5)
CYTOKINES
***Immune system communicate through CYTOKINES - they are released with synapses of cell-cell contact
* *Cytokines include;
- proliferation
- differentiation (B2 cell to plasma cell)
- activation (of macrophages)
- directed movement
- death (of infected virus)
91
1. How do pathogens get into the body
2. What are the routes of possible entry (4)
3. What happens when pathogens gain entry into the body?
- acute?
1.
- Any pathogen has to cross skin/dry surface or epithelial/mucosal/wet surface
- Pathogens only get in if the surfaces are; cut, irritated or damaged.
- Dry skin, nasal cavity, GI tract, genital tract all lined by an epithelium and serve as a barrier to protect you form pathogens.
- MUCOSAL MEMBRANES are moist epithelium.
2. Skin, GI, UG tract, nasopharyngeal tract (NG)
3. INFLAMMATION - immediate acute reaction
- trigger innate mechanisms of immunity (minutes to 4 days)
- acquired mechanism develop if possible (4-7 days)
**Once insult is removed, tissue damage is repaired via process called WOUND HEALING
92
Identify 2 phases of the Immune response
1.
- simply see the molecules or cells as foreign (nonself)
- use of cell surface receptors which bind ligands in other cells or molecules
- binding occurs - SIGNALING EVENTS (from cell surface to nucleus) - cellular change (activation, cytokines release, mobilization or proliferation).
2.
- immune cell does something about what is seen
- in most cases, the foreign cell is eliminated
- involve neutralization, complement cascade, phagocytosis or cytotoxicity (innate and adaptive immunity)
1. Recognition phase
* *Bind epitope (on antigen) To paratopes (on antibody or immune effector molecule)
2. Effector phase
93
During an immune response, there is a complex interplay between stromal/connective tissues cells and cells of the immune system
1. How do these 2 cells communicate (2)
2. How can these cells be elicited or drawn from one anatomic site to another?
1. 2 ways
- direct cell contact
- elaboration of soluble mediators (cytokines)
2. Through chemical messengers called CHEMOKINES
94
1. What organisms are predominantly intracellular vs extracellular? What immunity does this usually translate to?
2. Identify immunity type
A. B cells, antibody production
B. T cells, helper and killer cells
3. What is the goal of immune system ? (2 main functions under this goal)
- HLA proteins
- innate vs adaptive immunity
1.
Extracellualr - bacteria - innate immunity
Intracellular - Virus - Adaptive immunity
**Innate and adaptive work together
2.
A. Humoral Immunity (B1-innate/make natural antibody while B2 is adaptive)
B. Cellular Immunity (CMI)
3. Goal - maintain HOMEOSTASIS
A. Recognize self vs Non-self (by HLA proteins?)
B. Effector functions; neutralization (antibodies), phagocytosis (phagocytes), complement cascade, cytotoxicity (killer cells)
95
Identify
```
A. A tissue (red fluid) comprised of both solid and liquid components
B. identify components
C. volume
D. % body weight
E. functions
F. site of? **
G. Transported by?
```
A. BLOOD
B.
*Solid - cells (RBCs, platelets, leukocytes)
*Liquid - plasma (contain various proteins - albumin, globulins, fibrinogen)
C. 5 liters
D. 7% of body weight
E. Function to transport O2, CO2, nutrients and cells
F. SITE of IMMUNE RESPONSE to BLOOD PATHOGENS
G. Transported by vessels of the circulatory system
96
What is the difference between plasma and serum?
Contents?
1. Serum; the remaining fluid left when plasma is clotted
2. Plasma; transparent yellow fluid containing 3 principle plasma proteins
- Albumin; most abundant. Produced by liver and maintains colloid osmotic pressure within the capillaries
- Globulins; divided into gamma globulins (antibodies **gammadelta yd T cells make innate immunity) and beta globulins (transport)
- fibrinogen; synthesized by liver for blood clotting
97
Which cell types (of plasma) have;
A. Amoeboid type movement
B. Transported by current
A. Leukocytes
B. RBCs and platelets
98
Development of immune cells
1. Hematopoietic stem cell give rise to? (3)
2. How do they further differentiate
1. Stem cell give rise to COMMON LYMPHOID PROGENITOR and COMMON MYELOID PROGENITOR and common erythroid megakaryocyte progenitor
2
A.
- Common LYMPHOID progenitor - B cell - plasma cell (adaptive immunity)
- Common LYMPHOID progenitor - T cell - effector T cell (adaptive)
- common LYMPHOID progenitor - NK/T cell precursor - NK cell (adaptive)
**Products of lymphoid - plasma cell, effector T cell, NK cell
B.
- common MYELOID progenitor - common granulocyte precursor - neutrophils, eosinophil, basophil (innate)
- common myeloid progenitor - unknown precursor - monocytes - dendritic cell and macrophage (innate)
- common myeloid progenitor - unknown precursor - mast cell (innate)
**Products of myeloid - neutrophil, eosinophil, basophil, dendritic cell, macrophage, mast cell
C. Common erythroid - platelets and erythrocytes
99
Innate vs Adaptive immunity
1. What cell types are typically innate
2. Which are typically adaptive
3. Which type become memory cells
4. Which innate cells can be adaptive
5. Which adaptive cells can be innate
6. Which cells turn off immune response that is critical in cancer
**LA and MI (los angeles and michigan) LA - lymphoid - adaptive. MI - myeloid - innate.
1. Innate - Myeloid precursors - neutrophils, eosinophils, basophils, macrophages, dendritic cells, mast cell
2. Adaptive - Lymphoid precursors - B cell, plasma cell, T cell, effector T cells, NK cells
3. Adaptive have memory cells
4. Dendritic cells can make adaptive immunity
5. NK cells (LAK) and gamma delta T cell can make innate immunity
6. Tregs - regulatory T cells (with the FoxP3 at end)
100
What 2 things function in blood clotting
)
1 cell (actual
1 plasma protein
1. Platelets (get activated at the site of a tear in the vasculature and seal up the wound)
2. Fibrinogen
101
Identify cell type
True cells with a nucleus (Eukaryotic), contain all
appropriate organelles.
• Are proliferative and responsive to cytokine and
chemokine signaling.
• Spherical in shape in the vasculature, amoeboid in
shape in tissue
• Range in size from 7-18um. Small lymphocyte is
7um, same size as an RBC.
• Classification can be confusing. Can be done by
morphology, origin, and/or function. Focus on origin
and function.
Leukocytes - WBCs
**they undergo proliferation once in target cell
102
Differentiate polymorphonuclear vs mononuclear and give examples
1. Polymorphonuclear
- single nucleus with a lot of twists and turns resulting in different morphologies
- e.g granulocytes (neutrophils, basophils, eosinophils)
2. Mononuclear
- refers to lymphoid cells - lymphocytes and monocytes/macrophages
- nucleus appears more classical
- rounded to horse-shoe shape
103
In terms of origin, leukocytes (everything - neutrophils, lymphocytes, eosinophils, monocytes, basophils - NLEMB) - come from a pluripotent stem cell located where?
**what 2 lineage does the stem cell give rise to and what classes are under the lineage? What immunity type?
1. Hematopoietic Stem cell located in BONE MARROW
2. Lymphoid lineage - lymphocytes (B cell, T cell, NK cells) - acquired immunity
Myeloid lineage - monocytes (macrophages and dendritic cell) and granulocytes (basophil, eosinophil, neutrophil) - innate immunity
104
1. The granules in granulocytes contains what 2 things?
2. Used for?
3. Nuclei shape?
4. Immunity type?
1. Enzymes and other soluble factors
2. Used for cell function
3. Appear to have multiple nuclei but in fact the nuclei are lobular in appearance (nonspherical)
4. Major component of INNATE IMMUNITY
105
Identify; cell type, lineage, percent of peripheral blood, granules color
- function as effectors in the initial response to BACTERIAL INFECTION
- infiltrate areas of infection and ingest bacteria by PHAGOCYTOSIS
- Short life span of 2-3 days
- Dead neuts comprise “PUS”
NEUTROPHILS
- Myeloid linage
- 40-60% of peripheral blood
- neutral color granules or colorless (H&E stain)
106
Identify; cell type, lineage, percent of peripheral blood, granules color
- contain MBP (major basic protein - cause pores and kill parasite), peroxiadase and certain cytokines
- Numbers rise drastically during PARASITIC INFECTIONS, ALLERGIES AND HYPERSENSITIVITY
- are capable of phagocytosis but not very good.
**How do they kill parasite?
EOSINOPHILS
- Myeloid linage (granulocyte)
- 1-4% of peripheral blood
- granules stain red
**Kill parasite and possibly harm normal tissues by releasing granular contents on the surface
107
Identify; cell type, lineage, percent of peripheral blood, granules color
- granules help MEDIATE INFLAMMATORY RESPONSE
- Lie close to vasculature
**what do granules contain?
**What has similar properties?
BASOPHILS (pollen/pet allergies)
- MYELOID (granulocyte)
- <1% of peripheral blood
- stain blue
**
Granules contain; heparin, serotonin and histamine
**MAST CELLS; found in tissues and have similar properties, may or may not Share origin
108
Identify; cell type, lineage, percent of peripheral blood, granules color
- DIFFERENTIATE INTO 2 TYPES of cells based on exposure to cytokines; professional phagocytes (macrophages) and APCs
MONONUCLEAR PHAGOCYTES
- Myeloid lineage
1. Professional phagocytes aka MACROPHAGE aka big eater; remove particulate matter (debris made by neutrophil). Help promote wound repair and innate immunity
2. APCs (antigen presenting cells); take up, process and present antigens to lymphocytes (B, T NK cells)
* *APCs are commonly called DENDRITIC CELLS or DCs (acquired immunity)
109
There are 3 monocytes cell types of the mononuclear phagocyte system
Identify
Predominantly found in blood
• 2-8% of total WBC in blood
• Kidney-shaped nucleus, extensive cytoplasm
• Weakly phagocytic; weakly microbicidal
• Differentiates to macrophage or dendritic cell
upon leaving blood (1⁰ function)
MONOCYTES
** precursor cell to macrophage or dendritic cell
110
There are 3 monocytes cell types of the mononuclear phagocyte system
```
Identify
• Predominantly found in tissue sites
• Rare found in blood
• Strongly phagocytic (1⁰ function)
• Strongly microbicidal (1⁰ function during
infection)
• Promote tissue repair (1⁰ function following
tissue injury)
```
MACROPHAGE (professional phagocyte)
- phagocytosis and kill microorganisms
- activate T cells and initiate immune system
111
There are 3 monocytes cell types of the mononuclear phagocyte system
```
Identify
• Predominantly found in tissue sites
• Rare found in blood
• Strongly phagocytic (1⁰ function)
• Activates T helper cells (1⁰ function during
infection
```
DENDRITIC CELL
- activation of T cells and initiation of adaptive immune responses
112
What serve as the bridge between innate and acquired immunity?
HOW?
MONOCYTES
1. Viral infection or tumor; monocytes - APCs, DCs - lymphocytes (B,T,NK cells) - acquired immunity
2. Wound; monocytes - macrophages - phagocytosis and wound repair (innate immunity)
113
Identify cell type
- very sticky and spread out when trying to eat something
- they have different names depending on where they are
**What are 6 different names and the locations? PLLBKL
MACROPHAGES
1. PERITONEUM; resident cells
2. Liver; kupffer cells
3. Lung; alveolar cells
4. Brain ; microglial
5. Kidney; intraglomerular mesangium
6. Lymph node or spleen ; sinusoidal
114
Identify cell type
* Come from monocytes
* Turn on adaptive immunity
* Engulf debri, process it to peptides, present peptides to T cells to destroy tumor or bacteria - cytokines activate T cells - activate clonal proliferation - get rid of organisms.
??They do not proliferate in the tissue, they proliferate in bone marrow. Stop proliferating when they get out ??
Antigen presenting cells (Dendritic cells)
- A heterogeneous population of cells which have
immunostimulatory capacity. DCs take up
antigen from the environment, process it to
peptides, and present the peptide to T cells.
- Following the interaction, in the presence of
appropriate cytokines, T cells become activated,
clonally proliferate, and perform effector
function.
**Like macrophages, DCs can take different
names depending upon where they are located.
* *DC has a long extension to increase surface area to present antigens to lymphocytes
* *Need to look at surface to differentiate DC (CD4, CD8 etc)
115
Identify cell types and derivatives
1. Important in EARLY recognition and destruction of VIRUS infected cells and tumors
2. Make antibodies - HUMORAL IMMUNITY
3. Both helper and cytotoxic are responsible for eradicating virus infected cells - CELL MEDIATED IMMUNITY (CMI)
**NK, T cells, B cells are all derived from the same LYMPHOID progenitor
1. NK cells (Natural Killer)
2. B cells lymphocytes
3. T lymphocytes. Derived from thymus ** kill viral infected cells
**Lymphocytes are used in early days of a viral infection. They discriminate viral infection from others
116
Identify cell type - (and lineage)
- first cells used in immunotherapy for cancer
- first line of defense against virus and tumors
2. What do they release to mediate immune reactions?
3. How do they kill cells? (2)
NK cells (lymphoid lineage)
- CD3-CD16+CD56+ cells
• First line of defense against virus and tumors.
• These cells RELEASE CYTOKINES (GAMMA INTERFERON)
which mediate immune reactions.
• NK cells can kill cells by DIRECT CYTOTOXICITY using mechanisms similar to CTL-T cells (kiss of death?). Can also bind and kill antibody coated target cells (opsonization).
• NK cells + lymphokine IL-2 results in a killer cell with
a broader range of reactivity. Lymphokine activated
killer cell (LAK) - Innate immunity. First cell used in Cellular
Immunotherapy trials in cancer.
**LAK cells first used for cancer but wasn’t DURABLE so went on to T cells
117
1. After a B cell immune response, some B cells that become plasma cells remain as??
2. The process of B cells making antibody is called?
3. Classes of B cells (2)
- which is innate response
- which is adaptive
1. MEMORY CELLS (also T cells - killer and helper cells)
2. HUMORAL IMMUNITY; B cells have antibody they secrete as a cell surface receptor on their surface.
3. B1 and B2
- B1 - innate (can turn into cancer)
- B2 - adaptive - become PLASMA CELLS and then memory cells when you clear pathogen (make antibodies?)
118
Identify the T cell types
1. Stabilize T cells
2. Helper T cell
3. Cytotoxic T cell
4. Most numerous T cell
5. T cell associated with MUCOSAL SURFACES** and may be involved in gut immunity
**What is T cell proportion in normal person vs HIV
1. CD3
2. CD4+
3. CD8+
4. Alpha beta (Ab) - adaptive immunity (CD3 found here)
5. Gamma delta (yd) - can do innate immunity
* * You normally have more 4 than 8. If you have more 8 than 4 - HIV infection.
- T lymphocytes or T cells are divided into helper
(CD3+CD4+CD8- αβ+ T cells) and cytotoxic
(CD3+CD8+CD4-αβ+ T cells). Helper cells are further
differentiated into T-helper subsets depending upon
the cytokines they release.
- αβ+ (alpha beta)T cells are the most numerous of the
T cells. αβ designates the T cell receptor. 90-95% of
T cells are of this kind.
- 5-10% of T cells are more primitive and express the
γδ (gamma delta)form of the T cell receptor. These
cells are associated with MUCOSAL SURFACES and may
be involved in gut immunity.
119
Identify
1. carry oxygenated blood
from lungs to heart - distributes to
tissues and organs of the body. Remove carbon
dioxide and take it back to the lungs for
exchange. ***Transports nutrients, hormones,
cytokines and effector cells.
2. Vessels pick up fluid at the
venule end of capillary network and return it to
the heart via a system of small vessels called
lymphatics. Fluid is returned to the heart via
the thoracic duct.
3. Filter circulatory system
4. Filter lymphatic
1. Circulatory system
2. Lymphatic system
- Lymphatics collect fluids (lymphatic fluid) and transport stiff back to the heart through thoracic duct. This is how cells circulate throughout the body
- Filter vascular system
3. Spleen - filter circulatory system? In lower organisms, spleen store and produce RBCs and give more oxygen. My spleen is not as muscular as a horse that’s why my back hurts when i run
4. Lymph nodes - filter lymphatic system?
120
Immune system is divided into diffuse vs encapsulated
Identify each with examples
- is MALT encapsulated?
- is thymus/bone marrow encapsulated?
- is spleen/lymph node encapsulated?
- is bone marrow primary or second lymph organ?
- what’s the difference between primary or secondary lymph organ?
1. Diffuse or non-encapsulated
◦ Tissue associated with mucosal epithelial
surfaces (wet surface), also called mucosal associated lymphoid tissue or MALT. Can change names based on where it is.
```
2. Encapsulated
◦ Primary lymphoid organs- site of immune cell
PRODUCTION
- Bone marrow make B cells
- thymus make T cells
```
◦ Secondary lymphoid organs -site of immune cell
FUNCTION.
- spleen filter circulatory system
- lymph nodes filter lymphatic system
-bone marrow; can slow have immune response (BOTH PRIMARY AND SECONDARY lymphoid organ)
121
Identify the following process of production in primary lymphoid encapsulated organs
1. granulocyte development (neutrophils,
eosinophils, basophils) and monocytes originate in
the BONE MARROW.
2. lymphocyte development begins in
the BONE MARROW for both B and T cells. Further
differentiation and tolerance induction for:
1. Myelopoiesis
2. Lymphopoiesis
- BOTH B cell and T cell originate from bone marrow and thymus resp. During development in the FETAL LIVER.
122
1. What cell is educated n the bone marrow to recognized nonself
2. What cell is educated in thymus for antigen specificity
3. What do both cells undergo after exposure to antigen
1. B CELL
2. T CELL
**
• Educated to recognize non-self
• Those that recognize self are killed so they don’t escape. People get diabetics etc because some cells escape
3. Both cell types undergo antigen specific clonal
proliferation following exposure to antigen.
123
What distinct tissue sites do the following cell interactions occur?
1. Cell development (2)
2. ACTIVATION by antigens and microorganisms (2)
3. DESTRUCTION of antigens and microorganisms
1. Thymus and bone marrow
2. Lymph nodes and spleen
3. Site of infection
**All cells moving around that’s why you need lymphatic system
124
Largest organ in the body, lined by a dry epithelium (stratified squamous keratinized epithelium)
• Barrier to infection, first line of defense.
• Designed to prevent the entrance of environmentally derived pathogens into the body. Tough durable and self renewing.
• Epithelial cells, which make up the skin, are joined together by tight junctions, prevent entrance of materials into the body.
• Commensal bacteria are located on the surface of the skin. Adults possess 10 TIMES more microbial cells than human cells.
SKIN
125
- Lines all surfaces of the body, skin (dry) and all
tracts (moist) including respiratory, gastrointestinal (GI), urogenital (UG), nasopharyngeal (NG).
* Differs in shape: flattened, cuboidal or columnar.
* Infections occur only when organisms can cross the epithelium and colonize, release toxins or compete for nutrients and destroy normal tissue.
Epithelium
126
Identify 4 types of MALT (wet mucosal surface)
**They are lymph node-like regions in subepithelia of all mucosal tissue sites
**They can be in form of what 2 structures?
1. GALT; gut-associated lymphoid tissue
2. BALT; bronchial/tracheal associated lymphoid tissue
3. NALT; nasal-associated lymphoid tissue
4. VALT; vulvovagina associated lymphoid tissue
** 2 structures
- DIFFUSE structures of intestinal lamina propria
- ORGANIZED structures; tonsils, peyer’s patch, appendix
• Sometimes the mucosal tissue can organize itself to a structure called a follicle (if there are frequent infections?) e.g peyer’s patches - germinal center?
