The Immune System Flashcards
What are some examples of myeloproliferative neoplasms?
Polycythaemia vera
- An excess of red blood cells in circulation
Essential thrombocythaemia
- An excess of platelets
Idiopathic myelofibrosis
- Too few RBC, too many platelets and WBC
What are the clinical signs of polycythaemia?
Breathing difficulties Dizziness Excessive bleeding Splenomegaly Headache Itchiness Red colouring (esp. face) Shortness of breath Symptoms of phlebitis
What is thrombosis?
The pathological manifestation of haemostasis
Causes restriction or blockage of blood vessel, causing hypoxia and tissue damage
Thrombi can also dislodge, leading to embolisation
What is haemostasis?
In a physiological state, maintain fluid blood flow within the vasculature
On the vessel injury/trauma, limit/arrest bleeding by forming a clot at the site of injury while maintaining blood flow in the rest of the vessel
Removal of the blood clot following wound healing
What are the risks of Rhesus disease?
Does not effect first Rh+ baby of an Rh- woman but will effect subsequent pregnancies as anti-Rh antibodies will now exist
85% of the population is RhD+; 15% RhD-
Therefore probability of RhD- mother having an RhD+ baby is high
Range in effect - mild anaemia of the newborn to intrauterine death at 18 weeks of gestation (hydrops fetalis)
Kernicterus can also occur in neonates as a complication of severe jaundice which can cause brain damage in severe cases or in mild cases deafness
Relatively rare - alleviated through routine antenatal serology and administration of prophylaxis (anti-D therapy)
- anti-D immunoglobulin will bind and neutralise any RhD+ cells preventing the development of maternal antibodies
What are some features of blood?
One of the four main body fluids (intracellular, interstitial, blood and lymph)
Heart, blood and blood vessels form the circulatory system
Adults have ~5L of blood (4-6L, M>F)
What are the functions of blood?
Transportation:
Supply of oxygen to tissues
Supply of nutrients such as glucose, amino acids, fatty acids
Removal of waste products including CO2, urea and lactic acids
Messenger function - transport of hormones, signalling of tissue damage
Regulation:
Regulation of body temperature
Maintain pH (7.35-7.45)
Hydraulic functions - colloidal osmotic feature
What is the composition of blood?
Two major components: Liquid component - 55% - Plasma Formed elements - 45% - Red blood cells - White blood cells - Platelets
Describe blood plasma.
Yellow/straw coloured liquid portion of blood
Considered the extracellular matrix (ECM) of blood - keeps cells in suspension
Consists of :
Water - 92%
Plasma proteins (albumin, globulins, fibrinogen, prothrombin) - 7%
Other substances (electrolytes, nutrients, hormones, gases, waste products) - 1%
Serum is plasma devoid of clotting factors (achieved by drawing blood in the absence of an anti-coagulant)
What will you see in a test tube if blood is centrifuged?
55% - yellow/straw liquid top layer of plasma
White layer (buffy coat) of white blood cells
45% - red blood cells, red bottom layer, densest layer
What are the formed elements of blood?
Red blood cells - 99%
White blood cells and platelets - <1%
Where are blood cells produced prenatally vs postnatally?
Prenatally:
Yolk sac, then liver and spleen, then bone marrow
Postnatally:
Bone marrow
- Tibia and femur stop production around 20/25
- Production carries on in ribs, sternum, vertebrae and pelvis
Where do RBCs and WBCs migrate to after haematopoiesis?
RBC - blood
WBC - peripheral tissues and lymphoid organs
What are the cytokines and growth factors which tightly regulate haematopoiesis?
Erythropoietin (EPO) - megakaryocyte erythroid progenitor -> erythrocytes
Thrombopoietin (TPO) - megakaryocyte erythroid progenitor -> platelets
Interleukin (IL) - various
Granulocyte colony stimulating factor (G-CSF) - myeloblasts -> basophil/mast cells, neutrophils, - monocytes -> macrophages
Granulocyte macrophage colony stimulating factor (GM-CSF) - most of myeloid progenitor and granulocyte macrophage progenitor
Stromal cell factor (SCF) - various myeloid
What are the cytokines and growth factors which tightly regulate haematopoiesis?
Erythropoietin (EPO) - megakaryocyte erythroid progenitor -> erythrocytes
Thrombopoietin (TPO) - megakaryocyte erythroid progenitor -> platelets
Interleukin (IL) - various
Granulocyte colony stimulating factor (G-CSF) - myeloblasts -> basophil/mast cells, neutrophils, - monocytes -> macrophages
Granulocyte macrophage colony stimulating factor (GM-CSF) - most of myeloid progenitor and granulocyte macrophage progenitor
Stromal cell factor (SCF) - various myeloid
EPO TPO IL G-CSF GM-CSF SCF
What is erythropoiesis, where does it occur and what drives it?
Process of RBC formation
Occurs in bone marrow
Body produces approx 2.5x10^6 RBCs every second
Hypoxia is detected by cells in the kidneys
Kidney cells release EPO into the blood
Erythropoiesis is regulated by EPO binding to the erythropoietin receptor on progenitor cells
EPO-receptor is a kinase linked receptor
What is the pathway of erythropoiesis?
Stem cell (hemocytoblast)
Committed cell (proerythroblast)
Developmental phase:
- Early erythroblast (phase 1 - ribosome synthesis)
- Late erythroblast (phase 2 - hemoglobin accumulation
- Normoblast (phase 3 - ejection of nucleus)
- Reticulocyte
Erythrocyte
What is hemoglobin (Hb)?
Multi subunit polypeptide
4 globin polypeptide chains - 2 alpha and 2 beta chains, each associated with a Haem molecule
Haem molecule composed of porphyrin ring linked to a molecule of Iron as a cofactor
Hemoglobin molecules bind O2 for transportation around the body and CO2 for removal
Iron gives RBC their characteristic colour
What are the features of a red blood cell?
Bi-concave shape related to function
- Optimum O2 transfer
- Flexibility for travel throughout small capillaries
Lifespan of ~120 days
Do not possess repair processes, aged cells are removed by macrophages in the spleen
Abnormal RBCs can be named by their morphology e.g sickle cell anaemia
What is the process of RBCs being broken down by the spleen?
Hemoglobin is separated into Iron and porphyrin ring
Iron transported to bone marrow via transferrin for new RBC production
Porphyrin ring excreted via digestive or urinary tract
- Bilirubin - urine
- Biliverdin - faeces
What is the role of platelets/thrombocytes?
Haemostasis
Initiate blood clotting at site of injury
What is the role of platelets/thrombocytes?
Haemostasis
Initiate blood clotting at site of injury
What is thrombopoiesis and how is it regulated?
Production of platelets
10^11 produced every day
30% are stored in the spleen
Lifespan of 7-10 days
1 megakaryocyte can produce 5000 platelets
Regulated by thrombopoietin (TPO) produced by liver and kidney
Secrete components to aid in repair of vasculature - platelet derived growth factor (PDGF)
What are the steps of thrombopoiesis?
