Week 8 Flashcards
What are the four major antibiotic resistance mechanisms? Describe them
Prevent access to target - drug can’t get in or drug gets in but cant get pumped back out before affecting target
Modify or protect antibiotic target - target is altered structurally OR target is overexpressed (so you need much more drug)
Modification/inactivation of antibiotics - drug is inactivated before affecting target.
Modify expression of Bactrian factors needed to activate the antibiotic (prodrug)
Name three antibiotic tolerance mechanisms
Biofilm formation
Spores
Persister cells
What are two ways bacteria may restrict access of antibiotic to target?
Alter envelope to inhibit uptake
Boost expression of efflux pumps (gene amplification)
The ____ mutation in ____ rRNA protects against macrolides like erythromycin
A2580G
23S
Tetracycline binds ____ rRNA in ____ subunit, and distorts the ___ site
16S
30S
A
Bacteria may grow resistant to tetracycline by protecting their ribosomes via ___ or via ___ of a ____ that pertubs the ____ in 16S rRNA that is involved in tetracycline binding
Mutation
HGT acquisition
GTPasse
Helix
*basically, an enzyme is activated to unwind an rRNA so that it is not recognized by tetracycline. Note that this make the ribosome less effective, but it is resistant to tetracycline
How does vancomycin kill bacteria?
Prevents normal cross-linking of peptidoglycan.
Vancomycin binds to D-Ala-D-Ala preventing cross linking
Describe vancomycin resistance
Involves four enzymes that prevents vancomycin from preventing peptidoglycan cross-linking
The multiple enzymes that are required for resistance is what caused bacteria to take so long to grow resistant to vancomycin.
Enzymes are VanH, VanA, VanB, VanX
VanX will reform peptidoglycan cross-linkage
How do some bacteria grow resistant to penicillin by modifying the target?
Penicillin normally binds to transpeptidase to prevent cross-linking.
Point mutations in transpeptidase causes resistance. (Note, however that transpeptidase will be less effective with these point mutations)
What are two ways in that bacteria can grow resistant to antibiotics by modifying or inactivating the antibiotic?
Inactivation by hydrolysis
Inactivation by steric hinderence.
B-lactamases do this
How do B-lactamases work?
Break a bond in the B-lactam ring of penicillins to disable the molecule.
How do B-lactamase inhibitors work? (Like clavulanic acid)
Clavulanic acid is a suicide molecule. It interacts with B-lactamases to be cleaved but remains attacahed to B-lactamase to inactivate it.
**note that bacteria have also developed anti-B-lactamase inhibitors
What are three ways in which antibiotics can e modified for deactivation? What enzymes accomplish this? How is it that these modifications are detrimental?
N acetylation
O phosphorylation
O adenylation
Aminoglycoside inactivating enzymes
Modifications disrupt hydrogen-bonding network used to bind 16S RNA
Some bacteria can gain resistance by modifying expression of bacterial factors needed to activate the antibiotic (prodrugs). Give two examples of prodrugs
Metronidazole is activated by the bacterial factor flavodoxin in H. Pylori and P. Gingivalis
Isoniazid is activated by the bacterial catalase/peroxidase enzyme KatG in. MTB. Isoniazid will then continue to inhibit the synthesis of mycolic acid required for MTB cell wall
In what two ways is resistance acquired?
Mutation
Horizontal gene transfer
Note that E. Coli has a one in a billion chance of becoming resistant to streptomycin. But because of fast growth rates and high numbers, this probability becomes meaningful
What four things are contributing to the spread of resistant bacteria?
Overuse of antibiotics
Overpopulation
Poor hygiene
Travel
Name four pathogens that are becoming increasingly resistant
Extended-spectrum B-lactamase producing enterobactericae
Carbapenem-resistant enterobacteriacae
Clostridium difficile
Super neisseria gonorrhea
What are the three different priority pathogens in the list for R&D of new antibiotics?
Priority 1: Critical
Priority 2: High
Priority 3: medium
What are the three bacteria that are classified under priority 1 (critical)?
Acinetobacter baumannii, carbapenem-resistant
Pseudomonas aeruginosa, carbapenem-resistant
Enterobacteriaceae, carbapenem-resistant, ESBL-producing
What are 6 bacteria that are considered priority 2 (high)?