127
Mechanisms associated with mucosal immunity include what 2 things
1 antibody type
1 T cell type
1. Gamma delta T cells (line mucosal tissue of gut)
2. IgA antibodies - is a specific antibody (found in SEROMUCOUS SECRETIONS such as saliva, colostrum, trachobroncheal and urogenital secretions)
**Both not found in other locations
(Remember **yad is found in mucosal surfaces - yd T cells and IgA antibody)
128
Thymocytes or precursor T cells (that recognize nonself) pass from cortex of thymus to medulla and undergo differentiation
**These lymphocytes that possess reactivity against self (recognize self) are eliminated in process called?
** 2 types of tolerance inductors?
CENTRAL TOLERANCE INDUCTION
- T cells that recognize self are killed while those that recognize non-self are proliferated (leave site of differentiated)
* 2 types of tolerance inductors; 1 in bone marrow and 1 in periphery
129
What is an important reservoir for neutrophil?
• Start pumping out neutrophil within hours to get to site of infection (very active organ)
• Important reservoir for NEUTROPHIL (short-lived cells 6-8 hours that is released immediately you get infection)
- both primary and secondary lymphoid organ
BONE MARROW
Site of B cell development
• A primary lymphoid organ
• Site of granulopoiesis
• Site of immune reactions, thus, considered a
secondary lymphoid organ as well as primary
lymphoid organ.
• Found in all long bones
130
2 parts of spleen
1. Sponge that blood filters through (it is filled with macrophages)
2. Germinal centers are here (where immune responses occur)
3. Which has;
- rich in T cells (surround central arterioles)
- rich in B cell
- naive B cells (not encountered antigen)
- B cells proliferating in response to foreign antigen
1. Red pulp
- rich in macrophages which filter blood of foreign material and aged RBCs
2. White pulp ; multiple immune cell types necessary to initiate adaptive immune response
3. WHITE PULP
- periarteriolar lymphoid sheath (PALS) - area surrounding central arterioles - rich in T cells
• lymphoid follicle - outpocketing of lymphocytes from PALS - rich in B cells
• primary lymphoid follicle - naïve B cells (B cells that have never encountered antigen)
• secondary lymphoid follicle - outer mantle of naïve B cells with a germinal center containing activated B cells (B cells proliferating in response to foreign antigen)
131
1. Identify structure
- secondary encapsulated lymphoid organ
- gets large during infections de to increased immune responses
- occur in chains or groups
2. Function?
3. 2 GROUPS of lymph nodes
1. Lymph nodes
2. strategically placed to FILTER LYMPH derived from venous end of capillary beds
3. Groups of lymph nodes found in groups in distinct anatomic locations
- visceral or deep; pre vertebral and mesenteric
- somatic or superficial; mandibular, popliteal, axillary, pelvic
132
1. What iis the sites of adaptive immune response to tissue-borne antigens
2. What is filtration units for lymphatic drainage (enter lymph node)
3. How do naive lymphocytes (T and B cells) enter node (never encountered an antigen)
4. How do all lymphocytes exit?
5. Cortex of lymph nodes contains ?(2)
6. Medulla of lymph nodes is rich in? (2)
1. Lymph nodes
2. AFFERENT lymphatic vessels
- Normally: lymph return excess interstitial fluid/proteins to the blood
- During infection: lymph also carries microorganisms and antigens from tissue site
- Dendritic cells migrate from tissue to lymph nodes at ALL times
3. through specialized post-capillary venules - HEV - high endothelial venule
4. Exit via a single EFFERENT lymphatic to blood
5. Cortex - follicles (B cell in cortex) and T cell rich regions (paracortex)
6. Medulla - macrophages and plasma cells
133
Describe the routes of infection for pathogens across dry skin and moist/mucosal epithelium
Dry (3 route of entry)
moist (3 route of entry)
* *Describe
- mode of transmission
- pathogen types
- disease
Moist (mucosal surfaces)
1. Airway
A. Mode of transmission; inhaled droplet and spores
B. Pathogen/Disease; flu, meningitis, anthrax
2. GI
A. MoT; contaminated food and water
B. Pathogen/disease; salmonella - typhoid fever, rotavirus - diarrhea
3. Reproductive tract
A. MoT; physical contact
B. Pathogen/disease; syphilis, HIV - AIDs
Dry (external epithelial)
1. External surface
MoT; physical contact
Pathogen/disease; athlete foot
2. Wounds and abrasions
MoT; puncture wounds, minor skin abrasions, handling infected animals
Pathogen/disease; cutaneous anthrax, tetanus
3. Insect bites
MoT; mosquito, tick
Pathogen/disease; yellow fever, Lyme disease, malaria
134
What is the first line of defense against infection (mechanical, chemical and microbiological) in;
1. Skin
2. Gut
3. Lungs
4. Eyes/nose/oral cavity
- which is shared? (4)
- what gives an immediate response to bacterial infection?
1. Skin
- mechanical; tight junctions
- chemical; beta DEFENSINS, CATHELICIDIN, fatty acid
- microbiological; Normal MICROBIOTA
2. Gut
- mechanical; tight junctions
- chemical; alpha DEFENSINS, CATHELICIDIN, low pH, pepsin
- microbiological; Normal MICROBIOTA
3. Lungs
- mechanical; tight junctions, cilia
- chemical; alpha DEFENSINS, CATHELICIDIN, pulmonary surfactant
- microbiological; Normal MICROBIOTA
4. Eyes/nose/oral cavity
- mechanical; tight junctions, cilia
- chemical; beta DEFENSINS, CATHELICIDIN, enzymes in tears and saliva (lysozyme)
- microbiological; normal MICROBIOTA
* **DEFENSINS - immediate response to bacteria infection
- beta defensins in SKIN and eyes/nose/oral cavity
- alpha defensins in GUT and LUNGS
135
What happens if epithelial barrier is breached? (If the epithelium is crossed)
* virus, bacteria, parasites, fungi
- bacteria gain access to?
1. VIRUS infects epithelial cells or gains access to
target cells it can infect
2. BACTERIA gain access to submucosa
3. PARASITES colonize host tissue and/or organ
4. FUNGI take up residence
**Pathogen expands at the expense of the host and tissue damage occurs
136
Identify
1. A PHYSIOLOGIC process by which vascularized tissues respond to injury
2. 2 types
A. Resolves itself after a few days
B. continual infiltration that damage cellular tissue e.g chronn’s disease- can lead to cancer - prostate, pancreatic, breast, lung, GI cancer
3. What 2 things work together to eliminate any foreign agents causing physical stress
- one has 4 types
1. Inflammation
2.
A. Acute inflammation
- crucial to maintaining the health of an individual
- rapid, short lived (minutes to days), same response to injury
- characterized by accumulation of fluid, plasma proteins and neutrophils
B. Chronic inflammation
- lead to unwanted tissue destruction
- of longer duration and includes all above followed by influx of lymphocytes, macrophages and possibly ending in fibroblast growth ** May result in granuloma
3.
- soluble mediators (defensins, complement, lipid mediators, pro-inflammatory cytokines -IL1, IL6, TNFalpha)
- cellular components
137
1. Hallmark signs of inflammation (4)
| 2. What are preceding results to inflammation? (4)
1. REDNESS, SWELLING ‘W/ HEAT, PAIN
2.
- increased blood flow
- vascular permeability
- leukocyte migration into tissues and accumulation at inflammatory foci (lymphocyte, monocytes, granulocyte)
- activation of the leukocytes to destroy the foreign invader
138
1. What is the progression of leukocytes migrating into the infected tissues? (Which enter first, second, third)
2. What is the progression of immune response (3)
3. What happens when inflammation is resolved?
- remove what?
- 3 steps to normalcy
1. Neutrophils - Monocytes - Lymphocytes
2.
1. Immediate innate response - phagocytosis, complement
2. Need help - induced innate response (complement, cytokines, chemokines, neutrophil influx, DC migration)
3. Still stubborn - Adaptive response
3.
- removal of debris
- wound healing (angiogenesis - new blood vessels, connective tissue repair, restoration of tissue or organ function)
139
Identify stages in infection and differences in innate immunity (contents)
- normal (3)
- immediate innate (3)
- induced innate (6)
1. Normal/constitutive
- normal flora (microbiota)
- local chemical factors (defensins)
- phagocytosis (only in lung)
2. Immediate innate immunity
- antimicrobial peptides
- phagocytosis (resident macrophages - peritoneum?)
- complement cascade
3. Induced innate immunity
- complement
- cytokines
- chemokines
- macrophage activation
- neutrophil influx
- DC migration to LN
140
Innate immunity is initiated by effector cell types that can distinguish self from non-self in order to respond to microbial infections
1. WHat are the receptors on effector cells that can distinguish self from non-self?
2. What are molecules associated with groups of pathogens that are recognized by cells of the innate immune system?
1. Receptor is PRR (e.g TLRs)
- Pattern Recognition Receptors
- it is germline encoded (innate)
3. PAMPS (pathogen associated molecular patterns)
- small molecular motifs conserved with a class of microbes
- they are recognized by TLRs (toll-like receptors) and other pattern recognition receptors (PRRs) in both plants and animals
**INNATE immunity provides protection against a WIDE VARIETY of pathogens (bacteria, virus, fungi, parasites)
* *Genetic defects in innate immunity are very rare and usually lethal due to increased susceptibility to infections
- • Chemotherapy destroys myeloid - destroy neutrophils (innate) so you are more susceptible to infections (decreased immune system))
141
What happens when PRR bind PAMP (innate immunity)
Innate vs adaptive response (paper)
Release CYTOKINES which go to bone marrow which release NEUTROPHILS to go help. (Immediate innate)
In adaptive
- Dentritic cell upregulate SIGNAL
- Specific helper Tcells (CD4+) make cytokines and convert Th 0 to any Th needed (with help of DC)
- Th1 (CMI - intracellular virus and bacteria - suppress humoral - gamma interferon and IL2)
- Th2 (humoral - suppress CMI - extracellular bacteria - IL4 and IL10)
- Th17 (IL17 - fungi, stimulate inflammation)
- Th9 - parasitic infection
- Th22 - IL22 - tissue repair and skin
TfH - follicular helper cell - help antigen specific B cells
Tregs - regulatory T cell
142
Initiation of the acute inflammatory response
1. How does bacteria initiate inflammation ‘
- has 3 components in plasma
2. What do injured cells release? What does this release interact with (2)?
1. By activation of the PLASMA PROTEASE SYSTEM (plasma proteases)
- complement
- kinins; bradykinin - smooth muscle contraction, vasoconstriction and increased permeability of small blood vessels
- clotting/fibrinolytic proteins; amplifies inflammatory response by activating Factor 12
* *Do this through recognition of degraded bacterial cell wall products or toxins
2. Injured cells release DEGRADATION PRODUCTS - which interact with the PLASMA PROTEASE system - as well as the PRO INFLAMMATORY CYTOKINE system e.g TNF, IL1, IL4, IL6, IL8, TGF-beta
143
1. What is the goal of vascular effects/changes immediately following insult?
- what vessels are affected first? Second?
2. The results of vascular changes lead to what physical changes? (2)
3. How does the vascular endothelial cells change in normal state vs inflammation?
1. Vascular changes result in a NET INCREASE IN BLOOD FLOW TO THE AREA
- arterioles first then capillaries then venule
2. Heat/calor and Redness/rubor
3. Vascular endothelial cells
- Normal; act as semi-permeable membrane, restricting blood flow to extravascular space
- Inflammation; endothelial CELLS lining CAPILLARIES CONTRACT so that capillary bed can expand - WIDEN GAPS btw the cells - permit passage of blood components
144
What are vascular changes as a result of inflammation? 2 each
1. Blood vessels
2. Vessel wall and blood flow
3. Swelling/exudation
4. Other tissue
Summary of events (3)
1. Changes in caliber of blood vessels
- initial vasoconstriction
- persistent vasodilation
2. Slowing of circulation and stasis
3. Fluid and cellular exudate
4.
- Increase fluidity of ground substance
- mast cell degranulation (histamine and serotonin)
* ***Summary
1. Expansion of capillary bed - increased blood flow
2. Edema expands ECM - deposit fibrin and other plasma proteins
3. Inflammed - neutrophil emigration
145
1. Identify 5 soluble mediators of inflammation (with examples)
- pore forming protein that kill bugs?
- prostaglandin, PAF?
- IL1, IL6, TNF
- histamine
- complement, bradykinin, clothing factors
1.
A. Defensins; pore forming proteins that destroy bacteria
B. Plasma protease system
- COMPLEMENT; maintain inflammation, chemoattractant, kill bugs and serve as opsonins
- clotting proteins; fibrinogen and platelets
- kinins (bradykinin); vascular permeability
C. Lipid mediators; prostaglandins and leukotrienes and PAF)
D. Pro-inflammatory cytokines; IL1, IL6, TNF-alpha, TGF-beta
E. Pro-inflammatory mediators, histamine released by basophils and mast cells following binding of the complement to cell - open up vasculatour to get more cells (heat, redness, pain)
146
Identify 4 major functions of soluble mediators
All work together to;
1. Maintain inflammation
2. Signal for additional cells to come in
3. Kill a bug (defensins and complement cascade) - result in a MAC complex - pore in the bacterial cell wall - osmotic lysis
4. Bugs with capsules are resistant to phagocytosis. Soluble mediators (complement) bind to the bug and ingest it
147
1. Is fever good or bad?
| 2. What agents cause fever? (4)
1. it is good because ;
• it is a PROTECTIVE MECHANISM controlled by the hypothalamus (some organisms die in high temperature environment). Don’t let it get out of hand though
```
2. Pyrogens
IL1
IL6
TNF alpha
Prostaglandins
```
148
IDENTIFY THE pro-inflammatory soluble mediator
- a decarboxylated derivative of
histidine.
- A vasoactive amine stored in mast cell
and basophil granules.
- Released when antigen
binds to IgE on mast cells.
- Diffuses rapidly
through tissues and into the bloodstream.
- Promotes acute inflammation through increased
vascular permeability.
HISTAMINE
149
THERE ARE A LOT OF redundant function of cytokines
1. What is the overal goal
2. What are cytokines
- secreted by?
1. TO MAINTAIN INFLAMMATION
2. Molecules secreted by cells (LEUKOCYTES) that act
locally in micro-environment by binding
receptors on target cells. Cytokine binding will
cause a change in the target cell.
150
Identify the 3 cytokine types that have a wide spectrum of biological activates to help coordinate the body’s responses to INFECTION (pro-inflammatory cytokines)
How (3 functions)
IL1 beta
IL6
TNF alphA
1. Activation of COMPLEMENT opsonization (liver- C reactive proteins, mannose binding lectin)
2. Phagocytosis (bone marrow endothelium - mobilize neutrophil)
3. Decreased viral and bacterial replication (hypothalamus - increase blood temp, fat, muscle)
**high temp decrease viral and bacteria replication
151
Match the following acute-phase proteins with the functions
1. C reactive proteins, mannose binding lectin, lipopolysaccharde binding protein
2. Complement components C3, C4, C9, factor B
3. Granulocyte colony-stimulating factor (GCSF), serum amyloid A, secreted phospholipase A2
4. Fibrinogen, plasminogen, tissue plasminogen factor
**How are the acute-phase proteins synthesized?
1. Pathogen recognition (activate complement opsonization)
2. Pathogen elimination
3. Inflammatory response
4. Coagulation
**Bacteria induce MACROPHAGES to produce IL6 - acts on hepatocytes (liver cells) to induce synthesis of acute-phase proteins
152
Identify cytokine type
major inflammatory cytokine released by
monocytes and activated MACROPHAGES.
IDENTIFY ACTIVITIES (4)
IL1
**Other pro-inflammatory cytokines (IL1, IL6, TNF alpha, TGF beta - inhibit)
```
Numerous activities including
1. INCREASED BLOOD FLOW
2. FEVER
3. enhanced expression of adhesion molecules and
4. stimulation of CYTOKINE PRODUCTION
from other leukocytes.
```
153
Identify cytokine type
| derived from
MACROPHAGES. Distinct but related peptides.
Similar actions to IL-1. Also can induce APOPTOSIS.
2
TNF- alpha and TNF-beta
154
IDENTIFY CYTOKINE TYPE
activities related to ALLERGIC INFLAMMATION including BASOPHIL DEVELOPMENT and EOSINOPHIL CHEMOTAXIS.
**It also participates in the cell fusions seen in granulomas.
IL4
- help in promotion of B cell growth and induction of class switching to IgE and IgG
155
Identify cytokine type
released by T cells and endothelial cells, promotes
monocyte differentiation and increases platelets in
circulation and increases fibrinogen production by the
LIVER.
IL6
• IL 6 work on liver to release agents like C reactiove protein - serve as opsonate for opsonization (to coat bacteria make it edible - bind to bug that make it bind to phagocyte).
156
IDENTIFY CYTOKINE TYPE
1. made by T cells and NK cells, up-
regulates MHC proteins and also activates macrophages to
make them more efficient killers of bacteria.
(Innate immunity) **
2. made by macrophages and B cells, stimulates
proliferation of NK cells, T-helper and T-cytolytic cells
(CTL) - turn on adaptive immunity (pro-inflammatory cytokine)
1. Gamma interferon
| 2. IL12
157
A. Identify 3 ways of activating complement proteins
1. First to act
2. Second
3. Third to act (antibody)
Does C3b binding need help or not (in each pathway)
1. Alternative pathway
- C3b bind without help (independent)
- PATHOGEN SURFACE creates local environment conducive to complement activation
2. Lectin pathway
- MANNOSE-BINDING LECTIN binds to pathogen surface
3. Classical pathway
- C3b need help to bind (antibody helps)
- ANTIBODY binds to specific antigen on pathogen surface (IgM, IgG, IgA)
158
The complement system (summary)
1. How many proteins?
2. Function of 3 pathways?
3. 3 pathways lead to activation of ?
4. Same goal of all pathways which is?
5. Same effector mechanisms (3)
6. End result
1. 30 proteins (soluble and membrane)
2. Sensor molecule for infection
3. Complement activation
4. Same goal - cleavage of C3 to C3a and C3b. C3b is covalently bound to surface components of pathogens
5.
- recruitment of inflammatory cells (broadly effective)
- opsonization of pathogens, facilitating uptake and killing by pathogens (broadly effective)
- perforation of pathogen cell membranes (limited effectiveness)
6. Death of pathogen
159
1. 2/4 main molecules part of complement system
2. Which acts as an OPSONATE to bind the bug which bind to phagocyte
3. Which serve as chemoattractant to bring in neutrophils
4. Which bind to bug and initiate other complement proteins (C6, C7, C8, C9) - FORM PORE - cause osmotic lysis of bug which is normal job of defensins and NK cells
5. What prevent pore formation and how?
1. C3a/b and C5a/b
2. C3b (promotes phagocytosis by neutrophils and macrophages)
- complement activation lead to C3b deposited on bacterial cell surface
- CR1 on macrophage bind C3b on bacterium
- endocytosis of bacterium
- Macrophage membrane fuse to phagosome
- lysosomes fuse with phagosome - phagolysosome
3. C3a and C5a (complement use this to promote inflammation)
- C3a and C5a are ANAPHYLATOXINS (increase vascular permeability - allow for migration of monocytes and neutrophils)
- C5a is a CHEMOTAXIN
4. C5b
5. CD59
- binds to C5b678 complex and prevent recruitment of C9 to form the pore
160
Complement activation involves 3 distinct phases
1. detects the presence of infection via innate or adaptive molecules
2. rapidly and extensively amplifies complement activation AND promotes
cellular inflammation and opsonization
3. leads to damage of microbial membranes
1. Recognition phase
2. Enzymatic phase
3. MAC (membrane attack complex)
- binding of C5b leads to membrane damage via MAC
161
Identify what process this is
- Imbalance of intracellular ions
•Accumulation of intracellular water accumulation
**When is this process effective?
The physiological mechanism of cell lysis
•Subsequent cell lysis. (effective only on FEW BACTERIAL SPECIES)
162
Which is worse
1. deficiency in MAC vs opsonization
2. Deficiency in complement predispose you to infection via? (2)
1.
