Tissue Culture Many cell types can be grown in vitro

Many cells can be grown under special conditions as pure cell types (hepatic cells, fibroblasts, etc.) 1) Grown for study of cell biology.

2) Grown for production of molecules such as monoclonal 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?

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

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 MUST be 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.

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.

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.

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

Cells in the body grow in a low oxygen, high carbon dioxide environment

Tissue culture incubators are large chambers with heaters to keep the cells at 370C and a supply of carbon dioxide to keep the CO2 in 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 .

Cell passaging
Under ideal conditions, cells will increase in number until they touch each other and stop growing. Some cells can be maintained for long periods of time at a confluent state where they have stopped growing and have become quiescent. Other types of cells, especially cancer cells, are best maintained in a state of less-than-confluent so that they continue rapid growth. To transfer cells to other flasks, cells are taken up from their attachment to the plastic flasks and allowed to float freely in the media and transferred.

Confluence: Cells cover dish floor. All cells are in full contact with other cells. Less than confluent. Fully confluent

Cell Passaging: Trypsin Cells can be freed from attachment with the protease Trypsin to digest the cell surface proteins attaching to the dish. This causes them to become round and easily removed by tapping on the side of the culture flask.

Tissue culture media

Synthetic 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 effect of growth factors and hormones can be tested. Only a few cell types can be grown on this type of media Most cell type require growth factors and hormone that we dont know how and how much to supply

Tissue culture media Media supplemented with serum

Most animal and human cells are grown in defined media containing nutrients and vitamins (and a red ph indicator dye) that is supplemented with animal serum. Most common serum is calf serum also known as bovine serum Some cells require higher amounts of growth factors and hormones, these are given the more expensive Fetal calf serum or Fetal Bovine Serum

Tissue culture media: Serum

Serum is the fluid remaining after blood has clotted. The blood clot, which contains all the red blood cells, is removed and the serum is filtered sterilized and frozen. Mixed at 5-10% with a defined media. Serum contains and growth factors and hormones that cells need for growth. Different batches of serum can be different, for example, one batch of serum may contain antibodies that bind to a cell type and another batch may not have those antibodies. Also, serum must be tested to for the presence of viruses and other pathogens.

Tissue culture media: Antibiotics

Most media contains antibiotics against bacterial and fungal contamination. Media may also be given anti Rickettsial antibiotics for bacteria living within tissue culture cells Neutralizing anti-virus antibodies may also be added if a virus is used in the lab and the risk of virus contamination is high

Tissue Culture Hoods

Air flow over working space is filtered to be free of dust that contains bacteria.

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.

Inverted Microscope
Cells 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.

Inverted Microscope

Liquid Nitrogen Cryopreservation: Cell Freezing

Tissue culture cells can be stored in liquid nitrogen at -196oC, where all metabolic processes are stopped. At higher temperatures, even as low as -800C, the high ionic and osmotic concentration inside cells allows some movement of water molecules and organic processes that destroy the cells over time. Frozen cells should not be stored even briefly at -200C Cells are frozen in special media that contains molecules like DMSO to act as a Chaotropic, or, water denaturing agent to prevent the formation of water crystals (ice) that would otherwise grow until they ruptured the cell.

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

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.

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 to develop into a normal animal or person.

In Vitro Fertilization
Eggs and sperm collected and mixed together in tissue culture.

Successful fertilizations begin to develop into embryos.

Embryos can be collected and implanted into female, or frozen for later implantation

Embryos that are frozen have a survival rate of about 70%. Healthy ones can be selected with a mircopipette for implantation.

Stem Cells
Most cells have differentiated into a particular cell type: That is, liver cells and skin cells have exactly the same DNA, but they used different genes to make different proteins so the cell types are different. Totipotent: A cell like a fertilized egg cell having the ability 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 with mitosis only the cell type it already is.

Induced Pluripotent Stem cells

Much 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.

Immune System
Defends against microbes and parasites Removes dead and damaged cells and tissues Allergies and autoimmune diseases Chronic inflammation: heart disease and cancer Transplant tissue rejection Septic shock and anaphylactic shock Provides molecules useful in Biotechnology and Medicine

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 the bacteria - The bacteria lack or modify the DNA sequences the enzyme recognizes

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 poor resistance 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?

Immune System and History

The ancient Greeks were almost destroyed by a infectious disease plague that had symptoms that do not match any known disease today They said the plague came down the Nile River from central Africa. Why is central Africa a source of infectious disease? Can new infectious disease plagues appear today?