Enterococcus faecium, vancomycin-resistant
Staphylococcus aureus, methicillin-resistant, vancomycin intermediate and resistant
Helicobacter pylori, clarithromycin-resistant
Campylobacter spp. Fluoroquinolone-resistant
Salmonella, fluoroquinolone-resistant
Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone resistant
Name three bacteria that are considered under the priority 3 (Medium)?
Streptococcus pneumoniae, penicillin-non-susceptible
Haemophilus influenzae, ampicillin-resistant
Shigella spp. Fluoroquinolone-resistant
Antibiotic tolerance contributes to ___ and ____ infections. What are some methods bacteria use to tolerate antibiotics?
Chronic
Recurrent
Spores
Biofilms
Persister cell formation
Intracellular niches
> ___% of human infections may involve biofilms
65
What are six ways in which biofilm formation enhances the ability of bacteria to tolerate antibiotics?
Extracellular matrix
Altered protein expression
Decreased metabolism
Increased stress resistance
Increased gene exchange
Persister cell formation
What are persister cells?
Dormant bacteria that are insensitive to many stresses and antibiotic treatment.
Persister cells can remain dormant during antibiotic treatment, but after become reactivated
Note that antibiotics may not kill all of the dormant bacteria, but they can lower total bacteria numbers to regain control (sometimes)
B cells are not stockpiled, but constantly replaced. About how many B cells are replaced each day? What is the mature half-life?
30 billion per day
50-100 days
Name the different stages of B cell development in the bone marrow, starting with stem cell
Stem cell
Early pro-B cell
Late pro-B cell
Large pre- B cell
Small pre- B cell
Immature B cell
At which stage of B cell development is the heavy chain rearranged?
Early pro-B cell
At which stage in B cell development do you have negative selection?
Immature B cell
What is the function of immature B cells?
They move around the periphery from lymphoid organ to lymphoid organ, looking to be activated by an APC. Once activated, they will colonally expand into a large number of plasma cells and memory cells
What are memory B cells
Respond to subsequent infection.
Also produce antibodies directly after expanding to prevent immediate reinfection
What is an early pro-B cell?
RAG proteins activated to Rearrangement of D-J heavy chain on BOTH chromosomes
No immunoglobulin production yet
What is a late pro-B cell?
V-DJ rearrangement of heavy chain on first chromosome, then second. If neither work, the cell will die.
Note that about 50% will die
Still no immunoglobulin being produced
What is the first checkpoint in immature B cell development?
Between late pro-B cell and large pre-B cell
Checks to see if heavy chain is functional
What is a large pre-B cell?
VDJ is rearranged and heavy chain is being produced
What is a small pre-B cell?
Heavy chain is still being produced
V-J rearranging of light chain
What is the second checkpoint in immature B cell development?
Between small pre-B cell and immature B cell
Checks to see if light chain is functional
How do stromal cells drive B cell development?
Signaling factors, adhesion molecules, and growth factors are physically attached to the surface of bone marrow stromal cells
As a developing B cell moves along the stromal cell, the signaling factors change, causing changes in the B cells’ RAG gene expression and other factors
Pro-B cells are programmed to die in the absence of survival signals from the stromal cells
What is a way you can get dysfunctional rearrangement when producing the heavy chain?
Most problems occur with junctional diversity. A stop codon could be added in
What is a pre -BCR (pre-B cell receptor)? What is expressed along with it?
The heavy chain used as a receptor. Expressed in the ER
A surrogate light chain is expressed with it
What is a surrogate light chain?
Essentially a placeholder protein
Binds to heavy chain to ensure a light chain can bind to the heavy chain
Allows heavy chain to bind two other proteins, Igbeta and Igalpha
Since antibodies really have no ____, they cant signal. Thus they bind to ___ and ___ to send all of the signals from the pre-BCR. This is how it functions as a first checkpoint. When the heavy chain interactis with Igbeta, will cause a signal to pass the first checkpoint because it produced a functional heavy chain. This will turn off the ____ expression, initiates ____, and leads to ____
Cytoplasmic tail
Igalpha, igbeta
RAG
Cell division
Allelic exclusion
What are the four functions of igbeta signaling?
