Immunology

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What is used to treat rapidly proliferating T/B cells?

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Depends on the de novo purine synthesis - so we should stop that - we can use: methotrexate, mycophenylate, azathioprine

Stop the de novo pyrimidine: leflunomide

other drugs are:
Cyclophosphamide, antimetabolites

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Cyclophosphamide

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prototype alkylating agent: crosslinks DNA with DNA and DNA with protein.

used for autoimmune with unwanted B cells

highly toxic but managable; effects of Acrolein, a toxic metabolite that damages the bladder mucosa

cyclophosphamide: Derived from mustard gas (WWI) and this can wipe out the entire bone marrow!
Giving mesna we can reduce bladder cancer
Cyclophosphemide given to 13yr old – she can’t have children

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Cyclosporin

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Early activation inhibitor - suppress a Ca-dep signalling path that when activated generates IL-2 and high affininty IL-2. This normally activates T-cells to proliferate!

Cyclosporin combines with cyclophillin and inhibits calcinuerin, trapping NF-AT in the cytoplasm and preventing transcription of early T-cell activation genes

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Tacrolimus

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Early activation inhibitor - suppress a Ca-dep signalling path that when activated generates IL-2 and high affininty IL-2. This normally activates T-cells to proliferate!

Tacrolimus: binds FKBP and also inhibits Calcinuerin

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Sirolimus

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The “late” activation pathway is a calcium independent step involved in moving activated T cells into the G1 phase of the cell cycle.

Sirolimus=Rapamycin binds to FKBP, but instead of inhibiting production of cytokines, the interaction inhibits the cell cycle proteins --> block the growth factor induced signal transduction in T-cells

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Antibodies that target the cross talk between T cells and Antigen presenting cells

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Anti-Lymphocyte globulins (Broadly immunosuppressive --> high risk of opportunistic infection)
Anti-CD3, Anti-CD52
Anti-LFA-1 or Anti-ICAM-1
Anti-CD25 (IL-2 receptor - Daclizumab = Zenapax)
Anti-TNF-a (Infliximab and adalimumab = antibodies and Etanercept = inactivates soluble and membrane-bound TNF- a.
It has a lower FcgR affinity and causes less ADCC than the anti- TNF monoclonal antibodies )
CTLA-4 Ig (CTLA-4 Ig, a fusion protein of CTLA-4 and the Fc piece of IgG has been developed and is FDA approved.
CTLA-4 Ig is soluble and the addition of the Fc of IgG endows it with a long half-life in the circulation.
CTLA-4 Ig binds to B7.1 and B7.2 on APC, blocking their ability to provide costimulatory signals to T cells’ CD28.)

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ADCC

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Antibody dependent Cellular cytotoxicity : antibody-dependent immune reaction mediated by LGL cells (large granular lymphocytes), i.e., killer cells and NK-cell lymphocytes. The ADCC reaction is very sensitive. It plays an important role in hypersensitivity and occurs at antibody concentrations that are well below those leading to complement lysis.

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Glucocorticosteroids as immunosuppressant

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Panimmunosuppressive

-Inhibit phospholipase A2 thereby prevent cleavage of arachidonic acid and production of lipid mediators of inflammation.
-Increase synthesis of I kb, thereby suppressing transport of Nf kb into the nucleus.This decreases proinflammatory cytokine production and secretion.
-Suppresses leukocyte adhesion molecule expression. Leukocyte trafficking and immunosurveillance are thereby altered.
-Promote a TH2 cell phenotype, stimulate IL-10 production and an increase in CD25+ T Regulatory cells.

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biggest side effect of immunosuppressants

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renal failure

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IL-2 = CD?

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CD25 - used by the T-reg

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B7 binds more to CTLA-4 or to CD28?

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CTLA-5 and that stimulates:

IDO = indoleamine 2,3 dioxygenase, an enzyme that catabolizes
tryptophan, inhibits T cell proliferation and predisposes T cells to Apoptosis.

normally the interaction (say from teh B7 of a B-cell to the CD28 of the T-cell) causes proliferation of the B and T-cell (via CD40 now)

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Adalimumab and Infrliximab vs Etanercept

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The monoclonal antibodies to the TNF-a binds and inactivate the soluble cytokine. They also mediate the ADCC of cells with TNF-bound. There is a complication with pneumonia with this.