• DEFECTS IN OPSONIZATION are WORST (more severe than MAC - membrane attack complex)
• Susceptible to autoimmunity, bacterial and viral infections
2. Deficiencies in complement predispose patients to infection and autoimmune diseases
• Deficiencies in complement predispose to infection via: (1)
ineffective opsonization and (2) defects in lytic activity (defects in MAC).
• Opsonization and lytic function of complement protect
against both bacterial and nonbacterial pathogens, such fungi, viruses, and mycobacteria.
• Defects in opsonization are more severe than defects in
membrane attack complex.
• Incidence of autoimmune diseases is associated with
DECREASED CLEARANCE of IMMUNE COMPLEXES.
163
Identify process
1. Complement promotes phagocytosis by neutrophils and macrophages
2. How?
- bacteria engulfment by neutrophils is mediated by? (2)
- how is bacteria killed?
1. Opsonization
2
- FC receptors and complement C3 receptors
- granules fuse with phagosome - release toxic oxygen metabolite - kill bacteria
164
Identify the most important inherited disorder of the complement system
- primary clinical manifestations
- functional defect (6)
1. C3 deficiency
Manifestations
- bacterial infections
- SLE like syndrome (systemic lupus erythematosus)
Functional defect
- Classical + alternative c activation
- I;C clearance
- opsonization
- anaphylatoxins
- chemotaxin
- MAC
165
Identify how the following helps out in the cellular response to inflammation
NK cells/CLT cells
- Tail has?
- granules do what (2)?
Innate (but intracellular pathogens)
1. Direct cell lysis; Kiss of death - specific directed killing technique
2. NK and CTL have same killing techniques - tail has LYTIC GRANULES (contain enzymes) - bind to virus infected cell - release granules that 1) induce apoptosis 2) form pores that release enzyme to breakdown cell
* *NK Cells receptors recognize changes at the surface of human cells that are caused by viral infection (INTRACELLULAR PATHOGEN)
* * Macrophage receptors recognize the cell-surface carbs of bacterial cells but not those of human cells (EXTRACELLULAR PATHOGEN)
166
Identify how the following helps out in the cellular response to inflammation
Mast cells (basophils)
**what receptor has high affinity for the Ig/immunoglobulin here
- How do mast cells induce inflammation?
- origin?
Innate
- mediate/induce inflammation by release histamine and serotonin
- generate IgE
- Origin of tissue mast cells debated, some think they are
simply blood derived basophils, or, they come from a
self replicating pool of mast cells in the tissues.
• Very similar to eosinophils in terms of function
• Present at site of inflammation
• Growth factors are IL-3, IL-5 and GMCSF.
• Recruited to the site of IgE mediated allergic reactions
• Have HIGH AFFINITY FC RECEPTOR for IgE
• Release histamine IL-4 and IL-13 upon IgE binding and
activation.
167
Identify how the following helps out in the cellular response to inflammation
Macrophage
- **what do they release at site of infection? (3)
- release inflammatory cytokines at site of infection (IL1, IL6, TNF alpha) - IL6 increase temperature, IL1And TNF alpha increase blood flow
- Also release chemokines - recruit neutrophils (IL8 )
- Also release IL-12 - turn on adaptive responses and fight virus (also recruits and activates NK cells).
- Clear up debris and allow for wound healing
* *NK Cells receptors recognize changes at the surface of human cells that are caused by viral infection (INTRACELLULAR PATHOGEN)
* * Macrophage receptors recognize the cell-surface carbs of bacterial cells but not those of human cells (EXTRACELLULAR PATHOGEN)
168
Identify how the following helps out in the cellular response to inflammation
Eosinophil
2. types of receptors?
3. location
4. What do helper T cells release during allergic response? Why?
- have granules with MBP? (Major basic protein - arginine) - form pores in the WORM (big parasites).
- Also work in autoimmunity and allergic reactions - normal tissue damage in allergy (basophil in allergy is bad).
- Also secrete proteins involve in AIRWAY TISSUE REMODELING.
- synthesis of mediators such as prostaglandins, leukotrienes and cytokines. Major role in mediating inflammation and recruiting
other leukocytes to the site.
3. Small numbers in blood. Mos found in connective tissue of resp tract, gut and urogenital tract
2. Possess numerous receptors
- IL-5
– Fc gamma and Fc alpha
– C3 complement receptors
4. During allergic tissue reactions, helper T cells release IL-5, IL-3
and GMCSF which can induce eosiniphil degranulation.
169
Identify how the following helps out in the cellular response to inflammation
- what 2 respond by phagocytosis
MACROPHAGES
| NEUTROPHILS
170
As an immediate response during inflammation, resident tissue macrophages respond rapidly by 2 major functions
1. Phagocytoze microbes
2. Release cytokines/chemokines
How?
By
Bind bacteria to what respective receptors?
1. Phagocytic receptors (FC and C3)
- binding bacteria to PHAGOCYTIC RECEPTORS on macrophages to induce their engulfment and degradation
2. PRR (pattern recognition receptors
- bind bacteria (PAMP) to SIGNALLING RECEPTORS (PRR/TLR) on macrophages to induce synthesis of inflammatory cytokines
- **TLR is key to linking innate and acquired immunity (expressed by cells of innate but can release IL12 which activate adaptive immunity)
171
Recognition of pathogens
Differentiate PRR vs PAMP vs DAMP
- PAMP not found on what cells?
- which is found on dead host cells?
- which is found on pathogens?
- what link innate to acquired immunity?
- Which is for necrotic tissue
1. Pattern recognition receptor (PRR)
– Key example of PRR are Toll like receptors (TLR). Recognize PRR at cell surface or in endosomes. Key to LINKING INNATE AND ACQUIRED immunity. – Expressed by cells of the innate immune system
2. Pathogen Associated Molecular Pattern(PAMP)
– Found on PATHOGENS (Virus, bacteria, parasites)
– Bacterial carbohydrates, (lipopolysaccharide, LPS), mannose,
nucleic acids (bacterial DNA or viral DNA or RNA), bacterial
peptides (flagellin), peptidoglycans (gram + bacteria), fungal
glucans and chitins)
– These are molecules NOT FOUND ON EUKARYOTIC CELLS.
3. Damage Associated Molecular Pattern (DAMP)
» Cellular components released from DEAD HOST CELLS.
- acts as endogenous danger signals
- for necrotic tissues (not as much inflammation like PAMP - more like wound healing)
172
1. How many known TLR (PRR type) are there?
- why that many?
2. What is the ligand for PRRs
3. When binding occur, one of the following is initiated (4)
1. 10
- broad microbial recognition (turn on immunity)
2. PAMPS
- pathogen associated molecular patterns (unique to microbes - bacteria, fungi, virus, parasite)
3.
A. Phagocytosis
- increase expression of receptors for binding antibody (Fc) and complement (C3)
B. Cytokine release (inflammatory); secretion by macrophages and DC of cytokines
- proinflammatory (IL1, IL6, TNF alpha)
- IL12 (turn on adaptive immunity)
C. Inflammation
D. Activation of T lymphocytes; enhanced ability of DC to induce adaptive immunity
- antigen presentation to T cells is improved
- increased expression of MHC molecules
- Induces expression of costimulatory molecules on DCs.
173
What is DANGER SIGNAL?***
Litigation of TLR to PAMP
(Release inflammatory cytokines or interferons)
- some PRR (TLR) are cell surface structures and some are found in endosomes
** You will see when we discuss adaptive immunity, binding of a TLR to a PAMP by an antigen presenting cell will result in the expression of costimulatory moecules and the subsequent activation of effector T cells.
174
What are the different consequences of PAMP detected in
1. Cell surface
2. Endosome
1. Cell surface
- phagocytosis
- ingestion of pathogen
2. Endosome (PAMP bound by TLR 9, CPG) - intracellular pathogen
- Adaptive immune response
175
Identify the 2 types of cell death
1. Premature death from cell injury
2. Pre-programmed (natural occurring)
**causes?
- which is beneficial? Detrimental?
- which result in loss of cell membrane integrity and release of products to extracellular space
- 3 steps that cell use to program its own death*
- which is responsible for granulocyte death and also
lymphocyte death during developmental processes
including TOLERANCE INDUCTION in the thymus.
1. Necrosis
- premature death of cells in living tissue by AUTOLYSIS
- caused by factors external to tissues ; infection, toxins, trauma that result in unregulated digestion of cell components (poisoning and anoxia)
- DETRIMENTAL
- result in loss of cell membrane integrity and release of products to extracellular space
- better to remove necrotic tissue with surgery (debridement) because leukocytes release microbial damaging substance that cause GANGRENE - damage surrounding tissues
2. Apoptosis
- beneficial
- steps; nuclear DNA degradation, nuclear degradation and condensation, phagocytosis of cell debris
- responsible for granulocyte death and also
lymphocyte death during developmental processes
including TOLERANCE INDUCTION in the thymus.
176
Inflammation with no infection
1. What types of inflammation is this?
Myocardial infarction, Alcohol-induced pancreatitis, Ischemia reperfusion injury, Closed blunt tissue injury, Gout (uric acid crystal - arthritis)
2. What induce the acute inflammatory response in necrotic tissues injury?
3. In necrosis, what does PRR bind to that create an endogenous danger signal?
1. STERILE INFLAMMATION
2. Necrosis NOT apoptosis
3. DAMP (damage associated molecular patterns)
- Act as endogenous danger signals
- Promote and exacerbate acute inflammatory
response
-Necrotic injury?- get DAMP (not as much inflammation like PAMP - more like wound healing
177
Identify how the following helps out in the cellular response to inflammation
Neutrophils (part 1)
**Identify 3 sources of neutrophils
**what stimuli do the neutrophils respond to for them to be released?
1) neutrophils already CIRCULATING in peripheral blood.
2) neutrophils STIMULATED to differentiate and mobilize
from precursors in the bone marrow (send cytokine signal for this)
3) neutrophil STORES in venules (called MARGINATION).
The neutrophils are said to respond to CHEMOTACTIC
STIMULI (chemokines and cytokines) which are released
by macrophages and endothelial cells. COMPLEMENT
activation also leads to the recruitment of neutrophils
and other effector cells to the area of insult.
178
4 Steps of neutrophil response (Names)
1. Neutropils are first observed to roll slowly along the blood vessel wall (Neutrophil stores in venules)
2. More definitive sticking. Mediated by what receptors (2)
3. Pass through gaps in endothelial cells?
3. Pass through basement membrane to inflammatory site - from interior of blood vessels to extravascular space
(What do all immune cells use to migrate)
1. MARGINATION
2. SELECTINS (ICAM1) AND INTEGRINS (LFA1)
3. DIAPEDISIS
4. EXTRAVASATION
- overall efflux of materials from blood vessel
- ALL IMMUNE CELLS use CELL ADHESION MOLESULES to extravasate and migrate to site of infection (selectin, INTEGRINS, immunoglobulin superfamily -ICAM, VCAM)
179
All immune cells in blood use cell adhesion molecules to extravasate and migrate to site of infection
Identify types (with examples)
1. family of cell adhesion molecules with binding
specificity toward CARBOHYDRATES on glycoproteins
and glycolipids
2. family of cell adhesion molecules with binding
specificity toward LIGANDS on CELLS or EXTRACELLULAR MATRIX
3. family of cell adhesion molecules with binding
specificity toward LIGANDS ON CELLS
1. SELECTINS
• E-selectin (in endothelial cells)
• L-selectin (in leukocytes)
• P-selectin (in platelets and endothelial cells)
2. INTEGRINS
- LFA-1
- VLA-4
3. Immunoglobulin superfamily
• ICAM-1, ICAM-2
• VCAM-1
180
1. When neutrophil finally get to infection site, what do they do (3)
2. Use of FC and C3 receptors to do what?
3. STEP 2 initiates what?
4. What bind to microbial structures - PAMP
5. What does danger signal enhance (3)
6. How are microbes effectively killed (combined effects of what receptors? (2))
1. opsonize, phagocytize and kill microbes
2. Enhance phagocytosis by opsonization. OPSONIZED microbes bind tightly to neutrophil opsonic receptors
• Ab-coated particles to Fc receptors
• C3-coated particles to C3 receptors
3. Phagocytosis is initiated by opsonization
4. Pattern recognition receptors bind to microbial structures
5. PRR signaling enhances phagocytosis, degranulation, oxidative burst
6. Microbes are effectively killed by the combined effects from OPSONIC RECEPTORS and PATTERN RECOGNITION RECEPTORS
181
1. What 3 things ingest foreign material by phagocytosis
2. Ingested material is then digested in?
3. The accumulation of the neutrophils the ingestion and digestion of materials that occurs, and the subsequent clean-up by macrophages results in a material called
1.
- neutrophils
- tissue macrophages
- infiltrating monocytes
2. Phagolysosomes
3. Inflammatory exudate or pus.
182
How is innate response associated with viral infection? 2
**Because normally, an intracellular pathogen like virus is the key to trigger an adaptive or acquired immune response
Immediate innate response mediated by
1. Type 1 interferons (cytokines)
2. NK cells
** ALPHA and BETA interferon, inhibiting viral
replication and binding to neighboring cells as well
as eliciting and activating NK cells to deliver a
“lethal hit”.
183
1. Any cell infected with virus release what 2 types of interferons?
2. Identify 3 functions of the interferon type
3. What cell type secrete this interferons (innate cell that activate adaptive immunity?)
1. INF alpha and INF beta
2.
A. Induce resistance to viral replication in all cels (prevent viral replication)
B. Increase expression of ligands for receptors on NK cells
C. Activate NK cels to kill virus-infected cells (for kiss of death - direct cell lysis)
3. Plasmacytoid DENDRITIC CELLS
184
What soluble mediators help to resolve/stop the inflammatory response (4)
1. down regulates neutrophil superoxide production.
2. released by macrophages and T cells; inhibits the
production of pro-inflammatory cytokines such as GAMMA-
INTERFERON.
3. suppresses hematopoiesis, inhibits neutrophil accumulation and
inhibits production of pro-inflammatory cytokines.
4. produced by adrenal cortex. Mediate
immunosuppression, down-regulate inflammation.
1. IL4
2. IL10
3. TGF-beta (transforming growth factor)
4. Glucocorticoid
**must be short lived so as not to inhibit responses to other inflammatory stimuli
185
If acute process (of inflammation) only leads to PARTIAL elimination of invader - response can persist and lead to? (2)
1. Development of ACQUIRED IMMUNE RESPONSE
2. Development of CHRONIC INFLAMMATION
- more neutrophils migrating and start eating normal tissue - become tumor cells
186
Identify what this is
- soluble mediators of immunity
- small proteins that are released by immune effector cells
- have specific effects on how leukocytes interact with one another
2. What do they do during immune responses?
3. Are they always present?
4. Do you need receptor?
1. CYTOKINES
2. Cytokines mediate communication between cells during immune responses
3. They are generally transcribes and translated AS NEEDED
- although some can be stored as a secretory product
4. Cytokines act only on cells bearing specific receptors on target cells ( expression of cytokines and their receptors is highly regulated)
- e.g IL2 (LAK) - bind to receptor and allow for clonal proliferation
187
Cytokines are soluble mediators of immunity released by immune effector cells (e.g helper T cell)
**Identify 5 functions of cytokines
1. Proliferation (clonal expansion)
2. Differentiation
3. Activation
4. Cell movement
5. Cell death and survival
188
* *What are signalling molecules in the immune system
1. How do cytokines differ from hormones released by the endocrine system?
2. 3 pathways cytokines exert their function
Cytokines - signalling molecules
1. Unlike hormones, their action is GENERALLY on cells in their immediate vicinity or micro-environment
2.
A. Endocrine; long distance
B. Autocrine; within self - same cell
C. Paracrine; neghbouring cells - close proximity
* * the effects of cytokines are local, between
cells. For instance, within a synapse
189
1. Cytokine release lasts how long?
2. Is it constitutively expressed?
3. Chronic release of cytokines lead to?
4. Over production of cytokines to pathogens called? What cures this?
1. Cytokine release lasts from few hrs. to days.
2. Generally not constitutively expressed, but
expressed following activation. Production is
short lived and tightly controlled.
3. Do not want chronic or extended cytokine
production. Chronic release and exposure of
normal tissue to cytokines can result in
deleterious consequences (rheumatoid arthritis,
chronic inflammation).
4. Over production of cytokines to pathogens can
result in what is called “ Cytokine storm”. If this
occurs in the lungs, can result in death (Vascular
leak syndrome).
- over production leads to rapidly proliferating T cells and NK cells
- treat with CAR-T cell therapy
190
Describe the 4 families of cytokines
1. Most numerous - stimulate B cell and T cell
2. work on bone marrow to induce hematopoiesis
3. Produced by leukocytes and fibroblasts
4. Made by macrophages and T cells
**made by?
Function?
Clinical importance?
1. Interleukins (btw WBC - IL1 -IL40)
- made by leukocytes
- diverse functions include B and T cell growth and activation
- clinical; IL2 to melanoma and renal cell cancer
2. CSF - colony stimulating factors
E.g GMCSF - stimulate stem cells (important in immunotherapy)
- made by variety of cells and stimulate granulocyte, monocytes and dendritic cell production and differentiation
- GMCSF, GCSF, MCSF; Granulocyte monocytes colony stimulating factor
- Clinical; immunotherapy
3. Interferons
- gamma interferon important in adaptive immunity (VIRAL INFECTIONS) - help host recognize pathogen.
- T cell makes gamma interferon.
- Alpha - against renal cancer.
- Beta - against MS
4. TNF (alpha and beta)
- made by macrophages and T cells
- functions; promote inflammation, apoptosis, angiogenesis and T cell activation
191
1. Explain the concept of redundancy and pleutropy in cytokines and give example each
2. How do they do pleitropy?
3. Explain Synergy and its action in class switching?
1. Redundancy; different cytokines have same effects/function.
E.g; Incase there is mutation in IL2. IL4 or IL5 can take over in B cell proliferation
2. Pleiotropy; one cytokine can have different effect on different cells.
E.g
IL4 is growth factor but also work in activating B cell, T cell and mast cell (multiple effects - proliferation of diff cells).
**How? Parts of the receptors can be shared with different cytokines - may not be strong binding but you get binding
3. Cytokines can synergize with each other
E.g
IL4 and IL5 work together to induce class switch of B cell to IgE
192
CYTOKINE TYPES (immune responses)
1. Pro-inflammatory cytokines (4)
2. Anti inflammatory (3)
3. Inhibit virus replication (2)
4. Macrophage activating cytokines (1)
5. B cell activating cytokines (4)
6. T cell activating (4)
7. Eosinophil and/or mast cell activating cytokines (4)
1. Inflammation (16nfmc)
IL1, IL6, TNF alpha, many chemokines
2. Anti-inflammation (1r10gfb)
IL1 ra, IL10, TGF-beta
3. Prevent virus replication (interfere)
IFN alpha and beta
4. Macrophage (gam-y interfere for Mac’s life)
IFN- gamma/y
5. B cell (B 456-21)
IL4,IL5,IL6,IL21
6. T cell (24-12y interfere for T)
IL2, IL4, IL12, IFN-y
7. Eosinophil/mast cell (Em 345-13)
IL3, IL4, IL13, IL5
193
Cytokine types (subset of lymphocytes that secrete them)
How does this work and give types
- what is precursor?
- which inhibit which
- CMI? humoral? Helper cytokines?
- inflammation
- parasite? Fungi?
- skin or tissue repair?
