1+2 - Investigations into immune system function

Question 1

Important white blood cells

Answer 1

lymphocyte, monocyte, eosinophils, basophils, neutrophils

Question 2

Lymphocytes

Answer 2

B cells, Tc cells, Th cells

Question 3

B cells

Answer 3

Cells manufactured in the bone marrow that create antibodies for isolating and destroying invading bacteria and viruses.

Question 4

Tc cell (CD8)

Answer 4

Effector form of a cytotoxic T cell; it induces apoptosis in infected or cancerous “self” cells

Question 5

TH cells (CD4)

Answer 5

Helper T cells
MHC II complex
- recognizes APC and able to release cytokines and attract other cells to the area

Question 6

B cell receptor

Answer 6

The antigen receptor on B cells: a Y-Shaped, membrane-bound molecule consisting of two identical heavy chains and two identical light chains linked by disulfide bridges and containing two antigen-binding sites.

Question 7

T cell receptor

Answer 7

Unlike antibodies, T cell receptors are never produced in a secreted form.

Question 8

immunoglobulins

Answer 8

bind with specific antigens in the antigen-antibody response

Question 9

Antibody elimination of pathogen

Answer 9

1. The pathogen is opsonised = faster phagocytosis
2. Antibodies recruit complement proteins
3. causes formation of membrane attack complex
4. A hole forms in the pathogen’s membrane.
5. pathogen destroyed

Question 10

Acquired immune response

Answer 10

1. activated T helper cells release cytokines, activating Tc and B cells.
2. Tc cells trigger infected self cells to undergo apoptosis
3. B cells stimulate antibody production

Question 11

HIV Virion

Answer 11

Consists of RNA surrounded by core proteins and capsid
RNA serves as template for DNA synthesis
Outer envelope contains glycoproteins that are needed to attach to CD4 cells

Question 12

how does HIV infect T helper cells

Answer 12

• GP120 binds to CD4
• GP41 binds CXCR4 on the surface of TH HIV destroys T helper cells, B and T cells.
• mediated immunity lost

Question 13

AIDS (acquired immune deficiency syndrome)

Answer 13

increased susceptibility to opportunistic infections

Question 14

example infections

Answer 14

• toxoplasmosis
• tuberculosis (mycobacterium tuberculosis)
• pnuemonia (pneumocystis jiroveci)

Question 15

methods for detection of HIV

Answer 15

what might be detected?

1. antibodies to HIV
2. Viral RNA
3. Fall in T helper cell numbers

Question 16

1. Antibodies to HIV (‘seroconversion’)

Answer 16

• develop 2-8 weeks after infection (prior to this = ‘window period’)
• Often bind to p24 or gp41
• Detection via ELISA, immunochromatography, latex particle agglutination,

Question 17

2. Viral RNA

Answer 17

Useful in the ‘window period’ before antibodies to HIV are detectable in serum.

The genome of HIV is RNA

RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection

Question 18

3. FALL IN T HELPER CELL NUMBERS

Answer 18

Flow cytometry and Fluorescence-Activated Cell Sorting can be used to identify T Helper cells on basis of CD4

Question 19

ELISA

Answer 19

1. A HIV antigen bound to well
2. Wash off anything that hasn’t bound
3. Patient serum added. Any antibodies bind to the antigen.
4. Wash
5. Anti-human antibody added. This binds human antibodies. An antibody also has an enzyme attached.
6. Wash
7. E Substrate added. Enzyme converts substrate to a coloured product which is detected by absorbance of a certain wavelength of light

Question 20

chromogenic substrate

Answer 20

Antibody conjugated (linked) to: ALKALINE PHOSPHATASE (AP) ENZYME Substrate may be BCIP (5-bromo-4-chloro-3-indolyl phosphate)

• Can use different enzymes conjugated to antibody
• purple product detected by light abosrbance

Question 21

Immunochromatography

Answer 21

• Detection of antibodies to HIV
• Serum, plasma or whole blood added to absorbent pad
• Any anti-HIV antibodies bind HIV antigen in test
• HIV test line = antibody which binds antigen/antibody complex
• Control line binds HIV antigen

Question 22

Latex particle agglutination

Answer 22

1. Latex particles covered with HIV antigen e.g. gp41
2. Add blood which may contain Anti HIV antibodies
3. Visual clumping if HIV present

Question 23

PCR

Answer 23

1. REACTION Double stranded DNA is DENATURED (separated) when heated to 95oC
2. Primers added specific to sequence on either side of target sequence ANNEALING (54oC) (A-T; C-G)
   DNA polymerase and DNA building blocks (base + sugar + phosphate) joined to make copy of target sequence. EXTENSION

Question 24

benefit of pcr

Answer 24

amplifies a target DNA sequence

Question 25

window period detection of HIV using RT-PCR

Answer 25

1. DNA copy (cDNA) made of viral RNA using Reverse Transcriptase enzyme 2. DNA copy of cDNA made by DNA polymerase 3. PCR reaction to amplify DNA

Question 26

FLOW CYTOMETRY AND FLUORESCENCE-ACTIVATED CELL SORTING (FACS)

Answer 26

Can be used to distinguish TH cells from other blood cells by virtue of the CD4 on the TH cell surface. Therefore, identify number of T helper cells in patient’s blood. Useful in monitoring HIV infection

Question 27

Flow Cytometry/FACS

Answer 27

Blood cells mixed with antibody specific for CD4, which is engineered to contain a fluorescent label. Cells are streamed through a flow cell Measures physical and chemical characteristics of cells as they flow one cell at a time through a laser beam. The light scatter is a characteristic of the cells and their components.

