407 Innate Immune S 2014
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Tissue Culture
Many cell types can be grown in vitro
Many cells can be grown under special conditions aspure cell types (hepatic cells, fibroblasts, etc.)
1) Grown for study of cell biology.
2) Grown for production of molecules such asmonoclonal antibodies.
3) Used to grow viruses or other pathogenic organisms.
4) Used to test the effect of pharmaceuticals on cells.
For example: How does drug X affect liver cells?
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Tissue Culture: Two basic types of cell cultures
#1 Normal cells or Primary cultures from healthy
tissue:Normal cells, must be grown attached to special dishes.
Replicative Cell Senescence: primary culture cells grow
for awhile and then die, even with ideal conditions.
#2 Immortalized cells: Usually from cancer tissue.
Hela cells are the most famous example.
No replicative cell senescence
Not normal, but do many things same as normal cells
Can be grown attached to dishes or, some can be grown
in liquid, growing free in solution like bacteria
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Culture dishes: Cells can be grown in dishes like petri
dishes with many dishes with many wells.
Plastic surface is specially treated as most cells MUSTbe able to attach to a surface in order to stay alive.
Bacteria petri dishes cannot be used for cell culture
because they are not treated for cell attachment.
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Tissue culture flasks that can be sealed are the most
widely used dishes. Allow air to flow thru vents in caps
or thru loosely tightened cap. Flasks are mostly empty
with cell only growing on bottom surface.
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If large numbers of cells are needed for industrial
production, roller bottles are used and cells are grown
on entire surface area.
The constant rolling motion keeps cells bathed in a thin
layer of media for maximum gas exchange.
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Shaker incubation is used to grow cells types like HeLa
cells that are so transformed that cells can even grow
free in liquid suspension.
Only a few cell types, (like Hela) will grow free in
solution without attachment to a solid surface
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Cells in the body grow in a low oxygen,
high carbon dioxide environment
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Tissue culture incubators are large chambers with
heaters to keep the cells at 370C and a supply of carbon
dioxide to keep the CO2in the tissue media the same as
is found in body tissues.
Often the floor of the incubator is a pan of water to
keep the media in the flasks from evaporating from the
constant flow of dry CO2.
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Cell passaging
Under ideal conditions, cells will increase in numberuntil they touch each other and stop growing.
Some cells can be maintained for long periods of timeat a confluentstate where they have stoppedgrowing and have become quiescent.
Other types of cells, especially cancer cells, are bestmaintained in a state of less-than-confluent so that
they continue rapid growth.
To transfer cells to other flasks, cells are taken up fromtheir attachment to the plastic flasks and allowed to
float freely in the media and transferred.
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Confluence: Cells cover dish floor. All cells are
in full contact with other cells.
Less than confluent. Fully confluent
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Cell Passaging: Trypsin
Cells can be freed from attachment with the protease
Trypsin to digest the cell surface proteins attaching tothe dish. This causes them to become round and easily
removed by tapping on the side of the culture flask.
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Tissue culture mediaSynthetic or Defined Media
All of the ingredients in defined media are known,carbohydrates, amino acids, lipids, vitamins, insulin,etc. etc.
Allows complete control over cell media so the effectof growth factors and hormones can be tested.
Only a few cell types can be grown on this type ofmedia
Most cell type require growth factors and hormone that
we dont know how and how much to supply
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Tissue culture media
Media supplemented with serum
Most animal and human cells are grown in definedmedia containing nutrients and vitamins (and a redph indicator dye) that is supplemented with animal
serum.
Most common serum is calf serumalso known asbovine serum
Some cells require higher amounts of growth factorsand hormones, these are given the more expensiveFetal calf serum or Fetal Bovine Serum
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Tissue culture media: SerumSerum is the fluid remaining after blood has clotted.
The blood clot, which contains all the red bloodcells, is removed and the serum is filtered sterilizedand frozen. Mixed at 5-10% with a defined media.
Serum contains and growth factors and hormones thatcells need for growth.
Different batches of serum can be different, forexample, one batch of serum may contain antibodiesthat bind to a cell type and another batch may nothave those antibodies. Also, serum must be tested tofor the presence of viruses and other pathogens.
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Tissue culture media: Antibiotics
Most media contains antibioticsagainst bacterial and fungalcontamination.
Media may also be given antiRickettsial antibiotics forbacteria living within tissueculture cells
Neutralizing anti-virus antibodiesmay also be added if a virus isused in the lab and the risk of
virus contamination is high
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Tissue culture hoods can be exposed to UV light to
destroy the DNA and RNA and Proteins in bacteria and
fungus and viruses to keep the working area sterile.
