Therapeutics & Investigations 2 (Part 3) Flashcards

Question

Describe Hyperacute rejection

Answer 1

- The antibodies will bind to endotheial cells - Complement fixarion • Promotes the transfer of cells • Internalise damaged cells - Accumulation of innate immune cells - Endothelial damage, platelets accumulate and then thrombi will develop

Answer 2

- Inflammation results in activation of organs resident dendritic cels - T cell response will develop as a result of MHC mismatch - Based on the immune cells in the donor transplant - inflammed kidney - Immune cells in the kidney have been activated

Answer 3

- In transplants, both the MHC protein and the peptide in its binding groove might be foreign

Answer 4

- In transplant, both the MHC and the peptide can be foregin - Recipients immune system can detect that MHC - Eg. With the kidney = dendritic cells which are tissue white blood cells, transplanted kidney ==> all of the cells become weary - DCs are pro-bound to migrate to the lymph nodes where the lymph nodes reside • In order to trigger T cell immunity - T cells will identify these DCs - binding of the MHC molecule

Answer 5

- Alloantibodies recruit inflammatory cells to the blood vessel walls of the transplanted organ - bind to the endothelial cells ○ Causes them to be internalised by the recipient APC ○ Start to process the donor MHC molecules - Increasing damage will enable the immune effectors to enter the tissue of the blood vessel wall, and to inflict increasing damage

Answer 6

- In transplants, both the MHC protein and the peptide in its binding groove may be foreign

Answer 7

- The donor derived cells will die - Membrane fragments containing donor MHC are taken up by host DC - Donor MHC is presented into peptides which are presented by host MHC - T cell response is generated

Answer 8

- This was previously called bone marrow transplant - has now been renamed because the source is often peripheral blood; rather than bone marrow - Often autologous • To transfer biological material from one part of one person to another part of that same person • Irradiate the immune system = put the stem cells back in and migrate to the bone marrow - Until 1980 only HLA identical siblings could be used as donors due to the risk of rejection or graft vs. host disease

Answer 9

- HSCs can find their way to bone marrow, after infusion and regenerate there - They can be cryopreserved with little damage - Become a large number of different cell types = ALL start from pluripotent hematopoitic stem cells

Answer 10

- Having a replacement immune system being transplanted into you altogether - When transplanted tissue is immune cells themselves - Have the risk of the donor immune cells attacking the host = GVHD (graft vs. host disease) - This can be lethal = best approach is prevention - Removing the T cells from the transplant reduces GVHD - Remove immune cells from the recipient so they won't be attacked or the HSTs transplanted so that they won't be attacked - Prevent GVHD using immunotherapy

Answer 11

- Patient could have residual chemotherapy - the new immune system will think that things that are self are actually not theirs. - Sometimes mismatch + donor leukocytes can be beneficial - removing original leukaemia - Graft versus leukaemia response - Development of GVL may prevent disease relapsing (might think self cancer cells are foreign and therefore kill them - which is the sort of outcome that we want )

Answer 12

- Essential, to maintain non autologous transplant - Immunosuppressants for transplant can be: Ø General immune inhibitors (e.g. corticosteroids) Ø Cytotoxic ○ Kill proliferating lymphocytes, e.g. mycophenolic acid, cyclophosphamide, methotrexate Ø Inhibit T cell activation ○ Cyclosporin, tacrolimus, rapamycin - Immunosuppressives might need to be maintained indefinitely - Creating a more toloregenic system - cytogenic which will kill lymphocytes and there are other agents which inhibit T cell activation

Answer 13

- Breakthrough transplant drug • Essential for the maintenance and the differentiation of t cells - Blocks T cell proliferation and differentiation - Next generation therapies less toxic and effective at lower doses

Answer 14

1. Steroids (prednislone) 2. Cytotoxic (mycophenolate motefil) 3. Immunosuppressive specific for T cells (e.g. cyclosporin A, FK506)

Answer 15

- Our immune systems are not meant to be inhibited indefinitely. People on immunosuppressive therapy = greater risk of cancer. - There is currently no immunosuppressive that is able to prevent transplant rejection whilst maintaining other immune responses - Transplant patients are more susceptible to infection + malignancy • Immediate risk, e.g. CMV - Immunosuppressive drug toxicity can lead to organ failure - either of the transplanted organ, or healthy organs - Vital for a patients outcomes after transplanation • e.g. cyclosporin nephrotoxicity in kidney transplant

Answer 16

- Transplant rejection results from genetic differences between the donor and the recipient - Variability between MHC proteins is the major genetic difference in transplant rejection - Host attacks transplant - rejection (hyperacute, acute, chronic) - Transplant attacks the host - GVHD (Graft vs. Host Disease) - Immunosuppression can prolong the survival of transplanted organs

Answer 17

- They correspond to the normal stages of lymphoid development - The state or presence of a malignant tumour, cancer

Answer 18

- This is characterised by infiltration of blood and bone marrow Ø Corresponding normal cell = Ø B cell precursors in the bone marrow

Answer 19

- This is characterised by infiltration of blood and bone marrow

Answer 20

- Tumours of lymph nodes and other secondary lymphoid organs Ø Corresponding normal cell = Ø B-cells in the secondary lymphoid organs

Answer 21

- Foci of malignant cells in bone marrow

Answer 22

Ø B cell precursors | Ø That are in the bone marrow

Answer 23

Ø T cell precursors Ø In the bone marrow Ø OR in the thymus

Answer 24

Ø From B cells in the secondary lymphoid organs

Answer 25

Ø This corresponds to the mature circulating B cells

Answer 26

Ø Immunoglobulin secreting plasma cells | Ø That are in the bone marrow

Answer 27

HSC: haematopoietic stem cell [START point] CMP: common myeloid progenitor, Ø These go on to produce - Neutrophils - Red cells - Platelets CLP-:common lymphoid progenitor Ø Go on to produce Pre T, and Pre B cells Pre-T: T-cell precursors Pre-B: B-cell precursors Imm-B: immature B-cell [end product] - which is produced when there is immunoglobulin gene rearrangemen

Answer 28

- The secondary lymphoid organs are lymph nodes, spleen, peyers patches etc. - Stem cells in the bone marrow give rise to pre B and Pre T - Pre T = migrate to the thymus where they do their PCR gene rearrangement - B cells do all of this in the bone marrow before they then go to the secondary lymphoid organs - Immature lymphocytes will wait, looking for an antigen and looking for a reason to do something

Answer 29

- Immunoglobulins will make 2 x heavy chain and 2x light chains - Light chains can either be kappa or lambda = EITHER , not both. - Series of cells in normal person, will be light - Mutations happen in one cell (shown by the circled cell) = then all of the malignant cells descend from this one abnormal chain Ø E.g. if it is making one type of light chain, you will see so many in this type - See cells that are all making one light chain = clear sign of clonal disease and malignancy

Answer 30

- Usually non specific symptoms of bone marrow suppression | - Symptoms of organ infiltration, more often in advanced disease

Answer 31

- Commonest leukaemia in children that are less than 10 years old - generally no longer a cause of death - But majority of patients are over 40 years old, overall.