* all come from precursor - Th0
* Dendritic cell tell Th0 to become Th1, Th2 or Th17 cells
1. Th1 - for cell mediated immunity (CMI), (virus or intracellular bacterial) - INF y and IL 2 are helper cytokines
2. Th2 - If you have bacteria infection (humoral immunity)- release cytokines - IL4 and IL10 (table)
* *Th1 inhibit Th2 and vice versa
3. Th17 - fungi, stimulate inflammation. Secrete IL17
4. Th9; parasite infection
5. Th22; tissue repair and skin
6. TfH; help antigen specific B cells
194
What are cytokine receptors? Describe action using IL2 receptor
- how many chains?
- similar affinity? Functions?
- do diff cytokines share same receptor?
2. What 2 things follow after cytokine bind to receptor
3. Haploid or diploid?
4. How do cytokine receptor signal? (What pathway)
5. IL2 examples; how many chan? Affinity?
- binding component?
- transducing component?
- 2 or more chains (alpha, beta, gamma)
- IL-2R shares a common receptor with IL-15, IL-4, IL-7, IL-9 and IL-21
- IL-2R and IL-15R share both beta and gamma chains
(next slide)
2. Binding - internalization - reexpression of new receptors
3. Most cytokine genes exist as single copies in the haploid
genome.
4. JAK/STAT pathway. Others are IRAK, G-protein, TRAFFIC-dependent
5. IL2; 3 chains that have different affinity and functions (a,b,y)
• focus on high affinity (all 3 chains combined - alpha, beta, gamma)
- binding component; alpha chains
- transducing; beta and gamma chains
195
Identify
- cytokines that induce cell movement along a gradient (42 known types)
- direct cell movement, migration from one anatomic space to another
2. Action? (2 types of mediators?
3. How are they similar to cytokines? (2)
4. Receptors xteristic (can it be shared?)
CHEMOKINES
- induce chemoattraction
2. Act as inflammatory mediators and mediators of constitutive
leukocyte migration
3. PLEIOTROPY and REDUNDANCY characteristics
4. Specific receptors - can be shared by different leukocytes and tissue cells
196
There are 2 types of chemokines
Identify
- which is not expressed in normal tissues? Other is expressed in what organs?
- which is constitutively expressed?
- which was the only known chemokines for years?
- which is usually redundant?
- which mediate lymphocyte recirculation?
**How are they both regulated? (What expression)
Function?
1. Inflammatory chemokines
- not expressed in normal tissue
- Turned on by infection /insult
- Stimulate INFLAMMATION
- Usually, but not always, redundant
- For years, only known chemokine type
- Activity regulated by chemokine expression
2. Lymphoid Chemokines
- Expressed in lymphoid organs
- Mediate LYMPHOCYTE RECIRCULATION
- Maintain lymphoid structures
- Act in lymphoid tissue development
- Constitutively Expressed
- Action regulated by receptor expression
197
Explain how cytokines can be used as Biomarkers
- how are Th1 and Th2 related to different stages of cancer (which do you want more of in cancer pt?)
- how to measure cytokines? (Which is measured in bulk from fluid? Which is visualized?)
* Monitor levels of IL6
* Cytokine levels can change with disease state
* Want Th1 in cancer patient (less Th2) - Th1 is CMI (fight virus and tumor)
* Cytokines can be very helpful in measuring and seeing disease changes (staging disease)
• Cytokines measured with use of ELISA or visualized with ELISPOT
- measured in bulk from serum and culture fluids (ELISA)
- visualized as secreted (ELISPOT)
198
Identify
- molecules produced by B cells which bind to antigen
* *must antigen be bound or free?
- Made where (what immunity? Function where?
- what 3 forms?
ANTIBODIES
- antigen can be free in solution or bound to a cell
* Protein molecules
* Made in adaptive/acquired immunity (by B lymphocytes) but function in innate immunity e.g mother give baby antibodies in breast milk.
* Making antibody is a complicated process.
* Monomeric, dimeric or pentameric
199
Immunologically, a molecule foreign to a host may have one or both of the following attributes (a foreign molecule in a cell is 1 of 2 things)
- to induce immune response you must? (3)
Induce immune response or bind to antibody
1. Immunogenicity; capacity to induce an immune response. Require molecule to;
- be foreign to immunized host (except in autoimmunity)
- posses certain molecular weight
- certain degree of molecular complexity
2. Antigenicity; ability to bind with antibodies or cells of the immune system. Highly specific binding - paratope bind to epitope
**remember; Epitope - antigen and Paratope - antibody
200
The binding of epitope (antigen) to paratope (antibody) involve what forces (4)
** Which force/bond is NOT INVOLVED
4 types
1. Attraction between opposite charges
2. Hydrogen shared with electronegative atoms (N, O, F)
3. Fluctuations in electron clouds around molecules oppositely polarize neighboring atoms
4. Groups interactly unfavorably with water - pack together to exclude water molecules
WEAK Forces (4); bind antigen to antibody
1. Electrostatic forces (opposite charges)
2. Hydrogen bonds (NOF - electronegative atoms)
3. Van dear Waals forces (neighboring atoms)
4. Hydrophobic forces (exclude water molecules)
NOT INVOLVED
- COVALENT BONDS not involved
201
Location for action
Tell the difference between how a B cell vs T cell act based on location
- which needs to be in specific place (you have to isolate the cells to treat someone)
- which can be anywhere (just give plasma to treat someone- no need to isolate )
- what makes antibodies? What helps?
* T CELL has to be somewhere to do their job. In treatment, you have to isolate T cells
* B CELL can be anywhere (in spleen, etc) and just be making antibodies. In treatment, you Dont have to collect B cells just give plasma
* Majority of antibody need help (T cell help B cell to make antibodies)
202
THERE are 5 types of immunoglobulins
- identify in order
1. first one you make. Pentameric (5 antibody hook together so 10 binding sites). MAJOR JOB IS TO FIX COMPLEMENT - alternative or classical way (previous lectures)
2. secondary response. EAT THE BUG - isotype switching
3. Dimeric secretory antibodies (found in breast milk), PROTECT YOU
4. **unique - has receptor found on MAST CELLS - so can induce response immediately if you see pathogen, pollen dust etc (inflammation, redness etc
5. play a role in eliminating B-lymphocytes generating self-reactive autoantibodies.
1. IgM - pentameric (10 biding sites)
- fix complement
2. IgG
- neutralization, NK cells, OPSONIZATION for phagocytosis
- also used in complement but IgM is main
- cross PLACENTA
- diffuse into EXTRAVASCULAR sites
3. IgA - dimeric (4 binding sites)
- cross EPITHELIUM (dimeric part - 20%)
- diffuse into EXTRAVASCULAR sites (monomeric part -80%)
- neutralization
- SEROMUCOUS SECRETION e.g saliva, colostrum. UG and trachobronchal secretions
4. IgE
- mast cells / basophils
5. IgD
- basophils
203
Identify 2 recognition molecules in ACQUIRED IMMUNITY (receptors)
**Which bind MHC molecules presenting peptides on APCs
1. BCR - B cell receptor; surface of B cells
- antibodies are a secreted form of the BCR and provide soluble effector mechanism for B cells
2. TCR - T cell receptor ; surface of T cells
- bind MHC molecules presenting peptides on APCs
204
Relationship of BCR and antibodies
- where do B cells encounter antigen
- what does antigen bind to
- what happen after cytokin release? Interaction with T and DC cells
• B cell encounters antigen in the environment
• Antigen binds to the BCR
• BCR: antigen is internalized
• Following cytokine release and interaction with T cells and DCs, a modified form of the BCR is transcribed and translated and
secreted, we call this antibody or immunoglobulin.
• The secreted antibody provides a number of
different effector mechanisms
205
Identify the following
1. Generation of antibodies that is considered part of acquired immunity
* *Involves what? **what is required in some cases for B cell activation
2. When B cell precursor is activated by antigen, the B cells undergo?
3. Activated antibody producing cells are called?
4. A portion of B cells form?
5. Where do plasma cells make antibody? (General name in 3 places )
6. When do antibodies provide their effector function (what type of immune response)
1. Humoral Immunity
* *Involves recognition of a specific antigen. **T cell sometimes required for B cell activation
2. CLONAL PROLIFERATION
3. Plasma cells
4. memory cells
5. Germinal centers
- cortex of lymph nodes
- white pulp of spleen
- mucosal tissue
6. Adaptive immunity?
206
Identify 5 functions of antibody
1 and 2 by IgG
3. mainly by IgM but also IgG
4. IgG
5. Make NK a better Kiss of death
1. neutralization of foreign cells
2. neutralization of toxins
3. scaffold for complement activation
- fix or activate complement
4. facilitate phagocytosis (opsonization)
-
5. antibody dependent cellular cytotoxicity (ADCC). Antibodies bind to Fc receptor found on NK, macrophages and/ or neutrophils, in some cases, eosinophils, mast cells and basophils.
- NK cells have FC receptors that antibodies bind to. Make NK a better killer
207
Properties of antibodies
- what do antibodies bind ?(5)
- how do they bind? (Free? Bound to other cell?)
- binding site of antibody? Binding site of antigen?
- does antigen have multiple binding site?
- what does one binding of paratope to epitope do to another binding?
- Antibodies are proteins that can bind to other
proteins. Antibodies also bind lipids, carbohydrates,
DNA and a number of synthetic molecules.
• Can bind to these molecules either free in solution or
when the molecule is bound to another cell.
• Specific antigenic determinants on molecules are
called epitopes. The binding site on the antibody is
the paratope.
• A single antigen may have multiple epitopes. Some
may be overlapping.
• The binding of an antibody to one epitope may block
the binding of a second antibody to its epitope. The
binding of one antibody may cause a conformational
change in the antigen and either ENHANCE OR INHIBIT
the binding of another antibody to its epitope.
208
Structure of immunoglobulin
1. what chains?
2. how are heavy chains linked together ?
**3. What makes up a hyper variable region? (2) aka?
4. A typical antibody has how many antigen combining sites
5. What ends are the variable regions?
6. Identify 2 regions of antibody molecules
1. light and heavy chains (2 each - identical)
2. Heavy chains linked by DISULFIDE BONDS
3. . ANTIGEN COMBINING SITE (Hyper variable region)
- heavy chain variable region
- light chain variable region
4. 2
5. AMINO ACID END - variable region
Carboxylic terminus - constant region
6.
• An antigen combining region (Fabs) - diversity region
• An effector region (Fc) - commonality region
209
2 regions/ends of antibody (2 name each)
1. this provides the specificity in antigen binding. This is the part of the molecule that binds to the foreign antigen.
2. this provides the link for the effector or immunologic function. That is, it binds to Fc receptors on phagocytes or NK cells (ADCC). It also serves as a binding site for complement components.
1. Recognition end aka diversity end aka hyper variable end
- • diversity in B cells and T cells is if very important to recognize antigen in time - if bug mutates (some survive, some don’t) e,g flu outbreak
- 1 antibody 1 unique binding site 1 unique antigen ( diversity in overall response to foreign antigens)
2. Effector end aka commonality end
- same from antibody to antibody (depending on class)
- Similar structure allow binding to FC receptors or complement molecules
210
What 2 processes/method allow for diversity in antibody (variable light and heavy chains, amino acid region)
1. During development (GERMLINE METHOD); as B cells begin generation - pull them out randomly and make proteins (RECOMBINASE ENZYMES - you have this if you have adaptive immunity). Achieve diversity by pulling all the different genes from molecules and make BCR (B cell receptors)
2. MATURATION METHOD; Once B cell is secreting antibody, mutation event can occur. Change one of the amino acid (either get bad protein or good protein/antibody). SOMATIC HYPERMUTATION
211
Differentiate somatic recombination from somatic hypermutation
- which occur in developing B cells?
- which occur in activated B cells?
- occur in bone marrow
- VJD segment?
- which generate variant antibodies?
Somatic recombination: DNA recombination that occurs
between gene segments in the immunoglobulin loci in
developing B cells. It generates a complete exon composed of
a V gene segment and a J gene segment (and a D gene
segment at the heavy chain locus) that encodes the variable
region of an immunoglobulin polypeptide chain. This occurs in
the bone marrow.
• Somatic hypermutation: mutation that occurs at high
frequency in the rearranged variable-region DNA segments of
immunoglobulin genes in activated B cells, resulting in the
production of variant antibodies, some of which have higher
affinity for the antigen. This occurs after antigen has been
encountered. Affinity can be improved or decays. B cells with
improved affinity are selected for. Thus, the B cell antibody
response evolves, matures over time; effector function
directed at pathogen improves due to more stable binding.
212
1. The recombination events (in somatic recombination or germline method of antibody diversity) is mediated by what enzyme ?
2. 2 of the component proteins are only made in what cells?
3. These proteins are encoded by what genes?
1. V(D)J recombinase
2. T and B cells
3. RAG 1 and RAG 2 genes (recombination activating genes)
213
Identify primary vs secondary response to an antigen
- day 4, 5, 15
- what Ig do you make first (why)
- which IgM comes next? What day?
- does IgM work in phagocytosis? (Why or why not)
- what Ig is low in secondary? Which is high?
1. Primary
• first time you see antigen (4 days)
• Day 5 - make IgM first cause its a pentamer and best to fix complement (but does’t work in phagocytosis - no FC receptor)
• Day 15 - IgG . Total antibody high, plateau and then decline
2. Secondary
• 50 years later
• Low IgM
• Total antibody and IgG boost rapidly and decline slower
• Quicker to initiate e.g vaccine importance
214
5 classes of antibody
1. Similar to one another in what basic structure?
2. Ones that have slight variation is structure called?
3. Which antibody associated with cell membrane?
4. Which is secreted
5. Which is transported through cells and have function on mucosal surfaces (seromucous)
1. Basic Y structure. Each have basic structure of 2 heavy chains and 2 light chains
2. Subclasses
3. IgD
4. IgG
5. IgA
215
Antibody types
1. Neutralization and sense killing by NK cells -
2. Opsonization;
3. Shortest half life (2 days)
4. Sense Mast cells
5. Sense Basophils
6. Activate Complement
7. Transport across Epithelium
8. Transport across Placenta
9. Diffusion into Extravascular sites
10. Which has longest half life (21 days)
1. Neutralization - IgG best
2. Opsonization; IgG best (for phagocytosis)
3. Shortest half life ; IgE
4. Mast cells - IgE (always link the two)
5. Basophils - IgD, IgE
6. Complement - IgM best (also IgG)
7. Epithelium - IgA
8. Placenta - IgG
9. Extravascular sites - IgG and IgA
10. Which has longest half life - IgG
216
Identify antibody type
- Makes up 10% of the immunoglobulin pool
• A pentamer, essentially 5 IgG like molecules make up a single one
- FIRST ANTIBODY made in the primary response.
- seen in response to complex infectious organisms.
• Fixes complement, does not mediate phagocytosis (no Fc receptor) or cross the placenta.
• How many antigens does a single one bind?
IgM
IgM bind 10 antigens
217
Identify antibody type
* 15-20% of the immunoglobulin pool
* 80% is monomeric although 20% is found in the dimeric form.
* Major immunoglobulin found in SEROMUCOUS SECRETIONS such as saliva, colostrum, trachobroncheal and urogenital secretions.
* Fixes complement and mediates phagocytosis
IgA
- third to respond (MGAE)
- cross epithelium (dimeric form)
- cross extravascular sites (monomeric form)
218
Identify antibody type
- Major immunoglobulin found in PLASMA
- Typical 4 chain polypeptide
- 4 subclasses,
- characterize the SECONDARY antibody response.
- can fix complement
- can mediate phagocytosis
- cross the PLACENTA
IgG
**cross extravascular sites
219
Identify antibody type
* Scarce in plasma
* Found on surface of mast cells and basophils
* Major role in the response to PARASITES
* Common in allergic states
* Shortest half life, 2 days
* mediates allergic reactions.
IgE
220
Identify antibody type
• Less than 1% of the immunoglobulin pool
• Found predominantly on the surface of mature naïve B
cells
• Function is not well understood
• May serve a role in the differentiation of B cells to plasma
cells
• The specificity of the IgD molecule found on the surface
of B cells is the same as the antibody which is secreted.
• Secreted IgD may have a role in the respiratory tract in
eliminating bacteria.
****Major ROLE??????
IgD
**** • IgD may play a role in eliminating B lymphocytes generating self-reactive autoantibodies
221
Identify the following types of differences
1. reflect amino acid differences which make an IgG different from IgA for instance, IgE different from IgA etc.
2. are slight differences in amino acid sequence which make IgG from one individual slightly different from IgG in another individual.
3. are amino acid differences found in the VARIABLE REGIONS which make up the antigen combining site.
1. Isotypic differences
2. Allotypic differences
3. Idiotypic difference
222
Antibody effector functions (4)
1. Neutralization (of foreign cells and toxins)
2. Bind complements (IgM mainly and IgG, IgA minor)
3. Opsonization
- FC on antibody bind FC receptor on phagocytes
4. ADCC; bring NK cell into close proximity to FC receptor to enhance direct cell lysis - kiss of death
223
What granulocyte kills IgE coated worms?
- how do they kill?
- what enzymes present in the granules are responsible for anti-helminthic activity?
- **WHat is the 1 component unique to this granulocyte that helps kill parasites?
Eosinophil
• The major role of the eosinophil is believed to be against parasites,
particularly parasitic worms.
• Eosinophils kill by ADCC [antibody dependent cell-mediated cytotoxicity] by binding to parasite - specific IgE via cell surface Fc receptors.
• When eosinophils bind to IgE on the surface of a worm, the cell is triggered to degranulate. The contents of the granules cause
damage to the worm's tegument. There are many HYDROLYTIC ENZYMES present in the granules responsible for the anti-helminthic activity.
• One component which is unique to the eosinophils - and highly toxic to worms - is a substance known as MAJOR BASIC PROTEIN (MBP).
224
Identify specific antibody function
1. neutralize bacterial toxins or prevent bacteria or virus from binding to cells and possibly entering them. Immune complexes formed. These complexes adhere to RBCs, are
transported to RES and removed by macrophages in the spleen, liver and lymph nodes.
2.
• Some bacteria resist binding by phagocytes
• Antibodies are produced against cell surface
molecules on pathogens.
• Antibody binds via antigen combining sites
• Fc of antibody binds to the Fc receptor on
phagocytes
• Phagocytosis occurs
1. Neutralization
| 2. Opsonization
225
Identify specific antibody function
• Sometimes, antibodies provide
their function by serving as a bridge between an effector cell such as an NK cell or macrophage and a target cell (tumor for instance).
- Antibody bound to mast cells, when bound by antigen, crosslinks the antibody and results in granule release.
- These granules contain histamine and heparin which are mediators of inflammation.
ADCC or antibody dependent cellular cytotoxicity.
226
IgE and allergic cascade
- how is production of IgE antibodies stimulated
- how long does it take for IgE to be produced?
- some IgE antibodies attach to what inflammatory cells?
- what happens to unattached IgE
- what happens with reexposure?
- During allergic response, T-cells and DCs interact to send a signal to B-cells (B-lymphocytes) and stimulate production of IgE antibodies
- IgE antibodies, specific to the allergen, are produced within a FEW WEEKS after exposure and released into the bloodstream.
- These IgE antibodies may attach to receptors on inflammatory cells such as MAST CELLS.
- Unattached IgE antibodies remain free floating in the bloodstream.
- When an allergic individual is re- exposed to an allergen, CROSS-LINKING to IgE bound on the mast cells may occur.
227
Back to micro (primerano)
Identify 4 main components/functions of BACTERIA cell structure
- cause disease
- movement
- adherence
- prevent phagocytosis
1. Cause disease (cell structures ARE VIRULENCE FACTORS)
2. Flagella (movement of cell to nutrient)
3. Pili (allow cell adhere to other host cells)
4. Capsule (glycocalyx) - gives resistance and smooth/shiny appearance of bacteria
228
Identify bacteria morphology (size range and types)
1. Size (for mycoplasmas and bacillus)
2. Different forms like; Straight (escherichia), club shaped (corynebacterium), spore forming (bacillus), spiral (spirochaeta), filamentous, branching (actinomyces), comma (vibrio)
3. Usually spherical. Can have; pairs (neisseria), chains (strep), tetras (sarcina) and grape-like clusters (staph)
4. Very short rods
5. Example of wall-less forms
1.
