MTII Flashcards
How did Metchnikoff propose cell-mediated immunity? What was the reception to the theory
He published his observations of starfish larvae phagocytes surrounding a thorn.
Her opposed that the body was defended against invasion by such phagocytes in a process analogous to inflammation
- his proposal was not well received
Who proposed cell mediated immunity and when
Ilya metchnikoff in 1882
Why was Metchnikoff theory of cell mediated immunity not well received
“Cellularists” were unpopular until the mid 1950s. His proposal was not well receive in the medical community because at the time inflammation was considered to be a harmful process. Also, cell mediated biology was only properly studied wig microbiology techniques - not used till 1950
Who proposed antibody-mediated immunity
Emil Behring
How was antibody mediated immunity published? How was it received
He published observations of “serum factors” could be used to confer immunity to Diptheria. He proposed that the body was defended against invasion by these serum factors (later called antibodies).
- his proposal was very well received
Who proposed the first model for an antibody function and the first cellular model for immune system function
Paul Ehrlich
What was the first model for an antibody function
An antibody and an antibody generating substance (antigen) interacted like a lock and key
What is the side chain theory
All antibody-producing cells are pre programmed to display protein “side chains” (antibodies) against all possible antigens
- antigen binding to a specific antibody induces a cell to synthesize this “selected” antibody to the exclusion of all other antibodies
How were Ehrlich proposals received
Very well
Why is the side chain theory considered a selective theory
The antigen “selects” the appropriate, off the shelf antibody molecule
What did Sir Almroth Wright propose. What did his observations indicate
He noticed that bacteria were only phagocytosed in the presence of serum
- he proposed that bacteria needed to be coated with certain serum factors he named opsonins (which means like relish, salsa)
They indicated That cell mediated immunity and antibody mediated immunity are equally important for protections against microbial infections
What did sir Peter Medawar show regarding transplant rejection
He showed that transplant rejects was a cell mediated process
- this revitalized interest in cell mediated immunity
- which brought money and researchers into immunology
What is the clonal selection theory
Proposed by Jerne, talmadge, burnet
- Individuals continuously produce numerous short-lived, clonally derived lymphocytes
- each clone arises from an individual lymphocyte with a particular, pre programmed antigen specificity - Antigen binds to one of these clonally derived lymphocytes and activates that particular lymphocyte
- lymphocytes proliferate and differentiate into effector cells and long lived memory cells - If no antigen binds to a particular lymphocyte, that lymphocyte will die within a few days
- this intentional high turnover rate allows the immune system to continuously re program and test antigen specificities
Where is the immune system
The lymphatic system
- all immune system cells originate in the lymphatic system. Most of them travel back and forth through the circulatory system and the lymphatic system
What are the functions of the lymphatic system
- Fluid recovery system
- the closed circulatory system is quite leaky- roughly 01% leakage rate (or about 3 L/day)
- the open lymphatic system returns this fluid to the bloodstream
- roughly 01% leakage rate (or about 3 L/day)
- Fat absorption system
- specialized lymphatics called lacteals (=milky ones) project into the villi in the intestines- lacteals contain milky looking lymph containing absorbed fats called chyle (=juice)
- lacteals allow fats to be slowly metered into the circulation. Fats are first packaged into chylomicrons, which are then folded into the circulatory system via the thoracic duct
- lacteals contain milky looking lymph containing absorbed fats called chyle (=juice)
- Immune response system
What are the components of the lymphatic system
Lymph
Lymphatics
Central lymphoid organs
Lymph
Clear extra cellular fluid that leaks from the circulatory system
- lymph does not contain RBCs or most blood proteins
Lymphatics
Thin walled, vein like vessels
Much more permeable to incoming fluids than blood capillaries
- collect lymph from tissue interstices
- the flow of lymph depends on skeletal muscle compression of the lymphatics. One way valves in lymphatics direct the flow. The “pooling” of lymph in your legs and feet is one of the reasons you get so tired during trans-pacific flight (also explains “Hong Kong feet”)
What are the central lymphoid organs
The bone marrow
The thymus
What do the central lymphoid organs do
(General or primary lymphoid organs)
- provide sites for the generation and maturation of lymphocytes
The bone marrow
(B for B cell)
- site for hematopoesis (=create blood)
- site for B cell “maturation”
- all blood cells – and that means all Immune System Cells – are produced by hematopoietic stem cells in the bone marrow
- B cells are produced in specific sites in the bone marrow and undergo an intensive negative selection against self specificity and positive selection for reactivity (“mature”) in other sites in the bone marrow
The Thymus
Site for T cells “maturation”
- T cells are produced in specific sites in the bone marrow but undergo an even more rigorous maturation in the thymus
What are the peripheral lymphoid organs
Spleen
Lymph nodes
What do the peripheral lymphoid organs do
(Secondary lymphoid organs)
Provide sites in the periphery for antigen lymphocyte interactions
Spleen
Filtering station for blood
- immune system cells passing through the spleen encounter antigens that have been concentrated from systemic infections
- immune responses to systemic pathogens usually originate from the spleen
(When most people say the pledge of allegiance they’re placing their right hand over their spleen, spleen is size of clenched fist)
Lymph nodes
Filtering station for lymph draining from a particular region
- Immune system cells passing through the lymph nodes encounter antigens that have been concentrated “downstream” from localized infections
- immune responses to localized infections usually originate from the lymph nodes
Immune system cells
Many leukocytes
Inflammatory cells
Lymphocytes
Leukocytes
Are enlisted personnel of the immune system
Macrophage
Note - not found in the blood
- commandos and antigen presenting cells (APC) of the immune system
- phagocytose and destroy antigens in their role as commandos
– and/or –
- phagocytose, process, and then return to base to present antigens to CD4+ T Cells in their role as APCs
(Macrophage frequently initiate an immune response by presenting antigens to appropriate lymphocytes; they just as frequently are the cells that are enlisted to eliminate the antigen. Monocytes aare the circulating precursors of macrophage)
Inflammatory cells/Granulocytes