Infectious Diseases Constantly Evolving and Invading new Hosts HIV Monkey Pox

Immune Systems
Innate Fast
Physical barriers and recognition of a limited number of microbe molecules Production of a limited number of anti-microbe molecules Stable genes for receptors such as Toll Like Receptors. (TLR) Responds in same way every time

Specific or Adaptive Slow

Can develop recognition to almost any microbe Variable genes encode B and T cell receptors Has memory and responds differently to repeated infections or after vaccination

Innate Defenses
Physical and Microbial barriers

Specialized proteins
Phagocytic cells Cell-intrinsic defenses Inflammation and fever

Innate Immune system Barriers

Barriers prevent infections by preventing infectious agents from gaining access to tissues or cells Physical barriers: Thick layers of dead skin, basal lamina, mucus layers and tight junctions between cells all can prevent infections. Microbial barriers: Specialized communities of microbes in body openings and within female reproductive tract and intestines help to prevent establishment of populations of pathogens

Innate Defenses: Physical Barriers

Epithelial cell layers prevent microbes from reach body tissues ---skin ---respiratory ---gastrointestinal ---urinary and vaginal

Outer layers of skin mostly protein

Basal lamina: Beneath epithelial layers is dense connective tissue

Tight junctions: Hold epithelial cells together. Intestinal epithelial cells both absorb nutrients and protect tissue from intestinal contents

Celiac Disease: Allergy to wheat gluten

Some food allergy diseases are caused by loose tight junctions in intestines letting proteins enter the blood

Innate Defenses Chemical barriers: Mucus

Some epithelial layers secrete highly hydroscopic molecules to form mucus barriers to prevent bacteria from directly touching tissues and by trapping and washing away bacteria and viruses

Innate Defenses Chemical barriers: Mucus

During pregnancy the cervix forms a mucus plug as a barrier to maintain a sterile uterus

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 intestines have similar communities of protective bacteria Eliminating these bacterial communities by excessive antibiotic use can allow fungal infections to develop.

Recent publication in January 2013: New England Journal of Medicine Title or Article: Duodenal Infusion of Donor Feces for Recurrent 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 identified hospital transmitted infection. It causes serious and life threatening diarrhea. A solution made from healthy donor feces contains many different species of bacteria, protozoa and fungi

New England Journal of Medicine A controlled experiment: 1) Vancomycin therapy and bowel lavage and infusion of donor feces by nasoduodenal tube 2) Vancomycin therapy and bowel lavage 3) Vancomycin therapy Targeted 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

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 complements adaptive immune system

Defensins
Large 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: Cationic and hydrophobic sides

Some form membrane pores in bacteria and fungi

Some defensins may enter cells and have other functions

Defensins: Found in storage granules of some phagocytes

Some Viruses have cell-like membranes that are targets of defensins

Some viruses such as HIV and influenza need membrane membrane fusions for infection to occur

Some Defensins may work by blocking membrane membrane fusions necessary for virus infection

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 or carbohydrate structures or patterns

DAMP:
Damage Associated Molecular Patterns
Cells under stress or dying for any reason release the same molecules. These molecules can signal that an infection 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 Pathogen Associated Molecular Patterns Recognized by Pattern Recognition Receptors
Innate immune system proteins that bind to multiple molecules essential for microbe viability.this makes it hard for microbes to evolve new, unrecognized molecules. This is like recognizing fish scales, most fish cannot evolve away from them
Sugars, such as those found in microbe membranes proteins, such as those in bacterial flagellins peptidoglycans of bacterial cell walls Lipopolysacchride (LPS) nucleic acid molecules such as dsRNA or CG repeats

PAMP: PRR Pathogen Associated Molecular Patterns Recognized by Pattern Recognition Receptors

Complement proteins:
Complements function 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 cascade to 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

Activation Classical pathway Activated when antibodies bind microbes Functions Signaling: Activate adaptive system and inflammation

Opsonization: Coating Lectin and Alternative bacteria and facilitating pathways: recognize and phagocytosis bind to bacterial, fungal membranes sugars All three pathways merge Membrane Attack at C3 Complexes to form pores to destroy target cells

Some Pattern Recognition Receptors such as Mannose Binding protein bind sugars found on the surface of bacteria and activate the complement system.

Lectin binding pathway: Lectins (a specific sugar 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.

