Clinical microbiology and Immunology
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how do CD8+ cytotoxic T lymphocytes (CTL) function?
by killing their target cells
what does CTL stand for?
cytotoxic T lymphocytes
when does a CTL become activated?
when its TCR recognized the correct combination of MHC class 1 and peptide on the target cell
what happens when a CTL becomes activated?
peptides from intracellular pathogens will be presented on MHC class 1 to CD8+ cells
is it better to have a stronger or weaker CTL response?
is is better to have a stronger response because it leads to better immunity against that virus
what are HIV controllers?
people who are infected with the HIV virus but never progress to AIDS
what were special about "controllers" that allowed them to evade the HIV virus?
they had a specific allele of an MHC class 1 gene called HLA-B that could present certain HIV peptides that activate CD8+ T cells to kill the HIV infected cells
which family does Cytomegalovirus (CMV) part of?
what type of DNA does CMV have?
double stranded DNA
what percentage of the global population is infected with CMV?
how does a fetus develop CMV congenital disease?
occurs when a pregnant woman becomes infected for the first time (or infected with a new strain) during pregnancy. The absence of protective maternal antibodies leads to uncontrolled growth and vertical transmission to the fetus
what are the clinical signs of CMV congenital disease?
spot - like hemorrhages in the skin, hepatosplenomegaly, pneumonia, retinitis, neurological abnormalities
what is a MHC?
cell surface proteins that signal infected cells to T cells to kill infected cells
what do viruses do to prevent Tcells from destroying infected cells?
viruses inhibit the expression of MHC class 1 on the surface of infected cells
steps for peptide loading of MHC class 1
- 1. intracellular proteins degraded by proteasome
- 2. TAP transporter translocates peptides to the ER
- 3. MHC class 1 synthesized with peptides in the ER
- 4. MHC+peptide complex is moved to cell's surface through the golgi
what are the 4 CMV proteins that are synthesized to prevent MHC class 1 expression on the cell surface?
what does the US2 protein do to MHC class 1?
reroutes MHC class 1 a-chains to the proteasome for degradation
what does the US3 protein do to MHC class 1?
retains MHC class 1 in the ER
what does US6 protein do the MHC class 1?
blocks TAP-mediated translocation of peptides into the ER
what does US11 protein do to the MHC class 1?
reroutes MHC class 1 a-chains to the proteasome for degradation
Viruses have limited genomes, why would they devote so many genes to down regulating MHC class 1 proteins?(4 explanations)
- 1.To cope with MHC polymorphisms: US2 binds to HLA-A proteins but not HLA-B or HLA-C proteins
- 2.Cell-type specific adaptaEon: US11 is more effecEve at targeEng MHC class I to the proteasome in DCs than is US2
- 3. cooperation
- 4.Temporal regulaEon: CMV proteins are expressed at different Emes in the replicaEon cycle
what are the direct methods for identifying microorganisms?
- molecular methods
- immunologic tests
what are the indirect methods for identifying microorganisms?
what stains are used for microscopy?
gram stain and acid fast stain
what produces monoclonal antibodies (mAb)
what is an epitope?
the part of the antigen that is recognized by the immune system
why are monoclonal antibodies useful for identifying microorganisms?
they recognize a single epitope and fluoresce
what are the 2 types of immunofluorescence?
- fluorescent-antibody (FA) technique
- indirect fluorescent anibody (IFA) technique
how does the indirect fluorescent antibody technique work?
fluorescent molecule binds to the antibodies that attach to infected cells
what factors have aided to the number, sensitivity and specificity of serological tests?
- 1. immune cell surface antigens
- 2. lymphocyte biology
- 3. production of monoclonal antibodies
- 4. development of sensitive antibody-binding reporter systems
what is serotyping?
use of serum antibodies to detect and identify other molecules. It can be used to differentiate servers or serotypes of microbes that differ in antigenic composition of a structure or product.
what are agglutinates?
visible clumps or aggregates of cells or particles
what is complement fixation?
binding of complement to an antigen antibody complex to see if there is any antibodies in the serum
what are the steps for complement fixation?
- 1. heat the serum to destroy existing amount of complement proteins in it
- 2. a measured amount of complement proteins and antigens are then added to the serum
- 3. the antibodies, antigen, and complement complex are formed (if there are no antibodies, the complex does not form)
- 4. sheep red blood cells and antibodies are added to the serum
- 5. if there are free complement proteins, they will bind to the antibodies and cause lysis and turn the solution pink. IF NOT, the redblood cells will sink to the bottom of the solution
what is the Enzyme-linked Immunosorbent Assay (ELISA)
used to detect antigens in a sample
how is the reaction of the enzyme linked immunosorbent assay visualized?
the addition of chromogen visualizes the reaction
what are the steps for indirect ELISA?
- 1. antigen is absorbed into the well
- 2. serum samples with unknown antibodies are added to the well. the antibodies stick to the well if it matches with the antigen
- 3. the well is washed out to remove unbound antibodies
- 4. another antibody with a linked enzyme is added to attach to any bound antibody
- 5. a colorless substrate is added
- 6. enzymes on the second antibody hydrolyze the substrate which releases a dye
- 7. wells that are positive for the antibody develop color
what are the steps for the direct ELISA test?
- 1. antibody is absorbed into the well
- 2. antigen is added
- 3. if complementary, it will bind to the antibody already in the well
- 4. and antibody with an enzyme on it is added to bind to the antigen
- 5. substrate is added
- 6. enzyme reaction with substrate produces a color change
what is the difference between indirect and direct ELISA tests?
- INDIRECT= antigen is absorbed into wall and the enzyme linked antibody attaches to the first antibody
- DIRECT = antibody is attached to the wall and the enzyme linked antibody attaches to the antigen
what are the steps for agglutination?
- 1. use beads with antibodies attached to them
- 2. plate them on a flat surface
- 3. add serum with antigen
- 4. if the antigen matches with the antibodies, the solution should form clumps
what is immunoprecipitation?
detects soluble Ag reacting with Ab because the binding of Ag to Ab forms a lattice that precipitates
what is flow cytometry?
- forces suspension of cells through a laser beam and measures amount of light scattering of fluorescence
- it can detect heterogeneous microbial populations with different response to antimicrobial treatments.
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