Skin should not be exposed to the UV light.
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Inverted MicroscopeCells grow on the bottom of flask. This puts them too
far away for visualizing with a standard microscope.
An inverted microscope has the objective underneath
the flask, next to the cells.
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Inverted Microscope
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Liquid Nitrogen
Cryopreservation: Cell Freezing
Tissue culture cells can be stored in liquid nitrogen at -196o
C,where all metabolic processes are stopped.
At higher temperatures, even as low as -800C, the high ionic andosmotic concentration inside cells allows some movement ofwater molecules and organic processes that destroy the cellsover time.
Frozen cells should not be stored even briefly at -200C
Cells are frozen in special media that contains molecules likeDMSO to act as a Chaotropic, or, water denaturingagent toprevent the formation of water crystals (ice) that wouldotherwise grow until they ruptured the cell.
Id l G L PV NkT
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Ideal Gas Law PV = NkT
P = Pressure in atmospheres
V = Volume in liters
N = number or particles in the gas (moles)
K = a constant relating temperature and energy
T = Temperature
Liquid nitrogen is stored at high pressure (694
atmospheres) if kept at room temperature.
High pressure: 694(V) = (Nk) T
Low pressure: 1(V) = (Nk) T/694
F ll t d i ll t b hill d b li id
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Frozen cells are stored in small tubes chilled by liquid
nitrogen in large vacuum flasks or thermos bottles.
Warm nitrogen in high pressure tanks is transferred to
thermos at atmospheric pressure at low temperature.
I Vit F tili ti (IVF) d
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In Vitro Fertilization (IVF) and
Embryo Cryopreservation
Whole animals cannot be frozen and recovered
alive.
Embryos of just a few cells can be frozen and
recovered alive. The thawed multi-cell
embryos can then implanted in the uterus todevelop into a normal animal or person.
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In Vitro Fertilization
Eggs and sperm collectedand mixed together intissue culture.
Successful fertilizationsbegin to develop intoembryos.
Embryos can be collectedand implanted intofemale, or frozen for
later implantation
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Embryos that are frozen have a survival rate of
about 70%. Healthy ones can be selected with a
mircopipette for implantation.
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Stem CellsMost cells have differentiated into a particular cell
type: That is, liver cells and skin cells have exactlythe same DNA, but they used different genes to makedifferent proteins so the cell types are different.
Totipotent: A cell like a fertilized egg cell having theability to develop into an organism
Pluripotent: A stem cell that has the ability to
differentiate into any other cell type.
Unipotent: A cell that has the ability to produce withmitosis only the cell type it already is.
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Induced Pluripotent Stem cellsMuch scientific work is not being invested in trying to
make pluripotent cells from a persons own tissue.These are being used by science to study genetic
diseases in vitro: producing CNS cells with disease in
laboratory for study for example.
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Immune System
Defends against microbes and parasites
Removes dead and damaged cells and tissues
Allergies and autoimmune diseases
Chronic inflammation: heart disease and cancerTransplant tissue rejection
Septic shock and anaphylactic shock
Provides molecules useful in Biotechnology andMedicine
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Immune Defenses: Found in all life forms
How can a bacteria defend
itself from a virus?
Restriction enzymes
- Cut up bacterial virus DNA
but not the DNA from thebacteria
- The bacteria lack or modify
the DNA sequences theenzyme recognizes
I S d Hi
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Immune System and History
Infectious disease causes 1/3 of deaths today
Infectious disease is a major factor in history:
Native American population fell 95% after the Spanish
landed in North AmericaNative Americans had poorresistance to Old World diseases.
What would the world be like today if Native
Americans.like Asians and Africans. had been
biologically able to resist invasion by Europeans?
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Immune System and History
The ancient Greeks were almost destroyed by ainfectious disease plague that had symptoms that donot match any known disease today
They said the plague came down the Nile River fromcentral Africa.
Why is central Africa a source of infectious disease?
Can new infectious disease plagues appear today?