- Mycoplasma; 0.2 x 0.2 micron
- Bacillus; 3 X 10 micron
2. Rods
Straight (escherichia), club shaped (corynebacterium), spore forming (bacillus), spiral (spirochaeta), filamentous, branching (actinomyces), comma (vibrio)
3. Cocci
Can have; pairs (neisseria), chains (streptococcus), tetras (sarcina) and grape-like clusters (staphylococcus)
4. Coccobacillary form
5. Mycoplasmas
229
Identify morphology (shape, type) of following examples
1. Streptococcus pyrogens
2. Staphylococcus aureus
3. Both strep and staph pneumonia
4. Clostridium perfringes
5. Escherichia coli/PMN
6. Vibrio cholera
7. Bacillus subtilis
8. Proteus vulgarisms
1. Chains (cocci); forms long stain and is gram positive
2. Clusters (cocci)
- golden smooth edges
3. Strep forms chains while staph form clusters
4. Bacilli - spore forming rods
- rectangular box car appearance
5. Slender glaci rods
- straight rod
6. Curved rods (comma shape)
7. Irregular rough edges
8. Green pigment that swarm without forming colonies
230
Identify
- component of a microbial cell or virus which enhances its ability to cause disease
**what 3 things can occur
Virulence factors
- colonization
- invasion
- tissue damage
231
1. What bacteria structure is required for movement of bacteria cell, toward a nutrient or away from a toxic substance?
2. What is the process called?
3. What is the primary component of #1
1. Flagella
2. CHEMOTAXIS
3. Flagella primarily composed of one protein - FLAGELLIN
232
IDENTIFY 4 surface distributions in the structure of bacterial flagella (diff structure of flagella)
1. Monotrichous - vibrio cholera
- 1 flagella, 1 pole
2. Lophotrichous - bartonella bacilliform
- Multiple flagella, 1 pole
3. Amphitrichous - spirillum Serpens
- multiple flagella, 2 poles
4. Peritrichous - Escherichia coli
- Multiple flagella, Multiple poles
233
Identify bacteria structure
- not as long as flagella
- thin rigid appendages composed of proteins (called?)
**function? (Give examples)
PILI (fimbriae)
- composed of proteins called PILINS
**Function; allow cells to adhere to host cell or other bacterial cells
E.g
- Neisseria pili required for adherence to host epithelial
- Gram neg F pilus is required for conjugation
234
1. Identify bacteria structure
- thick viscous layer to thin amorphous slime layers
- gives colony a smooth or shiny appearance on agar
- usually polysaccharide, but occasionally protein (give example of one with gamma glutamyl polypeptide)
2. What is the function? (E.g?)
3. What other organism can have this bacteria structure? (E.g?)
1. CAPSULE (glycocalyx)
- bacillus attracts capsul is y-glutamyl polypeptide
2. Function - confers RESISTANCE to phagocytosis (prevent bacteria from being eaten up)
- streptococcus pneumonia capsule
3. FUNGI can have capsules too
- e.g Cryptococcus neoformans from spinal fluid
235
Identify 3 cytoplasmic contents of prokaryotes/bacteria
- do they have membrane bound organelles?
1. discrete area containing the bacterial chromosome
and plasmid DNA; bacterial DNA is organized by histone-
like proteins (NOT ACTUAL HISTONE DNA)
2. sites where nutrient macromolecules (usually polysaccharides like glycogen) are stored as large complexes visible with microscope
3. sites of protein synthesis; actively growing
cells are packed with this
Lack membrane bound organelles
1. Nucleoid
2. Cytoplasmic inclusion bodies
3. 70S ribosomes
(**remember eukaryotes have 80S ribosome)
236
Identify
1. Heat-resistant, dehydrated multi-layered cells that are formed within a bacterial cell
2. Formed by what process? Induced by? (Is this process protective or destructive? Why or why not?
3. When do these cells germinate into growing cells?
4. These cells are rich in what compound? Function of compound?
* *do all organisms form spores?
* *does gram stain work on spores? (What color are they on gram stain)
1. Endoscopes
2. Sporulation - protective process
- induced by nutrient starvation
3. When adverse conditions wane
4. Calcium- dipicolinate
Function; stabilize and protect DNA from damage)
* *sporulation is a protective process because when there is starvation, endospores are formed - they are heat resistant and contain calcium-dipicolinate that stabilize DNA and prevent damage.
* *Not all organisms form spores
* *Gram stain don’t work on spore - WHITE COLOR
237
Identify bacterial cell structure (3 NAMES)
- equivalent to what (in another organism)
- site of active transport (nutrient uptake)
- respiratory chain components and proton pumps(electron transport)
- site of synthesis for what 4 things?
**what enzyme is required for active transport
Plasma membrane aka cytoplasmic membrane aka inner membrane
**equivalent to eukaryotic plasma membrane
Site of synthesis for; PHOSPHOLIPIDS, LPS (in gram -),
PEPTIDOGLYCAN, and CAPSULAR POLYSACCHARIDES
***PERMEASES are required for active transport
238
Identify bacterial cell structure that has many functions and consists of 3 (some textbook say 4) things. Some functions are;
1. Give Structural rigidity/packaging of cytoplasmic contents
2. Permeability barrier
- Struture consist of? (3) - sometimes a 4th
- identify 5 other functions (just think of the functions of the 3 components)
1. Bacterial cell envelope
- plasma membrane
- cell wall
- intervening material (peri-plasmic space)
- capsule (some texts)
2. Functions (7)
- Structural rigidity/packaging of cytoplasmic contents (cell wall)
- Permeability barrier
- Nutrient uptake mediated by transport proteins (plasma membrane)
- Energy production via electron transport in the plasma
membrane
- Attachment to host cells mediated by pili and teichoic
acids (gram + only)
- Escape from immune recognition (capsule - give resistance against phagocytosis)
- Antibiotic target (peptidoglycan synthesis - in plasma membrane)
239
Bacteria cell wall
* *Bacteria can be divided into 2 major groupd based on?
* *what are the 2 major groups
Reaction to GRAM STAIN
1. Gram positive
2. Gram negative
240
Identify the difference in gram stain between + and - using. The following stains
1. Unstained
2. Crystal violet
3. Iodine
4. Decolonization (alcohol-acetone)
5. Safranin
***What major component makes the difference?
1. Unstained
Gram + clear
Gram - clear
2. Crystal violet
Gram + violet
Gram - violet
3. Iodine
Gram + violet
Gram - violet
4. Decolonization (alcohol-acetone) ***Diff starts here
Gram + violet
Gram - clear
5. Safranin
Gram + Purple
Gram - Red
* *PEPTIDOGLYCAN makes the difference
- thick peptidoglycan in gram + (trap the crystal violet/iodine complex) so cells are not decolonized by alcohol
- thin peptidoglycan in gram - (CVI complex escape so cells get discolored)
241
Summarize the 4 steps to gram stain of bacterial cell wall and each outcome
1. Stain cells with Gram stain (crystal violet)
2. Treat cells with a mordant dye, Gram’s iodine which forms a complex with crystal violet.
- both G+ and G- would appear blue under the microscope
3. Wash cells with alcohol.
- Alcohol has the effect of dehydrating the cell wall esp.
peptidoglycan layer.
- CVI is effectively trapped inside in gram+
4. Counterstain with safranin (pink-red).
- G+ cells are blue-purple and G- cells are pink-red
242
Identify 3 key features of Gram POSITIVE cell envelop
1) Thick (multilayered) peptidoglycan overlaying plasma membrane (horizontal plane)
2) Teichoic acids, lipoteichoic acid (vertical)
3) Plasma (Cytoplasmic) membrane
243
Identify bacteria structure
- made in the plasma membrane
- only found in BACTERIA
• Has a peptide and sugar component (consist of glycan polymers cross-linked by peptide chains)
• Backbone - linked by beta 1,4 linkages
• Decorated with peptide chains coupled to M
****pentapeptode bridges are present in only what type?
PEPTIDOGLYCAN (look at picture in slide)
- Complex macromolecule consisting of glycan
polymers which are cross-linked by peptide chains.
- Mesh or network or sacculus
- Only found in bacteria
- Glycan chains consist of alternating acetylated sugars
N-acetylmuramic acid (M, hexagon)
N-acetylglucosamine (G, large circles)
β-1,4 linkages (heavy vertical lines)
- Peptide chains (small circles) coupled to M
pentapeptide/pentaglycine bridges present only in GRAM POSITIVES
244
GRAM + peptidoglycan subunit structure
1. Tetrapeptide (of L-alanine, D- glutamic, L-lysine (or meso DAP) and D-alanine) is covalently linked to? Via?
2. Covalent linkage between tetrapeptides allows glycan backbones to be linked together and forms?
3. In gram +s, terminal D-alanine of tetrapeptide is covalently linked by?
4. β-1,4 linkages are cleaved by what enzymes?
- an enzyme that is widely distributed in human tissues and secretions and is part of the host defense
**Cross linking occurs in what 2 dimensions?
1. Carboxylic group of M via amide linkage
2. Sacculus
- continuous, rigid coat
3. Glycine pentapeptide (in 3D)
4. Beta - 1,4 linkages are cleaved by LYSOZYME
(You make lysozyme in tears that degrade peptidoglycan)
**Cross linking occurs in both HORIZONTAL and VERTICAL dimension
245
Identify 3 functions of peptidoglycan?
1) Allows diffusion of small molecules to the plasma membrane
- peptidoglycan is porous so allows for diffusion of small molecules and keeps large molecules out
(2) confers rigidity and shape to bacterial cell
(3) interferes with phagocytosis and is mitogenic
(Cell wall offer resistance against phagocytosis)
246
Biological activities of peptidoglycan
1. What is the product of peptidoglycan degradation in the host?
2. Product in #1 cause the following to happen
A. what stimulates immune response to an antigen?
B. stimulate cell division
C. fever causing
D. sleep inducing
1. MDP - muramyl dipeptide
```
2.
A. Adjuvant
B. Mitogen
C. Pyrogens
D. Somnagen
```
247
1. What are COMMON SURFACE ANTIGENS that promote attachment to specific receptors on cell surfaces (2)
- they are both possible virulence factors
2.
A. are water-soluble polymers of either ribitol (5C) phosphate or glycerol (3C) phosphate that are covalently linked to peptidoglycan. Polysaccharides. PHOSPHODIESTER LINKAGES
B. are teichoic acids which terminate in a fatty acid that serves to anchor the molecule in the plasma membrane.
1. TAs (teichoic acids) and LTA (lipoteichoic acids)
- TA can cause sepsis with both G+ and G- (replace LPS in gram -)
2.
A. Teichoic acids (TA)
B. Lipoteichoic acids (LTA)
248
Identify 4 gram negative - cell envelopes key features
- 2 things in between 2 membranes
1) Unique OUTER MEMBRANE which contains LPS
(outermost layer)
(2) THIN PEPTIDOGLYCAN overlaying plasma membrane
(3) Periplasmic space
(4) INNER (PLASMA) membrane
** Gram negative more complex ; has 2 lipid bilayes (outer and inner membrane - with thin layer of peptidogycan and periplasmic space sandwiched between the 2 lipid bi-layers
249
Identify struture
1. found in gram negative outer membrane (outer leaflet)
2. sometimes called an endotoxin. Why?
3. what 3 proteins are also present in outer membrane
4. Structure is an amphipathic macromolecule that consist of what 3 covalently linked structures?
1. LPS - lipopolysaccharide
* * Outer Membrane is a lipid bilayer composed of phospholipids and LPS
2. LPS is sometimes called endotoxin because
of its potent biological/clinical activities
3.
- Porins; allow passive diffusion of small charged molecules
- Braun’s lipoprotein; attach outer membrane to thin peptidoglycan
- omp proteins; stabilize outer membrane (specific receptor attach bacteria to host).
4.
- lipid A (hydrophobic)
- Core
- O antigen (hydrophilic)
250
LPS is an amphypathic macromolecule present in the outer leaflet of outer membrane. LPS consists of 3 covalently linked sections
Identify 1
- glucosamine
- beta hydroxymyristate
- fatty acids
- hydrophobic why?
- sugar backbone with?
- responsible for what activity?
1. Lipid A
• Hydrophobic so it anchor LPS to outer leaflet
• Sugar backbone with fatty acid (Lipid A is a phosphorylated disaccharide with esterified fatty acids)
• Responsible for ENDOTOXIC ACTIVITY
251
LPS is an amphypathic macromolecule present in the outer leaflet of outer membrane. LPS consists of 3 covalently linked sections
Identify 2
- KDO (ketodeoxyoctonate)
- phosphoethanolamine
- Heptose
- Glucose, galactose
- N-acetylglucosamine
- how many sugars?
- KDO structure?
- bottom and top parts linked to what?
**functions?
CORE polysaccharide
* contains 9-12 sugars
* KDO - unusual 8 carbon ring (may have additional sugar attached) - 2-keto-3-deoxyoctulosonic acid
* Bottom part linked to LIPID A
* Top pat linked to O ANTIGEN
****Core polysaccharide essential for LPS STRUCTURE and BACTERIAL VIABILITY (Panel C)
252
LPS is an amphypathic macromolecule present in the outer leaflet of outer membrane. LPS consists of 3 covalently linked sections
Identify 3
- hydrophilic
- polysaccharide chains; repeating units of species-specific monosaccharides e.g galactose, rhamnose, mannose and abequose in S typhimurium LPS
- is it the top most part or bottom?
**Function?
O ANTIGEN
- Top most path
- long linear polysaccharide (50-100 repeating units0 attachéd to the core
• Form basis for serotyping of bacterial strains so used for bacterial identification ( panels A and B)
253
1. What bacteria component is one of the most powerful immune stimuli
2. Clinical effects? (6) - how?
3. DIC results from severe cases of what bacteria type?
1. LPS
2. LPS is shed into bloodstream during bacteremia
and has a number of biological activities
A. Sleep (somnagen)
B. Fever (pyrogen)
C. Leukopenia
D. HYPOGLYCEMIA
E. Hypotension and shock (can be severe and
prolonged),
F. Because high levels of LPS activate a complement
cascade, disseminated intravascular coagulation
(DIC) can occur and in severe cases death from
organ failure.
3. DIC - Gram negative bacteria (LPS only found in gram negative)
254
Identify the 2 bacteria components that are AMPHIPATHIC
1. Induce synthesis of IL1, TNF and other cytokines - some are PYROGENIC. Also activate COMPLEMENT and COAGULATION
2. Causes COAGULATION but NOT FEVER
1. LPS (lipopolysaccharide)
| 2. LTA (lipoteichoic acid)
255
Identify other outer membrane structures and function (in gram negative bacteria cell envelope)
1. protein complexes that form channels that
allow passive diffusion of small (less than 600 MW)
CHARGED molecules.
- Found where?.
2. covalently anchors outer membrane
to peptidoglycan. (shown as coils)
- found where?
3. Stabilize outer membrane and act as specific receptors that allow attachement of bacteria to host
1. Porins
- Found only in OUTER MEMBRANE of g- bacteria.
• Porin is an essential protein - allows small charged nutrient molecules into gram negative cell (to get to outer membrane?)
2. Braun lipoprotein
• Allow for attachment of gram negative peptidoglycan to outer member (inner leaflet)
- found in INNER LEAFLET OF OUTER MEMBRANE
3. OMP proteins
• OMP (outer membrane proteins); specific receptors allow for attachment of bacteria to host (infection)?
256
Identify 6 functions of outer membrane (only in gram negative)
(a) serve as PERMEABILITY BARRIER to hydrophobic or large molecules (like lysozyme and some antibiotics) - block their entrance into bacterial
(b) maintain STRUCTURAL RIGIDITY of the bacterial cell
(c) protects peptidoglycan and plasma membrane
• Host make lysozyme that degrade the peptidoglycan - so outer membrane protect peptidoglycan
(d) SIEVE for small water-soluble molecules (Porins)
(e) presents sites for HOST CELL and PHAGE ATTACHMENT (omp proteins)
(f) possesses LPS and other molecules that serve as
MOLECULAR SIGNALS to host organism
257
Identify structure (Be specific)
- thin layer (1-2 layers)
- Glycan (M-G-M-G-M) polymer is cross-linked via
direct covalent bond between terminal D-ala of one
tetrapeptide and either lysine or DAP of another
tetrapeptide
- NO PENTAPEPTIDE/PENTAGLYCINE BRIDGING
- less crosslinking so more porous than counterpart
- floats within periplasmic space
GRAM NEGATIVE PEPTIDOGLYCAN
258
Identify structure (Be specific)
- thick layer
- PENTAPEPTIDE/PENTAGLYCINE BRIDGING
- more crosslinking so less porous than counterpart
- no periplasmic space
GRAM POSITIVE PEPTIDOGLYCAN
259
1. How are contents of periplasm released in gram negative bacteria?
2. Perplasmic space consists of variety of what enzymes? (Give examples)
3. Gram negative pathogens also have what enzymes - virulence factors that cause pathogens (3)
4. Is plasma/inner membrane of G- similar to plasma membrane of G+?
1. By SHEDDING OF OUTER MEMBRANE
2. HYDROLYTIC ENZYMES ( proteases, lipase, nucleases and components of sugar transport systems)
3.
- Collagenases
- hyluronidase
- beta lactamases
4. YES
260
SUMMARY OF DIFF - identify the differences between G+ and G- which the following features
1. Outer membrane
2. Peptidoglycan
3. LPS
4. Endotoxic Properties **
5. Teichoic acids
6. Spore formation
7. Exotoxins production - virulence factors**
8. Capsule - help evade phagocytosis
9. Lysozyme
1. Outer membrane
G- PRESENT
G+ ABSENT
2. Peptidoglycan
G- THIN
G+ THICK
3. LPS (outer leaflet of outer membrane)
G- PRESENT
G+ ABSENT
4. Endotoxic Properties **
G- PRESENT (LPS)
G+ SOME (LTA)
5. Teichoic acids
G- ABSENT
G+ PRESENT
6. Spore formation
G- NONE
G+ SOME STRAINS
7. Exotoxins production - virulence factors**
G- SOME STRAINS
G+ SOME STRAINS
8. Capsule - help evade phagocytosis
G- SOME STRAINS
G+ SOME STRAINS
9. Lysozyme;
G- RESISTANT**** - OUTER MEMBRANE protect destruction
G+ SENSITIVE
261
How are bacterial species classified (2)? Part 1
1. E.g Cell wall and lipid profiles
2. E.g Growth on carbon sources and enzymes
Other 3 ways?
3. Rapid ID without growing the organism
4. Binding to specific receptors in bacterial cell wall
5. Basic stuff (color, shape,size)
**are these methods phenotypic or genotypic
1. Cell structure
A. cell wall: gram + or gram –
B. Lipid profiles (LPS, LTA and mycolic acids)
2. Biochemical typing (e.g. growth on carbon
sources, enzymes +/-)
A. Allows subdivision below species level
B. API strips
C. Oxidative enzymes: e.g. catalase + or -
3. Serotyping , antibodies to specific antigens (e.g O157/H7)
A. Allows rapid identification without growing the organism
B. O157 refers to the g- O antigen; H is a flagellar antigen
4. Phage typing - susceptibility to lysis by specific bacteriophage
- binding to specific receptors in bacterial cell wall
5. Morphology - color/shape
***PHENOTYPIC - by general appearance
262
Identify genotypic classification of bacteria (3)
1. Southern blotting
2. Restriction enzyme
3. Phylogenetic analysis
**what do phylogenetic trees tell you?
1. DNA hybridization (e.g. Southern blotting)
2. Ribotyping (restriction enzyme analysis of
16S rRNA genes)
3. Sequence Analysis of 16S rRNA
most reliable for phylogenetic analysis
**
- 16S rRNA gene has been sequenced in many
organisms.