Mediators of inflammation
Inflammatory cells tells you what the cells do; Granulocytes tell you how you they look
Neutrophils
Inflammatory cell
- neutrophils (aka PolyMorphoNuclear leukocytes or PMNs)
- The infantry of the immune system
- phagocytose and destroy antigens
(They are by far the most numerous inflammatory cells. Pus is the battlefield carnage of dead neutrophils and microbes. It’s greenish color is due to a neutrophil enzyme)
Mast cells
Inflammatory cells
Mast cells are not found in the blood
The sentries of the immune system
- alert immune system cells to the presence of antigens
(Mast cells release chemo attractants and vasoactive chemicals when they encounter an antigen. This process is descriptively termed degranulation)
Eosinophils
Inflammatory cells
- specialized anti parasitic commandos of the immune system
- destroy parasites
(They are very important for defense from helminth infections - parasitic worm - but for us that live outside the tropics, they are a pain in the form of allergic reactions like hay fever and asthma)
What are lymphocytes. What are examples of them
The officers of the immune system
- “Leukocyte” refers to ANY white blood cells (including lymphocytes). “Lymphocyte” refers only to these officers of the immune system with restricted access to the lymphatic system
- examples= B cells, T cells, Natural Killer cells
B Cells
(B cells mature in the Bone marrow)
B cells are field officers for antibody-mediated immunity
-B cells make antigens
- B cells are also Antigen Presenting Cells (APC)
- activated B cells produce antibody molecules
- antibodies bind to antigens and tag them for disposal
T cells
(T cells mature in the Thymus)
CD4+ T cells
- they are commanding officers for both cell-mediated immunity and antibody-mediated immunity
- they “help” activate both T cells and B cells
- there are 2 subsets of CD4+ T cells
- TH1 Inflammatory T cells command and coordinate cell-mediated immunity by two pathways:
- Enlist/activate macrophage and inflammatory cells
- which eliminate bacteria-infected cells and large pathogens
- Activate Cytotoxic T cells (CD8+)
- which eliminate virus infected cells
- TH2 helper T cells command and coordinate antibody-mediated immunity
- Activate B cells
- which produce antibodies that tag pathogens for elimination
Natural Killer cells
They are licensed to kill - they act like cytotoxic T cells but do not need permission from a TH1 inflammatory T cell
- NK cells eliminate virus infected cells
Cytokines
Cytokines (=cell movers) are hormone-like factors important for choreographing immune responses
- macrophage secrete inflammatory cytokines:
- IL-1, IL-6 and TNF-alpha (IL=interleukin TNF= tumor necrosis factor)
- these inflammatory cytokines recruit and activate inflammatory cells for inflammation
(Inflammation is a process that utilizes activated inflammatory cells –mast cells and neutrophils– and macrophage to deliver blood plasma and additional inflammatory cells to extra vascular tissues)
- inflammatory cytokines are mediators of innate immunity
(Innate immunity – nonspecific immunity or natural immunity – is a fast, reliable, but non-specific first line defense system. An innate response is the same regardless of the pathogen. Innate immunity is the immunological equivalent of nervous reflexes. It’s primitive.. Bent also very reliable and effective)
CD4+ TH1 inflammatory T cells secrete Type 1 Cytokines
IL-2, TNF-Beta, IFN-gamma (IFN=interferon)
- these type 1 cytokines can recruit and activate macrophage and inflammatory cells for immune mediated inflammation
- these type 1 cytokines can activate antigen-specific CD8+ cytotoxic T cells for cytotoxic T cell mediated cell killing
Type 1 cytokines are mediators of cell-mediated responses in adaptive immunity
CD4+ TH2 Helper T cells secrete Type 2 cytokines
IL-4, IL-5, IL-10
- these type 2 cytokines activate antigen-specific B cells and favor an antibody-mediated response
Type 2 cytokines are mediators of antibody-mediated responses in adaptive immunity
(Adaptive immunity –specific immunity or acquired immunity – is a slow, cranky, but highly specific defensive system. An adaptive response is specific for a particular pathogen. Adaptive immunity is the immunological equivalent of higher brain functions (“thinking”). It’s quite advanced and quite effective… But it’s easily messed up)
The immune response
There are two separate but interconnected and overlapping sub systems involved in an immune response: innate immunity and adaptive immunity
Innate immunity
(Non-specific immunity or natural immunity)
Fast, reliable, but non specific first line defense system
- similar response regardless of the pathogen
.
Four important innate immunity defense mechanisms:
- Fever - mediated (in part) by inflammatory cytokines
- Complement - mediated by functionally linked plasma proteins
- Natural killer cells
- Acute inflammation - mediated by mast cells, macrophage and neutrophils
Adaptive immunity
(Specific immunity or acquired immunity)
Slow, cantankerous, but highly specific defense system
- different responses for different pathogens
Adaptive immunity can be cell mediated or antibody mediated
- cell mediated immunity
- immune mediated inflammation
- TH1 inflammatory T cells secrete Type 1 Cytokines that enlist macrophage and neutrophils
- cytotoxic T cell mediated response
- TH1 inflammatory T cells secrete Type 1 cytokines that help activate CD8+ T cytotoxic T cells
- antibody mediated immunity
- TH2 helper T cells secrete Type 2 cytokines that help activate B cells
Innate immunity defense mechanisms
Innate immunity involves the interactions of cells and factors derived from the bloodstream and from tissues. The effector functions of innate immunity are intricately interwoven with one another and with adaptive immunity
Fever
Fever creates an environment unfavorable for the growth of many bacteria. Since metabolic activity increases roughly 7% for each degree increase in temperature! fever can dramatically enhance innate immunity.
Endogenous pyrogens
Fever inducing substances produced INSIDE the body
- IL-1, IL-6, and TNF-alpha (Inflammatory cytokines)
- secreted by macrophage
- prostaglandins
- produced by the hypothalamus
(Aspirin reduces fever by inhibiting the enzyme required for prostaglandin synthesis)
Exogenous Pyrogens
Fever inducing substances produced OUTSIDE of the body
- Lipopolysaccharides (LPS)
The Lipopolysaccharide-induced cytokine cascade
- minor localized infection
- macrophage release small amounts of TNF-alpha
- localized infection
- serious localized infection
- macrophage release larger amounts of TNF-alpha
- systemic response
- Septicemia (infection of the bloodstream)
- systemic macrophage release of massive amounts of TNF-alpha
- Septic shock
(A patient with septic shock will usually die within a few hours)
Complement
It is a highly regulated cascade of functionally linked proteins in the plasma. Activation of complement results in the opsonization or lysis of the intruding pathogen, or results in acute inflammation.