Answer 32

1. Bone marrow morphology • Infiltration by undifferentiated blast cells 2. Immunophenotyping Ø B cell surface markers (or T markers for T-ALL) ○ See that there are too many white cells, or too many of the same type of cell. Ø Look for Light chain restriction Ø TdT positive ○ Tdt = enzyme ○ Made in early pre B cells at the stage when they are messing around with Ig genes to make a new antibody ○ Marker for early lymphoblasts ○ If you see a lot of them = they are leukaemic cells. 3. Cytogenetics

Answer 33

1. Induction 2. Intensification, adding an extra stage 3. CNS directed chemotherapy • Cells can lurk behind the blood brain barrier • Do a direct injection behind the spinal cord - (can kill the patient if the dosage is too high.) - Maintenance, continuation of low level chemotherapy - Radiotherapy to CNS

Answer 34

- Children = >90% cure - Adults have much lower survival rate, due to Ø Differentiated cell of origin Ø Different oncogene mutations Ø Older patients = do not tolerate intensive treatment, it also depends on how fit they are ○ Adults biologically do not have the same disease for the reasons above

Answer 35

- Presentation = with enlarged lymph node(s) - Epidemiology = Ø Peak incidence is in young adults ○ There is a second smaller peak in older people Ø Possible associations with Epstein Barr Virus (EBV) aka Human Herpes Virus 4 (HHV4) ○ HHV4 =

Answer 36

- Presence of large Reed-Sternberg cells = pathognomonic • IF there is this cell present, there is DEFINITELY Hodgkins Lymphoma - These are malignant B cells - But typically 99% of cells are reactive to non-malignant cells - Multinuclear

Answer 37

- Presence of large Reed-Sternberg cells | Ø Giant cells that are found in those with Hodgkins lymphoma

Answer 38

- Chemotherapy, +/- radiotherapy - Prognosis = 5 year survival • About 50-90% depending on age, stage and histology • Especially good results in young adults

Answer 39

- These are all of the remaining lymphomas - Can be characterised into Low grade, High grade - T cell lymphomas - EBV (HHV4) - driven lymphomas in immunosuppressed patients

Answer 40

- Many lymphomas carry chromosome translocations, involving the Ig heavy chain or light chain loci - Ig genes are highly expressed in B cells Ø Each Ig gene has a powerful tissue specific enhancer, near to the constant "C" segment - OMA = lump in the lymph nodes. Tumour in the lymph nodes.

Answer 41

- Most cases of follicular lymphoma will carry t(14;18) (q32;q31) - This juxtaposes the BCL-2 gene on chromosome 18, with the IgH locus on chromosome 14 - - Causes overexpression of BCL-2 protein - BCL-2 is an apoptosis inhibitor - the cells do not die when they are suppsoed to.

Answer 42

- B cell lymphoma 2 is encoded in humans by the BCL2 gene - BCL2 = apoptosis inhibitor; inhibits the process of programmed cell death Ø Regulator proteins, which regulate apoptosis Ø By either inducing pro apoptotic, or inhibiting anti-apopotic apoptosis ○ BCL-2 = oncogene (therefore cancer causing)

Answer 43

- Some cases of high grade lymphoma carry t(8;14) (q24;q32) - This juxtaposes the MYC gene on chromosome 18, with the IgH locus on chromosome 14 - MYC = powerful oncogene • Once overexpressed it drives the cells into the cell cycle.. • Slow growing lymphoma they are not cycling faster, but are just failing to die. - Can also get MYC, or BCL-2 translocations to one of the Ig light chain loci

Answer 44

- Presentation = enlarged lymph nodes - Histology Ø Normal tissue architecture partially preserved = in cancer terms low grade is that the cells and tissues are not THAT changed from normal ○ High grade = the cells are VERY changed from the norm. Ø Normal cell of origin recognisable ○ Used to name lymphoma - follicular lymphoma, mantle cell lymphoma etc. ○ Named by comparing it to the normal tissues

Answer 45

- Histology - Immunocytochemistry - Cytogenetics - Light chain restriction - PCR • For looking for the clonal Ig gene rearrangement • For chromosome translocations - e.g. (t(14;18) Ig Bcl-2) ○ For these, look directly at the DNA

Answer 46

- Look at PCR + amplify the DNA = there are lots of differently sized band - The bands that you get = single size - Get single monoclonal band - These are polyclonal therefore they are not malignant.

Answer 47

Ø Chemotherapy Ø Glucocorticoids (like prednisolone) • Because they cause cell death in lymphocytes Ø Radiotherapy • Targeting particular lymph nodes Ø Monoclonal Ab therapy (mouse) • Rituximab against human CD20 (anti CD20)- this is a B cell marker • This antibody will attack B cells = this is effective treatment

Answer 48

Ø Chemotherapy Ø Glucocorticoids (like prednisolone) • Because they cause cell death in lymphocytes Ø Radiotherapy • Targeting particular lymph nodes Ø Monoclonal Ab therapy (mouse) • Rituximab against human CD20 (anti CD20)- this is a B cell marker • This antibody will attack B cells = this is effective treatment

Answer 49

- Creation of human antibodies against the mouse immunoglobulin - Relatively indolent (lazy and does not grow particularly fast) - Responds well to therapy ; But is hard to cure - patients can be managed, but are never actually cured. If the disease starts to come back then you could re treat it.

Answer 50

- Presentation = enlarged lymph nodes - Histology shows: Ø Loss of normal tissue architecture Ø Normal cell of origin hard to determine ○ Cells have changed a LOT from normal

Answer 51

- Histology - Immunocytochemistry - Cytogenetics - Light chain restriction - PCR Ø For clonal Ig gene rearrangement Ø For chromosome translocations

Answer 52

- Chemotherapy - Glucocorticoids - Radiotherapy - Monoclonal Ab therapy Ø Rituximab - anti CD20

Answer 53

- Prognosis • This is variable depending on type, stage and other factors e.g. health of patient • Overall long term survival is about 65%

Answer 54

- These are rare (Most lymphomas are B cell) - Usually CD4 cells - Often present with skin infiltration, - it is not that clear why e.g. Ø Sezary syndrome Ø Mycosis fungoides

Answer 55

- Starts off looking like a fungal infection of the skin | - But may be a T cell lymphoma

Answer 56

- Found in Japan, Caribbean, UK citizens of Caribbean origin = this is where the disease is found - Associated with retrovirus HTLV-1 human T cell leukaemia/lymphoma virus 1 infection • Predisposes you do developing this but is not a guarantee

Answer 57

- EBV, or Human Herpes Virus 4 (HHV4) Ø Will directly transform B lymphocytes in culture Ø Cells transform & start growing. - This is due to viral oncogene LMP1 - Over half of all normal individuals carry latent EBV infection • Experience in early childhood • If you are exposed when you are older; get glandular fever (infectious mononucleosis) - develop cytotoxic T cells and immunity - Do not develop lymphomas, due to effective immune surveillance by cytotoxic T cells - Do not eliminate virus from the body - lurking somewhere but is not actually a problem.

Answer 58

- The endogenous latent EBV might transform B cells - No longer eliminated by cytotoxic T cells - Develop high grade lymphoma • Situations when people are immunosuppressed • Infection with HIV aids • Giving transplant = immunosuppression so that they do not reject the graft.

Answer 59

- Lymphoma usually regressed on withdrawal of immunosuppression - t cells come back - But the patient might lose the graft; therefore this is a difficult clinical balance

Answer 60

- Lymphoma might regress on successful HAART (HIGHLY active anti retroviral therapy) - Not successful in eliminating virus - But good at stopping it progressing into AIDS

Answer 61

- Most often as incidental finding on FBC - Persistent infections • Due to immunosuppression - suppression of the bone marrow and low neutrophil count. Large number of malignant B cell but not normal B cells. • Low IgG, suppression of normal B cells - Lymph node enlargement - Symptoms of bone marrow suppression Epidemiology = 85% of cases are over 50 years old

Answer 62

- FBC : lymphocytosis - Immunophenotyping Ø Cell surface markers Ø Light chain restriction - Cytogenetics, chromosome translocations

Answer 63

Ø Huge amount of mature lymphocytes seen in CLL

Answer 64

- This is the presence of a single, monoclonal Ig in the serum / plasma

Answer 65

Ø Monoclonal gammopathy of undetermined significance (MGUS) • Low levels of paraprotein • Low levels of paraprotein occasionally seen in lymphoma, or chronic lymphocytic leukaemia Ø IgM paraprotein seen in Waldenstroms macroglobulinaemia

Answer 66

- There are THREE aspects of myeloma that all give rise to different clinical features 1. Suppression of the normal bone marrow, blood cell and immune cell function 2. Bone resorption, and release of calcium Ø Stimulate bone resorption Ø Pushing out lots of immunoglobin = there is so much of it that it causes problems 3. Pathological effects of the paraprotein

Answer 67

- Anaemia - Recurrent infections - Bleeding tendency

Answer 68

- Myeloma cells produce cytokines (especially IL-6) • Which stimulate bone marrows stromal cells, to release the cytokine RANKL • This will activate osteoclasts which will produce 1. Lytic lesions of bone 2. Bone pain 3. Fractures - Presentation of this = unexpected fractures. - Calcium released from the bone causes hypercalcaemia - Multiple symptoms including mental disturbance = therefore need for psychiatry in high blood calcium