Complement Activation

C3b binds to glycoproteins on pathogen surfaces and then binds the final complement pathways

Opsonization: Complement proteins coat microbes to promote or complement or help the activity of antibodies to stimulate phagocytosis

Complement also forms Membrane Attack Complexes to destroy bacterial cells

Complement: Membrane Attack Complex

Complement: Membrane Attack Complex

Sialic Acid: A terminal residue on human cell- surface carbohydrates protects cells from complement attack.

Nisseria Gonorrhoeae
Escapes the innate immune system by coating itself with sialic acid to escape attack by complement Causes a very serious bacterial sexually transmitted disease that can cause female sterility, increase chances of HIV transmission and cause kidney failure and meningitis. Cephalosporins, have been the only effective antibiotic: Now a drug resistant strain has been detected in Japan.

NOD and Toll Like Receptors (TLRs)

NOD and NOD-Like Receptors (NLRs) found within cells mutant forms linked to Crohns diseasea chronic inflammation of the intestine believed to activate transcription of cellular defense genes Toll Like Receptors (TLRs) transmembrane receptor proteins found on cell surface and on endosomes within cells recognize PAMPs of Virus, Fungus and Bacteria 10 TLRs currently known

Toll Proteins: Proteins found widely in life. First discovered in Drosophila: Flys without the gene for Toll often died of fungus infection.

Toll Like Receptors: Recognize PAMP and begin complex immune response
PRR recognize indicators of infection A) Begins Cell-intrinic defenses within infected cells and also trigger inflammation and macrophages to come to the area to phagocytose bacteria B) Dendritic cells recognize bacteria with TLRs and phagocytose them and then signal T-cells and Bcells of adaptive immune system to respond

Toll Like Receptors: Recognize PAMP and begin complex immune response

Toll Like Receptors

TLRs bind to a PAMP on one side of the membrane and send signals to the other side of the membrane to activate NF-kB and cause it to translocate to nucleus NF-kB or NF kappa B gene activation within the cell leads to cell defenses and the release of cytokines and interleukins that act similar hormones to simulate the growth and activation of the cell and other immune system cells. TLR10 is known as an orphan receptor: the gene sequence shows it is a TLR, but we dont know what it binds to.

Cytokines and Interleukins

Cytokines are molecules produced by nearly all cells types, especially epithelial cells and cells of the immune system Many different functions, especially related to immune system 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 to migrate to an area.

TLRs target binding

Cell surface binding is to different bacterial molecules such as LPS or flagellin proteins Endosome TLRs scan molecules that have been brought within 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

Lipopolysacchride: LPS
A molecule found in gram negative bacterial membranes. Recognized by the body as a molecular signal of the presence of bacteria (infection).

TLR 4 Responds to Lipopolysacchride (LPS)

LPS: Part of gram negative bacteria cell membranes.

Excessive exposure within blood causes over-reaction of immune system Septic Shock which 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 cause inflammation etc.

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 types that my look exactly alike under the microscope. For example: only CD8 cells have the CD8 molecules on their surface. Most cell types have more than one CD protein found on their surface. There are now hundreds proteins that have been given CD numbers.

How the proteins involved inLPS response were found Tissue culture cells tolerate LPS exposure

Mice injected with sterile solutions of LPS die of septic shock 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 the gene for TLR4

LPS binds to a receptor protein that then binds to a CD protein (CD14) to activate innate immune system responses such as inflammation.

Both (lipo)teichoic acid and LPS can be recognized by Toll Like Receptors

TLRs: Medical and Drug development

Activators: anti-cancer, anti-virals used to stimulate an anti-disease immune response Adjuvants: stimulate TLR to activate immune system to improve response to a vaccine

Inhibitors: antisepsis treatments, Crohns disease and other autoimmune disorders. Anti-inflamatories
Diagnostics: detection of genes forms (alleles) of TLRs with DNA testing to indicate genetic disease from either overactive TLRs or lack of TLR activity

Infection in blood: Sepsis

Can cause multiple organ failure and death due to lack of adequate circulation. Excessive cytokine release in response to molecules such as LPS in blood with sepsis can cause system wide blood vessel dilation and increased permeability of capillaries leading to hypotension: Septic Shock Death may result from myocardial infarction or heart attack as the heart exhausts itself trying to maintain an adequate blood pressure by increasing cardiac output

Septic Shock
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 reduce the immune response. Hydrocortisone also has the effect of helping to raise the blood pressure.