Checkpoint clearance
Turns off RAG proteins
Initiates cell division
Allelic exclusion
What is allelic exclusion? Why is this important?
Allows a functional heavy chain to produced by only one chromosome
This is important because it will the antibody specific for only one epitope
Why is it that large pre-B cells go to small pre-B cells?
Large pre-B cells go through a bunch of division (leading to smaller cells) (about 100 small pre-B cells per one large pre-B cell)
They replicate because you want to invest in a promising B cell
What three things occur in the transition from large pre-B cell to small pre-B cell?
Cell division (100 smalls produced)
RAG genes reactivated (for rearranging the light chain). **note that now they all share the same heavy chain, but they can each produce different light chains
Unique recombination per cell
What is different between heavy chain rearrangement and light chain rearrangement?
Light chain rearrangement can have a few attempts whereas heavy chain only has one chance to produce a functional chain
You have two chromosomes that can code for light chain: the ___ and ___. Usually the ___ rearranges first
Kappa
Lambda
Kappa
How many light chain recombination attempts possible per chromosome?
4-5
If pre-B cells cannot produce functional light chain, they will die. But approximately ___% of small pre-B cells survive
85
Describe the second checkpoint in B cell development
Functional antibody on cell surface (including light chain). Utilizes Igbeta signaling to promote the survival of these cells (absense of this signalling will lead to apoptosis)
What stage of B cell development does negative selection occur?
Immature B cell
What is negative selection?
Kills B cells that recognize self antigen
If immature B cells bind to self antigen in bone marrow they will be retained in bone marrow
If immature B cells do not bind to bone marrow, they will move to the blood and express IgD and IgM. Here they will be exposed to more self antigens. If they recognize self antigen in the periphery, they will become anergic
Exposure to self antigen ensures tolerance (meaning the inability to bind to self antigen). What are the two types of tolerance?
Central tolerance - tolerance gained in the bone marrow
Peripheral tolerance - tolerance gained in the circulation and secondary lymphoid tissues
What happens if an immature B cell recognizes self antigen in the bone marrow?
It is retained in the bone marrow and goes back into a small pre-B cell state to rearrange its light chain, so long there is genome left. If none left, it will die. Once it finally produces a good light chain, it will be released into periphary
Immature B cells migrate to lymph nodes via ___
HEV (high endothelial venules)
Chemokines attract B cells to ___ into the ____, then into the ____. Here, interactions with ____ and ___ drive the maturation of B cells
HEV
Lymph node
Primary follicle
Dendritic cells
Cytokines
Describe the process of B cell maturation (positive selection). B cells release ____ to stimulate the ____ to release ____ . Once B cells are matured, they produce more ___ than ____
In the primary follicle, immature B cells interact with follicular dendritic cells.
Lymphotoxin
Follicular dendritic cells
BAFF (B cell activating factor)
Now B cells are mature. They then produce more IgD than IgM
What happens to an immature B cell if it doesn’t undergo positive selection within a few days?
It will become anergic
What happens B cells once they are considered mature?
They remain in the lymph node for a few days, then circulate in the body, going from lymph node to lymph node
What are the four steps of mature naive B cell activation in lymph nodes?
Antigen exposure in cortex
Interaction with T cells in T cell area
Migration, secondary lymphoid follicle formation, and colonial expansion in germinal centers
Plasma cell production and antibody secretion
Why is it that dysfunctional B cell development can result in cancers?
Due to mutations that lead to unregulated replication or excessive proliferation of precursor B cells
What are CD8 cytotoxic T cells?
Kill virus-infected cells and some intracellular bacteria
What are CD4 Th1 cells?
Activated infected macrophages. Provide help to B cells for antibody production
Targets extracellular bacteria
What are CD4 Th2 cells?
Provide help for B cells for antibody production, especially switching it IgE*
Targets helminths/parasites
What are CD4 Th17 cells?
Enhance neutrophil response
**promote barrier integrity (prominent in mucosal surfaces)
True or false… TCRs may be secreted
False… they are only expressed on the cell surface
TCRs primarily bind to ___
Polypeptides. Not so much polysaccharides or lipids like antibodies can
CD8 cells interact with MHC ___ whereas CD4 cells interact with MHC ___
1
2
What is the function of MHC1 and where is it found?