I f i Di
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Infectious Diseases
Constantly Evolving and Invading new Hosts
HIV Monkey Pox
Immune Systems
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Immune SystemsInnate
FastPhysical barriers and
recognition of a limitednumber of microbemolecules
Production of a limited numberof anti-microbe molecules
Stable genes for receptors such
as Toll Like Receptors. (TLR)
Responds in same way everytime
Specific or Adaptive
Slow
Can develop recognition to
almost any microbe
Variable genes encode B and Tcell receptors
Has memory and responds
differently to repeatedinfections or after
vaccination
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Innate Defenses
Physical and Microbial barriers
Specialized proteins
Phagocytic cells
Cell-intrinsic defenses
Inflammation and fever
Innate Immune system
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Innate Immune system
Barriers
Barriers prevent infections by preventing infectiousagents from gaining access to tissues or cells
Physical barriers: Thick layers of dead skin, basallamina, mucus layers and tight junctions betweencells all can prevent infections.
Microbial barriers: Specialized communities ofmicrobes in body openings and within femalereproductive tract and intestines help to preventestablishment of populations of pathogens
I t D f Ph i l B i
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Innate Defenses: Physical Barriers
Epithelial cell layers
prevent microbes
from reach body
tissues
---skin---respiratory
---gastrointestinal
---urinary and vaginal
O t l f ki tl t i
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Outer layers of skin mostly protein
Basal lamina: Beneath epithelial layers is
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Basal lamina: Beneath epithelial layers is
dense connective tissue
Tight junctions: Hold epithelial cells together.
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g j p g
Intestinal epithelial cells both absorb nutrients and
protect tissue from intestinal contents
C li Di All h l
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Celiac Disease: Allergy to wheat glutenSome food allergy diseases are caused by loose tight
junctions in intestines letting proteins enter the blood
Innate Defenses
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Innate Defenses
Chemical barriers: Mucus
Some epithelial layers
secrete highly
hydroscopic molecules
to form mucus barriers
to prevent bacteria fromdirectly touching tissues
and by trapping and
washing away bacteria
and viruses
Innate Defenses
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Innate Defenses
Chemical barriers: Mucus
During pregnancy thecervix forms a mucus
plug as a barrier to
maintain a sterile
uterus
Innate Defenses
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Innate Defenses
Microbial barriers
Mucus membrane secretions select for a community of
bacteria that cause the female reproductive tract to
be highly acid, this limits the growth of pathogens
Ears, skin, oral and respiratory cavities and intestineshave similar communities of protective bacteria
Eliminating these bacterial communities by excessiveantibiotic use can allow fungal infections to develop.
Recent publication in January 2013:
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Recent publication in January 2013:
New England Journal of Medicine
Title or Article: Duodenal Infusion of Donor Feces forRecurrent Clostridium difficile
Recurrent C. difficile infection is difficult to treat, and
failure rates for antibiotic therapy are high.
C. difficile infection is the most commonly identifiedhospital transmitted infection. It causes serious and
life threatening diarrhea.
A solution made from healthy donor feces containsmany different species of bacteria, protozoa andfungi
New England Journal of Medicine
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New England Journal of Medicine
A controlled experiment:
1) Vancomycin therapy and bowel lavage and infusionof donor feces by nasoduodenal tube
2) Vancomycin therapy and bowel lavage
3) Vancomycin therapyTargeted endpoint: end of diarrhea for 10 weeks.
Success rates for reaching targeted endpoint:
1) 81% (re-treatment improved success rate)
2) 23%
3) 31%
Innate Defenses: Specialized Proteins
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Innate Defenses: Specialized Proteins
Antibiotic peptides and proteins
Lysozyme
Dissolves bacterial cell wallsfound in tears
Defensins
Naturally produced antibiotic peptides found
widely in nature
Complement system
Molecules in blood that attack microbes and helps or
complementsadaptive immune system
Defensins
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DefensinsLarge family of antibiotic peptides found from plants to
humans
Different defensins have different functions and reduce
infection by bacteria, fungi and some viruses
Also found as venoms in some poisonous snakes
Perhaps dozens of human genesnot all active..difficult to study as the different defensins overlap
in functions but have potential as drug molecules
Defensins:
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Defensins:
Cationic and hydrophobic sides
Some form membrane
pores in bacteria and
fungi
Some defensins may
enter cells and have
other functions
Defensins:
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Found in storage granules of some phagocytes
Some Viruses have cell-like membranes
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Some Viruses have cell-like membranes
that are targets of defensins
Some viruses such as HIV and influenza need
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membrane membrane fusions for infection to occur
Some Defensins may work by blocking membrane
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membrane fusions necessary for virus infection
Innate Immune system Recognition of Infection
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Innate Immune system Recognition of Infection
DAMP: Damage Associated Molecular Patterns
Some human molecules can recognize molecules
associated with human cell damage
PAMP: Pathogen Associated Molecular Patterns
Some human molecules can bind to molecules found on
many different bacteria, fungus and viruses
These molecules often have repeated lipid orcarbohydrate structures or patterns
DAMP:
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DamageAssociatedMolecularPatterns
Cells under stress or dying for any reason release the
same molecules. These molecules can signal that aninfection is underway even if a pathogen has no
molecules the innate immune system recognizes.