What are the two complement pathways
The classic complement pathways
- major defense mechanism of adaptive immunity
- initiated by antibody binding to antigen
The alternative complement pathway
- major defense mechanism of innate immunity
- initiated directly by specific microbial antigens
What does the activation of either complement pathway result in
The formation of various cleavage products with different effector functions
Opsonization
C3b and C4b bind to pathogens and attract macrophage and neutrophils that will phagocytose the pathogens
- the pathogen gets an “eat me” sign taped to its back
Complement mediated cell lysis
C3b initiates a cascade reaction that results in the formation of a polymeric membrane attack complex that lyses pathogens
- the pathogen gets a hole punched in its outer membrane, envelope or cell membrane
Activation of inflammation
C3a, C4a, and C5a induce mast cell degranulation
- degranulation releases chemicals that initiate inflammation
C5a attracts neutrophils that will phagocytose pathogens
- the pathogen gets attacked (and probably eaten) by macrophage and inflammatory cells (particularly neutrophils)
Natural killer cells
They’re very closely related to Cytotoxic T cells. But cytotoxic T cells need to obtain a warrant from a TH1 inflammatory T cell before they can kill infected cells, NK cells are licensed to kill. The serve as a “bridge” between innate immunity and adaptive immunity
- seek out infectd cells (“target cells”)
- the endogenous apoptotic pathway is initiated in these cells
- the target cell commits seppuku (Japanese stomach stab suicide)
- antigen is eliminated (and so is the cell)
(NK cells are like ninjas, cytotoxic T cells are samurai)
Inflammation
Acute inflammation is a coordinated assault on pathogens that involves a variety of different cells and molecules. The activation of acute inflammation will usually either eliminate the pathogen, or initiate an adaptive immune response, with macrophage serving as liaison officers
Acute inflammation
(An innate immune response)
Activated macrophage secrete inflammatory cytokines (IL-1, IL-6, TNF-alpha) that recruit and direct inflammatory cells
- macrophage and inflammatory cells destroy infectious agents and release cytokines
The presence of large numbers of macrophage and inflammatory cells is a characteristics symptom of acute inflammation
Immune mediated inflammation
(This is an adaptive immune process)
Activated TH1 inflammatory T cells secrete type 1 cytokines (IL-2, TNF-beta, IFN-gamma) that recruit and direct macrophage and inflammatory cells
- TH1 inflammatory T cells, macrophage and inflammatory cells destroy infectious agents and release cytokines
The presence of large numbers of TH1 inflammatory T cells, macrophage, and inflammatory cells is a characteristic symptom of immune mediated inflammation
(Antigen-antibody complexes, as well as activated TH1 inflammatory T cells, can induce immune-mediated inflammation. The presence of antigen-antibody complexes is another highly characteristic symptom of immune-mediated inflammation)
What type of immunity are Antigen presenting cells in? What are some APCs
Adaptive immunity
- macrophage
- B cells
- Dendritic cells
Macrophage
Present microbial and particulate antigens to CD4+ T cells
- macrophage are big cells and they can phagocytose, process and present antigens from big stuff like bacteria and Protozoa to a TH0 CD4+ T cell
- an APC
B cells
Present soluble antigens (such as toxins) to CD4+ T cells
- B cells display antigen-specific IgD antibody molecules in their surface. If smells stuff binds to these surface IgD antibodies, the B cells will Endocytose these IgD antibodies, remove, process and present antigens from small stuff like toxins and viruses to a TH0 CD4+ T cell (and then recycle the IgD antibody to the surface)
Dendritic cells
Present virus antigens to both CD4+ T cells and CD8+ T cells
- dendritic cells are designed to be infected by viruses. Once infected, they can process and present antigens from viruses to both a TH0 CD4+ T cell and CD8+ T cell
Antigen presenting cells also determine the development of what cell
TH0 CD4+ T cell
If circumstances call for a cell-mediated response:
APC to TH0
The APC can nudge a TH0 CD4+ T cell to develop into an activated, Type 1 cytokine-secreting TH1 inflammatory T cell that will–
- initiate a cell mediated immune response
- inhibit the activation of TH2 helper T cells
If circumstances calls for an antibody-mediated response:
APC to TH0
The APC can nudge a TH0 CD4+ T cell to develop into an activated, Type 2 cytokine-secreting TH2 helper T cell will–
- initiate an antibody-mediated immune response
- inhibit the activation of TH1 inflammatory T cells
Cell mediated immune responses
Induced by foreign antigen produced inside cells
- such endogenous antigens would include viruses, some bacteria and parasites, plus cancerous cells
Activated TH1 inflammatory T cell secretes Type 1 cytokines that
- enlist the assistance of macrophage and inflammatory cells (this is called immune mediated inflammation)
- activate antigen specific CD8+ T cells to become cytocic T cells (This is called a cytotoxic T cell mediated response)
– WHERE a TH1 inflammatory T cell secretes Type 1 cytokines and the precise mix of WHICH type 1 cytokines are secreted determines whether immune-mediated inflammation or a cytotoxic T cell-,edited response is initiated
Immune mediated inflammation
Activated TH1 inflammatory T cells secrete Type 1 cytokines (IL-2,TNF-beta, IFN-gamma) that enlist and activate macrophage and neutrophils
- activated macrophage and neutrophils phagocytose and eliminate antigen
- activated macrophage phagocytose antigen and present it to (usually) CD4+ T cells
Immune mediated inflammation evolved to kill intracellular bacteria and fungal, protozoal and helminth infestations
Cytotoxic T cell mediated response
Activated TH1 inflammatory T cells secrete type 1 cytokines (IL-2, TNF-beta, IFN-gamma) that activate CD8+ cytotoxic T cells
- activated CD8+ cytotoxic T cells seek out infected cells (“target cells”)
- the endogenous apoptotic pathway is initiated in these cells
- the target cell commits seppuku
- antigen is eliminated (and so is the cell)
Cytotoxic T cells evolved to kill virus-infected cells and tumor cells
Antibody mediated immune responses
Induced by foreign antigen in body fluids
- such exogenous antigens would include viruses, bacteria, Protozoa, and helminths in blood, lymph,interstitial spaces and mucosal surfaces
Activated TH2 helper T cell secretes Type 2 cytokines (IL-4, IL-5, IL-10) that activate B cells
- activated B cell (now termed a plasma cell) produces and secretes antigen-specific antibody molecules
- antibodies bind to antigens and tag them for disposal
(WHERE a TH2 helper T cell secretes Type 2 cytokines and the precise mix of WHICH Type 2 cytokines are secreted determines the class of antibody secreted by an activated B cell)