Myeloid stem cell
Megakaryoblast
Megakaryocyte (have platelet precursor extensions coming off it)
Platelets
What is the function of WBCs/leukocytes and how are they classified?
Mediate the protective effects of blood through the generation of the inflammatory process
5 cardinal signs of inflammation - pain, heat , redness, swelling and loss of function
Classifications: Granulocytes vs agranulocytes Polymorphonuclear vs mononuclear Myeloid or lymphoid Innate or adaptive immune system
Which immune cells do you only find in the tissues?
Mast cell
Macrophage
Dendritic cell
Plasma cell
What are the cells of the innate immune system and what are their functions?
Neutrophil: Lifespan ~18 hours 60-70% WBCs Phagocytic - ingest and kill pathogens, debris and damaged cells Initiate inflammatory process
Eosinophil:
Lifespan 2-5 days
2-5% of WBCs
Phagocytic - protection against helminths
Basophil:
Lifespan 1-2 days
0.2% of WBCs
Involved in allergic reaction
Monocyte:
Lifespan 1-7 days
2-10% WBCs
Phagocytic and differentiate to become macrophages within tissues
Natural killer cell:
Lifespan 14 days
15% of WBCs
Killing of virally infected cells
What are the cells of the adaptive immune system and what are their functions?
Lymphocytes: Lifespan weeks to years 20-40% of WBCs B cell (CD19+) CD4+ T cell - helper (CD3+, CD4+) CD8+ T cell - cytotoxic (CD3+, CD8+) All have same morphology so flow cytometry used to identify markers
What is the haematocrit test and what do different results mean?
Centrifuging a blood sample of a specific amount to see the different proportions of plasma, RBCs and WBCs
Normal - ~55% plasma, ~45% RBCs, buffy coat of WBCs and platelets, F = 37-47%, M = 42-52%
Anaemia - Less RBCs, more plasma
Polycythaemia - More RBCs, less plasma
What is a full blood counts purpose and how is it carried out?
Determines properties of blood and blood cells
- Panel of tests investigating different elements
- Good indicator of general health
Use electrical impedance to quantify cell numbers and cell volume
WBC subsets are determined by flow cytometry due to differential cellular properties
What is included in a full blood count?
Hb - hemoglobin RBC - red blood cells MCH - mean corpuscular hemoglobin MCV - mean corpuscular volume Hct - haematocrit WCC - white cell count - Neutrophils (%) - Lymphocytes (%) - Monocytes (%) - Eosinophils (%) - Basophils (%) - Bands (%) (immature form of neutrophils) Platelets Reticulocytes ESR - erythrocyte sedimentation rate EPO (serum) - erythropoietin
What are some disorders of the blood?
Anaemia Myeloproliferative disorders - Polycythaemia (Primary form - polycythaemia vera (PV), Secondary form - essential thrombocythaemia (ET)) - Myelofibrosis or myelosclerosis Jaundice
How many blood groups are there?
8
A, B, AB, O
RhD positive or negative
What is the ABO system?
Blood group A - has A antigens on the RBCs with anti-B antibodies in the plasma
Blood group B - B antigens, anti-A antibodies
Blood group O - no antigens, anti-A and anti-B antibodies
Blood group AB - A and B antigens, no antibodies
O is the most common - 48%
As O has no antigens it can safely be given to any other group
What is the Rh system?
RBCs may have another antigen, a protein known as the RhD antigen
If present your blood group is RhD positive, if not it is RhD negative
e.g A-, B+ etc.
~85% of the UK population is RhD positive
36% - O+
O- can safely be given to anyone as it has no antigens
O- is the universal donor
Why must pregnant women be given a blood test?
If the mother is RhD negative but the child inherits RhD positive blood from the father it can cause complications
Rhesus disease
If the woman become pregnant with an Rh+ fetus again, her anti-Rh antibodies will cross the placenta and damage fetal red blood cells
How are blood types tested for?
Antibodies are added to blood samples and if agglutination takes place the RBC has the matching antigen
What is anaemia?
Anaemia is caused by a reduced number of red blood cells in circulation or a decreased amount of haemoglobin in the red blood cells
This results in reduced levels of oxygen delivery to tissues
This causes weakness, tiredness, inability to exercise and shortness of breath
In some cases this can lead to confusion, thirst and loss of consciousness
Anaemia may be significant before a patient appears pale
What are the signs and symptoms of anaemia?
Signs:
Pallor - especially of the conjunctiva
Tachycardia - pulse over 11 beats per minute
Glossitis - swollen and painful tongue (reasonably specific for vitamin B12 deficiency
Koilonychia - spoon nails (reasonably specific for iron deficiency)
Dark urine - in haemolytic anaemia
Symptoms:
Decreased work capacity, fatigue, lethargy
Weakness, dizziness, palpitations
Shortness of breath - especially on exertion
‘Tired all the time’
In children - decreased IQ, poor concentration, and sleepiness
Rarely - headaches, tinnitus, taste disturbance
More severe disease: Jaundice Splenomegaly Hepatomegaly Angina Cardiac failure Fever
What are the aetiological classifications of anaemia?
The bone marrow
- Suppression
- Infiltration
Problems with iron
- Deficiency at the bone marrow
- Excess absorption
- Ineffective incorporation into haem
Lack of vitamins
- Vitamins B12 and B6
- Folic acid
Anaemia associated with disease in other organs
- Liver
- Kidney
- Reproductive organs
- Connective tissues
- Thyroid
Anaemia due to blood loss
- Haemorrhage
- Trauma (road traffic accident, personal violence e.g. stab wound)
Haemolysis
- Due to antibodies
- Due to drugs
- Infections
Anaemia arising from gene mutation
- Haemoglobinopathy
- Membrane defects
- Enzyme defects
What is the lineage of erythrocytes?
Erythroid
Where do the stages of erythropoiesis take place?
Bone marrow:
Multipotential haematopoietic stem cell -> (regulated by stem cell cytokines e.g. SCF, IL-3 and TPO)
Common myeloid progenitor -> (EPO)
Proerythroblast (pronormoblast) -> (EPO)
Basophilic erythroblast -> (EPO)
Polychromatic erythroblast -> (EPO)
Orthochromatic erythroblast (normoblast) -> (EPO)
Bone marrow and circulating blood:
Polychromatic erythrocyte (reticulocyte) ->
Erythrocyte
How is EPO related to tissue oxygenation?
Blood levels of EPO are inversely related to tissue oxygenation
The level can increase up to 1000x in response to anaemia
What produces EPO?
Juxtatubular interstitial cells of the renal cortex produce 90% of the EPO in blood
They sense oxygen levels through oxygen-dependent prolyl hydroxylase
This regulates the stability of the primary transcription factor for EPO - hypoxia-inducible factor 1 (alpha) (HIF-1(alpha))
Hypoxia increase -> HIF increase -> EPO increase -> RBCs increase (BM) -> Hypoxia decreases -> HIF decreases -> EPO decreases
What are pure red cell aplasias (PRCA)?