Answer 69

- There are holes / areas of thinning in the skull. | - This is very typical of multiple myeloma = malignant B cells

Answer 70

- Plasma cytes = factories of producing Ig. They are making too much = is too much protein going out into the circulation. - Precipitates in kidney tubules, cause renal failure - Deposited as amyloid in many tissues - amyloid. This can be damaging to tissues. - 2% cases develop hyper viscosity syndrome = blood gets TOO thick • Increased viscosity of blood, which leads to stroke and heart failure

Answer 71

``` - Chemotherapy; not curative Ø Cytotoxic drugs Ø Glucocorticoids Ø Thalidomide analouges Ø Bortezomib - Allogeneic bone marrow transplant Ø Only is available for a small number of younger patients (less than 45 years old, not too ill) Ø Need to find an HLA (MHC) matched donor Ø But potentially curative ```

Answer 72

- Group of diseases - That are characterised by malignant overproduction of white blood cells; or their immature precursors- (there are too many white blood cells and not enough room for other things like RBCs, platelets.) - (Or their immature precursors)

Answer 73

- Varies between the type of leukaemia - There is suppression of normal haemopoiesis - But typically first presents with symptoms, due to the loss of normal blood cell production Ø Most common presentation = abnormal bruising Ø Or repeated, abnormal infection Ø Sometimes just anaemia These things are all affected by the overproduction of White blood cells ○ Less neutrophils = infection ○ Less RBCs= anaemia ○ Less platelets = abnormal brusing

Answer 74

- Abnormal bruising | - Or repeated, abnormal infection

Answer 75

- Involving cells of the lymphocyte lineages Ø Commonly B cell Ø And more rarely T cell

Answer 76

- Involving any of the non-lymphocyte blood cell lineages - Commonly neutrophils, or their precursors - But can be erythroid, platelet, basophil lineages etc.

Answer 77

- Undifferentiated leukaemia's | - Characterised by immature white cells (known as blast cells)

Answer 78

- Activation of oncogenes and inactivation of tumour suppressor genes - Might involve genes that are similar to other malignancies • RAS • MYC • P53 - tumour suppressor gene, mutated in lots of cancers and leukaemias. Involved in cell cycle regulation & stopping cycle when there is some damage ○ These are dominant oncogenes that are mutated in many cancers - But also some that are specific to leukaemia's

Answer 79

Ø Normal haemopoiesis = many cells contribute to blood Ø Mutation in one cell Ø Clonal haemopoiesis, all leukaemic cells derive from one mutant cell Ø Leuk = clonal disease Ø All of the leukamia genes start from this particular cell

Answer 80

1. Chemotherapy Ø With CYTOTOXIC DRUGS (which will mostly target dividing cells) 2. Stem cell and bone marrow transplants (SCBMT) 3. Disease specific agents, including oncogene targeted drugs

Answer 81

- Combinations of drugs, used to kill leukaemia cells - Optimised for type and subtype of leukaemia - Cytotoxic drugs = will mostly target dividing cells - therefore this relates to the cell cycle

Answer 82

- Cytosine arabinoside (ara-C, cytarab when used as a drug) - Cytosine analogue - Interferes with deoxynucleotdie synthesis - Prevents successful DNA replication - If cell is going to enter S phase, has to makke lots of deoxynucleotide triphosphates in order to divide - Means cell cannot make enough deoxynucleotides = kills cells that are trying to go through the cell cycle

Answer 83

- Binds to tubulin dimers - Inhibits microtubule formation = mitotic spindle is made up of tubulin • Cell cannot divide • Will go on and then die - So blocks the mitotic spindle

Answer 84

- This is when intense chemotherapy is given and total body irradiation - Wipes out leukaemia cells AND the normal stem cells - This reconstitutes bone marrow, by transplanted stem cells - Patients bone marrow recovers but there is no more leukaemia - Sometimes the foreign bone marrow will make T cells which attacks the leukaemia • Have to find HLA MHC matched donor.

Answer 85

- SCBMT = Stem cell, and bone marrow transplantation - There is a shortage of HLA (MHC) matched donors = need to have a match. - Very intense procedure - High mortality of the procedure for older, or sicker patients

Answer 86

- Large numbers of myeloid blasts (AML) or lymphoblasts (ALL) in the bone marrow - Therefore = "undifferentiated leukaemia's" - Lots of cells that all look the same = immature lymphoblast's = very suspicious and not what you should see in the bone marrow

Answer 87

Thrombocytopenia - Purpura (bruising) - Epistaxis (nosebleed) - Bleeding from the gums • Low platelet count Neutropenia - Recurrent infections Anaemia - Lassitude - Weakness - Shortness of breath

Answer 88

alpable bruising | P

Answer 89

Ø Yeast infection of the mouth which is rare in those who are wel

Answer 90

- What you see in Peripheral blood • Presence of blasts cells = should not usually see • Cytopenias = shortage of normal blood cells - What you see in Bone marrow aspirate • Over 30% of the blasts is diagnostic of acute leukaemia = too many blasts!

Answer 91

- there is maturation arrest - These are all immature - They have all become immature at the same stage - They have all got stuck at the same stage = this is maturation arrest - AUER rods = very diagnostic of acute leukaemias

Answer 92

- There is cell proliferation which produces blast cell pool, these cells then mature, carry out their function and die = this is the normal process - If you block cells from maturing they will continue to proliferate & blast cell pool will keep building up - In malignancy in general you do not actually have to have cells dividing faster if you stop them dying - Lifespan of mature neutrophil = 24hrs - There is high turnover, they are being made fast and dying fast - If you stop them dying off, they are still being made fast = enormous buildup of malignant cells

Answer 93

- Many of the chromosome translocations in acute leukaemia's: Ø Involve genes for transcription factors Ø Which control cell differentiation - Chromosome abnormalities also help to determine the prognosis and response to treatment

Answer 94

1. Chemotherapy 2. Combinations of drugs 3. Optimised for each type and sub type of acute leukaemia ALL = add chemo, or radio-therapy to the CNS Ø Blood brain barrier = main cytotoxic drugs to not get at that area Ø Brain might be an area where the leukaemia cells can stay and survive, and then come back from

Answer 95

- Phase 1 = remission induction Ø Circulation and bone marrow full of blasts Ø Give them chemo, barrier ursing and lood transfusion Ø Wipe out most of the blasts - Phase 2 = consolidation therapy Ø This is to kill residual leukemic stem cells Ø With oral leukaemia therapy = this makes a difference to the long term outcome

Answer 96

- Differentiated leukaemia's = here we see peripheral blood smear from patient with CLL - There are too many white cells, and they are all lymphocytes - Increased numbers of differentiated cells

Answer 97

- Large numbers of mature (clonal) lymphocytes in bone marrow and peripheral blood = lymphocytic - Therefore are also called chronic lymphocytic leukaemia

Answer 98

- Recurrent infection, due to neutropenia • + suppression of normal lymphocyte function • All of these lymphocytes in the circulation - But they are not particularly useful to have (if you happen to get a disease that these have antibodies against then this COULD be fine but there are very slim chances of this happening!) - Anaemia - Thrombocytopenia - Lymph node enlargement - Hepatosplenomegaly

Answer 99

- Controlled by regular chemotherapy to reduce cell numbers | - A few patients might die in

Answer 100

- Differentiated leukaemia's - Increased number of differentiated cells - Far too many white cells - Involve mature cells = there are also lots of immature cells. - Immature myeloblasts = circled - They are NOT all arrested at an early stage

Answer 101

- Anaemia - Night fever / night sweats due to the excess cells metabolising or de to cytokines - Spenlomegaly • Infection = not a major symptom. Although lots of NT might not be normal; there are a lot of them

Answer 102

Ø Very high white count (neutrophilia) Ø Left shift in blood and bone marrow • Put out development of neutrophils = left, there are the very immature cells and the get more mature to the right • = excess of immature cells, more than one would expect. Ø Presence of the Philadelphia chromosome Ø BCR-ABL gene rearrangement

Answer 103

- Controlled, but not cured by chemotherapy. Ø Cytotoxic drugs (original treatment) Ø Interferon alpha - this was found to be more effective, quite effective in extending lifespan. But the person will always feel like they have the flu Ø Imatinib ○ Anti oncogene, anti oncoprotein drug

Answer 104

- Survival on treatment, usually measured in years (4-5 years) - But eventually progresses to accelerated phase and then blast crises; disease turns into something that looks like an acute leukaemia and there are immature blasts - CML blast crises = refractory to treatment - Can try chemotherapy but does not work very well compared to normal AML.

Answer 105

- Resembles an acute leukaemia | - But very refractory to treatment

Answer 106

- Allogenic bone marrow or stem cell transplant curative - Autologous transplant sometimes tried - Most patients are over 50 years old. • Do not tolerate the transplant

Answer 107

- Chromosome 9 and 22 are both abnormal

Answer 108

Ø Philadelphia chromosome (Ph1) 22Q-

Answer 109

Long arm = slightly longer than it should be - therefore 9q

Answer 110

- 95% of cases of CML have a detectable Philedalphia chromsome (Ph') when karyotyped.

Answer 111

- BCR gene = chromosome 22 - ABL gene = chromosome 9 - BCR-ABL chimaeric gene (Philedelphia chromosome) ABL = this is where the break point iis. It is a dominant oncogene. There is a gene that is a bit of BCR and a bit of ABL. - Chimeric = made up of different pieces

Answer 112

- It is a protein tyrosine kinase - But activity is tightly regulated - BCR-ABL protein has constitutive (unregulated) protein tyrosine kinase activity - ABL = not a cell surface prtoein but is an important signalling protein in the cell. Mostly inactive unless it is specifically activated = not generally active.

Answer 113

- Proliferation of progenitor cells in the absence of growth factors • NOT regulated, has constitutive activity; independent of regulation. - Decreased apoptosis - Decreased adhesion of cells to the bone marrow stroma - BCR ABL can be put into bone marrow stem cells and this will give them leukaemia.