What are the 5 human antibody classes
IgG IgM IgA IgD IgE
IgG
The major antibody class in the circulatory system ("Joe immunoglobulin")
- unless otherwise specified, an antibody molecule is an IgG moleculeIgM
The first antibody class secreted by a B cell - IgM look like -- and acts like -- five IgG molecules arranged in a ring
IgA
The major antibody class secreted on mucosal surfaces - such mucosal surfaces would include the GI tract,respiratory tract, and the genitourinary tract
IgD
An antigen-specific receptor for a B cell recognition and activation
- this guy is supposed to stay on B cells but sometimes it falls off
IgE
The antibody class involved in allergies and helminth infestations - if you have Hay fever or a food allergy, you have lots of IgE molecules
Antibody effector functions
Neutralization (all antibody classes) is when antibody binding blocks the functioning of a pathogen or toxin
- the pathogen or toxin gets a “parking boot”
Opsonization (IgG only) is when antibody binding tags a pathogen for phagocytosis
- the pathogen gets an “Eat Me” sign
Complement fixation (IgG and IgM) is when antibody binding initiates the classical complement pathway - the pathogen gets the whole nine yards of complement-mediated lysis, opsonization and activation of inflammation
Antibody dependent cell mediated cytotoxicity/ ADCC (IgG, IgA, IgE) is when antibody binding tags virus-infected cells, tumor cells, or parasites (stuff too big to be osponized) for cell mediated killing
- the pathogen gets a “Kill me” sign
Primary antibody response
Requires relatively large amounts of the antigen
- antigen can be protein, polysaccharide, or lipid
- protein antigens tend to work best but there are exceptions
- 5 to 10 day lag; then a low-to-moderate affinity antibody response
- moderate amounts of IgM appear first
- lesser amounts of IgG appear later
Secondary Antibody Response
Requires astonishingly small amounts of antigen
- antigen must be protein
- polysaccharides and lipids tend not to work, but are exceptions
- 1 to 3 day lag; then a high affinity antibody response
- massive amounts of IgG (or, if necessary, IgA or IgE)
(Affinity (=relatedness) is the “fit” between an antibody and it’s cognate antigen. Affinity maturation (an amazing Darwinian natural selection process for best-fitting antibodies) generates antibodies that fit the antigen extremely well)
System redundancy
The immune system has built in redundancy. Most effector cells are capable of multiple effector functions. Most effective functions can be equally well performed by different types of effector cells. The different antibody classes can perform overlapping effector functions. And different cytokines act similarly on different cell types.
Systems redundancy is important
Smallpox “ingrafting”
Dried pus from smallpox pustule introduced to healthy person (usually a child) vid nose or small incision
- induce lifelong immunity to smallpox
- mortality rate of ~3%
- this mortality rate was acceptable because the disease had a 30% rate and if you survived you had disfiguring scars
Lady Mary Wortley Montague and ingrafting
Not your typical 18th century English ambassadors wife
Wrote a letter to a friend about how ingrafting worked
Used her connections to champion ingrafting in Europe
- first tried on inmates, success
- next orphans, success
She lacked diplomacy in medical establishment – they knew it still had a 3% mortality rate, and the fad lost favor
Edward Jenner
English country physician and amateur scientist
Avid observer of dairy maids, and noticed dairymaids always caught cowpox, but never smallpox
In 1796, Jenner used cowpox for ingrafting
- he used the crusts from the dairy Maids hands, inoculated a boy and he became immune to smallpox
- he inoculated the same boy with smallpox 20 times to check
Jenner introduced cowpox ingrafting throughout England
- had considerable influence in medical establishment
- cowpox ingrafting became widely accepted
- George Washington had continental army ingrafted
World health organizations campaign to eradicate smallpox
This wins possible for several reasons
1. humans are the only host for smallpox
- so three is no animal reservoir (this means an animal population chronically infected with the causative agent of a human disease that can serve as a source of future human infections)
- diseases with an animal reservoir can never be completely eradicated
- diseases without an animal reservoir can be eradicated via an effective vaccination program
2. There are no sub clinical smallpox infections
- so everyone with smallpox with manifest its symptoms
3. A safe and effective vaccine was available
- so no worry concerning side effects
4. Vaccine could be easily administered by non medical personnel
The disease was claimed as eradicated in Dec 1979
Passive immunity
(Acquired naturally in utero and via breast feeding. It can be acquired artificially via antibodies (globulin))
It results from the introduction of antibodies made by another persons or an animals immune system
- the immune system of the immunized individual is not activated and reminds passive
- no anamnestic response (immunological memory)
Ex) gamma globulin, snake anti venom, rhogam
Active immunity
(Can be acquired naturally by contracting the disease, or artificially by vaccine.)
It result from the introduction of an antigen
- the immune system of the individual is activated to produce antibodies (possibly activated T cells) and plays an active role in providing this immunity
- anamnestic response occurs within about a week
- “boosters” can speed up this response time
Active immunity is long term immunity
- it is frequently life long
Ex) plague vaccine, tuberculosis vaccine, cholera vaccine, malaria vaccine, flu vaccine, polio vaccine
All current vaccines are designed to activate what immunity
Antibody-mediated
What immunity is transient
Passive immunity
- these “foreign” antibodies will eventually be broken down by the immunized individual
- the half life of IgG is ~23 days, IgA ~7
Passive immunity via maternal antibodies
IgG antibodies are transferred across the placenta
IgG, IgM and (particularly) IgA antibodies are transferred via colostrum
(Maternal antibodies can interfere with a child’s ability to mount an immune response to some vaccines)
Passive immunity via preformed antibodies
Homologous antibodies
- antibodies produced in human volunteers
- ex) immune serum globulin (ISG), Rabies immune globulin (RIG) etc
Heterologous antibodies
- antibodies produced in animals usually horses
- can cause hypersensitivity reactions
- ex) Diptheria antitoxin, snakebite antivenom etc
What are the types of vaccines and what is the point of them
The point is to introduce an appropriate antigen into an individual such that an effective immune repose can be stimulated.