Etanercept: is a fusion protein Fc of IgG and 2 TNF receptors --> binds TNFa and inactivates it. Less good for ADCC. But no complication with pneumonia

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Prednisone

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Glucocorticoid - inhibit A2 - arachiodonic production of inflammatory

stops cytokine synthesis

stim. IL-2 - T-reg...

increased WBC - can't get out of blood stream

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what tumors do to protect themselves

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promote T-reg cells to supress immune around them;

tell macrophages to help them proliferate - to secrete endothelial growth factor

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infuence of infections on the cancer outcome

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microbial agents causing inflammation and immunity improve cancer out come. The antibodies can cross-react with the tumor cells later on.

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anti-idiotypes

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antibody or T-cell recognizes the bindingsite of a antigencombining site of B-cell- Ig's on B-cell surface - this will stop the B cell-lyomphoma.
with that unique tumor site of those B-cells.

making a T cell and B cell response to those antigens - you kill these B-cellomas

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Types of adoptive Immunotherapy
for tumor

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Antigen specific polyclonal CD 8+ T cells
Cloned CD 4+ T cells
T cells engineered to express Chimeric Antigen Receptors

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Important costimulation for T cell

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LFA-CD28
CD28-IL-2R
ICOS (regulation of the expansive phase)
Survival for CD8:
CD40-CD40L
41BB-41BBL (particular CTL)
"CD8's are really depressed - they just kinda wanna die"

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Epitope spreading

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CD4 cells with specific tumor-attacking signals - they fostered Ag-recognition by some of the other cells "hey we have a tumor here"

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Chimeric antigen receptors

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T-cell singalling on inside - outside with Tumor recognition; hope to transduce to active T-cells - but they often need help: transduced T-cells that were EBV ready - this was very clever because these T-cells are communicating with B-cells - this interaction stimulates Bcells to secrete survival signals for the T-cells keeping them alive for a longer time

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Gell and Coombs classification scheme

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1)mast cell - minutes
2)antibody
3)immune-complex
4)t-cell mediated - delay 48h

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Indirect Coombs vs Direct Coombs

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looks for Antibodies already present on the RBC's - add crosslinking anti-IgG --> clotting

indirect: Abs in serum capable of binding to RBC - (from other tissues hard to biops) use a "test" RBC from the lab.

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frustrated phagocytosis

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neutrophils trying to phagocytose cause the release of ROS - this is part of the type II hypersensitivity

Type II: Antibodies directed at specific tissue
1. Opsonization
2. Complement fixation
3. ADCC
4. Antagonist
5. Agonist (ex: myasthin. gravis)

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complexes are most efficient if?

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there is neither Ab excess nor Ab lack

ratio of Antigen:antibody

symptoms can come or go

also affinity changes

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III Hypersens

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soluable antigen, bystander injury, characteristic deposition - kidneys joint s and skin

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RBC shuttle

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gets rid of Ig-complexes
Complement coats the complex
the RBC have complement receptors (CR1) for C3b or C4b;
liver phagocytes grab the red cells - but the red cells should not die - the C3b is degraded to C3d - this releases the RBC

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missing late vs early component of the complement

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late component missing: not immunecomplex diesease (meningococcal diseases)

early missing: immune complex diseases - because they don't have the attachment spots for RBC to clear the complexes out of the circulation;

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II vs III

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II is tissue-specific!!!
III is just a bystander antibodies

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Angioneurotic edema looks like?

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Hypersensitivity I, but it's not! if it doesn't itch
drug reaction mediated form

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children "outgrow" alergies

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Infants may develop and LOSE 80-87% of allergies to common foods like the milk proteins by age 3 yrs.

One third of older children and adults, with time, also lose allergic reactivity to common foods.

BUT!
Allergies to peanuts, tree nuts, fish and crustacea (shrimp, etc.)
remain life long problems in ~80% of those who react to these allergens.