Answer 114

- Inhibition of BCR-ABL tyrosine kinase, • Causes apoptosis of the CML cells = was approved very quickly because it was so successful + huge drive from patients who wanted it. - Remission reduced more in patients (compared to other treatments) - Greater durability - remission lasts longer - Fewer side effects - But some patients can become drug resistant :( so new tyrosine kinase inhibitors are required.

Answer 115

- Drugs that specifically inhibit the BCR-ABL - Imatinib (Glivec, STI571) (selective tyrosine kinase inhibitor) • Specific tyrosine kinase inhibitor ○ Does not inhibit all tyrosine kinases • Inhibits BCR-ABL but not most other tyrosine kinases ○ Does inhibit some other tyrosine kinases ○ Most of them = it will leave alone

Answer 116

- There are 147 current species - Only 3 of them are obligate human pathogens Ø M tuberculosis Ø M leprae Ø M ulcerans - The maximum likelikhood phylogeny of Mycobacteriym genus

Answer 117

Ø M tuberculosis Ø M leprae Ø M ulcerans - Obligate parasites = cannot complete life cycle without exploiting a suitable host - If it cannot find a host then will not be able to reproduce - Advantageous for a parasite to preserve health of their host, when this is compatible with their nutritional and reproductive needs - apart from when the death of the host is needed for transmission

Answer 118

- Biological agent - That causes disease or illness to its host • But they can also be present, but not necessarily disease causing

Answer 119

- The pathogen is present - But it is not capable of causing a disease in the host • E Coli • Bacteroides Thetaiotaomicron ("good bacteria") ○ Lots of the organisms in our bodies do not actually cause any disease = e.g. putting e coli in gut = good but would be bad if you put it into a cut

Answer 120

- The pathogen is present - But only capable of causing disease, in another host E. Coli O157:H7 Ø This is subclinical in cattle ○ E.g. wouldn’t make a cow ill, but would make us as humans ill

Answer 121

- Is present, & is capable of causing disease to the host - But only causes damage in certain circumstances Ø Bacteroides Fragilis Ø Coagulase Negative Staphyloccus (CNS) - Not all positive samples, are diagnostic of active disease - Become pathogens when they get the opportunity to do so - Not enough to say organims is there ==> and person has the disease.

Answer 122

- A microbe - That is capable - Of causing a specific degree of host damage Ø Have to push this capability by defining it

Answer 123

- Looking at larger things = very easy to identify - Trichommas vaginalis Ø Causes majority of STDs - only swab and slide needed - Lots of UK children have strongyloides (threadworm) = sellotape and microscope 1. Trichomas vaginalis 2. Schistomasoma maroonni 3. Entemoeba histolytica Ø Stomach bugs caused by this 4. Stronyloides (threadworm) Ø 50% of children

Answer 124

Ø This is for looking at smaller things Ø Shapes visible Ø Rotavirus = D&V 1. Rotavirus (reovirus) from faeces 2. Rabies (Lyssavirus) from brain tissue 3. Hepatitis B (Headnavirus) from the liver 4. Tonsilitis (Adenovirus) from nasal secretion

Answer 125

- Able to see the shapes of bacteria = changes the way thatcell wall changes from one to another - These are already ways of categorising the cells - Gram positive = blue - Gram negatives = red colour 1. Gram negative bacillius = these are rod shaped, 2. Gram negative motile virus 3. Gram positive coccus Ø Streptococcus 4. Ziehl nielsen 5. Auramine 6. Gram negative diplococcus - If we know they are gram + or - = give antibiotics that work specifically on this type of organism - Or could explain why antibiotics are not working properly - Advantage is that this is very cheap

Answer 126

- Spiral shape is seen in gastric syphilis Ø Conjugated antibody is used to stick to this and it lights up - Have to use electron microscope = want to look at the inside of the cell then you can use a complementary antibody to do so. - Cannot see the actual viruses but you can see the stain

Answer 127

- Easy to perform - Rapid screening - Some parasites, have SPECIFIC morphology Ø E.g. Schistosoma mansonii - It is possible to do specific immunofluorescence staining

Answer 128

- It is NOT sensitive Ø e.g. mycobacterium tuberculosis Ø Screening sputum smears is required Ø At least 10,000 orgs per ml to be visualised and come up as positive - General stains are NOT specific - Labour intensive, which is expensive - Requires specialist interpretive expertise which is also more expensive

Answer 129

- This relies on the ability of the test system, to be able to grow the pathogen - Put in on plate and give it nutrients = put in condition that it likes to grow, if organism is able to grow in high temperatures like 37 degrees = looking for those that grow at 37 degrees in particular will help us find pathogens that will grow in humans Ø Selective temperatures = cows are at 42 degrees Ø Organisms would not usually grow at this temperature = campylobacter does, this is a way of identifying this orgnaism because things like e coli will NOT grow at 42

Answer 130

1. Non selective media - e.g. blood agar 2. Semi selective media - e.g. MacConkey Agar, DCA, CLED 3. Selective growth temperatures - e.g. campylobacter species

Answer 131

- Non lactose fermenting - Enterobacteria growing on Cysteine Lactose Electrolyte Deficient (CLED) Agar - From a urine sample - If the sample is also not going to be sterile, we want to grow this and also use a particlar agar • And we can make the agar "look" for certain things • e.g. an agar that selects "lactose fermenting" and "non lactose fermenting" and therefore separates out based on these characteristics - Non lactose fermenting organisms = behind causing disease, can work out due to the colour that they give.

Answer 132

- Deoxycholate agar (DCA) medium - Selective for Ø Shigella and salmonella - On a faecal sample - Grow well on this media, whilst e.coli will not be able to grow very well in this

Answer 133

- Shigella - Salmonella, on a fecal sample (but E.coli might not be able to grow very well on this medium)

Answer 134

- S aureus = grow at: • Aerobic • Anaerobic • Micro-aerophilic which is in between aerobic and anaerobic. Organisms may favour these conditions because that the situation in our lungs (getting o2 in and co2 out) Ø If it grows well in high co2 = this is what the bottom of the lungs are Ø This system can be used, to select out particular organisms Ø Rather than putting it on plate, put in incubator with CO2, & find organisms that ONLY favour the lung conditions ○ Grow respiratory pathogens by putting them inside a jar - Catalase possible = take peroxide and take off oxygen, which is why it is shown bubbling

Answer 135

Ø This is chocolated blood agar ``` - There are respiratory pathogens • Neisseria meningitidis • Neisseria gonorrhoea • Haemophilus influenzae • Brucella melitensis ```

Answer 136

Ø Organisms hate oxygen = oxygen kills them and is harmful to them. Ø They are still able to survive, as they permanently live INSIDE of the body Ø (where there is no O2 - this is their ecological niche, where they divide and where their metabolism is designed for) Ø Clostridium perfringes on blood agar is grown in anaerobic atmosphere

Answer 137

Ø Metabolic function, and sugar utilisation tests for identification of Enterbacteriae ○ e.g. Salmonella, Shigella, E.Coli Ø Inside = organism in each one and changes colour depending on the test Ø They go POSITIVE, or NEGATIVE = can see a pattern, and known that certain pattern is a certain pathoegn Ø Gone from taking a sample growing and isolating it, to looking at microscope Ø & THEN looking @ exact test to determine the species

Answer 138

- Antibiotic typing for bacteria - Circled = penicillin - T = tetracyclin, sensitity for that

Answer 139

E Test - This will tell us HOW MUCH to give - Different concentrations of antibiotics are at each level, and the organisms are not growing around the top Ø This helps us to know how much of the antibody that we should be giving

Answer 140

Ø Viruses are intracellular organisms = can divide inside the cells Ø Dependent on being intraccellular Ø Need cell lines to grow viruses, cell line that viruses can get into and to make something happen **Cell Lines Lecture, Semester 3** • Can also include how a cell line is produced and the advantages and disadvantages of cell lines Ø Unless you use Electron Microscopy, you will not be able to see the viruses Culture • Requires permissive cell lines ○ e.g. Vero cells (kidney epithelial), for Herpes Simplex • Cytopathic effect ○ Immunofluorescent staining of the culture

Answer 141

1. Culture and microscopy Ø Requires permissive cell lines ○ e.g. vero cells (kidney epithelial cells), for measles (Morbillivirus) Ø Cytopathic Effect Ø Immunofluorescent staining of culture 2. Direct antigen detection Ø ELISA ○ e.g. influenza virus

Answer 142

- ELISA = antibody **SEM 3 ELISA tests and the antibodies lectures, can be added to this section of the plan ** Ø This is an Antibody, that is against something that we are wanting to detect Ø Has different binding capacity again pathogen Ø Come with another antibody that sticks to the top = maker sends the media through Ø If there is flu present, it will go blue, and the more flu there is the more blue it will become - Electron microscopy, can see this virus - but not identify it as swine flu - The culture takes 3-10 days - Rapid ELISA for fluA antigen = 15 minutes, ELISA for Flu antibody

Answer 143

1. Cheap, simple and reliable agents 2. Sensitive Ø e.g. single organisms can be grown and identified 3. Validated specificity Ø e.g. "Gold standards" with multiple parameters Ø This is important = anything that is newly brough in has to meet or exced this gold standard 4. Direct in vivo measurement of the effectiveness of therapy Ø Antibiotic sensitivity 5. Easily archived Ø e.g. epidemiology - working out what happened in the past duing an outbreak

Answer 144

1. Some of the pathogens cannot be grown Ø Mycobacterium leprae 2. Some pathogens cannot be well differentiated by biochemistry alone 3. Slow, because the cultures need at LEAST overnight incubation • Viral = 3-10 days • Mycobacterial = 6-12 weeks ○ Slow and overnight culture = sometimes not good enough, e.g. if a child has meningitis you would want to know immediately 4. Some pathogens grow too slowly to aid rapid diagnosis Ø e.g. Mycobacterium tuberculosis 5. Labour intensive (expensive) 6. Requires specialist interpretive expertise (more expensive)

Answer 145

