Week 4 - Immunoassays + Antigen Presentation (APC) Flashcards
What do TH1 cells omit? Which cytokines?
IFN- gamma
IL12
What are the output of the elispot test?
Metal precipitation - spotiness of the plane
62 years old paitent, low heigine conditions of living.
Symptoms are: Weeks of coughing, Weight loss, chest pain, fever, blood in the spit.
Blood smear - High leukocyte count
Chest Radiogram - Reticulonodular Pattern
How can we be sure this is Tuberculosis?
Mycobacterium TB causes memory to be activated - TH1 cells will release IFN-gamma upon antigen contact.
Detection by ELISA quantiFERON.
Screening HIVspot/Indirect ELISA came back positive.
How would we confirm?
Western Blot
What do we detect in the indirect ELISA for HIV?
AntiHIV antibodies
Why is it Important to wash after the Enzyme linked Ab binds the First Ab in the Indirect ELISA for HIV?
Detection is compromised- too high Illumination
Western Blot HIV positive proteins?
p31 or p24 + gp160 or gp120
Pros of Lateral flow technology:
Fast ( exact time of measurement)
Inexpensive
No need for lab experience
Examples for what Lateral flow test detects:
Pregnancy - hCG
Helicobacter Pylori
HIV
How does Lateral flow test works?
Ag in Urine binds the Ab covered Latex Bid.
Ag-Ab Latex Bid flows to 2 specific Abs for conformation:
Anti-(Ab-Ag complex) and Anti-Antigen
IRMA
Immunoradiometric assay- Sandwich like ELISA. Use Radiolabeled antibody. Higher Signal = Detection
Immunohistochemistry :
General Antigen Detection - Based on clinical history, Abs are put in solution.
Could be Direct or Indirect (Stronger signal due to complex binding).
Could work for detection of Autoimmune diseases - Ag is the Ab.
FACS (which in diagnosis is practically FCM) is used for:
Immunophenotyping - Detection of cells by fluorescent labeling.
Part of routine Hematology test.
Antigen Recognition by T and C cells.
How are they different?
B Cells - Native Antigen
T Cells - Peptide portion (Epitope) only together with MHC
What is an Immunological Synapse?
T-Cells and APCs interaction
CCL19 and CCL21
Chemokines that attract APCs and T cells through their CCR7 receptors to Lymph nodes for Interaction
Examples for Endo- and Exogenous Antigens:
Which MHC kind is presenting them?
Endogenous Antigens - Viral or Tumor proteins: MHC Class I
Exogenous Antigens - Bacterial Infection proteins: MHC Class II
What are the additional genetic code appearing in chromosome 6 with MHC 1 and 2 genes region? (MHC 3)
Complement proteins and TNF-alpha
Human Leukocyte Antigens of MHC 1 and 2 (Gene Synonym)
MHC 1 : HLAA, HLAB, HLAC
MHC 2 : HLDP, HLDR, HLDQ
Structural difference between MHC 1 and MHC 2 Epitope binding segments?
Cells present on?
MHC 1: closed pocket - on all nucleated cells ( including APC)
MHC 2: peptide binding groove, open pocket- only on APCs (in certain conditions on other cells)
Amino acid bound length for MHC 1 and MHC 2:
MHC 1 - 8 to 9
MHC 2 - 20
What are the important amino acids in regard to MHC binding?
Anchoring Amino Acids - no matter the length of the peptide chain these are the ones that bind the MHC
Minor histocompatibility antigen: Name
H-Y
APC capability from High to Low:
Dendritic Cells
Macrophages
B cells (plasma)
Cells with no MHCs:
RBCs
Some forms Tumor cells
Cells with Low expression of MHC:
Myocytes
Fiberblast
Hepatocytes
How can Inflammatory cytokines effect the APC capabilities of cells:
Non- APCs could become not professional APC and express MHC-II
How many HLA can we find on a Dendritic Cells?
up to 12!
all the six, from mother and father..
Components of the Endogenous APC system + Function:
Proteosome-LMP2 - Viral or tumor proteins turned to antigen
TAP - Pumped to ER
HLA-A/B/C - bind Antigen and move to the surface as MHC-I
Proteosome capability that adds to the recognition complexity and variety of Antigens:
Peptide Splicing
How are the exogenous pathway and endogenous pathway of APC are separated?
Invariant (Ii) chain works as a stopper to separate the two pathways it blocks the peptide binding groove while in ER
Clip peptide is the remnants of Invariant chain - HLA-DM clears it,only when exogenous peptides for APC (in endosome of digested Antigens).
How does lipid Ag presentation happens?
On CD1 molecules, expressed on thymocytes, B cells and many other.
Not well understood.
What is Cross presentation?
Presentation of Exogenous antigen peptide by MHC 1 (by DC only).
Could work as a shortcut for Cytotoxic attack promotion.
What is the job of exosomes in Antigen presentation:
Cross dressing of DCs - sitting on their surface with Antigens from other cells.
MHC-1 expression enhancement is caused by:
IFN-gamma
TNF-alpha
Lymphotoxin
MHC2 expression modulation by:
IFN-gamma : Up regulation
Corticosteroids and Prostaglandins : Down regulation
Are RBCs arnt killed by NK cells for not presenting MHC1?
occurring in low amount. Not fully explained.
Most polymorphic parts of MHCs:
HLA : A, B and DRbeta
HLA G importance
Trophoblast cells express it as a monomorphic Antigen - NK cells tolerate it in mother.