.Reactive oxygen species (ROS) released by cells
under oxidative stress caused by infection
. ATP released to extracelluar space with cell death
.a severe drop in cellular potassium ion levels
PAMP: PRR
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PathogenAssociatedMolecularPatterns
Recognized by Pattern Recognition Receptors
Innate immune system proteins that bind to multiplemolecules essential for microbe viability.this makesit hard for microbes to evolve new, unrecognized
molecules. This is like recognizing fish scales, mostfish cannot evolve away from them
Sugars, such as those found in microbe membranes
proteins, such as those in bacterial flagellinspeptidoglycans of bacterial cell walls
Lipopolysacchride (LPS)
nucleic acid molecules such as dsRNA or CG repeats
PAMP: PRR
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PathogenAssociatedMolecularPatterns
Recognized by Pattern Recognition Receptors
Complement proteins:
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p pComplementsfunction of antibodies
About 20 defensive proteins produced in liver
Recognize microbial molecules or antibodies that have
bound to microbial molecules
Become activated and act in a proteolytic cascadeto
activate C3
Smaller proteolytic fragments may function as signaling
molecules to attract phagocytic cells to the site of
infection or to stimulate inflammation
Complement protein system
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p p y
Activation
Classical pathwayActivated when antibodies
bind microbes
Lectin and Alternativepathways:recognize andbind to bacterial, fungalmembranes sugars
All three pathways mergeat C3
Functions
Signaling: Activate adaptivesystem and inflammation
Opsonization: Coating
bacteria and facilitatingphagocytosis
Membrane Attack
Complexesto form pores
to destroy target cells
Some Pattern Recognition Receptors such as Mannose
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Binding protein bind sugars found on the surface of
bacteria and activate the complement system.
Lectin binding pathway:Lectins (a specific sugar
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binding protein) such as MBL, bind to sugars
found in bacteria and fungus in a particular
pattern activate complement pathway.MASP: Mannose associated serine protease.
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Complement Activation
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C3b binds to glycoproteins on pathogen surfaces and
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then binds the final complement pathways
Opsonization: Complement proteins coat microbes to
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promote or complement or help the activity of
antibodies to stimulate phagocytosis
omp emen a so orms em rane acComplexes to destroy bacterial cells
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Complexes to destroy bacterial cells
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Complement: Membrane Attack Complex
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Sialic Acid: A terminal residue on human cell- surface
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Sialic Acid: A terminal residue on human cell surface
carbohydrates protects cells from complement attack.
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Nisseria Gonorrhoeae
Escapes the innate immune system by coating itselfwith sialic acid to escape attack by complement
Causes a very serious bacterial sexually transmitted
disease that can cause female sterility, increasechances of HIV transmission and cause kidney failureand meningitis.
Cephalosporins, have been the only effectiveantibiotic: Now a drug resistant strain has beendetected in Japan.
NOD and Toll Like Receptors (TLRs)
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NOD and Toll Like Receptors (TLRs)
NOD and NOD-Like
Receptors (NLRs)found within cells
mutant forms linked to
Crohns diseaseachronic inflammation of
the intestine
believed to activate
transcription of cellulardefense genes
Toll Like Receptors (TLRs)transmembrane receptor
proteins found on cell
surface and on
endosomes within cellsrecognize PAMPs of Virus,
Fungus and Bacteria
10 TLRs currently known
Toll Proteins: Proteins found widely in life.
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First discovered in Drosophila: Flys without the
gene for Toll often died of fungus infection.
Toll Like Receptors: Recognize PAMP
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and begin complex immune response
PRR recognize indicators of infection
A) Begins Cell-intrinic defenses within infected cells
and also trigger inflammation and macrophages tocome to the area to phagocytose bacteria
B) Dendritic cells recognize bacteria with TLRs andphagocytose them and then signal T-cells and B-
cells of adaptive immune system to respond
Toll Like Receptors: Recognize PAMP and begincomplex immune response
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complex immune response
Toll Like Receptors
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Toll Like Receptors
TLRs bind to a PAMP on one side of the membrane and
send signals to the other side of the membrane toactivate NF-kB and cause it to translocate to nucleus
NF-kB or NF kappa Bgene activation within the cell
leads to cell defenses and the release of cytokinesand interleukins that act similar hormones tosimulate the growth and activation of the cell and
other immune system cells.