``` - The aim, is to detect a gene • Or gene products Ø That are pathogen specific - Nucleic acid amplification techniques (NAAT) - Polymerase chain reaction (PCR) ``` ``` Ø Influenza / H1N1 Ø Norovirus - D&V Ø MRSA • Testing for methicillin resistance, want to know fast and want to screen patients fast • Want to make sure that they do not have so they do not give to other patients Ø HIV Ø Heptatits B Ø Hepatitis C • Hepatitis = test in the blood Ø Mycobacterium tuberculosis Ø CMV • Cytomegalovirus has a cytopathic effect in pregnancies :( Ø EBV - Epstein barr virus ```

Answer 146

Describe the process of polymerase chain reaction - 2 DNA primers (18-20 base pairs) specific, for opposite DNA strands. This is used to AMPLIFY a DNA region, and there is exponential amplification - The product is visualised by fluorescent tags, or staining in gels for an amplicon of an exact size - DNA used to attach to specific part of the DNA Ø This will multiply only a certain part of the DNA Ø Amplifying a single gene, & a small part of it = able to go backwards and forwards within a few minutes and double the amount that we get each time - Gets lots of what we are looking for, specifically and going to make sample that size - Amplicon = will contain millions of copies of that particular gene. - Size is also important, and there are also specific primers have been used - Gene is only in a certain organism, in the right size and there is lots of it = this therefore shows that it is there

Answer 147

- Can measure the amount of signal that comes out - As the signal comes in = it will start to amplify the DNA and comes up more slowly Ø Can work this out, by doing a calibration curve to work out how much you put in in the first place (how many pathogens were there initially)

Answer 148

EACH SPOT = REPRESENTS A SINGLE GENE Ø Rather than just looking for one target = can look for several Ø Microarray, has spotted bits on the slide • Each spot contains a specific gene Ø If there are 5000genes in genome, can spot ALL of them onto the array Ø Can see it and the way that it works = can use DNA and RNA, can see the transcriptome and how it is working Ø Really expensive method, for just looking for ONE organism

Answer 149

1. Covers the whole genome 2. Strand dependent 3. Can be used for RNA and transcriptomics 4. Can look for microRNA

Answer 150

Ø Looking at pieces of cell wall and protein = this can be broken up Ø Takes a little bit of sample and chucks it, breaking it into atoms and pieces Ø Which produces a messy smear of atoms and pieces, across the detector Ø Little bits come off, and produces a huge series of peaks Ø Organism will only make a certain pattern = can identify lots of organisms very fast Ø But they are very expensive to buy and operate

Answer 151

- Looking for selected genes, or gene products That drive the disease process

Answer 152

- There are so many factors that we could look for - Could look for things in the cell walls, peptidoglycans - Want to look at the parts that are on the ouside = the parts that the bacterias are making

Answer 153

Ø Take latex beads and put the antibodies, onto these latex beads Ø Antibodies will stick, & that organism that is inside the sample, will stick to the latex bead Ø There is more than one antibody on the latex bead - this means the latex just agglutinates Ø CSF = latex beads with different types of an on it, and add the CSF and a A has clearly agglutinated Ø But B -G all have not agglutinated (only the very top left one has actually agglutinated seen by its irregular shape) Ø This can be done very quickly :) Ø Taking the CSF and put it on test = gives identification good enough for doctor to administer the correct medication / antibiotics

Answer 154

- Testing on a plate | - Testing for the thing that it makes, or for the gene that is made

Answer 155

1. Good specificity 2. Good sensitivity Ø See a very small amount of it 3. Easily automated Ø Put ona big machine and can easily see

Answer 156

Ø Human has to have made thee anitbody before you are able to see this = this takes 2 weeks and disease could have been transmitted to lots of other people before this time Ø Have early and a late one Ø Asked for flu typing = ask for one and then come back later, if the titre goes up = you have it Ø If titre is low = do not have it Ø If it stays the same, but is high then it means that the person HAD it 1. The serological response, is not rapid Ø Therefore not useful in acute infections 2. Single sera results, are meaningless Ø This is due to possible previous exposure 3. Some antibodies are cross reactive 4. Virulence is only INFERRED by presence of a biomarker Ø Infected into an animal model can prove virulence