Types Whole pathogen vaccines (attenuated or inactivated) Purified molecular Synthetic peptide DNA vaccines
Whole pathogen vaccines
You introduce the actual pathogen, or a close relative. They are very successful against viral diseases.
- attenuated vaccines (weakened version of pathogen)
- inactivated (dead pathogen is used)
Advantages of inactivated vaccines
Readily incorporated into other vaccines
Easy to ship and maintain (don’t need to maintain cold chain)
No danger of reversion to virulence (poison)
Disadvantages of inactivated agents
Generally require injection
- an injected vaccine must be axenic (without foreigners). In many underdeveloped regions, personnel who can do this are scarce
Generally require multiple doses
- complicated in underdeveloped areas
Limited cell mediated immune response (since no infection)
What advantages and disadvantages are identical to that of inactivated vaccines
Purified molecular vaccines
Synthetic peptide vaccines
Purified molecular vaccines
This is sophisticated and effective (whole pathogen is primitive but effective). You only introduce the purified immunogenic portions of the pathogen (i.e just the proteins the immune system “sees”). Influenza vaccines and early hepatitis B vaccines are examples
Synthetic peptide vaccines
Even more sophisticated. You are introducing synthesized immunogenic portions of the pathogen. The new hepatitis B vaccine was the first synthetic peptide vaccine approved for human use)
DNA vaccines
Incredibly sophisticated and shows great promise. DNA encoding the immunogenic portions of the pathogen is injected directly into the muscle tissue
What does attenuation generally require
Serial passage through a live host or through cell culture
Advantages of attenuated vaccines
A transient infection is established
- interferon production is stimulated
Only a single injection is required (huge in underdeveloped areas)
Stimulate antibody mediated and cell mediated responses
- an actual infection is established, so a normal immune response will be activated
Disadvantages of attenuated vaccines
Expensive to make, to ship, to store
- must maintain cold chain from start to fin ish, very sensitive to high temp
Residual virulence/virulence reversion
- they a unsuitable for immunodeficient patients (so HIV/AIDS patients or organ transplant patients)
Berries history/facts about Black Death
Europe was in an unusually vulnerable state
- the little ice age, population increased, marginal lands to farm
Plague was brought to Europe as a result of an early experiment in biological warfare
The Black Death spread along traditional European trade routes
- it killed about a quarter of the pop (25mil)
Surprising results of the plague
Resulted in accelerated downfall of an already collapsing feudal system
- labor was suddenly scarce
- land was suddenly abundant
Accelerated shifts in trading routes and trading powers
- like Great Britain, Spain, Portugal,etc
Changed thinking about disease
- no longer by an angry god but by “poisoned air” etc
Introduced the Halloween witch
- old widows counted the dead, used the stick of a broom to see if plague victim is not dead
What did Kitasato do regarding the plague and British medical authorities
He was a hot shot Japanese bacteriologist who had exclusive access to plague cadavers. He isolated what he thought was the causative agent of plague within 48 hours of his arrival in Hong Kong.
What did Alexander Yersin do about Kitasato’s claim
He was denied access to plague cadavers and was reluctantly given meager research space. He was skeptical of the “isolate” done in 48 hours. He bribed people to get samples from cadavers.
Yersin isolated the causative agent in 5 days, he published in French, fulfilled Kochs postulates and used them to make an effective antiserum treatment for plague
The bacterium was initially named bacterium pestis, remamed yersinia pestis
Endotoxin
=inside poison
Components (such as the lipid component of LPS) in the cell wall of gram negative bacteria that are released when the bacteria divide or die
Septic shock is caused by endotoxin
Exotoxin
=outside protein
Harmful proteins produced and release to the OUTSIDE by both gram positive and gram negative bacteria
Composed of two components
- A (active) component
- B (binding) component
Diptheria and anthrax are caused by exotoxins
Types of exotoxins
Enterotoxin
Neurotoxin
Hemolysin
Neurotoxin
=nerve poison
Exotoxins that harm nervous system cells
Tetanus and botulism are caused by neurotoxins
- Botox is a neurotoxin
Enterotoxin
=intestinal poison
Exotoxins that harm epithelial cells of the intestinal (enteric) tract
Cholera is caused by an Enterotoxin
Hemolysin
=blood (cell) splitters
Enzymatic exotoxins that lyse red blood cells
- alpha hemolysins partially break down hemoglobin
- beta hemolysins completely break down hemoglobin
Some species of staphylococcus and streptococcus characteristically produce hemolysins
What is the causative agent of the plague? Describe it
Yersinia petis
- gram negative, ovoid rods
- characteristic bipolar staining
- looks sort of like a diaper pin
- optimum growth 27C to 30C
- produces a capsule when grown in 37C
The Yersinia flexible gene pool
High and low pathogenicity Yersinia both have pYV (Plasmid Yersinia Virulence) in their cytoplasm
- highly conserved 70 Kbp plasmid that encodes for virulence genes
High pathogenicity strains of Yersinia also contain HPI (High-Pathogenicity Island) in their chromosome
- 35-45 Kbp pathogenicity island
- low pathogenicity Yersinia lack this pathogenicity island
Non pathogenic Yersinia do not have pYV or HPI
Animal reservoir of plague
Manchurian marmots seem to be the natural host
- rats are a common reservoir in developing countries
- ground squirrels and prairie dogs are reservoirs in the southwest US
(Most victims in the US are hikers who have been bitten by infected fleas from ground squirrels or from prairie dogs)
What is the mode of transmission of bubonic plague? Pneumonic plague?