Conditions affecting specifically erythropoiesis in the bone marrow
Most common congenital PRCA is Diamond-Blackfan anaemia, which occurs due to reduced proliferation of erythroblasts
Rare condition - occurring in ~5 live births/million
Acquired PRCA can be classified as either primary or secondary
Primary - idiopathic (no clear cause can be identified)
Secondary - acquired as a result to exposure to a pathogenic agents such as a drug or infection
What are pancytopenias?
Conditions affecting other cell types in addition to RBCs (white cells and platelets)
What can cause changes in the bone marrow leading to anaemias?
Congenital - Diamond-Blackfan syndrome, congenital dyserythropoietic anaemia
Infections - Viruses (parvovirus B19, hepatitis B virus, Epstein-Barr virus, mumps, cytomegalovirus, human immunodeficiency virus), bacteria (meningococcal, staphylococcal species)
Malignancy - Solid tumours (such as cancer of the thymus, stomach, breast, lung, thyroid and kidney), haematological tumours (leukaemias, lymphomas, myeloma, myelofibrosis, essential thrombocythaemia, Waldenström macroglobulinaemia)
Autoimmune disease - Systemic lupus erythematosus, rheumatoid arthritis and autoimmune haemolytic anaemia, Sjögren’s syndrome, autoantibodies to red cell progenitors, autoantibodies to Epo, T-cell mediated recognition of red cell progenitors
Other causes - Drug and chemicals (notably azathioprine, methotrexate, gold chloramphenicol, recombinant human Epo and co-trimoxazole), pregnancy, severe renal failure
Many of these conditions cause pancytopenia and aplastic anaemia in addition to red cell aplasia
How do HSCs maintain their population whilst differentiating?
When they replicate, one daughter cell is an HSC (self-renewal) and one is a progenitor cell (differentiation)
This ability may be damaged by insult (chemical, genetic etc.) which eventually leads to HSC exhaustion and pancytopenia
What is haemolytic anaemia?
Caused by the premature destruction (reduced lifespan from ~120 days) of functional erythrocytes either by intrinsic or extrinsic mechanisms
Anaemia develops if the bone marrow is unable to match the rate of destruction by production of new erythrocytes
What are the 2 broadly different reasons for erythrocyte destruction in haemolytic anaemia?
There is nothing fundamentally wrong with the erythrocyte, but they are destroyed by external pathological processes, such as drugs, toxins, autoantibodies or infection
There is something intrinsically wrong with the erythrocyte so it is destroyed, this can be due to damage, absence of certain enzymes or abnormal types of haemoglobin
What is auto-immune haemolytic anaemia (AIHA)?
Autoantibodies are found when a patients’ immune system produces antibodies that recognise their own erythrocytes as foreign and mediate their destruction
Idiopathic AIHA accounts for approximately 50% of cases
Secondary AIHA can result from many other illnesses
Most common causes of secondary AIHA include
- Lymphoproliferative disorders (e.g chronic lymphocytic leukaemia, lymphoma)
- Other autoimmune disorders (e.g. systemic lupus, erythematosus, rheumatoid arthritis, scleroderma, Crohn’s disease, ulcerative colitis)
Drug-induced AIHAs are rare
- Can be caused by a number of drugs, including alpha-methyldopa and penicillin
- Drug binds to macromolecules on the surface of the RBCs and acts as an antigen
- Antibodies are produced against RBCs which leads to complement activation
- This is one type of ‘penicillin allergy’
What is seen on abnormal blood films?
Schistocytes and polychromasia - Fragments of erythrocytes and larger, blue-tinged reticulocytes
Nucleated erythrocytes
Spherocytes - Some cells are very small and termed micro-spherocytes
Can be result of body upping production to maintain normal levels
What are haemoglobinopathies?
Autosomal co-dominant genetic defects resulting in abnormal structure of one of the globin chains of the haemoglobin molecule
Sickle cell anaemia is one of the most common haemoglobinopathies
Sickle cell attributed to mutations in the beta-globin gene
Sickle cell erythrocytes are an abnormal shape, which affects their passage through the circulatory system, their ability to carry O2 and increased haemolysis
Sickle cell anaemia can cause vaso-occlusive crises (vessel blockage), visceral sequestration crisis, aplastic and haemolytic crises
What is haemoglobin, its function and structure?
An iron-containing protein made in erythrocyte cytoplasm
Each erythrocyte contains ~640 million haemoglobin molecules
Designed to absorb O2 from areas of high O2 content (lungs) and release it where the O2 levels are low (tissues)
Consists of a protein part (globin) and a complex non-protein part (haem)
Haem group contains iron which binds to O2 and the globin part consists of 4 globin subtypes
2 alpha chains, 2 beta chains
Each chain includes an iron containing haem group
How can malnutrition and malabsorption cause anaemia?
Essential micronutrients required for various steps in regulating erythrocyte function, especially for the metabolism of haem
- Iron
- Vitamins B6 & B12
- Folic acid
These are obtained in the diet so malnutrition can cause different types of anaemia
Malabsorption may be cause by liver disease, gastric disease or abnormalities in the intestines
Each molecule of haemoglobin must have an atom of iron to which O2 can bind to in the lungs
Therefore a lack of iron directly impacts the amount of O2 that can be transported tot he tissues
How does iron-deficiency anaemia appear on a blood smear?
Characterised by hypochromic (pale) microcytes (small)
RBCs seem to be ‘empty’ with a lack of staining in the centre of the cell
RBCs are also significantly smaller (compare the size of RBCs to the white blood cell in the normal film
How do daily iron requirements change over lifetime?
Infants, adult males and post-menopausal females only require 1 mg
Adolescents require 2-3 mg
Menstruating adolescents require 3-4 mg
Menstruating adults require 2-3 mg
(20-25 mg lost per menstruation)
Pregnancy requires 3-4 mg - (500-1000 mg required overall)
Lactation (amenorrhea likely) requires 1.5-2.5 mg - (also required to repopulate stores
What intestinal factors may contribute to malabsorption?
The stomach: Achlorhydria (lack of HCl) Gastric atrophy Gastritis Alcoholism Gastric carcinoma
Upper and lower digestive tract: Duodenitis Coeliac disease Ulceration Crohn's disease Other inflammatory bowel disease Increased hepcidin levels
Surgery:
Resection of any of the above tissues
What are sources of iron?
High content: Dark-green leafy vegetables Iron-fortified cereals Whole grains Beans Nuts Meat Apricots Prunes Raisins Iron tablets
What is sideroblastic anaemia?
Characterised by failure of iron to be incorporated into haem in the erythrocyte precursor cells rather than lack of iron
Various forms of sideroblastic anaemia that are the consequence of mutations or deletions of genes regulating the expression of key enzymes involved in haem synthesis
A consequence of failure to incorporate iron into the correct areas result in the formation of iron-rich mitochondria which surround the nucleus of the erythrocyte precursor as granules
These cells are termed sideroblasts
What can make iron absorption more difficult?
Tea and coffee Calcium Antacids Proton pump inhibitors (PPIs) Wholegrain cereals (phytic acid)
Why is haem synthesised in erythrocyte precursors and not erythrocytes?
Synthesis requires a nucleus and mature erythrocytes do not have one
What are key regulators of haem production?