Answer 157

- Why look at the assembled product, when you can look at ALL of the pieces

Answer 158

- This is sequencing can show differences, between SINGLE bases in strains Or resistance mutations to antibiotics

Answer 159

WORKING OUT WHAT TESTS TO USE FOR IDENTIFYING THE MICROBES 1. Rapid 2. There is faster detection of pathogens, than traditional techniques 3. This allows appropriate, timely antimicrobial therapy and infection control interventions 4. Increased sensitivity over culture, and microscopy based techniques - in positive samples 5. Can be automated. Ø And has potential for Point of Care testing - take the molecular test and put it in the patients hand to do, if you want to do this then have to get over all of the disadvantages which are below

Answer 160

1. Expensive 2. Does NOT screen for unknowns + does not MISS anything 3. Requires expertise 4. Labour intensive 5. Possibilty of contamination by other bacteria and microbe 6. Require complex, and efficient methods for extraction of nucleic acid Ø Negative samples might still need gold standard culture - needs to AT LEAST be as good as the gold standard Ø Hospitalisation costs, accounted for 95% of health system costs among patients suspected of tuberculosis Ø In culture negative patients, PCR tests do not significantly decrease the time to tuberculosis exclusion

Answer 161

- Following the host transcriptomic profile, with microarrays Ø Above = this is during treatment Ø All of the genes working in different genes and how they work

Answer 162

- Metabolic profiling of disease - Breathe into tube and it diagnoses TB because it produces different types of gas - If you have TB, will metabolise this volatile compound - Breathing into tube can be good enough

Answer 163

Ø Do not have to use PCR Ø Can use fluorescence & see how much they auto-fluorescence Ø There are no chemicals, can put into bottom of the tube and see how much intrinsic fluoresce they produce and identify them just from their pattern Ø This can be made small = can be looked at easily from home

Answer 164

- Any new systems for detecting pathogens have to be the following: 1. Reliable, sensitive, specific and preferably rapid 2. Applied to the correct specimen 3. Must derive from a large reference database 4. Constantly updated with new species or variants 5. Must be as good, or better than the gold standard (direct culture)

Answer 165

- IMMUNO = there is use of antibody-antigen interaction - Either the antibody or antigen will be labelled, or tagged - To allow its detection - ASSAYS= measures (amount or concentration) of antibody or antigen - Very sensitive + specific • Hence = widely used in research and analytical labs - Immunoassays use a mixture of polyclonal or monoclonal antibodies

Answer 166

- There is an antibody mix; will bind to different epitopes - This is the typical immune response that we do when challenged with antigen - This antigen has 2 epitopes = mouse will mount immune response against the antigen - The B cell receptor will bind to one of the epitopes ○ Another B cell will bind to other epitope § Both of these B cells that have bound to the epitopes: § Will proliferate and produce clones § The clones will be able to bind to both of the epitopes

Answer 167

Ø There are immortal, derived from B cell tumour and do not produce antibodies Ø A lack of hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene ○ Hypoxanthinine-aminopterin-thymidine (HAT) selection Ø The B cells are isolated ○ They are fused using poly ethene glycol - ○ Get a mixture of cells, some are the original B cells, + some are myeloma cells

Answer 168

- In the HAT medium, myeloma cells die ○ HAT Medium = myeloma cells are not able to grow in this medium, lack the HPRT gene and the B cells are not immortal therefore they will die fast in culture - This is because they cannot make nucleotides ○ Because they are lacking the HGPRT gene - B cells will die, because they have a short life span - Only the hybridomas grow and proliferate and survive - There is dilution into individual cells ○ Most individual cells will divide ==> form clonal cells - Culture the cells individually = they will proliferate to form a clone of the cells = monoclonal antibodies - Identical to the original parent - Antibody of one specificity will be produced

Answer 169

- When they cannot make nucleotides | - Because they are lacking the HGPRT gene (hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene)

Answer 170

- Can be stored indefinitely, and then grown to produce monoclonal antibody when needed - Antibody genes can be clones from the hybdridomas. • Will allow antibodies to be engineered, for different applications - Polyclonal, or monoclonal antibodies can be produced which binds to Fc regions of particular antibody classes • e.g. to IgGs, IgAs, etc • These are anti isotypic antibodies - very useful for use in assays.

Answer 171

- One of the antibodies will be labelled - The labels are originally radioactive (but radioactivity = dangerous so this is not used as commonly anymore) • Radioimmunoassay = RIA - Commonly now enzyme - rather than being radioactive • e.g. horseradish peroxidase • Alkaline phosphatase (usually detected by coloured product) • Enzyme linked immunosorbed assay (ELISA) - Other alternatives are luminescent - Enzyme given substrate = produces a colour product - Antibodies that bind to antigens = enzymes

Answer 172

Enzyme linked immunosorbent assay 1. Direct • Often used to quantify an antibody 2. Capture or sandwich • Often used to quantify an antigen - Using these assays, the concentration of the analyte (anitbody or antigen) - In the sample can be calculated by comparison to analyte standards of known concentration of analyte (antibody or antigen) - In the sample can be calculated by comparison to analyte standards of known concentration

Answer 173

- The antigen gets immobilised on solid support - Test antibody solution added + incubated and non bound removed, by washing - Bound antibody detected by incubation with labelled 2nd antibody • i.e. anti immunoglobulin - And non bound removed by washing

Answer 174

1. Screening hybridoma supernatants | 2. Detect exposure to infectious agent

Answer 175

- The antigens might be present, in low concentration - Because antibodies have high affinity for antigen this technique = can concentrate the antigen - Need 2 antibodies, reacting with different epitopes on the antigen - One antibody is immobilised on solid support - Test antigen solution added, incubated, and non bound removed by washing - The bound antigen detected by incubation with the other antibody • Which has been labelled • And non bound removed by washing - Need 2 antibodies that bind different epitopes - On is mobilised on the solid support and then add the solution that you are trying to detect

Answer 176

Ø Coat the well with antibodies that react with a specific cytokine • Then add activated T cells • Once cytokines are secreted - will bind specifically to the antigen Ø Captured by those antibodies Ø Can then wash off the cells and come in with second labelled antibody Ø Add the enzyme, which can detect the colour formation 1. Cytokine specific antibodies are bound to the surface of a plastic wall 2. The activated T cells, are added to the well. These T cells are a mixture of different effector functions 3. Cytokine secreted by some activated T cells, is captured by the bound antibody 4. The captured cytokine is revealed by a 2nd cytokine-specific antibody, which is coupled to an enzyme - this gives rise to a spot of insoluble coloured precipitate

Answer 177

- Each of the small dots represents T cells | - Detecting IFN gamma

Answer 178

Characteristics 1. Can be used to detect antigens, or antibodies 2. Used to measure size of the protein being analysed 3. Can be used to calculate protein concentration 4. May show if protein has been degraded Ø There are protein standards that are of a known size and concentration; which form a "ladder" - labelled this way to allow detection in the western blot. Ø On the far left = Protein standards, of KNOWN SIZE and concentration "ladder" The ones to the right = the ones that we are trying to work out. Know what the concentrations of the standards are. Blot tells us if the protein has been degraded as there are other immunoreactivity bands in the well.