Bubonic plague =groin
- occurs when bacilli introduced by the bite of an infected flea multiply with lymph nodes
Pneumonic plague = breath
- occurs when bacilli spread from the bloodstream to the lungs
- respiratory droplets can then spread bacilli directly to another persons lungs
Progression of plague - plague in rodents
When a flea bites an infected rat
- bacilli (without a capsule) multiply within the fleas gut
- flea bites will introduce the bacilli
When an infected rat dies
- the 50 to 60 fleas found on this dead rat will run off in search of a. New warm home – humans can be accidental intruders in this cycle
Progression of plague in humans
Bacilli (without a capsule) are introduced by a flea bite
- bacilli can be filtered out of lymph by the lymph modes
- neutrophils successfully phagocytose and digest most bacilli
- some are phagocytosed by macrophage, which do not digest them
- bacilli can multiply within macrophage
- bacilli produce a capsule and so resist further phagocytosis
Bubonic plague in humans
Multiplying, encapsulate bacilli release necrotizing exotoxin (=death)
- lymph nodes downstream from the bite location enlarge and form eponymous buboes
Bubonic league can be treated successfully with antibiotics
- otherwise bacilli can access the circulatory sustenance via the lymph nodes, resulting in —-
Septicemia (septicemic plague)
- necrotizing exotoxin can cause subcutaneous hemorrhage anywhere on the body
- it is usually fatal (90% fatal if untreated)
- bacilli cqn enter thongs, resulting in pneumonic plague
Pneumonic plague
Occurs when bacilli enter the lungs
The only contagious plague
- it spreads very rapidly (via respiratory droplets) –‘100% fatality if untreated
Symptoms of plague
High fever and vomiting during the 1-5 incubation period
Bubonic plague
- buboes (enlarged lymph nodes)
- “Black Death” (subcutaneous hemorrhaging = bruising)
Pneumonic plague
- massive lung hemorrhage s
Diagnosis of plague
Blood cultures, sputum samples or bubo aspirated examined for bacilli
- identification is via characteristic bipolar staining, phage typing or fluorescent antibodies
Treatment of plague
Tetracyclines (tetracycline and doxycycline)
- highly effective for bubonic plague
- somewhat for pneumonic if administered within 15 hours
Aminoglycosides (streptomycin and gentamicin)
- effective in conjunction with tetracyclines
Recovery results in lifelong immunity
Prevention of plague
Elimination or reduction of animal reservoir
- usually rats (and their fleas)
Vaccine
- the primary series involves 3 doses, with 2 boosters at 6 month intervals, followed by boosters every year
Impact of the plague
Past epidemics have had an enormous impact on western culture and thinking
Plague is a problem in India
Pague kills approximately 5 individuals per year in the southwestern us
Why is it difficult to track tuberculosis through historical records
Any disease that involved coughing was classified as tuberculosis
Tuberculosis and the romantic age
The intellectual elite had tuberculosis, any woman who was anyone had it too.
The disease was used to enlist sympathy for literary or stages characters and created an aesthetic and poetic exit.
The Industrial Age and tuberculosis
With everyone moving to crowded US urban areas, it was considered low class and vulgar to have the disease (which was the truth, people would cough up blood and dead lung tissue)
Rich people used Sanitaria (fresh air, sunlight and healthy living) to cure the disease
Robert Koch and tuberculosis
He presented his findings to the Berlin physiological society in may of 1882
- one of the rare circumstances where both the general public and the scientific community at large realized a quantum leap of knowledge had occurred
Albert Calmette and Camille Guérin and BCG
They subcultured Mycobacterium bovis every 3 weeks (231 cycles)
- Bacille Calmette-Guérin (BCG)
BCG was first given (orally) to the children of Paris in 1921
- in 1928 the League of Nations declared it safe
A problem occurred in Germany in 1929
- 252 children received the BCG vaccine contaminated with virulent Mycobacterium tuberculosis
- 71 of the kids died (lack of BCG in US probably related to this)
BCG is one of the most controversial vaccines in use today
- it is variable in its protection (according to research studies)
- several theories on its variability
- genetic variability in BCG strains used in the vaccine
- genetic variability in populations receiving the vaccine
- interference from endemic non tuberculosis mycobacterium
- interference from endemic parasitic infections
What is the causative agent of Tuberculosis
Mycobacterium tuberculosis
- “gram positive”, non motile, slightly curved oboes
- obligate aerobes
- unusually resistant to drying
What is unique about mycobacterium tuberculosis
It’s mycobacterial cell wall
(Many of the characteristics of mycobacterial – unusually slow growth rate, resistance to digestion within phagolysomes, resistance to drying and acid fast staining – are due to the presence of waxes and mycolic acids in their cell walls)
- peptidoglycan layer
- arabinogalactan lipid complex (similar to outer membrane in G - bac)
- 6,6 dimycoyltrehalose (cord factor), lipoarabinomannan and mycolic acids
Animal reservoir of tuberculosis
Humans are the only natural host for mycobacterium tuberculosis
- birds are natural host for mycobacterium Avium, but that can cause tuberculosis in HIV/AIDS patients
- cows natural host mycobacterium bovis, but can cause tuberculosis in badgers, deer, elk, water buffalo and humans
Mode of transportation for tuberculosis
Inhalation of respiratory droplets or duet particles containing mycobacterium tuberculosis or Avium
- mycobacteria are small enough to stay airborne for several days
Ingestion of non pasteurized (“raw”) milk containing mycobacterium bovis
Progression of tuberculosis
- Respiratory droplets are inhaled
- droplets generated by coughing/sneezing; most stopped in upper respiratory tract, but some make it to alveoli - Mycobacteria in the alveoli are phagocytosed by alveolar macrophage
- Mycobacteria multiply inside the phagolysosome of these alveolar macrophage
- mycobacteria continue to multiply until the macrophage bursts
(They have a waxy coat that protects them from digestive enzymes) - More mycobacteria come on scene and phagocytose these newly released mycobacteria
- mycobacteria grow in these macrophage until they burst too
(The ability of an immune response to activate these infected macrophages determines the progression of the disease. If your immune system is up and running, this cycle is easily broken. If for any reason it’s not - especially cell mediated immune response - the cycle continues to amplify the mycobacteria) - CD4+ T cells come on scene
