Module 2 - first half mixed Flashcards

Question

Prophylaxis of GvHD

Answer 1

``` Methotrexate Corticosteroids Cyclosporin A CsA plus MTX FK506 ``` T-cell depletion Post-transplant cyclophosphamide

Answer 2

ATM (Ataxia telangectasia mutated) gene is on 11q Abnormalities of the ATM gene are among the most commonly occurring somatic mutations in cancer and generally have been associated with inferior prognosis. Major role in repairing ds breaks of DNA Associated with 17q deletions and/or TP53 gene mutations. Stressing cells and checking how they respond is used to assess ATM function.

Answer 3

Following DNA DSBs, the MRN complex senses and initiates DNA repair and recruits ATM. As ATM is brought to the site of DNA damage, it dissociates from homo-dimers into active monomers and is catalytically activated by auto-phosphorylation. Once @, ATM serves as a transducer and phosphorylates and activates other protein kinases. E.g. phosphorylation of checkpoint kinase 2 (CHK2), which in turn modulates its own substrates, > in cell-cycle arrest. ATM also @ p53, by direct ATM-mediated phosphorylation through CHK2 ATM also has effects on chromatin relaxation, which aids in repair, through phosphorylation of KRAB-associated protein 1 (KAP1) and others. Finally, ATM can result in prosurvival signalling that is balance with the apoptotic signals, including phosphorylation of AKT, and activation of NF-κB (not shown).

Answer 4

STEP ONE PCR. THEN YOU CAN DO THE REST •Agarose gel analysis/ Qiaxel segregate fragments within the product according to size/molecular weight - qualitative information only oQiaxel is preferred because it avoids the need for the bromide (carcinogen) and is much quicker •Gene scanning is more sensitive/ specific/qualitative oIt can determine sequences and hence is useful in segregating fragments that vary by only a few bases, which would otherwise have a similar weight and be missed on agarose gel/Qiaxel •Heteroduplex gel analysis is useful when one allele is mutated and the other is not oIt uses slow re-annealing which can generate mismatches in such cases oHowever this is laborious and you need a lot of sample material •Real time PCR uses 2 primers and 1 probe oThe probe is degraded by TAQ-polymerase after about 15 cycles, and then produces light oConcentration of your molecular target at the beginning of the reaction effects the number of cycles needed to produce light (cycle threshold. Hence you can use RQ-PCR to tell how much of your target was in a particular sample. (All samples reach the same plateau, but after a different number of cycles) •Sanger Sequencing is similar to heteroduplex but it will tell you what nucleotides are mutated, hence it is quantitivate, whereas heteroduplex can only tell you if a mutation is present or not (qualitative)

Answer 5

•You have to extract DNA/RNa from cellular (usually WCC) suspensions •Then you do PCR - you can use upto 70 cycles if you are looking for a rare molecular target •Primer design is important o 18-30bp long o Avoid repeats & complimentary sequences to prevent self-annealing o Limit C/G because they require more heat to denature (4degs as opposed to 2degs with A/T)

Answer 6

* Establish clonality in T/B cell proliferations * Demonstrate chimerism/ monitor treatment post BM transplant * PCR detection of chromosomal abnormalities/ fusion genes

Answer 7

``` Immunoglobulin class switching, also known as isotype switching, isotypic commutation or class-switch recombination (CSR), is a biological mechanism that changes a B cell's production of immunoglobulin from one type to another, such as from the isotype IgM to the isotype IgG. During this process, the constant-region portion of the antibody heavy chain is changed, but the variable region of the heavy chain stays the same (the terms "variable" and "constant" refer to changes or lack thereof between antibodies that target different epitopes). Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigens, but can interact with different effector molecules. Double-stranded breaks are generated in DNA at conserved nucleotide motifs, called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody heavy chains; these occur adjacent to all heavy chain constant region genes with the exception of the δ-chain. ```

Module 2 - first half mixed Flashcards

Immunophenotyping