Answer 179

- SDS is a good method, for seeing if protein is being degraded. - Take mixture and mix with SDS • Will bind to the protein • Gives the protein a negative charge - Load on gel ==> run gel on electric current, & it will migrate towards the positively charged electrode - Take gel and stain it with a dye like comassie blue; which will binds to proteins Linking this with the western blot - Then a western blot is used = transfer the proteins in the gel, onto cellulose gel - Then use antibodies that are specific to the antigen we want to detect - Then take nitrocellulose & incubate to the enzyme =single band that goes to the protein

Answer 180

- SDS PAGE/Western blotting are often used alongside ELISA • But both immunoassays say slightly different things about the protein • ELISA = might give a slightly false result, which is a disadvantage - In Western blotting Ø [Protein] can get measured Ø By comparing the intensity of band that was detected Ø To band from a protein standard of known concentration - If protein is degraded, may be more useful to use Western blotting to calculate protein concentration - As some of the degradation fragments might contribute to signal in ELISA if both coating and detecting antibody are able to bind to them

Answer 181

Ø Heterogenous population of lymphocytes gets mixed with antibodies Ø That are coupled to paramagnetic particles or beads Ø And poured over an iron wool mesh Ø When a magnetic field is applied, the coupled cells stick to the iron wool - unlabelled cells are washed out Ø The antibodies will be able to specifically bind to certain white blood cells Ø These white blood cells, have certain surface proteins Ø A magnetic field can be applied - so that the antibodiy is bound to the iron mesh. Ø Then you can stop applying the magnetic field and elute out the cells - Population of a certain cell type - This cell is then used to purify a particular subset

Answer 182

- The individual cells that are part of a mixed population are tagged - They are tagged by treatment with monoclonal antibodies Ø These monoclonal antibodies will then bind to surface molecules ○ & then they are labelled with fluorescent dyes - Mixed cells are then forced, through a nozzle • This forms a stream of single cells - Individual cells pass through a laser beam - Which scatters light and causes the dye to fluoresce + provides information on bound antibody and cell surface protein

Answer 183

- A cell sorter can separate specific sub populations of cells - Each of the spots corresponds to a certain cell - The dot plot creates various population = of what we think are B cells etc. • Antibodies bind to certain immunoglobulins • They all have different fluorochromes and this corresponds to the increasing fluorescence. - Some of the population do NOT flouresce. If they do not, then they are NOT T cells (B cells?) Parts of population flouresce specifically for IgM or IgD. Some will be more for one, than the other - In combining Flow Cytometry with Flow Activated Cell Sorting • When cells pass through single stream • They can isolate cell, one type for another = ○ e.g. looking for cells that JUST have IgG or IgM, or both

Answer 184

- Transplant compatibility (Transplant immunodeficiency + suppression lectures) - Immunodeficiency - Autoimmunity - Allergy - Malignancy

Answer 185

Ø Histocompatibility • Genetic differences between individuals are detected by the immune system ○ This leads to rejection of non-self Ø Major histocompatibility proteins (MHC) • Major players in transplant rejection (transplant and immunosuppression lecture) Ø The best transplants will result when the donor and recipient MHC = as similar as possible • MHC proteins are important • This is because the T cells = good at seeing MHC molecules as non self-molecules

Answer 186

- Binds fragments of intracellular proteins - T cell receptor (which is "self") - On cytotoxic T cells - will kill the foreign cell - With assistance from CD8 T cells

Answer 187

- Binds fragments of proteins which have been taken up by endocytosis - T cell receptor on Helper T cells (Th cells will see the MHC class 2, non-self-complex.) - With assistance from CD4

Answer 188

- In humans, the Major Histocompatibility Complex (MHC) is known as HLA (Human Leukocyte Antigens) - The MHC, is located on Chromosome 6 • Contains 3x MHC Class 1 Proteins • And 3x MHC Class 2 Proteins - Highly polymorphic - 100s of different variants

Answer 189

- In humans, the Major Histocompatibility Complex (MHC) is known as HLA (Human Leukocyte Antigens) - The MHC, is located on Chromosome 6 • Contains 3x MHC Class 1 Proteins • And 3x MHC Class 2 Proteins - Highly polymorphic - 100s of different variants

Answer 190

- Class 3 = is not actually involved in antigen presentation. - Class 3 is involved in coding proteins of other parts of the immune system. - This is just showing the structure of the human MHC (human leukocyte antigen - HLA).

Answer 191

Ø The MHC alleles of donor and recipient are identified • By using the Polymerase Chain Reaction (PCR) Ø This is really specific Ø We can even make specific primers, for certain genes • These will amplify certain genes specifically Ø D and L have the same gene which is shown by the specific amplification of the gene

Answer 192

- X linked disorder - Patients cannot generate mature B cells - B cells have the CD19 surface protein • This is the co-receptor for the B cell receptor

Answer 193

Ø HIV =deficiencies n certain blood cells. Ø Lymphocyte subset estimations are performed using monoclonal antibody to various markers • CD3 and CD4 and CD8 on whole blood • And analysed by flow cytometry Ø The percentages of cells, in each subset is determined using a FACs machine Ø The results are reported as percentages and absolute counts

Answer 194

- This shows the relationship between the MHC protein, with its attached flurochrome and the peptide. - T cell receptor does NOT bind to antigen • But binds to combination of antigen and peptide • It is now possible to observe this reaction - Expression of MHC protein - Mix the MHC + peptide of interest - Then take the MHC peptide molecule • And combine with biotin vitamin; which binds strongly

Answer 195

• These complexes bind specifically to the T cell receptor of appropriate MHC peptide specific T-cells - A fluorochrome, e.g. phycoerythrin (PE) is then added and visualisation can be done - via flow cytometry - By creating MHC complexes • Loaded with HIV antigen, can calculate for example what proportion of the CD8+ T cells will bind the antigen Binding of the amount = measured by flow cytometry.

Answer 196

- Neutrophils are found in acutely inflamed tissue • The Neutrophils Ingest pathogens • Neutrophils kill, using reactive oxygen species - like superoxide anions - The neutrophils die soon after phagocytosis = this will which will generate pus • (pus is just dead white blood cells) - Deficiency in neutrophil numbers- neutropenia, will lead to a high rate of infection - Deficiency in phagocyte function • Chronic granulomatous disease • Patients cannot form reactive oxygen species: ○ Therefore the patient will succumb to bacterial and fungal infections

Answer 197

- This test, is based on the principle that NON FLUORESCENT DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils - • Look at DHR (dihydrorhodamine) after it has been eaten and recognised as foreign. • After stimulation with PMA - Phorbol myristate acetate ○ When this is taken up, can get OXIDISED by reactive oxygen species (ROS) • That is produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123 - which is a green fluorescent compound ○ And can be detected by flow cytometry

Answer 198

- Green fluorescent compound | - Which can be detected by flow cytometry

Answer 199

- Electrophoresis - Nephelometry • This is an automated and rapid method • That is used to measure the serum immunoglobulin levels • It relies on the light scattering properties of antigen-antibody complexes - Nephelometry = measures light refraction - A laser is shone through a microplate well; with the analyte in solution - Goes through a ULBRICHT sphere and there is a light trap on the other side * when there is a change in solubility* - Particles will scatter light into the sphere = will then go into the light detector

Answer 200

1. Light beam with a wavelength of 840nm gets passed through a cuvette Ø Cuvette contains antigen-antibody complex 2. Nephelometry measures the amount of light scattering by an antigen-antibody complex 3. The amount of scattered light is proportional to the concentration of the antigen-antibody complexes in the sample, over a wide concentration range - Nephelometry is often used to study the amount of antibody - That are from different classes present in the serum e.g. IgA and IgG - Here, serum is mixed with anti isotype antibodies

Answer 201

- Allergen is mediated by IgE. | - Common allergens include house dust mites, cat, dog, trees, grasses, moulds, egg, milk, cod, soya, peanut

Answer 202

Ø The IgE will bind to allergen ==> then will bind to FC eRI receptors on certain cells of the immune system= (mast cells.) • When the allergen binds; the IgEs are brought together • This will causes mast cell degranulation Ø Release of histamine § This causes reddening of skin

Answer 203

• In allergic person: • IgE will bind to the allergen • Via the Fc region of the antibody, will bind to receptors on the mast cells ○ This will cause mast cells to DEGRANULATE and there will be release of the mediators, e.g. histamine ○ Which causes reddening and swelling of the skin

Answer 204

- Suspected allergen and the bound to a solid phase - If there are antibodies to the allergen then it will bind and come in with antibodies that specifically bind to IgE - Radioactivity is dangerous = replacing this increasingly with fluorescence - Suspected allergen bound to insoluble material. - Patient serum added - If the serum contains antibodies to the allergen, then there will be binding. - Radiolabelled anti-human IgE antibody is added, where it binds to those IgE antibodies, already bound to the insoluble material - The amount of radioactivity is proportional to the serum IgE for the allergen - Often fluorescence is used instead of radioactivity ○ e.g. ImmunoCap