Vitamins B6, B12/folate
Deficiencies have significant effects on erythrocyte function
Isolated B6 deficiency is rare in the absence of drugs, particularly the anti-tuberculosis agent isoniazid
B12/folate deficiency is relatively common
What are causes of vitamin B12/folate deficiency and how can it be replaced?
Malnutrition and malabsorption Poverty Pregnancy Drugs Pernicious anaemia: deficiency of gastric intrinsic factor Gastrectomy Small bowel disease Tropical sprue Fish tapeworm Antacids Bacterial overgrowth Pancreatitis
Replacement: Meat Salmon Milk Eggs Fortified breakfast cereals Soy products
How can a deficiency in B12/folate result in a reduction of erythropoiesis?
It is essential for DNA synthesis in erythrocyte proliferation
What are microcytic anaemias and indicators of them?
Iron deficiency:
Mean cell volume <80 (occasionally normal)
Low mean cell haemoglobin
Low ferritin (normal if acute phase response)
Low RBC count
Target cells and pencil cells on blood film
History of bleeding
Anaemia chronic disease: MCV - 70-80 (often normal) Normal MCH Normal or high ferritin Low RBC count No specific features on blood film History of medical disease
Thalassaemia/trait: MCV - Low, often 50-60 Low MCH Normal ferritin High RBC Target cells, poikilocytes, tear drop cells, nucleated red cells on blood film History of ethnic origin
Lead poisoning: MCV - Low or normal Normal MCH Normal ferritin Normal RBC Basophilic stippling on blood film History of exposure
Rare red cell disorders (e.g. sideroblastic anaemia, pyropoikilocytosis): MCV - Low Normal MCH Normal ferritin Low/normal RBC Blood film according to condition Congenital history
How does MCV denote anaemia type?
High - macrocytic
Normal - normocytic
Low - microcytic (further divided by RBC)
What are the different anaemias, by cell size?
Macrocytic: Megaloblastic anaemia Vitamin B12 deficiency Folic acid deficiency Liver disease Hypothyroidism Reticulocytosis
Normocytic: Hemolytic anaemias Bone marrow disorders Hypersplenism Acute blood loss Anaemias of chronic disease
Microcytic: High RBC - Thalassaemias (alpha and beta thalassaemias; or combination with other haemoglobin abnormalities Low or normal RBC - Iron-deficiency anaemia - Lead poisoning - Anaemia of chronic inflammation - Sideroblastic anaemia
Which parts of the bone marrow are responsible for haematopoiesis?
Red marrow - highly vascularised, this is where HSCs reside and therefore where haematopoiesis takes place
Yellow marrow - where adipocytes reside
What are primary lymphoid tissues?
The sites where lymphocytes differentiate to express antigen receptors
- Thymus (T lymphocytes)
- Bone marrow (B lymphocytes)
What are secondary lymphoid tissues?
Specialised sites for turning on the acquired immune response
- Lymph nodes
- Spleen
- Mucosal associated lymphoid tissue (MALT)
(Gut associated lymphoid tissue (GALT))
(Nasal associated lymphoid tissue (NALT))
etc.
Describe the structure of the thymus.
Capsulated (collagen and other extracellular proteins)
Compartmentalisation
- Capsule
- Layer of adipose cells
- Cortex of thymus (very dense lymphocytes)
- Medulla (looks paler on histology - less dense)
- Lobes separated by trabeculae
- Also contains vascular tissue
- Hassall’s corpuscles
Describe the lymphatic system.
A drainage system involved in fluid balance, returning to the blood
- excess interstitial fluid (approx. 3 litres//24h)
- plasma proteins
Lymph capillaries:
Originate as ‘closed tubes’ in almost all tissues (except CNS, epidermis and cartilage)
Capillary wall constructed of overlapping endothelial cells that respond to fluid pressure
Also include valves to stop backflow
Lymphatics:
Lymph capillaries join to form lymphatics
Superficial lymphatics follow superficial veins
- flow into lymph nodes in axillary (armpit), inguinal (groin) or cervical (neck) areas where they drain into deep lymphatics
Deep lymphatics follow main vessels
- lymph nodes either side of aorta (para-aortic) drain the paired organs, nodes lying anterior (pre-aortic) the gut etc
- lymph from 3/4 body drains into the left brachiocephalic vein via the thoracic duct
- lymph from the upper right quadrant enters the right brachiocephalic vein
What is a lymph node?
Important component of host defence
Filter lymphatics
LN represent the anatomical meeting place for cells of immune system an their exposure to antigen
- Arteries transport naive lymphocytes to LN
- Lymph transports antigen
What is a lymph node?
Important component of host defence
Filter lymphatics
LN represent the anatomical meeting place for cells of immune system an their exposure to antigen
- Arteries transport naive lymphocytes to LN
- Lymph transports antigen
Describe the structure of a lymph node.
Lymph drains into LN via afferent lymph vessels
Exit via efferent lymph vessels
Capsulated
Have lobes separated by trabeculae
Has an arterial and venous system to supply lymphocytes
Lymph flows through reticular tissue to germinal centres which are supplied by arteries and veins
Then drains into sinuses and out efferent vessels
LNs vary in size from a pin-head to 1-2 cm
Enlarge during immune response
Lymph nodes of the MALT
- Aggregates of lymphocytes in a reticular mesh
- No incoming lymphatic vessels
- May be isolated or form clusters (Peyer’s patches in ileum; tonsils)
What is the function of the spleen?
Largest lymphoid organ
Receives lymphocytes and antigens via bloodstream
Directs immune responses to antigens in the blood
Important for clearance of effete red blood cells
Hyposplenism or asplenia not fatal, but predisposes to infection (especially of capsulated bacteria)
Presence of Howell-Jolly bodies (erythrocytes with nuclear fragments) in peripheral blood is often diagnostic of hyposplenism/asplenia, as normally cleared by spleen
Splenomegaly common in chronic disease (malaria, leishmaniasis, Hodgkin’s disease)
What is the function of the spleen?
Largest lymphoid organ
Receives lymphocytes and antigens via bloodstream
Directs immune responses to antigens in the blood
Important for clearance of effete red blood cells
Hyposplenism or asplenia not fatal, but predisposes to infection (especially of capsulated bacteria)
Presence of Howell-Jolly bodies (erythrocytes with nuclear fragments) in peripheral blood is often diagnostic of hyposplenism/asplenia, as normally cleared by spleen
Splenomegaly common in chronic disease (malaria, leishmaniasis, Hodgkin’s disease)
What is the location and size of the spleen?
Left hypochondriac region of abdominal cavity
Normally 12 x 7 x 2.5 cm and 0.2 kg
Accessory spleen may be present in 10% of population
What is the microanatomy of the spleen?
Red pulp with round white pulp in it (bunch of grapes in red jelly)
Looks like a collection of many lymph nodes
Non-immune functions in red pulp
White pulp effectively the same as a lymph node
Spleen is capsulated but individual white pulp is not
How do lymphocytes know which compartment to migrate to?