MHC1 is found on most cells in the body. They are used to present self antigen. This will tell the T cells if they are healthy or diseased
What are the two different classes of TCRs? Which is most common?
Alphabeta (most common)
Delta gamma
What are some differences between alpha-beta T cells and delta gamma T cells?
Alpha beta: adaptive immunity, common in circulation (not tissues), developed and matures in thymus, positive and negative selection
Delta gamma: not involved in classic adaptive immunity, monitors tissue health (so its common in tissues not circulation), develops but does not mature in the thymus, limited positive and negative selection
True or false.. T cells may express both alpha-beta and delta-gamma TCRs
False… only one type
Delta chain genes are embedded within the ___ chain gene
Alpha
The alpha chain is similar to the ___ chain in antibodies in that it has the same ____ segments whereas the B chain is similar to the ___ chain in antibodies in that it has ____ segments
Light
V, J, C
Heavy
V, J, D, C
True or false.. TCR genes undergo genetic rearrangement and junctional diversity similar to B cells
True
Which has more diversity, TCRs or immunoglobulins? Why?
TCRs
Because they have a longer genome
Also note that TCRs have a LOT more joining segments
True or false.. TCRs can bind directly to soluble antigen
False… they can only bind antigen if it is presented b MHC
TCRs are not really able to signal on their own. They require ____ for signaling
CD3
What cells express MHC2?
APCs (macrophages, dendritic cells, B cells)
MHC1 will present ___ antigen whereas MHC2 will present ____ antigen. MHC1 is expressed on cell membrane by ___ antigen loading whereas MHC2 is expressed on cell membrane by ____ antigen loading
Intracellular
Internalized
ER antigen loading
Vesicular ER loading
What is the difference between MHC1 and MHC2 structure?
MHC1: alpha chain and B2 microglobulin
MHC2: alpha and beta chains
True or false… erythrocytes do not express MHC1 or MHC2
True
Describe the difference between MHC1 and MHC2 loading on the cell membrane
MHC1: intracellular antigen is broken down by proteasome. Antigen is put into ER where MHC1 exists. MHC1 is then loaded onto cell membrane
MHC2: after pathogen is phagocytosed and broken down in vesicle, another vesicle containing MHC2 fuses with it. Then MHC2 is loaded onto the cell membrane
in MHC1 antigen loading, the heavy chain of MHC1 is stabilized by ____ until ____ binds. Then ____ is released to form another complex, the peptide loading complex. A peptide delivered by ____ binds to the heavy chain, forming the mature MHC1. Then the MHC1 dissociates from the peptide loading complex and exported from the ___ to the ____
Calnexin
beta2-microglobulin
Clanexin
TAP
ER
Cell membrane
Intracellular peptides of ____ residues are expressed by MHC1. Peptides are trimmed before expression by a ____
8-10
Proteosome
How can crosspresentation of endocytosed antigen on an APC occur? Why is this bad?
The antigen can get into the ER where MHC1 is located
Most antigen is presented via MHC2
The presentation of antigen on MHC1 targets the APC for death
Describe MHC2 antigen loading
Internalized antigen in vesicles is fused with another vesicle containing MHC2. This is then sent to the cell membrane
How do APCs present the presentation of self antigen on MHC2?
Invariant chains block the binding of peptides to MHC2 in the ER. Then, in the vesicles, the invariant chain is cleaved, leaving behind CLIP* in the active site of MHC2. CLIP prevents the binding of peptides to MHC2 in vesicles. Then, once fused with a vesicle full of pathogen antigen, HLA-DM facilitates the release of CLIP to allow the binding of antigen in MHC2
What is CLIP?
Class 2 associated invariant chain peptide.
It prevents the binding of self antigen in MHC2
What are some things that T helper cells can do?
Stimulate macrophages to lyse the phagocytosed stuff
Stimulate B cells to isotype switch or produce antibodies
In the development of T cells, only about ___% will survive
2
T cells are developed in the ____. TCR development occurs within the ___. Selection occurs as thymocytes move from ___ to ___
Thymus
Cortex
Cortex to medulla
Why is it that even though the thymus degenerates with age (thus reduced T cell production) the T cell response doesn’t really diminish?