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Physical Urticarias

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Known potential stimuli:
Cold
Heat
Water
Pressure
Light
Vibration
Exercise

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ATOPIC DERMATITIS

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redness and eczema on face elbow and knees - (not like urticaria which can be on the whole body)

Pathophysiology
Genetic susceptibility to Allergic Disease and
a poorly characterized T cell defect and (sometimes)
loss of function mutations of proteins that enhance skin barrier functions
he had: tons of IgE and Eosinophils


Treatment
Allergen avoidance
Emollients
Cotton gloves at night to minimize scratching

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Acute Systemic Anaphylaxis

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Swelling and itching of mouth and throat
Difficulty swallowing or speaking
Difficulty breathing
Abdominal cramps
Skin rash or flushing
Nausea and vomiting
Feeling of impending “doom”
Hypotension
Syncope

treatment:
Epinepherine

Intravenous fluids

Antihistamines

Glucocorticoids (but takes 6h)

Airway management


long term Tx:
Allergy shots are not an option - they're
Dangerous!
Avoidance is the only option
Patients should be given (2) Epi-pens and taught how to use them.
Eat only food prepared under patient’s
Supervision.

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Hereditary Angioneurotic Edema

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5 types - 2 rare gene mutations, 2 rare aquired types and 1 common drug-induced type


Diffuse deep swelling (angioedema)
– but no redness or itching (no urticaria)

Often involves oral cavity or airway. May also involve GI tract

Sometimes precipitated by trauma
Lasts 24 hrs

Angioedema from C1INH deficiencyunregulated Complement activation?Yes, but that is not cause of angioedema ; C1INH has many other duties

C1INH doesn’t block Kallikrein

Unchecked Kallikrein produces too much Bradykinin

BRADYKININ.
causes EDEMA

ACE inhibitor: Too much Bradykinin produced (normally inhibited by ACE)

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Hemolytic Disease of the Newborn

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Mother is Rh negative. Father is Rh Positive.

During delivery newborn's RBC enter maternal circulation and immunize her against the Rh antigen.

The initial newborn will be unaffected.

With next pregnancy, anti-Rh antibodies cross the placenta and lyse RBC if fetus is Rh positive

Untreated anemia can lead to fetal heart failure
and potentially fatal hydops fetalis

Lysis of RBC also leads to hyperbilirubinemia
with deposition of bile related pigments in the brain.


use the tests:
The Direct Coombs test will tell you that the fetus' RBC are coated with IgG antibody.
The Indirect Coombs test will allow you to determine how much antibody is present in the mother's blood. This is done by making serial dilutions of her serum and testing each for its ability to cause agglutination of Rh(+) RBC.= the titer

Treatment
Pretreatment of the mother with RhoGam (300 mg of purified IgG anti-Rh Ab at 28 wks and just 72 hrs before delivery) will prevent most cases of hemolytic disease of the new born.

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Pemphigus Vulgaris

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55 y.o. Ashkenazi Jewish Lawyer develops a cough and later a sore on his right cheek.

Treated for Herpes simplex and Candidiasis( thrush) but lesions are spreading.

Biopsy of the affected skin reveal deposition of IgG antibody at dermal-epidermal junction.

Antibody titer to desmoglein-3 is increasing.

TX:
Prednisone 120 mg/day (very high dose)
Unsuccessful at controlling disease
But immunosuppressive enough to give him Pneumocystis pneumonia ( NO T-CELLS!, but still rxn!!)

Cyclophosphamide (Cancer agent - crosslinks the DNA) controls the disease
Antiproliferative chemotherapeutic agent
useful for controlling B cell mediated disease


HLA-DR4 -/DQ3 causes the inheritance

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vMyasthenia Gravis

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diplopia
Ptosis of eyelids and limited extraocular movements

lateral muscle ok, but the medial not strong enough

lid lag also manifestation of fast fatigue and muscle weakness -

Ig's that bind to Acetyl-choline receptor

These same IgG could cross the placenta giving baby myesthina gravis – at least for a while;

may require immunosupressants

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SLE

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systemic lupus erythematosus
type III (bystander injury whenever IC deposit, characteristic sites, frustrated phago- ROS )
Malar rash exacerbated by sun exposure
Anti Nuclear Antigen Abs (ANA)
Abs to dsDNA
Low C3
Fever
Arthritis

May develop glomerulonephritis

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Drug-induced serum sickness

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Type 3
Child admitted for pneumonia that resolves on penicillin
9 days after admission: puffy eyes, hives, wheezing, fever, swollen ankles, urticarial rash, diffuse adenopathy
ESR 30 mm/hr (NL <20), Low C3, high WBC (19,800/ml)
Day 10: Purpuric lesions, disoriented, edema, hematuria
So they realized he has drug induced hypersensitivity to the penicillin
Resolves on NSAIDS and prednisone
No IgE antibodies to PCN
but they would have found IgG
it took him 9 days! why so long? this secondary response should be 1d = he must not have a memory response!

so, it's called "serum sickness" = whooping cough - treated with horse antibody - but then they got sick from the horse Ig -there is a peak time where the ratio of Ag-Anti are right = fever, arthritis...

but why still no memory?