- 16S sequences are compared and phylogenetic
trees are built based on the number and types of
sequence differences.
- Phylogenetic trees tell how closely organisms
are related. • Structures with minor differences close together but the ones fa away have many differences
• Distance apart show evolutionary relatedness/changes
263
Identify 4 kingdom classification scheme
**what group does pneumocystis jirovecii belong to?
1. Kingdom Planta:
2. Kingdom Animalia:
3. Kingdom Protista: eukaryotic + unicellular (Protozoa, algae, fungi and slime molds)
4. Kingdom Prokaryota: all bacteria
- eubacteria has the most pathogens in this family
***Pneumocystis jirovecii - fungi (kingdom Protista)
264
Bacterial virulence factors are in what 2 broad classes of function ( 3 ways each)
1. Promote colonization and survival within host
- adherence
- invasion
- survival
2. Cause damage to host cells
- endotoxin
- exotoxins
- superantigens
265
Bacterial virulence factors - 2 broad classes
One is to promote colonization and survival within host by - adherence, invasion and survival
* *Identify 3 structures that do adherence (with examples)
1. Rod-shaped strutures.
- Tip mediates attachment by BINDING to a HOST RECEPTOR (either glycoprotein or glycolipid)
2. Bacterial surface proteins which mediate INTIMATE CONTACT with the HOST CELL (tightly bind to bacterial cell)
3. Formations of dense multiorganism layers on a surface. Significant in catheters, implants etc
1. Pili (fimbriae) ; e.g neisseria fimbriae bing GD1 ganglioside on host
2. Adhesins
- teichoic acids in gram positives
- streptococcus pyrogens adhesins (F protein) mediates brining to fibronectin on the host cell surface
3. BIOFILMS - grow on CATHETERS (can become resistant)
- Especially significant in implants, catheters, or indwelling devices.
- E. coli causing cystitis (bladder infection) in hospitals (via catheters) and Staph. epidermidis colonization of heart implants are serious clinical problems.
266
Bacterial virulence factors - 2 broad classes
One is to promote colonization and survival within host by - adherence, invasion and survival
1. What is induced phagocytosis?
2. Is intracellular existence desirable?
3. What is surviving phagocytosis
4. How does bacteria escape from phagocytic vesicle
5. **summary 3 ways of invasion and intracellular response
1. Entry into cells that are not naturally phagocytic (induced phagocytosis).
- Bacterial surface proteins (invasins) induce rearrangements in the actin cytoskeleton.
- This causes the formation of pseudopod-like
structures, which mediates bacterial engulfment.
2. Intracellular existence is desirable…
3. Surviving phagocytosis
- some bacteria produce catalase + SOD and have lysozyme resistant cell wall
4. Escape from phagocytic vesicles:
- Some pathogens are able to survive ingestion by
macrophages.
- Involves escape from the phagocytic vesicle +
advantages associated with an intracellular life
- A bacterial protein DISRUPTS the VESICLE MEMBRANE
to allow escape or it will form a pore.
5.
A. Entry into cells that are not naturally phagocytic (induced phagocytosis)
B. Surviving phagocytosis
C. Escape from phagocytic vesicles (by disrupting phagocytic vesicle)
267
Bacterial virulence factors - 2 broad classes
One is to promote COLONIZATION AND SURVIVAL within host by - adherence, invasion and survival
1. What metal is needed for oxidative phosphorylation?
2. This metal is present in low concentrations in the host. Identify 3 ways bacteria can overcome this limitation
**WHAT liberate intracellular nutrients like this metal?
1. IRON
2.
- Siderophores/enterobactins chelate iron effectively and bring it inside bacteria cell.
- Some bacteria have receptors for transferrin, lactoferrin etc
- CYTOTOXINS which damage/kill host cells act to liberate intracellular nutrients, including iron.
**Iron is an essential nutrient for bacteria but availability of iron is limited by role of lactoferrin, transferrin, ferritin, hemin.
268
Bacterial virulence factors - 2 broad classes
One is to promote COLONIZATION AND SURVIVAL within host by - adherence, invasion and survival
1. How do bacteria evade the immune system (2)
- what covers the cell helps bacteria evade complement/phagocytes
2. How do they evade the antibody response (2)
- carmelion xter lol
- coat bacteria with what?
1.
- CAPSULES and SLIME LAYERS cover entire cell; they are Network of polymers that helps bacterial cell evade complement and phagocytes.
2.
A. Antigenic switching; bacteria can change how they look
- H1/H2 pilus switching in Salmonella
- antigen variation in treponemes
B. Protein A of S. aureus and protein G of S.
pyogenes bind Fc portion of Ab
- coats bacteria with nonfunctional (nonimmunogenic) host protein
269
Virulence factors that damage host cells
1. Identify Toxic bacterial protein
- many (but not all) are secreted into
surrounding medium
- Some remain in periplasm.
-what the contrast of this?
2. Identify 3 major types
1. EXOTOXINS
Contrast with endotoxin = LPS in outer membrane of gram negative bacteria.
2.
A. AB toxins
B. Membrane disruptors / Hemolysis (pore formers)
C. Superantigens
270
Identify exotoxins type
- 2 subunits
- which subunit promotes binding to host cell
- which has enzymatic activity
**how do toxin enter? (2)
AB TOXINS
- B subunit promotes binding to the host cell
- A subunit has enzymatic activity
- Example: diptheria toxin ADP-ribosylates host EF-2
protein and shuts down protein synthesis
**Toxin entry through MEMBRANE TRANSLOCATION or ENDOCYTOSIS
271
IDENTIFY exotoxins type
1. Pore formers that allow water to enter and ions to escape
2. Give example (how does it work?)
- remove polar head from hydrophobic tail so it will destabilize the membrane
1. Cytolysins
- Cholesterol often the target
2. Phospoholipase
- Cleave hydrophobic portion away from charged
head group
- Destabilize membrane and cell lyses
**they are the 2 membrane disruptors (pore former and phospholipase)
272
Perturbation of Host defenses ; In some cases, host disease is not caused by virulence factors intrinsic to
the infectious agent, but result from the microbe’s ability to alter host
immune functions.
Give 2 examples
Host disease is caused by microbes ability to alter immune response;
1. Autoimmune activation; immune system responding to protein see it as a pathogen and attack the host cells e.g guillian barre syndrome
2. Elaboration Superantigen; toxic shock. They bind to receptors on T cells to activate it and elaborate cytokines.
273
Virulence factors
- effect of endotoxin is same as effect of what bacterial component?
LPS
274
IDENTIFY
1. Specific nutritional requirements for microbial growth? (6)
CNOSGT
1. carbon
2. nitrogen
3. oxygen
4. sulfur
5. growth factors
6. trace ions
CNOSGT
275
Identify Nutritional requirements of some bacterial species
1. Glucose, NH4+, MG2-, Mn2-, sulfate, Po4(3+) and other trace ions
2. Nothing extra - just basic nutrients
3. Tryptophan
4. Nicotinic acid, Thiamine, 10 amino acids
5. Nicotinic acid, Thiamine, Pantothenic acid, Pyridoxal, Riboflavin,
Cobalamin, Biotin, Folate, Guanine, Adenine, Uracil, 16 amino acids
6. Diff between nutrients needed for prototrophs vs auxotrophs
1. Basic nutrients/media
2. E. Coli
3. Salmonella typhi
4. Staphylococcus aureus
5. Leuconostoc paramesenteroids
6.
• Prototrophs - no additional requirements for growth (just basic media) - carbon, nitrogen and sulfur
• Auxotrophs - need additional requirements like vitamins, amino acids etc for growth
276
How are large and small molecules taken up in bacteria types/
- what covers bacterial cells
- macromolecules are good source of ?(3). They are too large to enter bacteria cell so what enzyme breaks them down?
- A selectively permeable envelope covers g+
and g- bacterial cells.
- Some small molecules are freely diffusible
while others require a specialized system for
uptake.
- Macromolecules in the environment are a good
source of carbon, nitrogen and energy.
Too large to enter… so exoenzymes break them
down
277
How do bacterial extracellualr proteins like exoenzymes/exotoxins and other virulence factors get outside/exterior of the cell
Aka what barriers do they have to pass through
G+ vs G-
They have to cross several barriers
1. Gram positive (cross cell membrane)
• Move from cytoplasm - cytoplasmic CELL MEMBRANE - peptidoglycan
2. Gram negative (cross inner and outer membrane)
• OUTER MEMBRANE - periplasm - INNER MEMBRANE
278
Identify system
- transports proteins to the gram positive
extracellular space or to the periplasm of gram
negative organisms.
- Gram negatives need a second system…
**location? How does it recognize precursor protein?
What does precursor protein bind to?
GSS - General Secretory System
GSS complex located in CYTOPLASMIC MEMBRANE recognizes precursor protein with SIGNAL PEPTIDE
Precursor protein passes through the complex
Signal Peptide is cleaved off and protein folds
into final shape
**precursor protein bind to sec-yeg
***anything that use GSS is called sec-dependent
279
What secretion types are sec dependent vs independent
1. Sec dependent; SecYeg move proteins from cytoplasmic space to the periplasmic space (Type II and V)
A. Type II
B. Type V
2. Sec independent; • Don’t use Sec Yeg - have their own protein complex (Type I, III, IV)
A. Type I
B. Type III (injectisome)
C. type IV
280
Secretions***
1. What type systems can transport proteins to the outside of the cell or inject them into host cell cytoplasm
2. Some of these systems require the GSS to deliver protein to
periplasm.
3. Others are multiprotein complexes that span the inner and
outer membrane.
4. acts like a molecular syringe which injects
effector proteins into the host cell cytoplasm upon contact
with the host cell surface.
Many effector proteins: invasins, phagocytosis inhibitors
1. Types I, II, III, IV, V, IV (gram negative)
2. Sec-dependent (Type II and V)
3. Sec-independent (type I, III, IV)
4. Type III system (present in Salmonella, Shigella, Yersinia
and Pseudomonas)
281
1. Why are some exoenzymes virulence factors?
3. Give 3 examples ***
- cytolysin
- exfoliative toxin
- lecithinase
1. because they
improve organism’s ability to INVADE, COLONIZE,
MULTIPLY and/or CAUSE DISEASE in the host. (receptors that bind to host cell, hemolysins, lipases, toxins)
2.
- Streptococcus; cytolysin
- Staphylococcus; exfoliative toxins
- Clostridium spp.; lecithinase
282
Identify transport system
Few molecules enter the cell passively.(give eg)
Low concentration nutrients…
Most substrates enter cell via specific transport mechanisms (against concentration gradient).
Examples: sugars, trace metal ions, vitamins,
amino acids
**IDENTIFY - energy independent CHANNELS that small molecules go through?
- most by Small molecule transport systems (porins?)
- few by diffusion (water, CO2, O2)
**Porins
283
Identify transport mechanism
glycerol
- Is it energy independent?
- do you need energy at any point?
FACILITATED DIFFUSION: Transport is mediated by
the glycerol facilitated protein found in the cytoplasmic/inner membrane:
- ENERGY INDEPENDENT, but subsequent steps may use
ATP.
- ATP-DEPENDENT kinase (hexokinase) catalyzes phosphorylation of glycerol (putting a CHARGE on it) and effectively
trapping the molecule inside the cell. (USE ENERGY)
284
Identify transport mechanism
- Most uncharged molecules larger than glycerol (lactose, galactose, glucose) and all ionized molecules
**How do substrate move? (3 types of transport)
Active transport
- In active transport systems, substrates are
moved against concentration gradient and
therefore ENERGY must be expended.
- Three general mechanisms: proton gradient active transport, ATP dependent active transport, group translocation
285
IDENTIFY transport mechanism
1. LACTOSE (lac Y permease)
- where is energy derived from?
- do they use more energy than group translocation?
- permease is found where
- is it shock sensitive?
2. GALACTOSE
- shock sensitive? Why or why not?
- more or less energy than group translocation?
1. Proton gradient active transport
• Move LACTOSE form periplasm into cytoplasm (part of lac operon)
- Required energy is derived from pumping of protons
- Uses more energy than group translocation.
- lacY permease found in cytoplasmic/plasma membrane
- SHOCK INSENSITIVE
2. ATP- dependent active transport
- Galactose transport is osmotic SHOCK SENSITIVE
because gal protein is in the periplasmic space.
Also uses more energy than group translocation.
286
Identify
• Type of active transport for sugars e.g GLUCOSE
Works by trapping a sugar-phosphate within the cell.
- consists of a complex of phosphotransferase
proteins (Enzyme I, Enzyme III and Enzyme II) which
transfer phosphate groups from PEP ultimately to sugar
as it crosses the cytoplasmic membrane
* *ONLY FOUND WHERE?
- TRANSFER PHOSPHATE GROUP from what to what?
- is it energy efficient? Why?
- how is phosphate group transferred (4)
- what membrane transported move glucose into cell in G6P form?
2. What are other functions of this transport mechanism? (2)
- what is key regulator in glucose effect
- PTS interact with ? So cell swim to nutrient
Group translocation (PTS)
- AKA PEP: Sugar Phosphotransferase system (PTS) a mechanism of the transport of glucose and other sugars.
-Only found in bacteria systems
• allows for effective uptakes of sugars - trap sugar-phosphate within the cell. PTS complex transfer phosphate groups from PEP to sugar
• TRANSFER PHOSPHATE GROUP from Enzyme I - HPR protein - Glc III (play role in glucose effect as KEY REGULATOR) - Glc II (membrane transporter that move glucose into cell in form of glucose 6 phosphate - more ENERGY EFFICIENT caus eyou make glucose 6 phosphate)
2. Other functions
- PTS components interact with chemoreceptors to tell
the cell to “swim” towards nutrients.
- PHOSPHORYLATED-IIIglc component of PTS serves as
key regulator in the glucose effect
287
SIDEROPHORES AND IRON UPTAKE (HOW?)
- action of siderophores?
- 2 types
- is siderophores a virulence factor?
• Siderophores chelate iron between carbonyl group and nitrous group?
A. Siderophores are macromolecules that bind iron in
the medium and transport it inside the cell.
B. Two types: a) ENTEROBACTIN (enterochelin) and b)
HYDROXAMATE (shown below).
C. Fe+3 is scarce in host tissues. So bacterial siderophores
compete for Fe bound to host lactoferrin and transferrin.
D. Neisseria have receptors for human siderophores (e.g.
transferrin).
E. Siderophores are one kind of virulence factor
288
1. All the genetic material of a bacterial cell is called?
2. Consist of 2 things
- single covalently circular molecule? plus any small self replicating molecule?
1. Genome
2.
A. Bacterial chromosome; single covalently circular duplex molecule
B. Plasmids; smaller self-replicating molecules
289
Arrange genome size of 5 organisms (humans, 2 virus, 2 bacteria, yeast, plasmid) from most to least genome size.
* *which has less repeated genes? Humans or bacteria??
* *Give 2 examples of repeated DNA
4. WHAT are SNVs? Use?
1. humans have the most (3x10^9 bp).
Most to least is; humans > yeast > Bacteria (B. Subtilis > mycoplasma >vaccinia virus> SV40 virus > plasmids.
2. Bacterial genomes contain very little repeated DNA, greater
than 99% is unique DNA.
3. Examples of repeated DNA:
rDNA genes and transposable elements.
4. SNVs; mutations occur randomly over generations and include single nucleotide changes called single nucleotide variants (SNVs)
- used to identify pathogenic subspecies or strains
290
Identify
- what has the ability to encode virulence factors (cluster of genes that contribute to virulence) **Give 5 examples that cause virulence
-
- are they mobile? Can they be transferred from species to species?
**Found mainly in what bacteria stain type?
PATHOGENICITY ISLANDS
- Clusters of genes that contribute to virulence
A. EXOTOXINS (Ab toxin, superantigen, hemolysis)
B. IRON UPTAKE (siderophores)
C. ADHESINS (TA)
D. INVASINS (induced phagocytosis), and
E. SPECIALIZED SECRETION PATHWAYS
- 10-200 kb region embedded in main chromosome
- Can be transferred from species to species
- P.I.’s are probably mobile elements
- So organisms can acquire whole set of virulence genes
in one step.
- Found mainly in GRAM NEGATIVE
291
1. What makes up the central dogma (prokaryotic version)
2. XTERS of bacterial chromosome replication
- fast or Slow?
- error? Why or why not?
1.
A. DNA replication (DNA - DNA)
B. transcription (DNA - RNA)
C. translation (ribosomes make proteins - initiation, elongation, termination)
2. rapid, few mutations in each generation (fidelity - through proofreading mechanism - reduce error by 10 fold)
- travel RAPIDLY and make ACCURATE copies
- low mutation rate (1 in billion base pairs); few mutations/variants in each generation
- DNA POLYMERASES accomplish fidelity through PROOFREADING MECHANISMS
292
Identify major features of chromosome replication
1. each daughter duplex DNA molecule is composed of one parental strand and one newly-synthesized strand.
- similar to what replication?
2. single site on chromosome where all DNA
replication is initiated.
3. Two replication forks begin at oriC and travel in
opposite directions around the circular chromosome, synthesizing the daughter strands as they progress.
4. required for DNA synthesis
- what polymerase and proofreading activity to they all share? Why?
5. What occur in the bacteria chromosome to allow for unwinding - enable replication and transcription (2)
1. Semi- conservative replication
- like eukaryotic replication
2. Single origin of replication (oriC)
- where replication starts
3. Bidirectional replication
4. DNA polymerases (DNA Pol I, II and III)
- All bacterial polymerases share 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease (proofreading) activity which is necessary for maintaining a lower error rate.
5. Supercoiling - negative helicity
293
1. Explain the concept of bacteria chromosome supercoiling. Why is it important?
- is negative superhelicity required? Why?
2. What abx target this mechanism? What is the result?
1. Bacterial chromosome has negative superhelicity which means that is can readily convert to a molecule with unwound helical regions.
** Negative superhelicity is absolutely required for DNA
replication and transcription.
2. QUINOLONES ; block introduction of negative supercoils
- block replication and cell growth
294
What are the steps in which DNA replication and cell division is coordinated
* *Image
- chromosome attached to?
- what 2 events coincide?
- what happens to chromosome as wall growth elongates cells?
1. Bacterial chromosome is attached to cytoplasmic membrane protein complex.
2. Initiation of chromosome replication coincides with
septum formation
3. As wall growth elongates cells, chromosomes are pulled into daughter cells
295
1. Identify process
- a functional polyribonucleotide tract (RNA) complementary to one of the DNA strands is synthesized.
* *DNA - RNA
2. Name 4 types of RNA molecules in a cell
A. Component of ribosome. Stability?
B. Translational adaptor molecules. Stability?
C. Script for translation. Short or long half life? Stability determines what?
D. Non-coding RNA
3. What single enzyme transcribes all 4 types of RNA in prokaryotes
1. TRANSCRIPTION
- how RNA is made (they have short half live and quick turn over)
2.
A. rRNA, 25S, 16S, 5S serves as components of
ribosomes (very stable).
B. tRNA, 30-40 different species, translational
adaptor molecules (very stable).
C. mRNA, 1000 - 3000 different species, serve as a
"script" for translation, short biological half-life
(T1/2=~minutes).
***mRNA stability partly determines the final abundance of
its protein product
D. microRNA (sRNA)
3. DNA-DEPENDENT RNA POLYMERASE
296
In prokaryotes, single enzyme, DNA-
dependent RNA polymerase transcribes all
four types of RNA - Called transcription apparatus.
1. IDENTIFY Auxiliary subunits of the holoenzyme (2)
1.
A. SIGMA (σ) FACTOR; help RNA polymerase bind to PROMOTER
- imparts initiation site specificity to holoenzyme, so that holoenzyme recognizes - 10 and -35 positions of the promoter.