B and T lymphocytes has different chemokines they are attracted to and these are sequestered into zones
Follicle (B area) - CXCL13
T cell zone - CCL19 and CCL21
What produces the chemokines which contribute to the compartmentalisation of lymphoid tissues?
Stromal cells (non-haematopoietic)
- Blood endothelial cells
- Fibroblastic reticular cells
- Lymphatic endothelial cells
How many systems are there for classifying blood types?
30
Based on a number of characteristics; cell surface and/or soluble antigens
2 main are ABO and Rh (Rhesus)
If undergoing longterm treatment people will be tissue typed more closely using the other systems
What are the A and B antigens?
Carbohydrate structures present on red cell membrane glycoproteins and glycolipids
The major carriers of A and B on red cells are the abundant N-glycosylated glycoproteins, the anion exchanger (band 3) and the glucose transporter (GLUT1)
Gene encodes glycosyltransferase
- Type A - N-acetylgalactosaminyltransferase
- Type B - Galactosyltransferase
- Type O (H) - no transferase
How are the ABO blood groups inherited?
Encoded by one gene on Chromosome 9
3 allelic variants
Isoagglutinogen (antigen) - i, IA and IB
Not Mendelian inheritance
IA and IB are co dominant while i is recessive to both IA and IB
4 blood groups; A, B, AB and O
Multiple genotypes giving rise to phenotype
What is the Rh blood group?
Most complex blood group system
5 major Rh antigens - D, C, c, E, e - most immunogenic is D
Encoded by two genes RHD and RHCE
Individuals can be homozygous for RHD (2 copies) or hemizygous (1 copy - other may be deleted), all express D antigen on RBC
Individuals where RHD is deleted have no expression of D antigen - both copies deleted
Individuals are RhD+ or RhD-
No anti-D antibodies present in blood
What are the risks of Rhesus disease?
Does not effect first Rh+ baby of an Rh- woman but will effect subsequent pregnancies as anti-Rh antibodies will now exist
85% of the population is RhD+; 15% RhD-
Therefore probability of RhD- mother having an RhD+ baby is high
Range in effect - mild anaemia of the newborn to intrauterine death at 18 weeks of gestation (hydrops fetalis)
Kernicterus can also occur in neonates as a complication of severe jaundice which can cause brain damage in severe cases or in mild cases deafness
What is the universal recipient and the universal donor?
AB+ - recipient
O- - donor
What are the genetic anaemias?
Haemoglobinopathies
- Sickle cell disease - change in quality of the beta globin chain
- Thalassaemia - caused by a reduction in quantity of the alpha or beta globin chain
What is the cause of sickle cell anaemia?
Mutation of the HBB gene - glutamic acid replaced by valine
Gene form termed HBS
Alters Hb affinity for O2
Under deoxygenated conditions Hb tetramers with a mutated beta-globin chain have the tendency to complex forming polymers which cause distortion of the RBC - ‘sickle’
Individuals with only one copy of mutated beta-globin gene are able to complex but less efficiently
2 A genes, Haemoglobin AA, no disease
A/S genes, Haemoglobin AS, SC trait
2 S genes, Haemoglobin SS, SC disease
What is the inheritance pattern of thalassaemia?
Similar to sickle cell
Multiple genes depending on the form of thalassaemia
A child can only have the disease if both parents have the disease gene
What is the incidence of sickle cell anaemia?
Most prevalent in Sub-Saharan Africa, Middle East and India
Most prevalent in individuals of African or Caribbean descent
What is the prevalence of thalassaemia?
Most prevalent in individuals of Mediterranean, South Asian, Southeast Asian and Middle Eastern origin
What disease has a relationship with sickle cell anaemia?
Malaria
Incidence correlates with endemicity of Plasmodium falciparum
Haemoglobin AS has a selective advantage in parts of the world with endemic malaria
Relative protection from dying from malaria: selective advantage
Sickle cell anaemia (SS) gives a worse outcome for malaria
AS population far higher than SS
How does sickle cell anaemia present?
Episodes of severe haemolytic anaemia
Vaso-occlusive episodes - crises with possible infarcts in bones, lung, spleen, brain
Infections
Sickle cell trait may present with minor symptoms
Debilitating condition - impacts on individual in a personal nature in addition to social/familial context
- impact on daily life, relationships, reduced lifespan; implications for having a family
What is the clinical management of haemaglobinopathies?
Genetic anaemias are a major disease burden, most common hereditary disorders in the world
Estimated that 1000 people in UK have thalassaemia, and between 13,000-15,000 people in the UK have sickle cell
Disease management :
Prophylaxis - reduce risk of infection e.g. pneumococcal vaccination, folic acid supplements, prophylactic penicillin, spleen size determination, lifestyle modification
Bone marrow or stem cell transplant may be curative but risky treatment, not a common approach
Management - may include blood transfusions at intervals
Prevention - genetic screening, antenatal screening of parents and postnatal screening of baby
NHS sickle cell screening programme
Education - genetic counselling and support to manage disease, Sickle Cell Centre
Why do we have innate defences?
Innate immunity may prevent pathogen establishment
Innate immunity may limit pathogen multiplication
Innate immunity provides protection from early death during the expansion phase of the acquired immune response
Innate immunity helps to ‘instruct’ the nature of the acquired immune response generated after infection
What are non-immunological barriers to infection?
Skin:
Mechanical - Epithelial cells joined by tight junctions, longitudinal flow of air or fluid
Chemical - Fatty acids, antibacterial peptides
Microbiological - Normal flora
Gut:
Mechanical - Tight junctions, flow of air
Chemical - Low pH, enzymes (pepsin), antibacterial peptides
Microbiological - Normal flora
Lungs:
Mechanical - Tight junctions, movement of mucous by cilia
Chemical - Antibacterial peptides
Eyes/nose:
Mechanical - Tight junctions
Chemical - Salivary enzymes (lysozyme)
What are the three pathways of the complement cascade?
Classical pathway:
Antigen:antibody complexes (pathogen surfaces)
MB-Lectin pathway:
Mannose-binding lectin binds mannose on pathogen surfaces
Alternative pathway:
Pathogen surfaces
What are the functions of protein classes in the complement system?
Binds to antigen:antibody complexes and pathogen surfaces -C1q Binding to mannose on bacteria - MBL Activating enzymes - C1r, C1s, C2b, Bb, D, MASP-1, MASP-2 Membrane-binding proteins and opsonins - C4b, C3b Peptide mediators of inflammation - C5a, C3a, C4a Membrane-attack proteins - C5b, C6, C7, C8, C9 Complement receptors - CR1, CR2, CR3, CR4, C1qR Complement-regulatory proteins - C1INH, C4bp, CR1, MCP, DAF, H, I, P, CD59
How do pattern recognition receptors identify pathogens?
By recognising pathogen associated molecular patterns (PAMPs)
What ligands are recognised by which Toll-like receptors (TLRs)?
TLR-1 dimer, TLR-2/TLR-6 dimer: Peptidoglycan (Gram +ve) Lipoproteins Lipoarabinomannan (mycobacteria) GPI (T. cruzi - parasite) Zymosan (yeast)
TLR-3:
dsRNA (virus)
TLR-4 dimer (plus CD14):
LPS (Gram-negative bacteria)
TLR-5:
Flagellin
TLR-9:
Unmethylated CpG DNA
What are the principles of phagocytosis?