TLR10 is known as an orphan receptor: the genesequence shows it is a TLR, but we dont know whatit binds to.
Cytokines and Interleukins
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Cytokines are molecules produced by nearly all cellstypes, especially epithelial cells and cells of the
immune system
Many different functions, especially related to immunesystem functions
Most cytokines are named as Interleukins(Inter+Between: Leukin, from leukocyte or white
blood cell). Currently IL-1 to IL-36 are known.
Chemokines: cytokines that attract white blood cells tomigrate to an area.
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TLRs target binding
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TLRs target binding
Cell surface bindingis to different bacterial moleculessuch as LPS or flagellin proteins
Endosome TLRsscan molecules that have been broughtwithin cells: virus associated molecules, dsRNA,
abnormally processed ssRNA, and unmethylated
CpG.
Gene activation caused by TLR directs immune
response to type of infection, B-cells for
extracellular infections, T-cells for within cells
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TLR 4 Responds to Lipopolysacchride (LPS)
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LPS: Part of gram negative bacteria cell membranes.
Excessive exposure within blood causes over-reaction of
immune system Septic Shockwhich may be fatal.
LPS binds receptor protein and forms a complex with
CD14 and associated proteins. TLR 4 then actives gene
transcription by way of NF-KB to activate innate
immune system response genes that causeinflammation etc.
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CD: Cluster of Differentation
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CD: Cluster of Differentation
A CD number is assigned to a protein after monoclonal
antibodies have been found that bind to that protein.
This is used to distinguish or differentiate cell typesthat my look exactly alike under the microscope.
For example: only CD8 cells have the CD8 molecules ontheir surface. Most cell types have more than one CD
protein found on their surface.
There are now hundreds proteins that have been givenCD numbers.
How the proteins involved inLPS response were found
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p p
Tissue culture cells tolerate LPS exposure
Mice injected with sterile solutions of LPS die of septicshock caused by excessive immune response
1. Mice were injected with toxic levels of LPS
2. Mice that survived were mutant in genes such as
TLR4 and did not respond to LPS
Genetics were used on the mutant mice to isolate thegene for TLR4
LPS binds to a receptor protein that then binds to a CD
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p p
protein (CD14) to activate innate immune system
responses such as inflammation.
Both (lipo)teichoic acid and LPS can be
recognized by Toll Like Receptors
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recognized by Toll Like Receptors
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TLRs: Medical and Drug development
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Activators: anti-cancer, anti-virals used to stimulate an
anti-disease immune response
Adjuvants: stimulate TLR to activate immune system toimprove response to a vaccine
Inhibitors: antisepsis treatments, Crohns disease andother autoimmune disorders. Anti-inflamatories
Diagnostics: detection of genes forms (alleles) of TLRswith DNA testing to indicate genetic disease fromeither overactive TLRs or lack of TLR activity
Infection in blood: Sepsis
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Can cause multiple organ failure and death due to lack
of adequate circulation.
Excessive cytokine release in response to moleculessuch as LPS in blood with sepsis can cause systemwide blood vessel dilation and increased permeabilityof capillaries leading to hypotension: Septic Shock
Death may result from myocardial infarction or heartattack as the heart exhausts itself trying tomaintain an adequate blood pressure by increasingcardiac output
Septic Shock
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Treatment generally addresses three medical issues at
once.
1) I.V. fluids and drugs such as norepinephrine to
cause vasoconstriction to increase blood pressure.
2) Antibiotics to treat the infection causing the sepsis
3) Low dose steroids such as hydrocortisone to reducethe immune response. Hydrocortisone also has the
effect of helping to raise the blood pressure.
Pyrogen: Gk,pyr,fire,genein,to produce
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y g , py , , g , p
Pyrogens: molecules that stimulate the immune systemto release molecules to cause fever and inflammation
Measured in EU or Endotoxin Units. One EU is equal to
approximately the same amount of LPS as is found inabout 100 E. coli
Presence of pyrogens can be tested on animals such as
rabbits, but this is expensive and considered cruel tothe animals. A more common test for endotoxin (LPS)is the Limulus amebocyte lysate (lAL) assay using .The blood of Horseshoe crabs (Limulus).
Limulus amebocyte lysate (LAL) assay
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Limulus amebocyte lysate (LAL) assay
The horseshoe crab blood cells
(amebocytes) respond to thepresence of endotoxin with aproteolytic cascade leading toblood clots.