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Classical path

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IC - C1 C4 C2 C3

(C4 and C3 missing = we have Immune complexes)
just C3 - just sticking via alternative

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Polyarteritis Nodosa

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Small-to medium-sized blood vessels
Skin, heart, kidneys, peripheral nerves, muscles and intestines.
Purpura, skin ulcers, muscle and joint pain, abdominal pain, and renal insufficiency due to vasculitis of renal arteries.
~30% hepatitis B antigen immune complexes.
Frequently fatal unless treated with immunosuppressives, usually prednisone plus cyclophosphamide.

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Rheumatoid arthritis

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type 4: Rheumatoid factor positive
IgM autoantibody to IgG
Common serologic test
Is not the causative agent

ESR is elevated 87 mm/hr (NL <20)

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Multiple Sclerosis

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Sudden loss of vision in one eye
Multiple brain lesions on MRI
Over next 6 years develops:
Weakness left side of face
Weakness in left leg and hand plus slurred speech
Ataxia and nystagmus

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Superantigens

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break the rule,
Since all T cells with TCRs bearing a particular V region can be activated…. regardless of which individual V(D)J rearrangement has occurred… superantigens can activate up to 20% of an individual’s T cells.


TCR recognition is not MHC restricted.
Superantigens are not presented in the peptide binding groove of MHC.
Superantigen recognition does not depend upon antigen processing.
CDR3 loops are not important for antigen recognition: superantigens bind to V regions
Therefore, superantigens can activate all TCRs bearing a particular V region.

need no costimulation;


Examples:
Food poisoning: staphylococcal enterotoxin (SEA, SEB, etc.)
Toxic shock syndrome (TSST)

mechanism: either viral/bacterial superantigens interact with the TCR - ("ha we lied - they can react with more than just peptide in a groove") and then SOOO many T cells are activated;

12h after surgery shock- TSS? or internal bleeding?

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T cells produce proinflammatory cytokines, including (Type IV)

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TNF-a, IL-1, IL-6, IL-17 and chemokines that attract other inflammatory cells

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Inflammatory cells release (IV)

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TGF-b, metalloproteinases and serine proteases

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T cells activate (what for type IV)?

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Macrophages and Mast cells

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Poison ivy

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Type IV: occurs after 2 days;
Patch Testing: The principal method
for identifying substances that elicit
T cell mediated Gell and Coombs type
IV hypersensitivity after prolonged
contact with the skin

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does Hyper I require IgE?

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NO, only Mast cells!!

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most hypersensitivities use ?

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more than one mechanism

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Maturing dendritic cells that migrate to a lymph node from peripheral tissues end up mainly in:

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T cell zones

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MHC polymorphims is generated by ?

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mutations? ? (HLA-A,B,C and HL DR DQ...)

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Antibody polymorphism is generated by

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somatic recombination of different exons; + junctional diversity (higher amount of diversity!)

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molecular biology of MHC I

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2 chains:
alpha: very long with a1, a2, a3 sites
beta: b2 microglobulin
binding site: a1 a2

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molecular biology of MHC II

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2 chains:
alpha: a1 a2
beta: b1 b2
binding site: a1 and b1

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Terminal deoxyribonucleotidyl transferase (TdT)

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Junctional Diverstiy enzyme

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Activation-induced deaminase (AID)

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enzyme that removes the amino group from the cytidine base in DNA.

AID is currently thought to be the master regulator of secondary antibody diversification. It is involved in the initiation of three separate immunoglobulin (Ig) diversification processes, somatic hypermutation (SHM), class switch recombination (CSR) and gene-conversion (GC).

AID does class switching and somatic recombination in germinal centers;

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Somatic Hypermutation

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SHM diversifies the receptors

process of mutation affecting the variable regions of immunoglobulin genes.

Unlike many other types of mutation, SHM affects only individual immune cells, and the mutations are not transmitted to offspring.