Attachment by pattern recognition receptors
Pseudopodia forming a phagosome
Granule fusion and killing (complete formation of the phagolysosome)
Release of microbial products
What is the respiratory burst?
When NADPH oxidase is activated and releases toxic oxygen radicals (superoxide and hydrogen peroxide)
Oxygen radicals may directly damage microbial membranes and/or activate microbicidal enzymes found in granules
How does the role of the phagosome differ between innate and adaptive immunity?
Innate: Pathogen killing 'Pathogen processing' 'Pathogen presentation' - TLRs 'Pathogen presentation' to cytosolic sensors - NODs/NALPs
Adaptive: Antigen degradation Antigen processing Antigen presentation - MHCII Antigen presentation via cytosol - MHCI
What are the microbicidal mechanisms of phagocytes?
Acidification
- pH~3.5-4.0, bacteriostatic or bactericidal
Toxic oxygen-derived products
- superoxide O2-, hydrogen peroxide H2O2, singlet oxygen 1O2., hydroxyl radical OH., hypohalite OCl-
Toxic nitrogen oxides
- Nitric oxide NO
Antimicrobial peptides
- Defensins and cationic proteins
Enzymes
- Lysozyme (dissolves cell walls of some Gram-positive bacteria), acid hydrolases (further digest bacteria)
Competitors
- Lactoferrin (binds Fe), vitamin B12-binding protein
What are the frequencies of phagocytes in blood?
Neutrophil - 40-75% Eosinophil - 1-6% Basophil - <1% Monocyte - 2-10% (Lymphocyte - 20-50%) Dendritic cells - <1%
What are neutrophils?
(Polymorphonuclear neutrophils; PMNs) Rapid responders Mobilised from BM by GM-CSF Found in 'depots' in tissue sites Highly phagocytic Produce multiple cytokines
How to activated neutrophils catch bacteria?
Neutrophil extracellular traps or NETs Contain: Chromatin Granule proteins Enzymes to degrade pathogen virulence factors
What do blood monocytes give rise to?
Tissue macrophages
Monocytes are phagocytes in their own right
HSC-derived monocytes and macrophage are:
A diverse and complex group of cells
Have varied differentiation pathways during inflammation
Share many functions
Relatively long lived
Host cells for many pathogens (e.g MTB)
Most tissue macrophages arise from non-haematopoietic precursors
What are the resident macrophages in the liver?
Kupffer cells
Embryonic yolk sac derived ‘resident macrophages’ have specific
- forms and functions in different tissues (inc. homeostasis)
- transcription factors and epigenetic mechanisms associated with development
- responses to environmental cues
What are dendritic cells?
Phagocytes specialised for interacting with lymphocytes
Complex subset of myeloid cells
- defined by specific TFs and cells characteristics
- can be sedentary or migratory
- are activated during innate immunity
- play an important role in initiating immune responses
How do cells move from blood to tissue?
Extravasation:
Monocyte binds adhesion molecules on vascular endothelium near sites of infection and gets chemokine signal
The monocyte migrates into the surrounding tissue
Monocyte differentiates into a macrophage and migrates to the site of infection
Pathways of cell recruitment
- links multiple aspects of immune cell biology
- an essential component of inflammation and immunity
- a key target for therapeutic manipulation
What are cytokines and chemokines?
The hormones of the immune system
Allow communication between a variety of immune and non-immune cells
>30 identified, with specific but overlapping functions
Mostly referred to as ‘interleukins’ hence IL-1, IL-2, IL-3 etc, but some use ‘functional names’ e.g ‘tumour necrosis factor’ (TNF)
A further heterogenous and complex family of small molecular weight chemotactic cytokines are known as ‘chemokines’ (>30)
What are key features of cytokine and chemokine regulation?
Mostly short acting ‘hormones’ but can have systemic effects e.g. in shock
May be released in a polar fashion at ‘synapses’
Recognised on target cells by specific receptors (with some redundancy)
Receptor expression is highly regulated to control the targets and duration of response
Subsets of cytokines/chemokines may be selectively co-regulated and help define cell subsets e.g Th1 and Th2 CD4+ lymphocytes
What aspects of immune functions can be affected by steroids?
Corticosteroid therapy:
Decreases IL-1, TNF-alpha, GM-CSF, IL-3, IL-4, IL-5, CXCL8
- decreases inflammation caused by cytokines
Decreases NOS
- decreases NO
Decreases phospholipase A2 and cyclooxygenase type 2, increase lipocortin-1
- decreases prostaglandins and leukotrienes
Decreases adhesion molecules
- reduced emigration of leukocytes from vessels
Increased endonucleases
- induction of apoptosis in lymphocytes and eosinophils
How does activation of a macrophage lead to different responses?
Classical activation (IFN-gamma, TNF, IL-1): Type 1 immunity - microbial killing, tissue damage, DTH
Alternate activation (IL-4/IL-13, IL-10, GM-CSF): Type 2 immunity - allergy, helminth responses, fibrosis and repair
What are natural killer cells?
Atypical ‘innate’ lymphocytes
What are the three stages of haemostasis?
1 - Vascular spasm - damaged blood vessels constrict, reducing blood flow in the damaged area
2 - Platelet plug formation - (primary haemostasis) platelets bind to the damaged vessel wall and form a platelet plug
3 - Coagulation - (secondary haemostasis) a stable clot forms by converting fibrinogen to fibrin
What are platelets?
Platelets are small (2-3µm) fragments of megakaryocyte cytoplasm
A healthy adult produces ~10^11 platelets/day, the physiological range is 150-400x10^9/L of blood
Platelets circulate in the blood and have a lifespan of ~5-9 days, old platelets are destroyed by Kupffer cells in the liver or by phagocytosis in the spleen
What are the secretory granules of platelets?
alpha-granules
Dense-granules
What is the ultrastructure of the platelet?
No nucleus, platelets contain all the factors required for their function
Critical components are:
- Membrane proteins
- Secretory granules
- Surface-connected open canalicular system (SCOCS)
Surface-connected canalicular system Microtubules alpha-granule Dense granule Glycogen Mitochondrion
What are alpha-granules?
Most prominent and numerous (50-80/platelet) and are 200-500nm in size
Contain:
- Adhesive proteins (fibrinogen, fibronectin, vWF)
- Platelet-specific proteins (PF4, PDGF)
- Membrane proteins (GPIIaIIIb, GPIb/IX/V)
- alpha-granule-specific proteins (P-selectin)
What are dense-granules?
2-7 dense granules/platelet, 2-300nm in diameter
Electron dense when stained with osmium tetroxide, dense core with a halo
Dense-granules contain:
- Vasoconstrictive agents (serotonin)
- Platelet agonists (ADP, ATP)
- Calcium and magnesium
What are the platelet membrane proteins?