T cells have long half lives. CD4: 4.2 years. CD8: 6.5 years
When they are activated, however, they may last for decades
True or false… T Cell progenitors dont commit until they reach the thymus. How do T cells tell if they are in the thymus?
True
They can tell if they are in the thymus by notch1 (T cell transcription factor complex) signaling and pax-5 signaling (B cell transcription factor)
What are the two main phases of T Cell development?
Double negative and double positive
What is the first thing that is rearranged in a developing T cell in a double negative stage?
Beta chain or delta gamma chains.
If delta gamma chains rearrange before the Beta chain rearranges, it will become a delta gamma T cell, if the Beta chain rearranges first it will become an alpha beta T cell (alpha beta is more common)
Beta chain will form a pre-TCR
What is the first checkpoint in T Cell development?
The formation of a pre-TCR from a beta chain
What happens once the T cell passes the first checkpoint by producing a functional ___?
Pre-TCR
It will go through a series of replication to have a bunch of cells with the same beta chains
After T cells have proliferated after producing functional pre-TCR (beta chain), what happens?
It enters the double positive stage by expressing both CD4 and CD8, while rearranging alpha chain (or delta gamma)
What happens if delta-gamma rearranges before the alpha chain rearranges?
The T cell will become a delta gamma T cell.
Note that the T cell has two chances to become delta gamma… before arrangement of beta chain and before arrangement of alpha chain
After rearrangement of the alpha chain, what happens?
It enters the second check point: forming a functional TCR
If it passes, it will form single positive T cells, which will then undergo positive and negative selection
Thymic location influences T cell development. They go from immature to mature as they progress from __ to ___ of the thymus
Cortex to medulla
What happens if at any point, the delta gamma chains rearrange first?
The T cell leaves the thymus with little selection
Once a T cell enters the double positive stage, what drives the cell to express both CD4 and CD8?
B chain
In regards to Beta chain rearrangement, each T cell gets ___ attempts at producing a functional beta chain because they get ___ attempts per locus and there are ___ genres
4
2
2
True or false… the Beta chain locus is organized the same way that immunoglobulin loci are organized
False… they are organized differently
Once beta chain is formed it binds o a surrogate alpha chain analogue to make the pre-TCR. Here, it may or may not bind to ____ to allow signaling. It will signal for ____ and ____. This is the first checkpoint. If no signaling occurs, it will ___
CD3
RAG function halts
Proliferation
Die
How do pre-TCRs recruit CD3?
They form a hero diner or super dimer to form a pre-TCR complex
When RAG is turned back on to rearrange the alpha chain in T cells, it also induces the expression of ___ and ___
CD4 and CD8
Why is it that a T cell can still become a delta gamma cell after it has already produced functional pre-TCR and is in the process of rearranging alpha chain?
The delta chain sits right in the middle of the alpha chain locus
What is the difference between positive and negative selection in the development of a T cell?
Positive selection : functional TCR
Negative selection: no self-antigen binding
Describe the process of positive selection in T cells
Epithelial cells in the thymus posses both MHC1 and MHC2. T cells have a few days to successfully bind to either of these MHC receptors. If they bind moderately or strongly, they live. If they dont bind, or bind weakly, they die.
Note that if they dont bind well, tehy can still go back and rearrange their alpha chain to bind better. But they only have a few days to do this.
This is the second checkpoint, to make sure they can bind to MHC
How does positive selection determine if it will be CD4 or CD8?
When T cell binds to the epithelial cell in the thymus, whichever it binds to first (MHC1 or MHC2) the strongest, it will become CD8 or CD4 accordingly
If it is destined to become CD8, it will shut down the expression of CD4 coreceptors
Describe negative selection in T cells
APCs present self antigen to T cells. If they bind strongly, the T cell will die.
Central tolerance occurs in the thymus and peripheral tolerance occurs in the periphery
Then mature naive T cells enter the blood stream going from lymph node to lymph node
Metronidazole is a prodrug that is activated by ____ found in ___ and ____
Flavodoxin
H. Pylori
P. Gingivalis
What are the different levels of sepsis? (6) briefly describe them.