Answer 205

- Autoimmune disease = is characterised by autoantibodies to nuclear antigens • e.g. binding to the DNA, RNA - Detection of autoantibodies is useful for diagnosis and monitoring disease activity Ø Like SLE - Systemic Lupus Erythematous Ø Autoimmune disesae that is characterised by production of autoantibodies Ø Causing a range of symptoms, often dermatological

Answer 206

Ø ELISA (quantitative) Ø Immunoflouresence (qualitative) • These are performed in parallel • To increase sensitivity Ø Immunofloursence on human epithelial cells Ø Tested with labelled anti IgG antibody Ø 98% of patients are anti-nuclear antibody positive - Specific antigens that form DNA or RNA and cover with serum, can bind f there is autoimmunity to the antigens and then colour formation is used Immunofloursnce can also be used in parallel Ø Use cell adult line, like the HeLa line Ø If person has autoimmunity = contain antibodies that bind to the human cell line and this is measured using fluorescence

Answer 207

POLYCLONAL ANTIBODY THERAPIES examples (can also be monoclonal) - Intravenous immunoglobulin (IVIG) = Ø blood product purified from the serum Ø Of between 1000-15,000 people / batch - Used to treat patients with antibody deficiencies at 200-400mg/kg/3 weeks at quite low doses - At HIGH dose, 2g/kg/4weeks, used as immunomodulatory agent in a number of immune and inflammatory disorders Ø Mechanisms of this are not fully understood Ø After a bite from a suspected rabid animal Ø Polyclonal antibodies isolated from the serum of individuals who have been immunised with the rabies vaccine injected into the wound site § Human Rabies Immunoglobulin (HRIG)

Answer 208

- Approx 45 monoclonal antibodies are licensed to treat diseased - Have their effects by binding or mediating the effects of other pathways • Cancer • Chronic inflammatory diseases • Transplantation • Infectious diseases • Cardiovascular medicine - Monoclonal antibodies can have their effects, either by: 1. Binding and blocking a process 2. Mediating immune responses a. Such as by the initation of complement b. Or antibody- dependent cell mediated cytotoxicity (ADCC) - Molecules, such as toxins or radionuclides can be joined to monoclonal antibodies • Antibody binds to cancer cell, which is then killed by toxin or radioactivity - The antibody is bound to pathogen - Bind to FC receptor on the surface of an immune cell which is normally and immuune cells - Stimulates the immune cell that activates it - The natural killer cell then goes on to kill that pathogen, by inducing apoptosis - Can also be used to specifically kill cancer cells - The antibody will then bind to the surface protein + toxin will kill cancer - Can use antibodies to identify other antibodies

Answer 209

- A resolved measure (diversity) of differences (variables) | - Have to find out how diverse pathogens are from each other

Answer 210

Have to look to see the measure of the diversity = which is known as "resolving" the diversity 1. Disease distribution time and place (When it was isolated) 2. Disease transmission (After isolation = suggests how it was transmitted. Can see if connected to a disease. Progression can change over time. 3. Disease manifestation 4. Disease progression

Answer 211

Ø Confirms outbreaks • Inside institutions (whether one patient got it from another or if those in same ward, have the same strain) • In the community and in the past ○ E.g. working out what has driven the geographical spread of important strains • In the lab Ø Identifying disease risks • Virulence shifts (∆ in infection incidence + why resistance strains rising.) • Reservoirs of infection (New infections or recrudescence = strain that stays in the body then returns) Ø Allows us to track the disease and then predict how it will behave

Answer 212

- Which variable (target) and how many variables that we should target - Have to work out the targets and the variables that we are looking for - Need to determine exactly what the differences are and how we are going to characterise things

Answer 213

1. Culture on selective media 2. O157 serotyping using the antibody on blue latex beads 3. PCR of the DNA for the verotoxin gene (stx2) 4. Phage typing

Answer 214

- Different types of phage added onto plate - Grid tells which are put on = the holes are the phages that have worked - Phage type = pattern - Taking variables and workiing out how different they are from each other

Answer 215

- Can look at different types of things that they might be looking for Factoral - Presence / absence of gene/bases ∆ - In the genome / gene relative to the location in the genome - Factoral based changes = presence or absence of A SINGLE BASE - where they change in the genome and where they are Functional weighting - Type of substitution (synonmyous / non synonymous) Temporal weighting - Mutation rate (the time since the last alteration)

Answer 216

- PCR - With RE regions primers - Generates multiple length amplifications

Answer 217

- Hybridisation of the labelled PCR products onto - 43 spacer specific oligonucleotides (between RE sequences) - Fixed on a membrane then visualise signal with RE probe

Answer 218

- A very simple, inexpensive and effective tool for Tuberculosis/ Mycobacterium research. - Spoligotyping = useful PCR-Based Method to Simultaneously Detect and Type Mycobacterium Tuberculosis Complex Bacteria. - Spoligotyping, which uses RLB (Reversed Line Blotting) Ø Offers alternative for typical Southern blotting when rapid results are required. - Can simultaneously detect and type M. tuberculosis complex bacteria in clinical samples (suspected nosocomial infections, outbreaks in prisons, etc.).

Answer 219

- Various strains found around the world & can form evolutional tree. - All of the organisms in TB came from one theoretical origin - Vertical differences = demonstrate the differences = shorter line means there are less differences

Answer 220

- Variable number of tandem repeats (VNTR) - The result is a profile of the number of specific repeats @ multiple genomic loci - Can apply same things to another part of DNA = aspects that could be used as variable - Tandem repeat units = happen and produce lots of copies, usually there to help the gene express / copy and for genomic rearrangement - They change in time

Answer 221

- Looking to see if amino acids are changing or not - Some amino acids will change even if the DNA has changed - Allows extra level of the way we measure molecular immunology - Phylogeny = tree of all of the various bacteria

Answer 222

- High sequence mutation rate - Might NOT correlate with pathogenicity (antigenic drift) - Can do it on something like flu = working out how much flu virus has ∆ • & point at which it is going to ∆ from 1 serotype → another = when is the DNA going to change - Can predict the next flu outbreak and predict WHICH flu virus is going to come next Ø So that can vaccinate everyone appropriately = PREVENTION EPIDEMIOLOGY Ø N1 ANTIGEN is also changing at slightly different rates. Ø Working out if H1 changes to H5 = this is when the new outbreak will occur

Answer 223

- We need to know when drift of antigen is going to occur, in all of the mutations happening - When it drifts so far then there is a shift - Shift = DNA changes = has caused the protein to change ○ Not as good = cannot be sure when it will shift and predict this - Drift = when the DNA IS GOING TO CHANGE INTO SOMETHING FUNCTIONAL - Turns from one functionality to another = protein functionality is important - Want to also know how fast this is going to happen.

Answer 224

Ø Replication rate Ø DNA or RNA polymerase proof reading fidelity Ø Selection pressure from the host or environment Ø Degree of redundancy in the genome Ø Transmission rate

Answer 225

- Hypervariable genes change MORE rapidly than conserved genes - The conserved genes are more likely to be associated with phenotype and virulence - Using variables that change @ different rates might bias to one variable - Some changes might revert BACK to older profile (convergent evolution) - Not all changes in genome = informative. • Too much hides differences • But too little discrimination can hide real variation

Answer 226

- Cutting the genome | - With low frequency cut restriction enzyme

Answer 227

- Separate the fragments on a cell - Then visualise, with a probe • Against repetitive element

Answer 228

Ø Rather than just looking at DNA = from probe POV, produces these individual bands Ø Produces a pattern - like a barcode Ø Looking to see if the pattern is the same as previously

Answer 229

1. Transmission - hospital acquired infection 2. Reservoirs of infection - contact tracing 3. Spread of emergence of resistance

Answer 230

1. Knowing the most appropriate variables 2. Quantitating variations and deriving diversity 3. Generating identities or clusters 4. Applying related data Ø Geographic location Ø Time of isolation Ø Incidence Ø Prevalence Ø Transmission rate Ø Severity of disease

Therapeutics & Investigations 2 (Part 3) Flashcards

(254 cards)