- Receptors for various agonists (ADP, thrombin, TXA2)
- GPIb-IX-V complex - GPIbα, GPIX, GPIβ, GPV (vWF interactions)
- GPIIb-IIIa complex - GPIIIa, GPIIb (vWF interaction, fibrinogen binding and platelet aggregation)
- GPIa-IIa complex - GPIa, GPIIa (collagen interactions)
- GPVI (collagen interactions)
What are the stages of platelet adhesion?
Following vessel damage:
- Platelets adhere to exposed collagen-associated vWF, the platelet is now ‘tethered’ to the sub-endothelium, shear stress is a critical component of tethering
- Blood flow then ‘rolls’ the platelet along the sub-endothelium promoting more GBIb/vWF associations, and creating firmer adhesion between the platelet and the sub-endothelium
- This firmer adhesion initiates platelet activation, resulting in granule release and conformation changes in specific adhesion molecules, leading to strong adhesion, shape change and further activation
How does the platelet plug form?
A platelet monolayer forms on the exposed sub-endothelial layer
The activated platelet monolayer release platelet agonists such as ADP, thromboxane A2 and adhesive proteins, such as fibrinogen, which recruits further platelets to the developing plug
As the newly-recruited platelets are attracted to the plug, they also change shape, allowing for greater platelet-platelet interactions, especially via fibrinogen cross-bridges, which bind to GPIIaIIIb
What is primary haemostasis?
Platelet activation and plug formation
What is secondary haemostasis?
Coagulation
What are coagulation factors?
Zymogens (inactive precursors of enzymes)
Cooperate in an integrated system of enzyme activation and inactivation steps
Considered a cascade as one event prompts the next as in a multi-level waterfall
What are the two basic pathways that initiate the coagulation cascade?
Extrinsic pathway - normally is triggered by trauma
Intrinsic pathway - begins in the bloodstream and is triggered by internal damage to the wall of the vessel
What are the two basic pathways that initiate the coagulation cascade?
Extrinsic pathway - normally is triggered by trauma
Intrinsic pathway - begins in the bloodstream and is triggered by internal damage to the wall of the vessel
What is the result of the coagulation cascade?
The production of a gelatinous but robust clot made up of a mesh of fibrin, an insoluble filamentous protein derived from fibrinogen, in which platelets and blood cells are trapped
What is the intrinsic coagulation cascade?
Damaged vessel wall:
- XII -> activated XII
- activated XII causes XI -> activated XI
- activated XI causes IX -> activated IX
- activated IX forms a complex with activated VIII
- activated VIII/IX complex causes X -> activated X
- final common pathway
What is the final common pathway of both the extrinsic and intrinsic pathway?
Final common pathway:
- activated X with activated V causes prothrombin (II) -> thrombin (IIa)
- prothrombin (II) -> thrombin (IIa)
- thrombin (IIa) causes fibrinogen (I) -> fibrin (Ia)
- fibrin (Ia) with activated XIII creates a cross-linked fibrin clot
What is the extrinsic pathway of the coagulation cascade?
Trauma to extravascular cells:
- III (tissue factor) causes VII -> activated VII
- activated VII and III form a complex
- activated VII/III complex causes X -> activated X
- final common pathway
What stages of the cascade do anti-thrombin (AT) and tissue factor pathway inhibitor (TFPI) inhibit?
Anti-thrombin:
- activation of XI (intrinsic)
- activation of IX (intrinsic)
- activation of X (intrinsic)
- prothrombin (II) -> thrombin (IIa) (final common pathway)
Tissue factor pathway inhibitor:
- formation of VIIa/III (extrinsic)
- activation of X (extrinsic)
What are some laboratory tests for platelet and coagulation disorders?
Platelet aggregometry
Coagulation test - prothrombin time (PT) and partial thromboplastin time (PTT)
How does platelet aggregometry work?
Take platelet rich plasma (or washed platelets)
Shines light through it to detector behind
Inactivated platelets are very cloudy, when agonist added platelets are activated and form clots (aggregates) which allows more light to shine through
Usually see small drop (shape change peak) before more light shines through due to platelets changing shape when first activated - makes it more cloudy
If platelets do not aggregate properly this shows an issue with clotting
Various diagnoses can be made depending on response to selected agonists
How is a coagulation test carried out?
Take plasma, no cells or platelets, and place in cuvette with little metal ball at bottom (for mixing)
Add different reagents which begin cascade, can be at different points of the cascade
Plasma becomes gelatinous - turn upside down and ball wont fall out
Time how long it takes for ball to stop moving - full coagulation
What does prothrombin time (PT) test in a coagulation test?
Tests the extrinsic pathway
What does partial thromboplastin time (PT) test in a coagulation test?
The intrinsic pathway
What conditions may PT and PTT indicate?
PT normal:
PTT normal - decreased platelet function, thrombocytopenia, factor XIII deficiency, mild deficiencies in other factors, mild form of von Willebrand’s disease, weak collagen
PTT prolonged - decreased or defective factor VIII, IX, XI or XII, von Willebrand disease, or lupus anticoagulant present
PT prolonged:
PTT normal - Liver disease, decreased vitamin K, decreased or defective factor VII
PTT prolonged - Decreased or defective factor I, II, V or X, liver disease, disseminated intravascular coagulation (DIC)
What are some bleeding disorders?
immune thrombocytopenia - antibodies to platelets leads to them being cleared
Glanzmann’s thrombasthenia - mutations leading to platelet dysfunction
Haemophilia - changes in clotting proteins
What is immune thrombocytopenia?
Thrombocytopenia - term used to describe a condition of low circulating platelets (<100,000/µl blood)
Primary (idiopathic) thrombocytopenia - condition where low platelet counts occur spontaneously for an unknown reason
About 60% of primary thrombocytopenia cases, antibodies to platelets proteins, usually GP-IIbIIIa or Ib/IX, are found in plasma - immune thrombocytopenia (ITP)
The IgG-coated platelets are then cleared by either splenic macrophages or Kupffer cells in the liver
Can cause extreme ITP, with platelet counts as low as 5000/µl
Mild ITP - can develop small haemorrhages called petechia, which appears as a rash
Severe ITP - can present with extensive hematomas and cerebral haemorrhage
What is Glanzmann’s thrombasthenia?
Affects platelet function
An autosomal recessive bleeding syndrome affecting the megakaryocyte lineage and characterised by a lack of platelet aggregation
Specific mutations lead to changes in expression and/or function of GPIIb and/or IIIa - severely affects function and platelet aggregation
There is a large number and range of mutations which can cause GT, the pathogenesis is also very broad, from bleeding gums to severe haemorrhage
What is Glanzmann’s thrombasthenia?
Affects platelet function
An autosomal recessive bleeding syndrome affecting the megakaryocyte lineage and characterised by a lack of platelet aggregation
Specific mutations lead to changes in expression and/or function of GPIIb and/or IIIa - severely affects function and platelet aggregation
There is a large number and range of mutations which can cause GT, the pathogenesis is also very broad, from bleeding gums to severe haemorrhage
What is haemophilia?
