407 Innate Immune S 2014

8/13/2019 407 Innate Immune S 2014

1/160

8/13/2019 407 Innate Immune S 2014

2/160

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?

8/13/2019 407 Innate Immune S 2014

3/160

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

8/13/2019 407 Innate Immune S 2014

4/160

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.

8/13/2019 407 Innate Immune S 2014

5/160

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.

8/13/2019 407 Innate Immune S 2014

6/160

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.

8/13/2019 407 Innate Immune S 2014

7/160

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

8/13/2019 407 Innate Immune S 2014

8/160

Cells in the body grow in a low oxygen,

high carbon dioxide environment

8/13/2019 407 Innate Immune S 2014

9/160

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.

8/13/2019 407 Innate Immune S 2014

10/160

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.

8/13/2019 407 Innate Immune S 2014

11/160

Confluence: Cells cover dish floor. All cells are

in full contact with other cells.

Less than confluent. Fully confluent

8/13/2019 407 Innate Immune S 2014

12/160

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.

8/13/2019 407 Innate Immune S 2014

13/160

8/13/2019 407 Innate Immune S 2014

14/160

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

8/13/2019 407 Innate Immune S 2014

15/160

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

8/13/2019 407 Innate Immune S 2014

16/160

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.

8/13/2019 407 Innate Immune S 2014

17/160

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

8/13/2019 407 Innate Immune S 2014

18/160

8/13/2019 407 Innate Immune S 2014

19/160

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.

8/13/2019 407 Innate Immune S 2014

20/160

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.

8/13/2019 407 Innate Immune S 2014

21/160

Inverted Microscope

8/13/2019 407 Innate Immune S 2014

22/160

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

8/13/2019 407 Innate Immune S 2014

23/160

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

8/13/2019 407 Innate Immune S 2014

24/160

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

8/13/2019 407 Innate Immune S 2014

25/160

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.

8/13/2019 407 Innate Immune S 2014

26/160

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

8/13/2019 407 Innate Immune S 2014

27/160

8/13/2019 407 Innate Immune S 2014

28/160

8/13/2019 407 Innate Immune S 2014

29/160

Embryos that are frozen have a survival rate of

about 70%. Healthy ones can be selected with a

mircopipette for implantation.

8/13/2019 407 Innate Immune S 2014

30/160

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.

8/13/2019 407 Innate Immune S 2014

31/160

8/13/2019 407 Innate Immune S 2014

32/160

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.

8/13/2019 407 Innate Immune S 2014

33/160

8/13/2019 407 Innate Immune S 2014

34/160

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

8/13/2019 407 Innate Immune S 2014

35/160

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

8/13/2019 407 Innate Immune S 2014

36/160

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?

8/13/2019 407 Innate Immune S 2014

37/160

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

8/13/2019 407 Innate Immune S 2014

38/160

Infectious Diseases

Constantly Evolving and Invading new Hosts

HIV Monkey Pox

Immune Systems

8/13/2019 407 Innate Immune S 2014

39/160

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

8/13/2019 407 Innate Immune S 2014

40/160

8/13/2019 407 Innate Immune S 2014

41/160

Innate Defenses

Physical and Microbial barriers

Specialized proteins

Phagocytic cells

Cell-intrinsic defenses

Inflammation and fever

Innate Immune system

8/13/2019 407 Innate Immune S 2014

42/160

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

8/13/2019 407 Innate Immune S 2014

43/160

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

8/13/2019 407 Innate Immune S 2014

44/160

Outer layers of skin mostly protein

Basal lamina: Beneath epithelial layers is

8/13/2019 407 Innate Immune S 2014

45/160

Basal lamina: Beneath epithelial layers is

dense connective tissue

Tight junctions: Hold epithelial cells together.

8/13/2019 407 Innate Immune S 2014

46/160

g j p g

Intestinal epithelial cells both absorb nutrients and

protect tissue from intestinal contents

C li Di All h l

8/13/2019 407 Innate Immune S 2014

47/160

Celiac Disease: Allergy to wheat glutenSome food allergy diseases are caused by loose tight

junctions in intestines letting proteins enter the blood

Innate Defenses

8/13/2019 407 Innate Immune S 2014

48/160

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

8/13/2019 407 Innate Immune S 2014

49/160

Innate Defenses

Chemical barriers: Mucus

During pregnancy thecervix forms a mucus

plug as a barrier to

maintain a sterile

uterus

Innate Defenses

8/13/2019 407 Innate Immune S 2014

50/160

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:

8/13/2019 407 Innate Immune S 2014

51/160

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


8/13/2019 407 Innate Immune S 2014

52/160


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

8/13/2019 407 Innate Immune S 2014

53/160

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

8/13/2019 407 Innate Immune S 2014

54/160

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:

8/13/2019 407 Innate Immune S 2014

55/160

Defensins:

Cationic and hydrophobic sides

Some form membrane

pores in bacteria and

fungi

Some defensins may

enter cells and have

other functions

Defensins:

8/13/2019 407 Innate Immune S 2014

56/160

Found in storage granules of some phagocytes

Some Viruses have cell-like membranes

8/13/2019 407 Innate Immune S 2014

57/160

Some Viruses have cell-like membranes

that are targets of defensins

Some viruses such as HIV and influenza need

8/13/2019 407 Innate Immune S 2014

58/160

membrane membrane fusions for infection to occur

Some Defensins may work by blocking membrane

8/13/2019 407 Innate Immune S 2014

59/160

membrane fusions necessary for virus infection

Innate Immune system Recognition of Infection

8/13/2019 407 Innate Immune S 2014

60/160

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:

8/13/2019 407 Innate Immune S 2014

61/160

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

8/13/2019 407 Innate Immune S 2014

62/160

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

8/13/2019 407 Innate Immune S 2014

63/160

PathogenAssociatedMolecularPatterns

Recognized by Pattern Recognition Receptors

Complement proteins:

8/13/2019 407 Innate Immune S 2014

64/160

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

8/13/2019 407 Innate Immune S 2014

65/160

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

8/13/2019 407 Innate Immune S 2014

66/160

Binding protein bind sugars found on the surface of

bacteria and activate the complement system.

Lectin binding pathway:Lectins (a specific sugar

8/13/2019 407 Innate Immune S 2014

67/160

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.

8/13/2019 407 Innate Immune S 2014

68/160

Complement Activation

8/13/2019 407 Innate Immune S 2014

69/160

C3b binds to glycoproteins on pathogen surfaces and

8/13/2019 407 Innate Immune S 2014

70/160

then binds the final complement pathways

Opsonization: Complement proteins coat microbes to

8/13/2019 407 Innate Immune S 2014

71/160

promote or complement or help the activity of

antibodies to stimulate phagocytosis

omp emen a so orms em rane acComplexes to destroy bacterial cells

8/13/2019 407 Innate Immune S 2014

72/160

Complexes to destroy bacterial cells

8/13/2019 407 Innate Immune S 2014

73/160

Complement: Membrane Attack Complex

8/13/2019 407 Innate Immune S 2014

74/160

Sialic Acid: A terminal residue on human cell- surface

8/13/2019 407 Innate Immune S 2014

75/160

Sialic Acid: A terminal residue on human cell surface

carbohydrates protects cells from complement attack.

8/13/2019 407 Innate Immune S 2014

76/160

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)

8/13/2019 407 Innate Immune S 2014

77/160

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.

8/13/2019 407 Innate Immune S 2014

78/160

First discovered in Drosophila: Flys without the

gene for Toll often died of fungus infection.

Toll Like Receptors: Recognize PAMP

8/13/2019 407 Innate Immune S 2014

79/160

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

8/13/2019 407 Innate Immune S 2014

80/160

complex immune response

Toll Like Receptors

8/13/2019 407 Innate Immune S 2014

81/160

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

8/13/2019 407 Innate Immune S 2014

82/160

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.

8/13/2019 407 Innate Immune S 2014

83/160

TLRs target binding

8/13/2019 407 Innate Immune S 2014

84/160

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

8/13/2019 407 Innate Immune S 2014

85/160

TLR 4 Responds to Lipopolysacchride (LPS)

8/13/2019 407 Innate Immune S 2014

86/160

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.

8/13/2019 407 Innate Immune S 2014

87/160

CD: Cluster of Differentation

8/13/2019 407 Innate Immune S 2014

88/160

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

8/13/2019 407 Innate Immune S 2014

89/160

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

8/13/2019 407 Innate Immune S 2014

90/160

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

8/13/2019 407 Innate Immune S 2014

91/160

recognized by Toll Like Receptors

8/13/2019 407 Innate Immune S 2014

92/160

TLRs: Medical and Drug development

8/13/2019 407 Innate Immune S 2014

93/160

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

8/13/2019 407 Innate Immune S 2014

94/160

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

8/13/2019 407 Innate Immune S 2014

95/160

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

8/13/2019 407 Innate Immune S 2014

96/160

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

8/13/2019 407 Innate Immune S 2014

97/160

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

8/13/2019 407 Innate Immune S 2014

98/160

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

8/13/2019 407 Innate Immune S 2014

99/160

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

8/13/2019 407 Innate Immune S 2014

100/160

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

8/13/2019 407 Innate Immune S 2014

101/160

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

8/13/2019 407 Innate Immune S 2014

102/160

8/13/2019 407 Innate Immune S 2014

103/160

Phagocytes: injest microbes into phagosomes

8/13/2019 407 Innate Immune S 2014

104/160

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

8/13/2019 407 Innate Immune S 2014

105/160

cytokines. They can function in innate or adaptive

immune systems and in tissue repair.