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V(D)J recombination

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This site-specific recombination reaction generates a diverse repertoire of T cell receptor (TCR) and immunoglobulin (Ig)

the V(D)J recombinase is encoded on RAG-1

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Class-Switch Recombination

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DNA is nicked and broken at two selected S-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases.[3][4] The intervening DNA between the S-regions is subsequently deleted from the chromosome, removing unwanted μ or δ heavy chain constant region exons and allowing substitution of a γ, α or ε constant region gene segment. The free ends of the DNA are rejoined by a process called non-homologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the antibody heavy chain.[5]

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Terminal deoxyribonucleotidyl transferase (TdT)

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Junctional Diverstiy enzyme

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Activation-induced deaminase (AID)

Back 62

enzyme that removes the amino group from the cytidine base in DNA.

AID is currently thought to be the master regulator of secondary antibody diversification. It is involved in the initiation of three separate immunoglobulin (Ig) diversification processes, somatic hypermutation (SHM), class switch recombination (CSR) and gene-conversion (GC).

AID does class switching and somatic recombination in germinal centers;

Front 63

Somatic Hypermutation

Back 63

SHM diversifies the receptors

process of mutation affecting the variable regions of immunoglobulin genes.

Unlike many other types of mutation, SHM affects only individual immune cells, and the mutations are not transmitted to offspring.

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V(D)J recombination

Back 64

This site-specific recombination reaction generates a diverse repertoire of T cell receptor (TCR) and immunoglobulin (Ig)

the V(D)J recombinase is encoded on RAG-1

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Class-Switch Recombination

Back 65

DNA is nicked and broken at two selected S-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases.[3][4] The intervening DNA between the S-regions is subsequently deleted from the chromosome, removing unwanted μ or δ heavy chain constant region exons and allowing substitution of a γ, α or ε constant region gene segment. The free ends of the DNA are rejoined by a process called non-homologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the antibody heavy chain.[5]

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opportunistic infections

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These infections include:

* Pneumocystis jirovecii, previously known as Pneumocystis carinii f. hominis
* Candida albicans
* Staphylococcus aureus
* Streptococcus pyogenes
* Pseudomonas aeruginosa
* Polyomavirus JC polyomavirus, the virus that causes Progressive multifocal leukoencephalopathy.
* Acinetobacter baumanni
* Toxoplasma gondii
* Cytomegalovirus
* Aspergillus sp.
* Kaposi's Sarcoma

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B cell problem -

Back 67

mainly pyrogenic bacterial infections as soon as the maternal IgG dissapear - (6 months)

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T cell deficiencies

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early - Viral and other intracellular microbial infections (Pneumocystis carinii, atypical mycobacteria, fungi)

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Innate immunity deficiencies

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variable pyogenic bacterial infections within days (LADS... )

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order of maturation of B cell clone

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mu
kappa
lamda

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linear or conformational epitopes or both?

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Antibodies recognize both but TCR only one

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H and beta have?
L and alpha have?

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V D J C (heavy and beta chain)
V J 1C (light and alpha chain)

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The bigger chain (H or b) recombines?

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The bigger chain (H or b) recombines first and is tested for functionality by pairing with an invariant surrogate L or a chain

Successful recombination of the first gene inactivates the other allele.
Unsuccessful recombination leads to trial recombination of the other allele

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increase in affinity of integrins on the T cell when?

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chemokines or antigen recognition

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integrens are not

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stored

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B7-1 and B7-2 are upregulated with?

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of “danger” signals, such as lipopolysaccharide, as well as cytokines, such as interferon (IFN)-g.

Activated helper T cells can induce expression of B7-1 and B7-2 on APCs via CD40L binding to CD40.

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What is the time-line and sequence of signals in T cells after contact with antigen and co-stimulatory signals?

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B.1) Nuclear localization of transcription factors c-Fos and c-Myc (min to hrs)
2) T cell surface CD40 ligand expression and IL-2 production (1-4 hrs)
3) High affinity IL-2 a receptor expression (3-12 hrs)
4) Then DNA synthesis (12-72 hrs)

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2 sources of second signals

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IL-2 from activated CD4 cells

APC can be activated and express B7

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TH17

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secrete IL-17 and IL-22 which mediate inflammation and help to control some microbes