Ameobocytes extracts are usedfor a very sensitive assay forthe presence of endotoxin.
This test is widely used in thepharmaceutical industry to testproducts before marketing.
LAL assay: endotoxin added to the assay activates a
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protease that digests an artificial chromogenic
substrate. The amount of protease activity indicates
the amount of endotoxin and is measured with photospectrometry.
Pyrogen-Free Manufactured ProductsDestruction or Removal of unwanted molecules
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Destruction or Removal of unwanted molecules
Equipment made of metal or glass can be heat treated
Plastics can be treated with acid or base or oxidationwith hydrogen peroxide
Solutions such as I.V. fluids can be subjected to
ultra filtration, distillation and ion exchangechromatography.
Solutions containing biologicals such as proteins mayneed individual protocols of production and mayrequire extensive testing
Internal DAMP Signals
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S g
Massive tissue damage such can cause septic shock-likeconditions even the skin was not broken and there isno bacterial infection.
Molecules such as CpG from dead mitochondrial cellscan stimulate TLRs the same as if they were frombacteria, this can lead to septic shock-likeconditions.
Apparently, the body uses this signal from dead tissuebecause in promotes healing by causing blood vesseldilation and recruitment of phagocytes.
Innate immune system: PhagocytosisKilling microbes b Ingesting or Eating them
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Killing microbes by Ingesting or Eating them
--Chemotaxisfollowing the concentration gradient ofChemokines released by other cells that have boundto the microbe to infection
--Adherence to microbes with TLRs and other bacterialrecognition molecules such as complement orantibodies of the adaptive immune system
--Ingest microbes into phagosomes within the cell
-- Phagosome fusion with lysosomes: phagolysosome
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Phagocytes: injest microbes into phagosomes
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Greek:phagein: To eat
Innate immune system cells designed to devour
and destroy:extra cellular matrix material and dead cells
for tissue remodeling and old RBCs
foreign cells and microbescan become activated and undergo a
Respiratory Burst..they increase their
oxygen use because they are using more ATP Macrophages can be activated by either pyrogens or
cytokines. They can function in innate or adaptive
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cytokines. They can function in innate or adaptive
immune systems and in tissue repair.
Phagocytosis
Surrounding and engulfing and destruction of microbe
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--Surrounding and engulfing and destruction of microbeby combinations of defensins, changes in ph,
proteases, and ROS added to phagosome
-- Reactive Oxygen Species (ROS) or Reactive oxygenintermediates (ROIs): HOCL, H2O2, Hydroxyl
radicals. These molecules break covalent bonds
--Some of these ROS seem to be toxic to the phagocyte
and to surrounding tissue and cause the damageassociated with prolonged inflammation
--Activates inducible Nitric Oxide Synthase(iNOS)
iNOS
i d ibl Nit i O id th t
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inducible Nitric Oxide synthetase
An enzyme which is turned on orinduced in activated
macrophages.
NO is toxic to bacteria and can act
as free radical and also acts as a
neurotransmitter to cause
smooth muscles in blood vesselsto relax so blood flow increases
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Macrophages
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Phagocytes: Macrophages
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Macrophages: Found as resident cells within tissues and
are one of the first cells to encounter a microbe.Develop in bone marrow as monocytes and thendifferentiate into macrophages in tissue.
Do much of the long term protection and do much ofthe digestion of dead and damaged self tissue
Special, tissue specific forms:
Microglial cells: found in CNS
..Dust Cells: in lung: alveolar macrophages
..Kupffer cells: found in Liver
Phagocytes: Kupffer cells
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Liver specific macrophages
engulf bacteria, debris
and damaged RBCs.
One of two functional celltypes in liver
Used to show the processof phagocytosis with soot
particles in India Ink
injected into mice blood
vessels
Phagocytes: Monocytes
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MonocytesDifferentiates into Macrophages in tissues
Migrate from bone marrow stem cells
Move into blood in response to chemokines released in
infections and follows chemokines to infection
Bone marrow production increases during infection
when more macrophages are needed.monocytes
leaving bone marrow stimulate the production ofmore monocytes
PhagocytesNeutrophilsor Polymorphonuclear leukocytes
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Always found in blood, travels to sites of infection
4,000,000 to 10,000,000 per ml of blood
Blood level increases rapidly when neutrophils arereleased from bone marrow to travel to infection
Produced by bone marrow stem cells
Additional cells to quickly help MacrophagesFast acting.but live for short time
Dead neutrophils make up most of Pus
Can release DNA to trap microbes in area
Bacteria trapped by DNA and nuclear proteinsreleased from dead neutrophils
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released from dead neutrophils
RBCs andMonocyte and Polymorphonuclear leukocytes
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y y p y
Pathogenic bacteria have evolved manyways to evade immune systems. But, it is
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y y ,
difficult for to evade all of our defenses.