Pyrogen: Gk, pyr, fire, genein, to produce

Pyrogens: molecules that stimulate the immune system to 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 in about 100 E. coli Presence of pyrogens can be tested on animals such as rabbits, but this is expensive and considered cruel to the 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

The horseshoe crab blood cells (amebocytes) respond to the presence of endotoxin with a proteolytic cascade leading to blood clots. Ameobocytes extracts are used for a very sensitive assay for the presence of endotoxin. This test is widely used in the pharmaceutical industry to test products before marketing.

LAL assay: endotoxin added to the assay activates a protease that digests an artificial chromogenic substrate. The amount of protease activity indicates the amount of endotoxin and is measured with photo spectrometry.

Pyrogen-Free Manufactured Products

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 oxidation with hydrogen peroxide Solutions such as I.V. fluids can be subjected to ultra filtration, distillation and ion exchange chromatography. Solutions containing biologicals such as proteins may need individual protocols of production and may require extensive testing

Internal DAMP Signals

Massive tissue damage such can cause septic shock-like conditions even the skin was not broken and there is no bacterial infection. Molecules such as CpG from dead mitochondrial cells can stimulate TLRs the same as if they were from bacteria, this can lead to septic shock-like conditions. Apparently, the body uses this signal from dead tissue because in promotes healing by causing blood vessel dilation and recruitment of phagocytes.

Innate immune system: Phagocytosis

Killing microbes by Ingesting or Eating them
--Chemotaxisfollowing the concentration gradient of Chemokines released by other cells that have bound to the microbe to infection

--Adherence to microbes with TLRs and other bacterial recognition molecules such as complement or antibodies of the adaptive immune system
--Ingest microbes into phagosomes within the cell -- Phagosome fusion with lysosomes: phagolysosome

Phagocytes: Macrophages, Monocytes, Neutrophils

Phagocytes: injest microbes into phagosomes 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 microbes can 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 immune systems and in tissue repair.

Phagocytosis --Surrounding and engulfing and destruction of microbe by combinations of defensins, changes in ph, proteases, and ROS added to phagosome -- Reactive Oxygen Species (ROS) or Reactive oxygen intermediates (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 damage associated with prolonged inflammation --Activates inducible Nitric Oxide Synthase (iNOS)

iNOS inducible Nitric Oxide synthetase

An enzyme which is turned on or induced 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 vessels to relax so blood flow increases

Macrophages

Phagocytes: Macrophages
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 then differentiate into macrophages in tissue. Do much of the long term protection and do much of the 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

Liver specific macrophages engulf bacteria, debris and damaged RBCs. One of two functional cell types in liver Used to show the process of phagocytosis with soot particles in India Ink injected into mice blood vessels

Phagocytes: Monocytes
Monocytes Differentiates 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 of more monocytes

Phagocytes
Neutrophils or Polymorphonuclear leukocytes
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 are released from bone marrow to travel to infection Produced by bone marrow stem cells Additional cells to quickly help Macrophages Fast 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 proteins released from dead neutrophils

Monocyte and

RBCs and Polymorphonuclear leukocytes

Pathogenic bacteria have evolved many ways to evade immune systems. But, it is difficult for to evade all of our defenses.

Start 2

Natural Killer Cells

Produced from bone marrow stem cells Have cell surface receptors that scan for abnormal cells and evidence of intracellular infection by bacteria and viruses Kills our own cells that are abnormally stressed, by infection or other processes such as becoming cancerous, by causing them to commit apoptosis or programmed cell death

Natural Killer Cell attacking another cell

Natural Killer cells Cell Killing

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 Perforins released towards cell 3 Perforins are believed to diffuse into target cell membrane and form complexes and create pores 4 Granzymes or 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

Cytokines signaling: Interferons

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 that produced the interferon to fight infection within the cell

Paracrine hormone: acts on nearby cells, even if cell is uninfected

Innate Immune system: Cell Intrinsic Responses

Intrinsic responses: proteins that respond to virus infection within the cell. Interferons: in response to the presence of large dsRNA or activation by the TLR pathways, stimulate the cell intrinsic responses. Interferon and interleukins leads to gene expression of over 300 genes. Sometimes this causes apoptosis. Goal is to make cells poor viral hosts by shutting down the protein synthesis virus need. Rnase L: degrades RNA inside of cell.both viral and cellular.

Below; Chikungunya virus (CHIKV) causing release of Interferons (INFs) and Interleukins example: IL-1B

Interferon and cell intrinsic responses can sometime reduce viral load (amount of virus) even before an effective adaptive immune system response. Graph showing CHIKV infection infection

Interferons are used as antiviral drug molecules 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, including severe depression.people with interferon therapy have committed suicide because they were not warned of the possible depression.