Infection - inflammatory response to microbes. Invasion of normally sterile tissues
SIRS - systemic response to a variety of processes
Sepsis = infection + SIRS criteria (2 or more)
Severe sepsis - sepsis + organ dysfunction
Septic shock - sepsis + hypotension despite fluid resuscitation
Multiple organ dysfunction syndrome - organ dysfunction that results in… Homeostasis cannot be maintained without intervention
What is SIRS? What are its four criteria?
Systemic inflammatory response syndrome
Abnormal temperature (>38 or <36)
Fast heart rate (>90 BMP)
Fast respiratory rate (> 20 rpm)
Abnormal WBC levels (>12,000 K/ul or <4000)
Define bacteremia
Presence of viable bacteria in the blood stream
Define septic shock
Sepsis-induced hypotension despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include but are not limited to…
Lactic acidosis
Oliguria
Acute alteration in mental status
What things may cause SIRS without infection?
Pancreatitis
Trauma
Burns
Other
What is it called when SIRS is a result of infection?
Sepsis
Describe the progression of sepsis
Infection leads to SIRS leads to sepsis leads to severe sepsis
Severe sepsis includes sepsis + at least one sign of organ failure and Shock!
Who gets sepsis?
Anyone can present with sepsis or severe sepsis
More likely in individuals with pre-existing diseases (immunocompromised)
What types of bacteria are most associated with sepsis?
Gram negative bacteira
Define the specifics behind hypotension
lower blood pressure than normal
<90 mmHG systolic (Reduction of SBP of 40mmHg from baseline)
Evidence of hypo-perfusion (lactic acidosis, low urine output, or change in mental state)
MAP=average pressure in a patients arteries during one cardiac cycle
What is the equation for MAP?
MAP = average blood pressure in one cardiac cycle
MAP = SBP - 2 (DBP)/3
What are the differentials diagnosis of shock/hypotension?
ENDSHOCK
Endocrine Neurogenic (anaphylactic) Drugs (distributive) Septic Hypovolemia Overdose (obstruction) Cardiogenic Kills
Define septic shock
Sepsis is the clinical syndrome that results from a dysregulated inflammatory response to infection
Patient with a continued hypotension after a fluid bolus
True or false… hypotension is often associated with organ damage/dysfunction
True
What is the difference between primary and secondary multiple organ dysfunction?
Primary - result of well-defined insult (renal failure from muscle breakdown of products (rhabdomyolysis))
Secondary - dot due to infection itself, but due to host’s response to the infection (possibly a dysregulation of the innate immune system)
Once the patient is known to have sepsis, you should see if they have severe sepsis (organ dysfunction ) by looking at signs and symptoms. What are the indications of organ dysfunction?
CNS - altered consciousness, confusion, phycosis, delirium
Respiratory system - tachypenea, hypoxemia, oxygen saturation, decreased ratio of arterial oxygen vs inspired oxygen
Liver - jaundice, increased liver enzymes, hypoalbuminemia, increased prothrombin time
Cardiovascular - tachycardia, hypotension, increased venous pressure, increased pulmonary artery occlusive pressure
Kidney - oliguria, anuria, increased creatinine
Hematological - thrombocytopenia, abnormal coagulation tests, decreased levels of protein C, increased D dimers
What are the three mechanisms of tissue injury? Describe them
Ischemia - insufficient O2 (often an uptake problem, not always a delivery problem)
Cytopathic injury - inflammatory mediators cause damage
Increased apoptosis - programmed cell death
What does PIRO stand for?
Predisposition
Insult - species infecting organism with special weapons
Response - activation of immune system
Organ dysfunction - collateral damage
True or false… the balance of pro-inflammatory and anti-inflammatory mediators is key for survival
True
How can you monitor tissue perfusion?
Clinical assessment of skin, urine output, MSE by using pH and lactate mechanisms (measuring rate of lactate clearance) - historical survival is <10% if lactate is > 10
What is the treatment for someone with sepsis?
Fluids** (not a slow steady flow, but a lot in a short period of time)
Pressure
Monitor tissue perfusion
Monitor physiology and fluid responsiveness
Note that animal model treatments worked, but then didn’t work in humans
As we age, the thymic tissue is displaced with what type of tissue?
Adipose tissue