S 2
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Start 2
Natural Killer Cells
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Produced from bone marrow stem cells
Have cell surface receptors that scan forabnormal cells and evidence of intracellular
infection by bacteria and viruses
Kills our own cellsthat are abnormally stressed,
by infection or other processes such asbecoming cancerous, by causing them tocommit apoptosisor programmed cell death
Natural Killer Cell attacking another cell
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Natural Killer Cell attacking another cell
Natural Killer cellsCell Killing
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g
1. NK cells recognizes an abnormal cell an forms an
immunological synapse with it.
2. Golgi apparatus moves like turret of a tank
and vesicles of Perforinsreleased towards cell
3 Perforins are believed to diffuse into target cellmembrane and form complexes and create pores
4 Granzymesor enzymes from granules in the NK cells
then diffuse into cell cytoplasm and stimulates
5 Apoptosis:a program of cell death that includes
DNA degradation
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Cytokines signaling: Interferons
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Released in large amounts in response to large dsRNA
molecules or unmethylated CG repeats in DNA
Interferon acts as:
Autocrine hormone: acts on the same cell thatproduced the interferon to fight infection within the
cell
Paracrine hormone: acts on nearby cells, even if cell is
uninfected
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Innate Immune system: Cell Intrinsic Responses
I i i i h d i
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Intrinsic responses: proteins that respond to virusinfection within the cell.
Interferons: in response to the presence of large dsRNAor activation by the TLR pathways, stimulate the cell
intrinsic responses.
Interferon and interleukins leads to gene expression ofover 300 genes. Sometimes this causes apoptosis.
Goal is to make cells poor viral hosts by shutting downthe protein synthesis virus need. Rnase L: degradesRNA inside of cell.both viral and cellular.
Below; Chikungunya virus (CHIKV) causing release of
Interferons (INFs) and Interleukins example: IL-1B
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Interferons (INFs) and Interleukins example: IL 1B
Interferon and cell intrinsic responses can sometimereduce viral load(amount of virus) even before an
ff ti d ti i t
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effective adaptive immune system response.
Graph showing CHIKV infection infection
Interferons are used as antiviral drug molecules
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Example: Chronic Hepatitis B infection..Naturally clears in 5-15% of patients a year.
..With interferon treatment 25-40% of patients appear
clear of infection after six months.
Interferon can have serious side effects, includingsevere depression.people with interferon therapyhave committed suicide because they were notwarned of the possible depression.
Interferons are used as antiviral drug molecules
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Chronic Hepatitis C infection.
Interferon treatment with an antiviral drug ribavirin
(Rebetol) has become the standard treatment option.
96-100% of patients who respond to this treatment have
undetectable levels of virus after 24 weeks
Innate Immune System: Inflammation
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Inflammation: limits the infection
Swelling Heat Redness Pain
Cytokines released from cells binding to PAMP or DAMP
can trigger many inflammitory effects including:
Mast cells release of histamineas signaling moleculefor increased blood vessel permeability of nearby
tissue
Mast cells also releases prostaglandins which increasevasodilation and promotes chemotaxis of neutrophils
Inflammation
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Dilation of blood vessels increase blood flow to infected
area
Capillary permeability also increases and fluid leavescapillaries and causes swelling in infected tissue
Fibrin from the blood clotting cascade forms fibersaround wound area to trap bacteria
Chemokines released into blood and travel to bonemarrow to cause release of phagocytes attractsphagocytes to infection site by chemotaxis
Inflammation: Phagocytes
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Distant effects: Phagocytes released from
attachment in bone marrow and enterbloodstream in response to chemokines
released at site of infection that travel to
bone marrow in the blood.
Local effects: blood vessel endothelial cells
respond to inflammation factors by dialatingand changing the glycoproteins on the
endothelial cell surfaces:selectins
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Emigration from blood vessel into tissue:
Monocytes and Neutrophils
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Monocytes and Neutrophils
The leukocytes begin to stick to selectins on bloodvessel walls and roll along: margination
Local presence of chemokines causes theleukocytes to adhere tightlyand begin migrating out of
blood vessels thru enlarged capillary pores:diapedesis
Leukocytes then migrateto site of chemokine release
(chemotaxis)
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InflammationRole in many diseases
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y
Chronic inflammation may have roles in heart manydiseases including heart disease, cancer andautoimmune diseases
Aspirin blocks prostaglandin synthetase . somedoctors in US recommend a childs aspirin every dayto reduce risk of heart disease and cancer. Recentevidence shows a 20% decrease in cancer deaths
among people who take aspirin daily.