Interferons are used as antiviral drug molecules

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

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 histamine as signaling molecule for increased blood vessel permeability of nearby tissue Mast cells also releases prostaglandins which increase vasodilation and promotes chemotaxis of neutrophils

Inflammation
Dilation of blood vessels increase blood flow to infected area Capillary permeability also increases and fluid leaves capillaries and causes swelling in infected tissue Fibrin from the blood clotting cascade forms fibers around wound area to trap bacteria Chemokines released into blood and travel to bone marrow to cause release of phagocytes attracts phagocytes to infection site by chemotaxis

Inflammation: Phagocytes
Distant effects: Phagocytes released from attachment in bone marrow and enter bloodstream 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 dialating and changing the glycoproteins on the endothelial cell surfaces: selectins

Emigration from blood vessel into tissue: Monocytes and Neutrophils

The leukocytes begin to stick to selectins on blood vessel walls and roll along: margination Local presence of chemokines causes the leukocytes to adhere tightly and begin migrating out of blood vessels thru enlarged capillary pores: diapedesis Leukocytes then migrate to site of chemokine release (chemotaxis)

Inflammation Role in many diseases

Chronic inflammation may have roles in heart many diseases including heart disease, cancer and autoimmune diseases Aspirin blocks prostaglandin synthetase . some doctors in US recommend a childs aspirin every day to reduce risk of heart disease and cancer. Recent evidence shows a 20% decrease in cancer deaths among people who take aspirin daily. C-Reactive protein used as a measure of systemic inflammation.

Inflammation: Endometriosis
Endometriosis: the most common cause of female sterility in the developed world

Believed to result from abnormal inflammation response in the endometrium lining of uterus Why would the uterus have such sensitivity to inflammation?

IUD: Intra-Uterine Device A birth control device widely used in China. Also used in US when other methods are not appropriate

Works especially well when made with copper.

Causes constant irritation and inflammation of endometrium: The cells lining the uterus. Prevents pregnancy because inflammation prevents embryo implantation

Innate Immune System: Fever

Fever: a metabolically elevated body temperature induced by the hypothalamus of the brain in response to 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 higher temperature. i.e. soil dwelling bacteria are not adapted to the higher body temperature.

Fever: Metabolic functions are 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 temperature can affect metabolic activity.

Dendritic cells and Macrophages: Phagocytes that present molecules to T-cells and Bcells of adaptive immune system: Antigen Presenting Cells (APCs)

Dendritic Cells: From Greek dendron for Tree. Contacts and recognizes bacteria with pattern recognition receptors (PRR) like TLRs 1) Releases cytokines to attract macrophages. 2) Phagocytoses microbes and migrates to lymph nodes to activate adaptive immune system to specifically recognize that microbe

Dendritic cells presenting microbe molecules to cells of the adaptive immune system for recognition.
The T-cells that recognize the antigen will migrate to the site of the infection.

Surgical modification of the innate immune system: Male Circumcision Circumcision: The removal of the foreskin, the skin covering the end of the penis. The most widely performed surgical procedure in the world and the only commonly performed surgery to remove healthy tissue. Has been done since the beginning of history and is shown in some cave paintings. Has cultural, hygiene and medical functions

Surgical modification of the innate immune system: Male Circumcision Estimated to have been performed on 30% of males worldwide, especially Christians, Jews and Muslims. Cultures vary, but very frequently surgery is done on infant children Some controversy exists as medical benefits have not been considered significant.

Now: recent studies in Africa have shown that circumcision in adult heterosexual men reduced HIV infection of the circumcised man by 60%. 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 the inner foreskin that is an HIV site of entry. Circumcision does not reduce virus transmission coming from an HIV-positive male.

Surgical modification of the innate immune system: Male Circumcision Traditional procedure involves cutting tissue and stitching. Infection can be a serious complication, in the developing world where sterile technique is imperfectly maintained. Also, trained surgeons are much in demand for many other surgeries

Current testing involves disposable medical devices that can be quickly applied in large numbers by teams of nurses and medical technicians PrePex Shang Ring (China)

PrePex
1) Ring device is placed over head of penis 2) Foreskin is folded back over it 3) PrePex: A rubber band is tightened over skin on top of plastic ring 4) The skin between plastic ring and rubber band dies from lack of blood without breaking the skin open and risking infection 5) Dead skin distal to the rubber band is cut away a few days 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 infections than traditional surgery.