- macrophage (which are antigen presenting cells) present mycobacteria antigens to these CD4+ T cells- As a result of complex interactions and signaling these CD4+ T cells become activated TH1 inflammatory T cells
- secrete type 1 cytokines (IL-2, TNF-beta, IFN-gamma)
- the tuberculin test assays for the presence of these mycobacteria specific activated TH1 inflammatory T cells
- As a result of complex interactions and signaling these CD4+ T cells become activated TH1 inflammatory T cells
- Type 1 cytokines - particular IFN-gamma - activate macrophage
- activated macrophage are able out destroy mycobacteria in their phagolysosomes
- activated macrophage secrete inflammatory cytokines (IL-1, IL-6 and TNF-alpha)
(Most of the pathology associated with tuberculosis is due to an overly aggressive immune response to the mycobacteria) - Type 1 cytokines activate mycobacteria specific CD8+ cytotoxic T cells
- these will kill non-activated, infected macrophage - Tubercle (=small lump) formation begins
- tubercles are accumulations of neutrophils, infected and activated macrophage, lymphocytes and connective tissue - Mycobacteria continue to grow in infected macrophage associated with the tubercle
- the bacilli within a tubercle can become dormant (they can be dormant for decades)
What are the three possibilities with tubercles and tuberculosis
- (Good) the tubercle will be “walled off” by calcification
- calcified tubercle and it’s draining lymph des is a Ghon complex
- and progression of the disease usually halts - (Bad) tubercle can grow, coalesce, spread and invade the circulatory or lymphatic system
- mycobacteria can spread to any tissue causing extra pulmonary tuberculosis or miliary tuberculosis - (Bad) tubercle undergoes caseous necrosis (=cheesy death)
- necrosis and liquification of the center of the tubercle releases enormous numbers of mycobacteria- mycobacteria can invade the bronchi
- mycobacteria can spread to other regions of the lungs
- mycobacteria can be coughed up and swallowed
- they can invade the gut
- mycobacteria can invade the bronchi
Symptoms of tuberculosis
Fatigue, cough, bloody sputum, weight loss
- hence the earlier name “consumption’
Diagnosis of tuberculosis
Larger tubercles and Ghon complexes will show up on X rays
Tuberculin test
- it assay a for the presence of these mycobacteria-specific activated TH1 inflammatory T cells
- positive indicates the individual has been exposed to mycobacteria
- positive result PLUS the presence of. Acid fast mycobacteria in sputum indicates active tuberculosis
Treatment of tuberculosis is pre antibiotic era
Sanitaria
- rest, good nutrition, fresh air
- heliotherapy (sun therapy)
- sunlight or a Finsen lamp (UV Lamp)
Basically a persons immune system could catch up, sun raised vitamin D levels seemed to help raise immune system levels
Post antibiotic era treatment of drug susceptible tuberculosis
Drug susceptible tuberculosis
- simultaneous administration of the four first line drugs: Rifampin (RIF), Isoniazid (INH), Pyrazinamaide (PZA), and Ethambutol (EMB)
Pat antibiotic era treatment of multi drug resistant tuberculosis
MDR-TB (multi drug resistant tuberculosis)
- resistant to Rifampin and Isoniazid
- administration of second line drugs
= cycloserine, ethionamide, streptomycin, amikasin, etc…
Post antibiotic treatment of extensively drug resistant tuberculosis
XDR-TB
- resistant to Rifampin and Isoniazid plus any of the fluoroquines plus at least 1 of the 3 injectable second line drugs: amikasin, kanamycin and capreomysin
Patient non compliance and tuberculosis
The drugs used to treat TB have nary side effects so once the patient started to feel better they stop taking them
- these is the drugs haven’t had time to eliminate all of the mycobacteria, just those highly sensitive to the agent. The patient thusly inadvertently is selecting antibiotic resistant mycobacteria to continue growing. Which means treating with stronger higher doses
What is DOTS
It was developed due to Tuberculosis patient non compliance
- DOTS (directly observed treatment short course) is a simple but effective three stage program for the elimination of TB and patient non compliance
- dots stages
- Deliver drugs to infected individuals
- Observe that these infected individuals actually take these drugs
- Document clearance of the mycobacteria by sputum sampling
Prevention of tuberculosis
Tuberculin test screening
- followed by treatment of infected individuals
Vaccination
- utilizes BCG
- it is attenuated mycobacterium bovis
- vaccinated individuals will test positive
- so vaccination hasn’t been used in North America
Impact of tuberculosis
~16000 individuals in USA have active TB
- TB cases have been on the decline in USA since 1993
~ 1/3 of people on the planet are infected with mycobacterium tuberculosis
- 1644000 die from it per year
TB is an opportunistic infection closely associated with AIDS
Who compare the distribution of cholera patients with distribution of water
John snow
What did snow hypothesize about cholera
It was caused by drinking “fecalized water”
Robert Koch and cholera
- epidemic broke out in Egypt in 1883
- he went there, realized French isolated platelets, not bacterium
- he isolated vibrio cholera from feces, contaminated linen, patient, water
What was the last outbreak that made purified drinking water a necessary idea
Cholera broke out in hamburg in 1892 because they didn’t treat ther water, the adjacent city did and so no outbreak
Oral rehydration therapy
Used to use an IV of saline to treat cholera
By 1960s known cholera was from a toxin
- cholera toxin deregulates a reversible secretory profess
- and it was in owe that glucose augments absorption four fold
- experiments on rats and dogs implied ORT would work
1971 Pakistan civil war place to try ORT
- lots of people in one place, doctors ran out of sterile saline
- reversed sterile saline solutions for patients in shock
- advised other patients to drink a glucose electrolyte solution
- IVT mortality 18%, ORT 3%
Can sari cloth filter out vibrio cholerae
No, when folded 8x it forms 20micrometer mesh size
- vibrio cholera is ~ 5 to 10 micrometers in length
- but they aren’t free living in aquatic systems
- they colonize core pods, aquatic crustaceans that measure ~150 micrometers in length
- vibrio cholera colored the surface of the animal it’s egg cases (which remained attached to the animal), and it’s gut
- but they aren’t free living in aquatic systems
- sari cloth removes these Core pods, so removes a big number of vibrio cholerae
Causative agent of cholera
Vibrio cholerae
- gram negative, comma shaped bacterium
- single polar flagellum
Vibrio flexible gene pool
Vibrio have numerous pathogenicity islands like
- RTX, VSP-1, VSP-2, SXT, VPI
Pathogenic vibrio are infected with bacteriophage CTXtheta (Cholera Toxin phage)
- the integrated CTXtheta prophase contains genes for cholera Enterotoxin