C-Reactive proteinused as a measure of systemicinflammation.
Inflammation:Endometriosis
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Endometriosis: the most common cause offemale sterility in the developed world
Believed to result from abnormal inflammationresponse in the endometrium lining of uterus
Why would the uterus have such sensitivity to
inflammation?
IUD: Intra-Uterine DeviceA birth control device widely used in China. Also used
in US when other methods are not appropriate
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in US when other methods are not appropriate
Works especially well whenmade with copper.
Causes constant irritationand inflammation ofendometrium: The cellslining the uterus.
Prevents pregnancy becauseinflammation preventsembryo implantation
Innate Immune System: Fever
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Fever: a metabolically elevated body temperature
induced by the hypothalamus of the brain in responseto Interleukin 1 (IL-1)released by macrophages
Cells, such as phagocytes, move and reproduce faster
and enzymatic reactions are quicker at higher
temperatures
Also, some bacteria may not function well at highertemperature. i.e. soil dwelling bacteria are not
adapted to the higher body temperature.
Fever:Metabolic functions are
temperature sensitive.
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temperature sensitive.
Siamese cats have a mutation
to an enzyme that makes
black hair color. In the
cooler parts of the body,the enzyme works well, in
the warmer parts it has no
activity. Even small
differences in temperaturecan affect metabolic
activity.
Dendritic cells and Macrophages:Phagocytes that present molecules to T-cells and B-
cells of adaptive immune system:
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cells of adaptive immune system:
Antigen Presenting Cells (APCs)
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Dendritic Cells: From Greek dendronfor Tree.
Contacts and recognizes
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bacteria with pattern
recognition receptors (PRR)like TLRs
1) Releases cytokines toattract macrophages.
2) Phagocytoses microbes and
migrates to lymph nodes toactivate adaptive immune
system to specifically
recognize that microbe
Dendritic cells presenting microbe molecules to
cells of the adaptive immune system for recognition.
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The T-cells that recognize the antigen will migrate tothe site of the infection.
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Surgical modification of the innate immune system:Male Circumcision
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Circumcision: The removal of the foreskin, the skin
covering the end of the penis.
The most widely performed surgical procedure in theworld and the only commonly performed surgery to
remove healthy tissue.
Has been done since the beginning of history and isshown in some cave paintings.
Has cultural, hygiene and medical functions
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Surgical modification of the innate immune system:Male Circumcision
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Estimated to have been performed on 30% of males
worldwide, especially Christians, Jews and Muslims.
Cultures vary, but very frequently surgery is done oninfant children
Some controversy exists as medical benefits have notbeen considered significant.
Now: recent studies in Africa have shown thatcircumcision in adult heterosexual men reduced HIV
infection of the circumcised man by 60%.
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HIV is able to infect the Dendritic cells in the mucosal
skin on the inside of the foreskinthey then carry the
virus to the lymph node where it infects other cells
Efforts are being made to circumcise 20 million adult
men in Africa by 2015 to fight the spread of HIV.
Problem: adult circumcision requires a medical team
including a surgeon. This has severely limited the
procedure in developing countries
Circumcision removes the mucosal layer of skin of theinner foreskin that is an HIV site of entry.
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Circumcision does not reduce virus transmission coming
from an HIV-positive male.
Surgical modification of the innate immune system:Male Circumcision
Traditional procedure
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Traditional procedure
involves cutting tissueand stitching.
Infection can be a serious
complication, in thedeveloping world wheresterile technique isimperfectly maintained.
Also, trained surgeons aremuch in demand formany other surgeries
Current testing involves disposable medical devices
that can be quickly applied in large numbers by teams
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of nurses and medical technicians
PrePex Shang Ring (China)
PrePex1) Ring device is placed over head of penis
2) F ki i f ld d b k it
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2) Foreskin is folded back over it
3) PrePex: A rubber band is tightened over skin on topof plastic ring
4) The skin between plastic ring and rubber band diesfrom lack of blood without breaking the skin openand risking infection
5) Dead skin distal to the rubber band is cut away a fewdays later after skin next to rubber band has healed
A two-nurse team can do over 100 procedures a day.
PrePex procedure has a lower rate of surgical infectionsthan traditional surgery.
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