B. RHO PROTEIN assists in transcription TERMINATION
- also serve as binding site for sigma factors
297
Mechanism of transcription
** RNA polymerase (RNAP) always synthesized RNA in what direction?
1. What bind to core polymerase? What is created?
2. Holoenzyme binds to specific sequences at what end?
3. What does RNAP holoenzyme do first to start synthesis of RNA?
4. Transcription end at what site?
5. RNAP transcribes all RNA types in prokaryote. Is this true for eukaryotes?
* *what is the sequence found most frequently in a survey of a large number of sequences?
- does increase in this sequence result in stronger promoter?
- What do weak promoters require?
**RNAP make RNA in 5’ to 3’ direction
1. σ subunit binds to core polymerase to create holoenzyme.
2. Holoenzyme binds to specific sequences at the 5' end of a gene: -35 and -10 promoter sequences
3. RNAP holoenzyme unwinds ds DNA to ss DNA and begins synthesis.
4. Transcription ends at sites called terminators.
5. NO.
**CONSENSUS SEQUENCE
- Promoter strength increases with increasing homology to the
consensus sequences. Weak promoters may require
ACTIVATOR PROTEINS and be regulable.
298
Identify antibiotics that inhibit transcription (and the stage)
1. binds to ß subunit of RNAP and inhibits the formation of the first
phosphodiester bond.
- used to treat what?
2. Blocks elongation + possible use in TB
3. Can’t use for microbial infection (too toxic for clinical use) - some use in cancer chemotherapy
* *how does it work?
1. Initiation - Rifampin
- synthetic version of RIFAMYCIN
- Used to treat disease caused by MYCOBACTERIA and by GRAM POSITIVE bacteria.
2. Elongation - STREPTOLYDIGIN
3. Elongation - ACTINOMYCIN D
- binds to double-stranded DNA and blocks the movement of RNAP holoenzyme.
- Can also inhibit DNA replication at higher concentration.
299
Machinery for translation
1. Identify the 2 subunits for prokaryotic ribosomes and function of each
2. Give examples of soluble protein factors that participate in translation
3. What molecule is considered an an adaptor in translational process? (Give 2 functions)
1. 50S composition ; 23S rRNA, 5S rRNA + 32 proteins
- primary function; PEPTIDE BOND FORMATION
- (23S RNA = ribozyme = transpeptidase/peptidyltransferase)
2. 30S composition; 16S rRNA + 21 proteins
- play role in TRANSLATION INITIATION and has site for tRNA binding
**Soluble protein factors; IFs, EFs and RFs
3. Adaptor in translation is tRNA. Each tRNA serve 2 functions
A. Codon recognition
B. Amino acid delivery
300
1. Identify
Sets of three ribonucleotides in mRNA which
define a codon
2. How many letters/word (bases/codon) are
needed to generate enough words (codons) for
all amino acids?
3. 1 set of #1 can encode how many amino acids and stop codons?
4. What do you do with the spare codons?
1. Triplet code
2.
- four nucleotides take one a time = 4 combinations, 4 amino acids
two = 16 combinations, 16 amino acids
Three = 64 combinations, 64 amino acids
3. A triplet code is sufficient to encode at least twenty amino acids and three stop codons.
4. Make duplicate (degenerate) codons
- buffer against deleterious codons (protect against mutations in 3rd position)
301
Identify the 4 features of triplet code
1. Almost universal
- codon CUU code for leuceins in all organisms except mycoplasma, tetrahymena and mitochondria
2. Degerate; different triplet codes code for same amino acid
3. 3 stop codons; UGA, UAA, UAG
4. All translation begins at codon AUG - encodes methionine (N-formylmethionine)
- all bacterial proteins initially have N-formylmethionine at their amino terminus
302
Translation - Initiation
1. What bind to 30S subunit
2. Rate of translation initiation i controlled by what?
1. Step 1 is binding of mRNA to 30S subunit
2. RATE OF TRANSLATION INITIATION is controlled by a sequence present in the 5' nontranslated region of all
prokaryotic mRNAs.
- Ribosome Binding Site or Shine-Dalgarno sequence.
- More base pair (between shine-dalgarno sequence and 16S ribosomal RNA?) make binding/translation more efficient
3. SHINE-DALGARNO SEQUENCE
- S-D sequence has homology to sequence present in the 3' end of 16S rRNA but varies from gene to gene.
- Efficiency of translation initiation improves with increases in
homology between 16S rRNA sequence and a given mRNA
sequence.
303
Translation elongation
1. WITH ASSEMBLY of 70S ribosome, what 2 binding sites are formed?
2. What site is occupied at beginning of cycle?
3.
A. What is the transfer of peptidyl residue to charged tRNA in A site called?
B. Deacylated tRNA must leave P site first and peptidyl tRNA moves to P site. Process called?
C. Energy consumed in what step?
1.
- A site; Accepts incoming charged tRNA
- P site; site where Peptidyl tRNA sits
2. Beginning of each cycle - peptidyl tRNA sit in P site, A site empty
- step 1; charged tRNA enters A site
• Step 2 (transpeptidation); form new peptide bond between amino acid 1 and 2 (1 sits on 2 to generate new peptidyl molecule)
• Original peptidyl tRNA is deacetyylated in P site - ribosomes now position over codons 2 and 3 (no more 1 and 2)
• Step 3 - translocation (energy consumed); deacetylated tRNA leave P site first. Pepdityl tRNA vacate A site and go to P site and start over
3.
A. Transpeptidaton
B. Translocation
C. Translocation
304
Identify abx that block translation
1. block assembly of 70S initiation complex and cause misreading by ribosomes that have passed through initiation stage.
2. block the binding of charged tRNAs to the acceptor site.
3. binds peptidyltransferase and blocks the peptidyltransferase reaction.
4. Blocks translocation step
1. Streptomycin and other aminoglycosides (gentamicin, neomycin, amikacin)
2. Tetracyclines (doxycycline, minocycline)
- bock step 1 (charged tRNA going not A site)
3. Chloramphenicol
- block traspeptidation step 2 (transfer of peptidyl residue to charged tRNA in site A)
4. Erythromycin
- peptidyl tRNA stuck in A site cause it cant move to P site
305
1. When can translation begin is prokaryotes?
2. What is coupling?
3. differentiate coupling in eukaryotes vs prokaryotes
1. As soon as nascent mRNA chain appears from RNA polymerase holoenzyme
2. Coupling is when transcription and translation occur simultaneously
3. COUPLING ONLY OCCUR IN PROKARYOTES
- Coupling can only occur in prokaryotes because in eukaryotes, nucleus separates processes of
transcription and translation.
- Shine-Dalgarno sequence found only in
prokaryotes.
- Many polycistronic transcripts for prokaryotes,
very few in eukaryotes.
306
Identify 4 energy consumption steps
1. Active transport
2. DNA and RNA synthesis
3. TRNA charging
4. Peptide synthesis (initiation and elongation)
5. REGULATION OF GENE EXPRESSION IS IMPORTANT TO THE CELL
307
Regulation of gene expression
- the abundance of a protein can be controlled at what levels in prokaryotic cells (5)
E.g
* where lac/trp operons work
* where shine dalgarno sequence works
1. Gene copy number
2. Transcription initiation (lac and trp operons)
3. Stability of mRNA
4. Translation initiation (shine dalgarno sequence)
5. Protein stability
308
Identify the following sites in prokaryotic genes
1. DNA site at which regulatory proteins like repressors bind
2. -35, -10 site for RNA polymerase binding, required for accurate, high level initiation of transcription
3. Genes encoding proteins which are involved in cellular metabolism (enzymes) or which constitute cell structures
4. Set of genes under one control - make equivocal amounts of related protein products close together
5. multiple genes scattered around with the same regulatory elements - Same equimolar amounts scattered around
1. Operator (O)
2. Promoter (P)
3. Structural genes
4. Operon e.g ilvGMEDIA operon
- operons have polycistronic organization; one continuous mRNA spans several structural genes
- polycistronic mRNAs can be translated into several different proteins because prokaryotic ribosomes can bind to internal shine-dalgarno sites
5. Regulon
-Structural genes that participate in a single pathway are present at different sites in genome.
- contain similar controlling elements (promoter/operator) so that
they are expressed coordinately.
- Examples: arginine, maltose
and SOS regulons.
309
What are the 2 ways to control transcription initiation? What 2 proteins involved? 2 outcomes each?
1. NEGATIVE CONTROL always involves a REPRESSOR PROTEIN
A. Negative inducible; ligand inactivates repressor
B. Negative repressible; ligand activates repressor
2. POSITIVE CONTROL always involves an ACTIVATOR PROTEIN
A. Positive inducible; ligand activates activator
B. Positive repressible; ligand inactivates activator protein
310
NEGATIVE transcriptional controls use a repressor
1. What regulatory proteins acts to prevent transcription
2. In what 2 ways can #1 act? Is operon on or off in each way? Give examples of each
3. Give example of something that uses both POSITIVE INDUCIBLE and NEGATIVE INDUCIBLE control? Why?
4. What does trans-acting mean?
1. TRANS-ACTING REGULATORY PROTEINS acts to prevent transcription - a REPRESSOR is the key trans-acting component
2.
A. Negative inducible; operon is turned OFF until repressor is inactivated by inducer E.g lacZYA operon - turned OFF until there is no more glucose (prefer to use glucose over lactose) - lacI is the repressor which will then be inactivated by an inducer to TURN ON TRANSCRIPTION
B. Negative repressible; operon is turned ON until repressor is activated by corepressor to TURN OFF TRANSCRIPTION
E.g trp operon where trpR is repressor and trp is the corepressor
3. Lac operon
- you only want to make lactose when stuff is present ? - Turn off lactose until all glucose is used up - we prefer glucose so use glucose first. Cells then begin to induce lactose by help of lac operon
- Repressor sense lactose but need a system to turn off the lactose; relies on cAMP binding protein (CBP) and cAMP. the cAMP level is determined by the glucose level
4. Trans-acting means freely diffusible
311
Positive control - use activator protein
Describe the 2 mechanisms
1.
A. Positive inducible;
- no transcription because apoactivator is inactive
- coactivator is usually a small molecule that binds to apoactivator to yield a functional complex
E.g any CATABOLITE ACTIVATION operon where CAP is activated by binding to cAMP. Include lacZYA, araBAD, galETK operons
B. Positive repressible
- activator protein is functional in the absence of inhibitor so transcription will occur
- in presence of inhibitor, activator is inactivated and no expression occurs
313
What operon uses 2 controls?
* Identify the 2 controls
2. This system is a source of ?
3. What are the components of the system (5)
A. repressor gene
B. promoter
C. operator
D. encodes ß-galactosidase which cleaves lactose into
glucose and galactose; also can convert lactose to
allolactose, the true inducer.
E. encodes a permease which facilitates the uptake of
lactose into the cell.
F. encodes transacetylase which may detoxify lactose
metabolites.
Lac operon
1. Positive inducible
2. Negative inducible
* *Needs both switches to be “ON” position for full expression
2. Allows utilization of lactose as carbon source. **Lactose is a glucose-galactopyranoside
3. Components
A. LacI repressor; repressor gene
B. LacP; promoter
C. LacO binding site for lacI; operator
D. LacZ; encodes ß-galactosidase which cleaves lactose into
glucose and galactose; also can convert lactose to allolactose, the true inducer.
E. lacY; encodes a permease which facilitates the uptake of
lactose into the cell.
F. lacA; encodes transacetylase which may detoxify lactose
metabolites.
314
What 4 regulator scenarios occur from the 2 control systems (positive and negative inducible)
1. Glucose (B)
2. Glucose + lactose (C)
3. Lactose (D)
4. Other sugar (not glucose or lactose) (E)
1. Glucose only present; lac operon turned OFF. Both switched off in panel B. Transcription off
- high glucose - low cAMP - CAP inactive - no transcription
- no lactose - lac operon remains bound to operator - prevent transcription by RNA polymerase
2. Glucose + lactose; MOSTLY OFF (some lactose enter?). Transcription. Mostly off
- lactose present - lac repressor release (inactive) because allolactose/inducer is present - transcription
- glucose present - cAMP low - CAP inactive - no transcription
3. Lactose; ON both on in panel D. Repressor and cAMP not bound. High level transcription
- lactose present - lac repressor inactive (allolactose present) - transcription
- no glucose - high cAMP - CAP active - transcription
4. Other sugar; lac operon and glucose OFF panel E. Repressor of lac operon, cAMP for glucose all bound so transcription OFF
315
1. What is the glucose sensing mechanism?
- what is the role of PTS system in sugar metabolism
2. Describe how glucose levels affect; glcIII, cAMP levels and ultimately transcription
1. PTS components phosphorylate glucose as it crosses the cytoplasmic membrane
- PTS transports sugars like glucose and mannose across the inner membrane
2.
• When glucose is low, Glc III activated/phosphorylated so it is high - active adennylate cyclase - produce cAMP so high - lacZYA is transcribe in presence of lactose (so use lactose instead)
• When glucose is high, High levels of Glc III not phosphorylated - turn off S1 and S2 - low cAMP levels (use glucose instead of lactose).
3. need promoter for RNA polymerase to bind to lac promoter to do transcription??
316
1. What does IIIglc (nonphosphorylated) turn off?
2. How is cAMP level determined
3. Phosphorylate IIglc activates what?
4. What happens when cAMP-CAP complex binds to site in lacP (promoter)
1. Turns off nonPTS sugar transport systems like lac permease and maltose binding protein (glucose level is high)
2. Level of cAMP determined by;
- adenylate cyclase; converts ATP to cAMP
- phsphodiesterase; converts cAMP to AMP
3. Adenylate cyclase (low glucose - PIIIglc - adenylate cyclase - high cAMP - CAP active (cAMP-CAP complex binds to site in lacP) - transcription of lac operon)
4.
- Cause HELIX UNWINDING at downstream sites
- FACILITATES BINDING of RNA polymerase and will increase transcription of lac operon
317
Lac operon regulation summary
1. What is lactose sensor
2. What is glucose sensor
3. Identify the 2 component regulators
1. Negative inducible (lacI + allolactose)
2. Positive inducible (cAMP + CAP)
3.
A. 1st protein component; membrane bound sensor kinase - detect environmental signals (oxygen and osmolarity).
B. 2nd protein component; response regulator (activator protein)- transcriptional activator or repressor
**In bordetella pertusis, a 2 component system controls expression of a large number of virulence genes
318
1. Identify sensing
- Sets of genes are activated when [bacteria cell] reaches a threshold.
- Bacteria produce a signaling molecule (S) which bind to cell wall receptors and
transduce a signal to activate gene expression (designated as the extracellular R (Regulator) in the figure).
- Activation of genes involved in the production of biofilms by P. aeruginosa is classic example
2. #1 is a type of what regulation?
1. Quorum sensing
- Bacteria sense quorum and produce a signal to activate gene expression
2. Global regulation
- the regulation of multiple metabolic pathways by a single regulator e.g. cAMP binding protein or quorum sensing
319
1. Why do mutations occur? (As consequence of 3things )
2. What is Darwin’s view on the origin of mutations
3. What does resistant bacteria on a plate with antibiotics show? (3)
1. Error in replication
2. Direct damage to genetic material
3. Error made by mutation repair system
2.
- mutations occur in absence of selective agents (e.g streptomycin)
- resulting mutant strains either THRIVE OR DIE depending on the nature of mutation and the environment
- BACTERIA BECOME RESISTANT DUE TO MUTATIONS (before exposure to antibiotics like streptomycin)
- you can detect bacteria resistant organisms by plating them onto medium containing the antibiotic
3.
A. Which generation mutation occurred
B. How many mutation occurred
C. How many times mutants double
320
1. What was Lucia-Delbruck fluctuation test about? Did It support of refute Darwin’s view that bacteria get resistant/mutation spontaneously - even before exposure to abx?
2. What are 2 take home points from the fluctuation test
3. Who was the other person that has a different view to bacteria mutation origin? What was the view?
1. Luria Delbruck flunctuation test; if Darwin is correct, you should have spot mutations. Proved that bacteria mutations actually occur spontaneously as Darwin predicted.
2. 2 take home points;
A. Mutations happen spontaneously
B. Selection has the effect of increasing or decreasing some variants (some colonies can be resistant while others may not) - JACKSPOT MUTATIONS
3. Lamarckian expectation
- electives agents (streptomycin) directs the formation of mutants
- bacteria resistance/mutation is solely based on the ability of streptomycin to induce specific mutation
- CONSTANT MUTATIONS SHOULD BE FOUND
321
Single nucleotide polymorphism (SNPs) aka single base changes occur in what 3 types of mutations
1. SILENT mutation; single base change in coding region (usually 3rd position) but NO AMINO ACID CHANGE - degeneracy of code
2. MISSENSE EVERY mutation; any change that cause SINGLE AMINO ACID SUSTITUTION in a peptide.
* Algorithms predict severity/pathogenicity of substitution
3. NONSENSE mutation; convert triplet code to stop codon
322
Identify the mutation types
1.
- Premature stop codon
- Insertion or deletion of any number of base pairs not
multiples of three.
- change context and often result in a
premature stop codon because of the abundance of
naturally occurring stop codons in the alternative
reading frames.
2. Transposable elements can insert at random sites in the genome
3. Represent loss of large segments of the chromosome
1. Frameshift mutation
2. Insertions
3. Large deletions
- large deletions are not revertible
- deletions can be caused by transposons
323
Summarize the 4 DNA repair mechanisms (found in both prokaryotes and eukaryotes)
* are they inducible?
- which have errors? No errors?
- which types are postreplication repair?
- which is induced by light? (1)
- which is induced by damage (2)
- which uses excinuclease and DNA ligase? Function of each?
- which uses photolyase? Induced by? Cleaves what bonds?
4 systems in prokaryotes and eukaryotes - most are inducible
1. Photoreactivation; pyramidine dimer is formed which lead to DNA damage. LIGHT induce enzyme called PHOTOLYASE which cleave bonds (cyclobutane ring) between the pyramidine dimer and restore the base. NO ERRORS because you restore original bases
2. Excision repair; dark repair (induced by damage not light). EXCINUCLEASE Recognize mispaired base that lead to improper hydrogen bonding - introduce nicks on both sides. After all that DNA LIGASE seals the nick. ERROR FREE (DNA polymerase is repairing and using proofreading mechanism - step 5)
* *Post replication repair - 3 and 4
3. Recombination repair; daughter-strand-gap repair. Thymidine dimer in one strand of duplex - cause damage in a daughter chromosome - gap forms in damaged region - move good piece from other daughter strand to the bad strand. **Dont really remove the damage but allow daughter cells to have intact strands (DNA polymerase covers the gap). ERROR FREE (DNA pol 1 proof reads)
4. Dimer bypass; similar to excision repair in that it is induced by damage (SOS). DNA pol III RANDOMLY inserts any bases in damaged daughter strand. ERROR PRONE (mutagenic) - advantage is that daughter cell gets completely closed
* *Both 3 and 4 are post replication repair
324
Why is recombinant repair error proof?
Recombination repair is said to be
error-proof because no incorrect bases are
incorporated in the patching process and
because of the high fidelity of DNA pol I.
325
Introduction of DNA repair systems
1. Identify the 2 trans-acting regulatory elements
A. Turns on damage repair
B. Cleaves and inactivates A above
2. This is an example of what regulation?
1.
A. LexA protein; repressor of the SOS regulon which includes din genes, uvrABC, umus and recA itself. TURN ON DAMAGE REPAIR
B. RecA protease; activated by DNA damage (ss regions, nicks, adducts).
- RecA protease then cleaves and inactivates lexA repressor protein
- Cleavage results in induction of genes in SOS regulon (inc recA gene)
- recA protein also plays an essential role in recombination repair.