Components of Exogenous APC system + Function:
Endosome with Ii and CLIP Remnants : transport of Extracellular Antigen peptide remnants (after acidification and digestion).
HLA-DR/DQ/DP : from ER to surface with Antigen.
How do viruses cause autoimmune diseases?
Viral peptides resembles (mimic) HLA of Human.
Immune response is formulated to it, attacks are initiated on own HLA.
MHC 3: What are the proteins coded in these genes?
-B factor
-C4
-C2
(“4 B or Not 2 B, MH see what is 3”)
Classical HLA of MHC 1:
HLA-A, HLA-B, HLA-C
Non-Classical HLA of MHC 1:
HLA-E, HLA-F, HLA-G
Classical HLA of MHC 2:
HLA-DP, HLA-DQ, HLA-DR
Non-Classical HLA of MHC 2:
HLA-DM, HLA-DO
What bonds anchor the peptide to the MHC on the C and N terminals?
Hydrogen Bonds
Antigen Presentation - Endogenous:
ERAAP
aminopeptidase associated with antigen processing in the ER
Antigen Presentation - Endogenous:
ERAD
ER-associated protein degradation system: pumps back the misfolded proteins/peptides to the cytoplasm
Antigen Presentation - Endogenous:
DRiPs
defective ribosomal products
What are the names of the subunits that the immunoproteasome (IFN-Gamma Induced) have?
LMP2
LMP10
LMP7
Proteasome-catalyzed peptide splicing (PCPS): the activity of proteasomes whereby two noncontiguous fragments of a polypeptide substrate or a protein are _____, thereby generating a spliced peptide with a _______not present in the original protein.
Proteasome-catalyzed peptide splicing (PCPS): the activity of proteasomes whereby two noncontiguous fragments of a polypeptide substrate or a protein are ligated, thereby generating a spliced peptide with a sequence not present in the original protein.
Transporters associated with antigen processing : What are the Proteins involved?
TAP-1: Peptide Binding
TAP-2: ATP Binding Cassette Domain
The short chain self lipids bind to ___ with low affinity (ER) and help folding of ___. Upon reaching the cell surface, they are exchanged by high binding exogenous lipids.
The short chain self lipids bind to CD1 with low affinity (ER) and help folding of CD1. Upon reaching the cell surface, they are exchanged by high binding exogenous lipids. (On dendritic cells)
MHC __ invariant chain (Ii) conversion to CLIP peptide:
Continuous shortening of the Ii invariant _ chain due to the effect of ______. Finally the ______-resistant CLIP peptide remains in the peptide binding groove of the MHC __ molecule.
MHC II invariant chain (Ii) conversion to CLIP peptide:
Continuous shortening of the Ii invariant γ chain due to the effect of proteases. Finally the protease-resistant CLIP peptide remains in the peptide binding groove of the MHC II molecule.
CD1 is expressed on many different cell types :
thymocytes, B cells, DCs, hepatocytes and epithelial cells
Activated TH produces IL-2 locally: ______to DCs (CD40-CD40L interaction) for cross-presentation of the antigen with MHC-I, ↑ costimulatory molecule expression, ↓ inhibitory molecule expression
Activated TH produces IL-2 locally: licensing to DCs (CD40-CD40L interaction) for cross-presentation of the antigen with MHC-I, ↑ costimulatory molecule expression, ↓ inhibitory molecule expression
MHC-I Regulation: IFN γ, TNF and lymphotoxin up-regulate it. How? When?
IFN γ is produced predominantly by phagocytic and ILCs - upon virus or bacterial infection.
TNF is produced after APC activation.
(Later IFNγ is produced by activated TH cells)
MHC II Regulation:
IFNγ - Effect
The expression of class II transcriptional activator (CIITA) - MHC-II expression by several cells ↑
MHC II Regulation:
Corticosteroids and prostaglandins - Effect
II MHC expression ↓
MHC expressions is ↓ in the case of certain tumor cells.
Why are MHCs Polygenic?
Different HLAs co-create the variations of the MHCs in different individuals
Why are MHCs Polymorphic ( on the level of a population)?
Its variants have >1% frequency: Each locus has several alleles.
Why is it helpful that there are Polygenic and Polymorphic traits to the MHCs?
The multiple different HLA allelic variants allow recognition of more antigens on the population level.
What is the medical implications of the fact there are a Polygenic and Polymorphic traits to the MHCs?
- Resistance or susceptibility to infections or autoimmune diseases
- Transplantation compatibility
HLA in Organ transplantation:
Significance? What is looked at first?
HLA is matched with appropriate recipient:
First HLA A, B, DR
Allotype identity is searched for
Professional APCs may express __ different types of MHC/HLA molecules at maximum.
Professional APCs may express 12 different types of MHC/HLA molecules at maximum.
HLA-G - Where is it located? What is the structure?
on the surface of trophoblasts - Role in the protection of the fetus.
Non-polymorphic with MHC-I-like structure
MHCs are __-dominantly inherited that means - ____ alleles are expressed!
MHCs are co-dominantly inherited that means - Both alleles are expressed!
RIA
Radio-immunoassay homologues to Competitive ELISA.
Radiolabeled antigen competes with unlabeled one for antigen binding - More antigen à weaker sign.
What is the large difference between ELISpot and ELISA?
load complete cells (not fluid), cells secrete soluble & insoluble antigen, need optimal condition of cell culture