Cholera Enterotoxin
CT has one A subunit and 5 B subunits
- the b subunits bind to a glycolipid on the surface of intestinal epithelial cells
- the A subunit enters intestinal epithelial cells
- the A subunit is cleaved to a fragment called A1
- A1 binds to adenylate cyclase, inducing high levels of cyclic amp (cAMP), resulting in – hyper secretion of water and chloride ions, and inhibition of the absorption of sodium ions
- the A subunit is cleaved to a fragment called A1
Animal reservoir of cholera
Vibrio colones crustaceans in aquatic and marine environments
- vibrio only causes diseases in humans
Cholera mode of transmission
Consumption of food and water contaminated with feces
Consumption of raw shellfish grown in contaminated waters
Progression of cholera
Vibrio must be ingested in remarkably large numbers (10^6)
- vibrio are extremely sensitive to stomach acid
Vibrio must colonize small intestine
- this requires mucinase production and the attachment to specific intestinal mucosal cell receptors
Vibrio must then produce cholera Enterotoxin (CT)
- CT b subunits binds to intestinal epithelial GM1 ganglioside
The resultant outpouring of fluids gives rise to profuse diarrhea
- this same outpouring of fluids washes vibrio out of gut
- so if the patient can keep hydrated cholera is self limiting
Symptoms of cholera
Fever, abdominal cramps, vomiting, severe diarrhea
“Rice water”
- small white flecks that help give rice water it’s name are pieces of the intestinal mucosa that have been sloughed off
Severe diarrhea includes electrolyte imbalances
- metabolic acidosis (due to loss of bicarbonate)
- hypokalemia (due to loss of potassium)
- hypovolemic shock (due to reduced blood volume)
If unchecked, the loss of water and electrolytes results in electrolyte imbalances that induce circulatory shock and death
- in severe cases, a healthy individual can be killed in 3 hours
Average fluid lost over ~4 day course of the disease is 30 liters
Oral rehydration therapy can keep a patient hydrated
- outpouring of fluids will wash vibrio out of the gut
- the cholera will be self limiting
Recovery frequently results in a protective slgA response
Diagnosis of cholera
Severe, watery diarrhea
Lab culture
- identification via agglutination reactions using vibrio-specific antiserum (serum containing antibodies against the bacterium)
Treatment of cholera
Tetracycline
- reduce the duration but do not eliminate the vibrio or ct
ORT
- in 1 L of water dissolve:
- KCl(1.5), NaHCO3(2.5), NaCl(3.5), glucose (20g=primary)
ORT takes advantage of glucose coupled sodium transport
Prevention of cholera
Be very wary of raw or undercooked shellfish
Clean (or chlorinated) water supply and proper sewage disposal
Cholera vaccines
Impact of cholera
~1000000 cases of cholera each year
- so ~100000 cholera related deaths per year
Lamp rheostat control
Like a dimmer switch, also “on/off” switch
Field diaphragm control
Online wdm closes the field diaphragm
Fine and coarse focus knobs
Raises and lowers stage and condenser in tandem
Condenser focus knob
Use this knob to raise and lower the entire condenser
- black plastic knob on the lower left
Condenser auxiliary lens lever
Silver knob on right
It should always be in place, so fully up except at 3.2 lens
Condenser venting screws
Physically moves the entire condenser lens
Condenser diaphragm control
Ninja lever
Opens and closes the condenser diaphragm
Mechanical state controls
Moves the stage
Gram stain procedure
Prepare smear, air dry, heat fix, crystal violet 1 min, rinse, iodine 1 mink rinse, ethanol 10drops/10sec, rinse, safranin 1 min, rinse, blot dry
Spore stain
Blah blah… Soak malachite green, pass through flame till steams, let sit 2 min, add a little more malachite green, remove paper towel with tweezers, rinse for 1 min, courier stain with safranin for 2 min, rinse
Calculating eyepiece reticle units
You calculate the number of microns per eyepiece reticle unit by dividing th number of microns spanned by the number if eyepiece reticle unite required to spam that distance
The distance at 10X lens is 25, a bacterium measures 3 eyepiece reticle units under this lens, now big is it in microns
100 microns/25= 4 microns at the yellow lens
Bacteria= 3.. So x 4microns= 12microns bacteria
Calculate bacteria measured at 3 eyepiece reticle units at 100x lens, 10x lens spans 25 eyepiece reticle units when being calibrated, what is size of bacteria
10x is 100microns/25= 4microns
So 100x lens is 4microns/10= 0.4 microns
Bacteria is 3 eyepiece reticle units
- 3 x 0.4 = 1.2 microns
What is negative staining used for
To show capsules
Hematocyter
Designed to count blood cells, but can also count bacteria, yeast, or any class culture in suspension.
- total volume is 3mmx3mmx0.1mm=0.9mm^3
Entire counting chamber marked off into 9 1mmx1mm squares
- outer 8 large squares used to count white blood cells
Center most square - fills field of view at 10x - further marked into 25 medium squares (0.2mmx0.2mm).
- used to count red blood cells
Each of the 25 medium squares are marked off into 16 small squares
- quick approx of rbc or platelet
How to count cells in a Hematocyter
Count 4 corner boxes (medium squares, so 25) and center most one and then average (so you multiply total by 25)
- cell touches top or right line in a medium squarer, it counts
- touches bottom or left, it doesn’t
How to calculate volume in Hematocyter
Total volume of each large square is 10^-4mL= 10^-4cc
How to estimate the total number of cells in the large square of Hematocyter
Divide the count by the volume
Say count is 750, the volume is… 10^-4mL
So… 750/10^-4= 7.5 x 10^6 cells per mL
IMViC test results
E.Coli = positive indole, positive Methyl red, negative VP and citrate Enteric = +,+,-,-
Indole = tryptone broth
- negative= no enzyme tryptophanase, no red layer (Emteric)
- positive= redd layer because has enzyme (E coli)
Methyl Red
- negative= butanediol fermenter, red to orange color (entero)
- positive= mixed acid fermenter, stays red(E. coli)
Voges-Praskeur
- negative= mixed acid fermenter! no color change (E. coli)
- positive= mixed acid fermenter! red color (entero)
Citrate
- negative. = not use ammonia salt as E, redwoods green (E. coli)
- positive= use ammonia salt as nitrogen source and citrate as carbon source, Malibu blue (entero)
Calculate pfu/ml
Divide count by dilution
So it’ll be like 42/10^-9 so… 42x10^9
Wright stain and neutrophil
Predominant inflammatory cell
Multilobed nucleus
Red amd blue granules in cytoplasm
Increase in neutrophils indicates bacterial or viral infection
Eosinophils and wright stain
Mostly red granules in cyto
Involved in allergic responses
Basophils and wright stain
Dark blue granules
- involved in extreme allergic responses (venom)
Mast cells are basophils like inflammatory cells