Phagocytosis

Surrounding and engulfing and destruction of microbe

8/13/2019 407 Innate Immune S 2014

106/160

--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

8/13/2019 407 Innate Immune S 2014

107/160

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

8/13/2019 407 Innate Immune S 2014

108/160

8/13/2019 407 Innate Immune S 2014

109/160

8/13/2019 407 Innate Immune S 2014

110/160

8/13/2019 407 Innate Immune S 2014

111/160

Macrophages

8/13/2019 407 Innate Immune S 2014

112/160

Phagocytes: Macrophages

8/13/2019 407 Innate Immune S 2014

113/160

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

8/13/2019 407 Innate Immune S 2014

114/160

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

8/13/2019 407 Innate Immune S 2014

115/160

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

8/13/2019 407 Innate Immune S 2014

116/160

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

8/13/2019 407 Innate Immune S 2014

117/160

released from dead neutrophils

RBCs andMonocyte and Polymorphonuclear leukocytes

8/13/2019 407 Innate Immune S 2014

118/160

y y p y

Pathogenic bacteria have evolved manyways to evade immune systems. But, it is

8/13/2019 407 Innate Immune S 2014

119/160

y y ,

difficult for to evade all of our defenses.

S 2

8/13/2019 407 Innate Immune S 2014

120/160

Start 2

Natural Killer Cells

8/13/2019 407 Innate Immune S 2014

121/160

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

8/13/2019 407 Innate Immune S 2014

122/160

Natural Killer Cell attacking another cell

Natural Killer cellsCell Killing

8/13/2019 407 Innate Immune S 2014

123/160

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

8/13/2019 407 Innate Immune S 2014

124/160

8/13/2019 407 Innate Immune S 2014

125/160

Cytokines signaling: Interferons

8/13/2019 407 Innate Immune S 2014

126/160

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

8/13/2019 407 Innate Immune S 2014

127/160

Innate Immune system: Cell Intrinsic Responses

I i i i h d i

8/13/2019 407 Innate Immune S 2014

128/160

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

8/13/2019 407 Innate Immune S 2014

129/160

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

8/13/2019 407 Innate Immune S 2014

130/160

effective adaptive immune system response.

Graph showing CHIKV infection infection

Interferons are used as antiviral drug molecules

8/13/2019 407 Innate Immune S 2014

131/160

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

8/13/2019 407 Innate Immune S 2014

132/160

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

8/13/2019 407 Innate Immune S 2014

133/160

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

8/13/2019 407 Innate Immune S 2014

134/160

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

8/13/2019 407 Innate Immune S 2014

135/160

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

8/13/2019 407 Innate Immune S 2014

136/160

Emigration from blood vessel into tissue:

Monocytes and Neutrophils

8/13/2019 407 Innate Immune S 2014

137/160

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)

8/13/2019 407 Innate Immune S 2014

138/160

8/13/2019 407 Innate Immune S 2014

139/160

8/13/2019 407 Innate Immune S 2014

140/160

8/13/2019 407 Innate Immune S 2014

141/160

InflammationRole in many diseases

8/13/2019 407 Innate Immune S 2014

142/160

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

8/13/2019 407 Innate Immune S 2014

143/160

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

8/13/2019 407 Innate Immune S 2014

144/160

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

8/13/2019 407 Innate Immune S 2014

145/160

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.

8/13/2019 407 Innate Immune S 2014

146/160

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:

8/13/2019 407 Innate Immune S 2014

147/160

cells of adaptive immune system:

Antigen Presenting Cells (APCs)

8/13/2019 407 Innate Immune S 2014

148/160

Dendritic Cells: From Greek dendronfor Tree.

Contacts and recognizes

8/13/2019 407 Innate Immune S 2014

149/160

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.

8/13/2019 407 Innate Immune S 2014

150/160

The T-cells that recognize the antigen will migrate tothe site of the infection.

8/13/2019 407 Innate Immune S 2014

151/160

Surgical modification of the innate immune system:Male Circumcision

8/13/2019 407 Innate Immune S 2014

152/160

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

8/13/2019 407 Innate Immune S 2014

153/160


8/13/2019 407 Innate Immune S 2014

154/160

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%.

8/13/2019 407 Innate Immune S 2014

155/160

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.

8/13/2019 407 Innate Immune S 2014

156/160

Circumcision does not reduce virus transmission coming

from an HIV-positive male.


Traditional procedure

8/13/2019 407 Innate Immune S 2014

157/160

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

8/13/2019 407 Innate Immune S 2014

158/160

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

8/13/2019 407 Innate Immune S 2014

159/160

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.

8/13/2019 407 Innate Immune S 2014

160/160

Documents

407 Innate Immune S 2014