Question 28

FLOW CYTOMETRY/FACS USING MORE THAN ONE FLUOROCHROME

Answer 28

Mix lymphocytes with range of antibodies (e.g. to CD4, CD8), each with different fluorochrome. • Antibodies engineered to have different fluorescent labels (fluorochromes). • Identification of more than one antigen in the same sample e.g. CD4, CD8. • Gives you number of Tc and TH etc

Question 29

FLOW CYTOMETRY/FACS EXAMPLE – PATIENT WITH HIV

Answer 29

Normal TH 30-50% of lymphocytes (Expect 500-1200 cells/ul 200-500 CD4+/ul= abnormal <200 CD4+/ul= AIDS CD4 cells labelled with FLUORESCEIN CD8 cells labelled with PHYCOERYTHRIN provides number so are able to quantify how far the disease has mobilised.

Question 30

Multiple Myeloma (MM)

Answer 30

cancerous proliferation of plasma b cells Osteoclasts dissolve bone. Activity up-regulated in MM. Fractures of long bones, ribs and vertebrae.

Question 31

presenting features of MM

Answer 31

Anaemia weakness, tachycardia Recurrent infection Bone pain/fractures

Question 32

DIAGNOSIS OF MULTIPLE MYELOMA (MM)

Answer 32

* Large amount of one IgG produced (PARAPROTEIN). * Detect via CELLULOSE ACETATE ELECTROPHORESIS. * Malignant plasma cells stay in bone marrow and activate OSTEOCLASTS * Bone broken down

Question 33

DETECTION OF ‘PARAPROTEIN’ IN BLOOD SERUM OF PERSON WITH MULTIPLE MYELOMA

Answer 33

``` Cellulose acetate electrophoresis 1. normal SERUM 2. multiple myeloma SERUM - PARAPROTEIN 3. normal PLASMA (Free light chains excreted in urine = BENCE-JONES PROTEIN ```

Question 34

difference between serum and plasma

Answer 34

serum is when you have had a blood sample and allow it to clot and spin it out, clot is pellet, serum is top layer in plasma you prevent clotting; spin serum, clot is pellet, plasma is top layer

Question 35

cellulose acetate electrophoresis

Answer 35

Blocking OH groups in cellulose with acetyl group (to form cellulose acetate) inhibits protein and water binding. Cellulose acetate made into paper. Proteins migrate through holes.

Question 36

CELLULOSE ACETATE ELECTROPHORESIS PH 8.6

Answer 36

Proteins have overall negative charge at pH 8.6 and migrate to ANODE. - NH2 not protonated at pH 8.6 to -NH3 + - COOH deprotonated at pH 8.6 to -COO-

Question 37

VISUALISATION OF PROTEIN IN CELLULOSE ACETATE ELECTROPHORESIS

Answer 37

* Immerse cellulose acetate in trichloroacetic acid * Protein reprotonated (-COOH and –NH3 + ) * Ponceau S stain added * Negative charge binds to + protein * Hydrophobic rings bind any hydrophobic groups in protein

Question 38

PERNICIOUS ANAEMIA

Answer 38

- Absorption of vitamin B12 requires a combination with INTRINSIC FACTOR (IF) made by gastric parietal cells - Pernicious anaemia = autoantibodies against gastric parietal cells so less B12 absorbed - Vitamin B12 needed to make DNA - Red blood cell production requires rapid DNA synthesis. - fewer RBC made, less O2 transport = FATIGUE

Question 39

INDIRECT IMMUNOFLUORESCENCE

Answer 39

FIXED GASTRIC PARIETAL CELLS ON GLASS MICROSCOPE SLIDE TEST BLOOD SERUM (Autoimmune antibody?) wash Add fluorescently-labelled Anti-human antibody EXAMINE WITH MICROSCOPE AND UV-LIGHT SOURCE Autoimmune antibody binds antigen in test sample Anti human antibody binds autoimmune antibody Fluorescent label on anti-human antibody detected

Question 40

TRANSMITTED LIGHT FLUORESCENCE MICROSCOPY

Answer 40

Enables detection of location of Anti-human immunoglobulin which has been labelled with fluorescein. Barrier filter – enables visible fluorescent light to pass to eye Exciter filter – maximum amount of appropriate wavelength light reaches specimen