2. REGULON AND GLOBAL REGULATION
326
GENETIC VARIATION is a consequence of what 2 things?
**which of the 2 is the more potent source of genetic variation?
1. MUTATION
| 2. RECOMBINATION - more potent source
327
Identify the 2 types of genetic recombination
1.
- Rec A dependent (also required recBCD proteins)
- must be DIPLOID or at least have transient diploid stage. All prokaryotes are haploid but gram + and - can form partial diploid by genetic exchange (transformation, transduction and conjugation)
2.
- Rec A independent
- diploid not required
- usually require specialized recombination enzymes (give 2 e.g)
1. Homologous recombination
- Rec A DEPENDENT exchange of genetic information between 2 genomes (chromosomes) with identical or nearly identical sequences
2. Site-specific recombination
- Rec A INDEPENDENT exchange of information between genomes with limited sequence homologous
- usually requires specialized recombination enzymes; TRANSPOSITION and PHAGE LAMBDA INTEGRATION
328
What are the biological consequences of recombination?
Aka result of mutation vs recombination
1. Mutation
- After a mutation event, the resulting mutant strain only differs at one or perhaps a FEW GENETIC LOCI from the parental strain.
2. Recombination
- After genetic exchange between two related organisms, the resulting progeny can differ from the parents at MANY GENETIC LOCI.
329
1. Describe the negative regulation of lac operon
2. Describe the concept of diauxic growth or sequential use
3. Describe the positive control of lac operon
- what determines cAMP level?
1.
- In glucose medium, lac repressor binds to lacO.
- Bound repressor overlaps lacP and physically blocks the progression of RNA polymerase. No transcription of lacZYA.
- In lactose medium, initially low level of lacZ gene product converts lactose to allolactose which causes a conformational change, so that the repressor can no longer bind to the operator.
- lacZYA can now be transcribed and translated at high levels, PROVIDED cAMP LEVEL IS HIGH.
2.
- Given a choice, E. coli preferentially metabolizes glucose
- After glucose is consumed, lactose metabolism is initiated.
- Since cells prefer to use glucose as carbon source, there is no
need to metabolize lactose until glucose has been exhausted.
- Sequential use is referred as diauxic growth.
3.
- CATABOLITE ACTIVATION required in order to turn off lacZYA transcription when glucose is present in the growth medium.
- RELIES ON cAMP binding protein (CRP) and cAMP
- cAMP level is determined by glucose level.
330
# Define the following
1. Specific allele of a given gene or set of alleles present in an organism (e.g E. coli strain NM522 has a number of mutant alleles and its genotype is proA4, proB45, thiA7)
2. Physical, biochemical, genetic or physiological xteristics that stem from a particular gene or sets of genes (e.g E. coli strain NM522 requires proline and thiamine for growth)
1. Genotype
| 2. Phenotype
331
1. What is the major requirement of an organisms to undergo homologous recombination?
2. How can prokaryotes achieve this requirement?
3. 3 possible genotypes are generated by recombination
- one from no crossing over (btw A and B)
- one from RECIPROCAL crossing over
- one from a GENE CONVERSION event at B
* *which 2 of the 3 occur from homologous recombination
1. MUST BE DIPLOID OR TRANSIENT DIPLOID STAGE
2. Prokaryotic ploidy
- all prokaryotes are haploid BUT
- Many Gram-positive and gram-negative organisms, can form partial diploids (called MERODIPLOIDS) by any of the three types of genetic exchange:
transformation
transduction
conjugation
3.
- both crossing over and gene conversion is a consequences of homologous recombination
332
Possible genotypes generated by recombination
1. What is formed if no crossover event occurs?
2. Single crossover event result in what?
3. In the first 2 events, no genetic information is lost in the products. This is referred to as?
4. In the case where cross over event lead to gain/loss of genetic information? What is this called? Why does this gain/loss occur?
1. No crossover - Parental type ascospores (PARENTAL DITYPE)
2. Single cross over - 2 parental and 2 recombinant type ascospores (TETRATYPE ASCUS)
3. RECIPROCAL RECOMBINATION
4. GENE CONVERSION - as a consequence of the resolution of the heteroduplex region
333
1. Define recombination type
- Exchange of genetic information between two
chromosomes with identical or nearly identical
sequences
- REQUIRES the recA and recBCD proteins. WHY/
- This reaction is analogous to SYNAPSIS in eukaryotes
and leads to the CROSSOVERS or CHIASMATA.
2. Molecular resolution of chiasmata leads to what 2 things? (2)
1. Homologous (generalized) recombination
- require recA to bind to single-stranded DNA and catalyze strain invasion reaction
2.
- gene conversion OR
- reciprocal exchanges
334
1. Identify recombination type
recA-independent exchange of information
between chromosomes with limited sequence
homology
2. What does it require?
3. Give 3 examples of this recombination type?
1. Site-specific recombination
2. Required SPECIALIZED RECOMBINATION ENZYMES
3. 3 examples
- TRANSPOSONS(integrate into genome sites randomly) e.g IS element, composite, TnA family
- SITE SPECIFIC PHASE VARIATION in salmonella (H1 antigen switch to H2 - mediated by recombinase)
- PHAGE LAMBDA integration ; integrate into specific site in E.coli during its lysogenic phase
335
Site specific recombination are independent of recA proteins but they use their own speecilized enzymes. 3 examples are; transposons, site specific phase variation in salmonella and phage lambda integration.
1. What are transposons and how do they integrate into a new genome site?
2. The mobility of transposons depend on what 2 things?
3. Identify the 3 classes of transposable elements
A. Transposase gene flanked by inverted repeats
B. Antibiotic resistance flanked by insertion elements
C. Transposase, resolvase + drug resistance flanked by inverted repeats
1. Transposons are DNA sequence elements (ranging from 500 bps to over 10kbp) that have ability to integrate into new sites in a genome in a NEARLY RANDOM manner.
2.
A. Enzymes encoded by genes within the element
+
B. Short sequences at either end of element
3.
A. Insertion (IS) element
B. Composite transposon; IS on either side
C. TnA family (multiple genes with inverted repeats on each end)
336
1. What are the hallmarks of site-specific recombination? (2)
2. These occur as a biological consequence of what?
A. INACTIVATE genes in some instances by inserting into coding sequences
B. ACTIVATE quiescent genes by providing a constitutive promoter (IS2, IS3)
C. Rapid spread of antibiotic resistance (they jump from one location to another and carry resistance with them)
D. Generalized recombination (when present on different chromosomes - portable regions of homology)
1.
A. Specific short sequences
B. Specialized recombination enzymes
2. Biological consequences of TRANSPOSITION
337
Site specific recombination are independent of recA proteins but they use their own speecilized enzymes. 3 examples are; transposons, site specific phase variation in salmonella and phage lambda integration.
1. Simple trasposons (IS elements) encode?
2. More complex COMPOSITE and TnA transposons encode? Give examples
3. How do you transfer antibiotic resistance?
4. How is composite transposons formed?
5. What are portable regions of homology?
1. The simple transposons (IS elements) encode only a transposase.
2. The more complex composite transposons and TnA
family transposons also have genes encoding
ANTIBIOTIC RESISTANCE determinants.
- Tn10 encodes tetracycline resistance,
- Tn3 encodes ampicillin resistance
3. Transfer resistance - JUMP from a chromosomal location to a plasmid (transferred by other stains by conjugation) **why you have rapid spread of resistance
4. Novel composite transposons can be formed when IS elements transpose to sites flanking chromosomal drug resistance gene.
5. Present on different chromosomes - IS elements and transosons can mediate GENERALIZED RECOMBINATION events - replicant fusion, resolution into 2 replicants, inversions
E.g in F mediated conjugation
**called PORTABLE REGIONS OF HOMOLOGY because elements are HOMOLOGOUS and MOBILE
338
Site specific recombination are independent of recA proteins but they use their own speecilized enzymes. 3 examples are; transposons, site specific phase variation in salmonella and phage lambda integration.
1. Some bacteria phenotypes change at frequencies higher than can be predicted by SPONTANEOUS MUTATION
Give examples
** what is this called
2. What does this result from? Medicated by?
1. Phase Variation (site specific)
E.g salmonella can produce 2 alternate forms of flagellar antigens (H1 and H2)
- if you grow say 1000 H1, some canswitch to H2 over time and go back to H1 production later if cloned out
**PHASE VARIATION
2. Phase variation results from site-specific inversion
of a promoter that controls transcription of H1 and H2.
- the more H1, the more switching occurs
**INVERSION is mediated by SITE-SPECIFIC RECOMBINASE
339
How does site specific inversion and phase variation work?
1. When promoter is ON (H1 or H2 made?)
2. When OFF?
3. What is the importance of antigenic variation?
1. ON
- H2 gene and H1 repressor TRANSCRIBED
- H2 MADE, H1 synthesis repressed
2. OFF
- H2 gene and H1 repressor NOT TRANSCRIBED
- H1 made constitutively
3. Antigen variation - observed in severe microbs - means to ESCAPE IMMUNE SURVEILLANCE (drive to survive)
340
1. A virus that infects a bacteria is called a? Are they parasitic?
2. What is the structure of #1 (2 main components)
3. How is a complex structure different from simple one?
1. Bacteriophage
- yes they are parasitic in that they can’t replicate without a host organism
2. Structure
A. Nucleic acid ; RNA or DNA, single or double stranded
B. Capsid; composed of proteins
3. Some phage have COMPLEX CAPSIDS (4)
- head
- tail
- baseplate
- tail fibers
4. Others have SIMPLE CAPSIDS that are icosahedral meaning that they have 20 sides
- no tail or baseplate
E.g MS2, OX174
341
1. Which factors determine the host range of bacteriophage? (What needs to be considered before a bacteriophage can infect an organism? - 2)
2. Give 2 examples that show host specificity
- which phage only infects E.coli? Salmonella?
3. What roles so phage receptors serve in host organism?
1. Host range depends on
- receptors on bacteria cell wall (e.g presence of siderophore receptor for T1 phage)
- presence of specific components of the host replication and transcription apparatus
2.
- phage lambda only infects E. coli
- P22 phage only infects Salmonella species
3. Phage receptors serve METABOLIC OR STRUCTURAL ROLES in the host organism (e.g siderophores/enterochelin is a receptor for T1 phage but also aids the host in iron scavenging)
342
Give 4 examples of coliphage and how they bind (phage type and receptor on host)
1. T1 phage - tonB/siderophore/enterochelin receptor
2. Lamba phage - maltose binding protein as receptor
3. MS2 phage - side of F pilus
4. Filamentous F1 phage - tip of F pilus
343
1. Identify the 2 modes of replication in a bacteriophage life cycle
- which only do lytic cycle?
- which can do lytic or lysogenic?
- give examples of each?
2. Differentiate between lytic and lysogenic
3. How do you break lysogenic state?
4. 2 benefits of lysogenic cycle
1.
A. Virulent phage ; only do LYTIC CYCLE
- invariable leads to lysis of host organism
- e.g T1, T4 phage
B. Temperate phage; can either do LYTIC OR LYSOGENIC
- e.g phage lambda and P22
2. - lytic lyse the cell
- Lysogenic; phage replicate and is integrated into genome but it is quiescent/DORMANT- phage coat proteins are not synthesized due to phage repression.
3. To break lysogenic state - activate recA protease that will cleave lambda repressor - infective phage particles can be made now (transcription and protein synthesis now activated)
4. 2 benefits of lysogenic
- escape deletion
- And for evolution
344
Lambda life cycle
1. Lambda phage injects its DNA into what host? (Remember maltose binding protein act as receptor)
2. WHERE DO linear chromosome circulate inside the cell
3. What determines whether phage enters lytic cycle of lysogenic phase?
4. Control over developmental pathways is mediated by what?
1. Phage injects its DNA into susceptible strain of E. COLI.
2. Inside the cell, linear chromosome circularizes at
its cohesive ends (COS sites)
3. MULTIPLICITY OF INFECTION determines whether phage enters lytic cycle or lysogenic phase.
4. Control over developmental pathways is mediated by PHAGE REGULATORY PROTEINS.
345
LAMBDA phage is an example of temperate phage (infection lead to host cell lysis or lysogeny)
Describe the lambda LYTIC CYCLE
1. How do circularized genome replicate?
2. What are concatamers?
3. What is fed into phage heads?
4. What is added only to filled capsid?
1. Circularized genome replicates by ROLLING CIRCLE mechanism, generating long concatamers.
2. CONCATAMERS consist of many linear unit length genomes that are covalently linked together.
3. UNIT LENGTHS are fed into phage heads.
4. PREFORMED TAIL is added only to filled capsid.
346
LAMBDA phage is an example of temperate phage (infection lead to host cell lysis or lysogeny)
Describe the lambda lysogenic CYCLE
1. How do circular lambda DNA intergrate into bacterial chromosome?
2. IS genetic information lost? What is this called?
3. 2 result of prophade
4. What is lysogeny synonymous with
5. What is not expressed in lysogenic phase? Why?
6. **what does superinfection immunity mean
1. Specific site called att
- occurs by SITE SPECIFIC RECOMBINATION
- prophage is DORMANT
2. No genetic info lost - RECIPROCAL RECOMBINATION
3. Some are DORMANT while others REPLICATE AUTONOMOUSLY LIKE PLASMIDS
4. Lysogeny is synonymous with DORMANCY not synonymous with integration into bacteria chromosome
5. In the lysogenic phase, phage replication and structural proteins are not expressed because transcription is blocked by lambda repressor.
6. No lambda phage can infect a lambda lysogen
- IMMUNITY TO SUPERINFECTION by identical or related phage
347
HOW do you break lysogenic state?
1. Mutagenesis of lysogenic bacteria activated what enzyme? Function of the enzyme?
- what happens to phage replication and structural proteins?
2. Because mutagenesis often lead to death of host and passengers, what will the phage do?
3. What are 2 benefits of temperate phage
1. RecA protease cleaves lambda repressor - phage replication and structural proteins are now synthesized - infective phage particles are made
**VIRUSES DESERTING THE SINKING SHIP (damaged bacterial cell)
2. PHAGE ESCAPE FROM THE HOST
3.
1. Phage escape deleterious agents and can safely spread to other environs as passenger
2. Lysogeny serve role in EVOLUTION of the bacterial genome
- phage that can’t excise will become part of bacteria genome
Called LYSOGENIC CONVERSION
348
These are examples of what process?
1.
▪Cholera toxin encoded by a filamentous phage, CTX.
▪Nonlysogenic strains of Vibrio are not virulent.
▪After lysogeny with CTX phage, they become fully virulent. ▪CT ADP ribosylates regulator of cAMP metabolism.
2.
Corynebacterium diphtheria synthesizes diphtheria
toxin only if prophage ß is present.
■ Nonlysogenic strains fail to synthesize toxin.
■ The advantage of the tox gene to phage is unclear.
■ But the lysogenized bacterial host has survival
advantage over the nonlysogen.
■ ***Diphtheria toxin ADP ribosylates eukaryotic
translational elongation EF2, resulting in
shutdown of mammalian protein synthesis.
LYSOGENIC CONVERSION
349
Identify 3 main mechanism of genetic exchange
* *what do these mechanisms allow for?
- which is naked DNA
- vehicle like phage involved
- cell to cell contact by F pilus
2. What is the significance of genetic exchange? (3)
* hint - how is antibiotic resistance spread?
1. Transformation; NAKED DNA involved followed by recombination (host genome) or self-replication (plasmid DNA) **Diff from transfection
2. Transduction; VEHICLE involved (PHAGE); generalized (lysogenic or lytic) and specialized (lysogenic)
3. Conjugation; CELL to CELL CONTACT mediated by F PILUS
**These mechanisms allow for spread of bacteria
2.
A. Homologous Recombination requires diploid or a merodiploid organism and Merodiploids are formed after all of the 3 forms of genetic exchange
B. Antibiotic resistance spread by TRANSFORMATION and CONJUGATION
C. Merodiploids allow for complement analysis - tell where mutations are in the same gene or different genes
- if mutant phenotype persist (same gene)
- if phenotype is normal (mutations are in different genes- intergenic complementation )
350
What is the difference between transformation and transfection
1. Transformation
- uptake of naked DNA into cells - recombination (either with host genome or self-replication)
2. Transfection
- uptake of naked phage DNA by cells - lysogeny or lysis
E.g lambda phage transfection into host
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Transduction is the process of transferring host cell DNA sequences to a recipient cell by bacteriophage
1. What are the 2 types?
- lysogenic or lytic
- lysogenic
- which acquire virulence factor?
1. Generalized; lytic or lysogenic phage. ANY BACTERIA CHROMOSOME DNA CAN BE CAPTURED RANDOMLY and introduced into host
- collection of phage particles called LYSATE; can be used to infect another bacterium
- this DNA can recombine with host genome by generalized recombination
2. Specialized; only lysogenic. After integration occurs - dormant for long time. ABNORMMALY EXCISED Phage capture adjacent bacteria.
- phage excise abnormally and take some bacteria chromosome - (Gal+ gene or bio)with it and leaving some of its DNA in host chromosome. New phage is SPECILIZED TRANSDUCING PHAGE.
- Only these 2 can be captured (Gal +gene or bio) and must be adjacent to site of integration. MECHANISM PHAGE ACQUIRE VIRULENCE FACTOR
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Conjugation is the transfer of DNA from a bacteria cell of one mating type (donor) to cell of other mating type (recipient)
1. cell to cell contact is mediated by what?
2. Donor mating type is determined by the presence of transmissible plasmid called?
3. Cells carrying the F plasmid designated?
4. Cells lacking the F factor ?
5. How do you get F’
1. F PILUS
2. F factor (F plasmid)
3. F+
4. F- ; recipients of DNA
**In mating, F+ and F- cells are mixed, conjugal pairs form and F plasmid DNA is transferred from F+ cell to the F- cell
• All become F bearing cells (by contact/ conjugal bridge) - replicate and transfer to host organ
5. F plasmid - donor. Could be excised and would generate F’
• Presence of F cause conjugal mating - you transfer F efficiently but bacteria chromosome inefficiently
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What are the functions of F plasmid? (4)
1. F plasmid CARRIES GENES for the synthesis of F
pilus, that brings the mating pairs together.
2. F plasmid also ENCODES GENES that enable its transfer to F- cells.
3. F replication origin uses host replication machinery.
4. F plasmid also carries SIMPLE TRANSPOSONS.
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WHAT ARE the 3 different states F plasmid can exist inside a cell?
1. F+ plasmid; F PLASMID HIGH FREQUENCY TRANSFER, bacteria F low frequency
2. Hfr; high frequency (similar to phage genome integrating to host genome). Generate new type of cell (you transfer bacteria stuff in linear fashion). BACTERIA CHROMOSOME (near site of integration) IS TRANSFERRED EFFICIENTLY while F plasmid transferred inefficiently. F plasmid is integrated by generalized/homologous or site-specific recombination mediated by IS elements .
A. Homologous; mediate Rec A event
B. Site specific; independent of Rec A
3. F’; (derived from Hfr). this occurs when there is an aberrant excision event of F plasmid and you take bacteria chromosome with you.
Type I excision - F plasmid left behind.
Type II - none left behind
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Identify the replication status and efficiency of transfer of F vs bacterial chromosome in the following
1. F+
2. Hfr
3. F’
1. F+
- Autonomous (self replicating)
- HIGH F TRANSFER
- low bacterial chromosome transfer
2. Hfr
- Intergrated replication (F plasmid integrate into chormosome - mediated by IS elements which are portable regions of homology)
- low F transfer
- HIGH BACTERIAL CHROMOSOME TRANSFER
3. F’
Type 1 excision; F plasmid left behind in bacterial chromosome
Type II excision; No F plasmid left behind
- HIGH* F TRANSFER (F’ carries some chromosomal genes and transfers those at high efficiency)
- low bacterial chromosome