A large group of bleeding disorders characterised b changes in clotting proteins
Recessive sex-linked X-chromosome disorder, meaning the vast majority of sufferers are male
Haemophilia A - occurs in ~1 in 5000 male births and is characterised by a deficiency in factor VIII
Haemophilia B - less common ~1 in 30,000 and is caused by a deficiency in factor IX
What are some anti-thrombotic drugs?
Aspirin
ADP receptor antagonists
What are some anticoagulant drugs?
Heparin
Warfarin
What are some anti-platelet drug targets?
Activation
- Thromboxane inhibitors
- PAR1 antagonists
- ADP-receptor antagonists
Aggregation
- alpha IIb beta 3-integrin inhibitors
Adhesion
How does aspirin work?
Thromboxane inhibitor - targets activation
Inhibits platelet cyclooxygenase, a key enzyme in thromboxane A2 (TXA2) generation
Thromboxane A2 triggers reactions that lead to platelet activation and aggregation, aspirin acts as a potent anti-platelet agent by inhibiting generation of this mediator
Effects last for the life of the platelet - approx 7-10 days
It is the first drug given to patients suspected of having acute coronary syndrome (ACS)
How is aspirin anti-thrombotic and anti-inflammatory?
Anti-thrombotic: Stimulated platelets Arachidonic acid -> PG-G/H via COX-1 PG-G/H -> Thromboxane via thromboxane synthase Aspirin inhibits COX-1
Anti-inflammatory:
Vasculature (Aspirin acetylation-dependent)
Arachidonic acid -> 15R-HETE via COX-2
15R-HETE -> 15-epi-Lipoxin A4 via leukocytes
Aspirin acetylates COX-2
What is aspirin effective against?
Ischemic stroke Transient ischemic attacks Chronic stable angina Unstable angina Coronary stenting Acute MI Previous MI Primary prevention of MI (from 65 years of age)
What is aspirin effective against?
Ischemic stroke Transient ischemic attacks Chronic stable angina Unstable angina Coronary stenting Acute MI Previous MI Primary prevention of MI (from 65 years of age)
What are the adverse effects of aspirin?
GI bleeding (~0.3% of patients taking aspirin for 5 years) Hemorrhagic stroke (~0.1% of patients taking aspirin for 5 years)
What is the dose of aspirin?
Low dose - 81 mg/day (this is the dose that maximally inhibits platelet activity and can be taken daily)
High dose - 325 mg/day (this is used for initial treatment of acute symptom e.g. MI)
What are the clinical uses of ADP-receptor antagonists?
Widely used to prevent blockage of coronary arterial stents
Also used to reduce thrombotic events (MI, ischemic stroke etc) in patients with recent MI, stroke or peripheral arterial disease
Clopidogrel is a pro-drug, that gets metabolised into it’s active form by hepatic CYP2C19 (a form of the P450 enzyme)
Some people carry mutations in CYP2C19, meaning they can not metabolise clopidogrel well, if this is the case alternative ADP antagonist drugs - prasugrel (Effient TM) and ticagrelor (Brilintia TM)
What are some examples of ADP-receptor antagonists?
Clopidogrel (Plavix TM)
Prasugrel
Ticagrelor
What are the adverse effects of ADP receptor antagonists
Generally well tolerated, similar side effects to those seen with aspirin treatment
Primary side effects of all ADP receptor inhibitor drugs is bleeding, although incidences of GI bleeding and intracranial haemorrhage are actually lower than aspirin
One potentially fatal side effect of clopidogrel is thrombotic thrombocytopenic purpura (TTP)
What is the primary goal of anti-coagulants and how are they classified?
Primary goal is to reduce the formation of fibrin
Two primary mechanisms of action:
- Inhibit the synthesis of clotting factors
- Inhibit the activity of clotting factors
Can be further separated into 2 groups:
- Direct thrombin inhibitors
- Direct factor Xa inhibitors
What is heparin?
A heterogenous natural polysaccharide
- Unfractionated heparin (UFH) is isolated from mammalian tissues, usually porcine intestinal mucosa or bovine lung
- Various procedures are used to fractionate UFH to low molecular weight heparins (LMWHs)
How does unfractionated heparin (UFH) work?
UFH binds to anti-thrombin (AT) causing a conformational change which increases its binding affinity for FXa and thrombin
The inactivation of thrombin is achieved by UFH and AT complex forming a ternary structure with thrombin
How does low molecular weight heparin (LWMH) work?
LMW heparin has the same pentasaccharide complex as UFH, but can only inactivate FXa
Most molecules of LMWH cannot form a ternary complex with thrombin
What are the clinical indications for use of UFH?
Prophylaxis of VTE
Treatment of VTE
Prophylaxis and treatment of atrial fibrillation with systemic embolisation
Treatment of acute and chronic consumptive coagulopathies (disseminated intravascular coagulation)
Prevention of clotting in cardiac surgery
Prevention of clotting in hemodialysis procedures
As an anticoagulant in:
- blood transfusions
- blood samples for laboratory purposes
What are the clinical indications for the use of LMWHs?
For VTE prophylaxis:
- after total hip and knee replacement surgery
- after abdominal surgery
- in high-risk medical patients
For the treatment of VTE
For long-term prophylaxis against VTE in pregnant women
For long-term secondary prophylaxis after VTE in cancer
For treatment of unstable angina
In thrombolytic therapy
For treatment of superficial thrombophlebitis
What is warfarin?
When it was first developed, was ruled out from clinical use due to concerns about haemorrhage and was instead used for rat poison
Now the most commonly used drug to prevent clotting following heart attacks, strokes and major surgery
How does warfarin work?
A vitamin K antagonist (VKA)
Many clotting factors (TF, FVII, FIX and FX) rely on the reduced form of vitamin K (KH2) for carboxylation of glutamic residues
Warfarin blocks the reformation of KH2 from the oxidised form (KO), thereby reducing the formation of clotting factors
What are the adverse effects of heparin?
Bleeding (especially with UFH)
Immune-induced thrombocytopenia
Severe neurological injury (especially in patients undergoing epidural anaesthesia)
Risks increased when drug is used in conjunction with other anti-platelet and anti-thrombotic drugs
What are the adverse effects of warfarin?
Bleeding (severe bleeding can be treated with vitamin K injection)
Foetal haemorrhage (warfarin can cross the placenta, drug should not be used while pregnant or breast-feeding)
Long term usage can result in osteoporosis
Where do B cells develop and where are they activated?
Bone marrow - mature B cells
They activate/proliferate in periphery lymphoid tissue - Memory B cell or a plasma cell
What are antibodies (immunoglobulins)?
Function as the B cell lymphocyte receptor for antigen
Soluble recognition molecules secreted by B lymphocytes
What is the function of antibodies?
High capacity for recognising fine molecular detail
Important functions:
- directly kill or neutralise microbes and/or their toxins
- help target phagocytes to microbes
- regulation of immune responses (indirectly)
- clearance of antigens and termination of immune response
How are antibodies involved in negative immune responses?
- mediate allergic responses
- cause immune complex diseases
- mediate autoimmune cell destruction
What are the 5 main classes (isotypes) of antibodies?
IgA IgD IgE IgG IgM
Classes differ in valency and function
Which Ig’s have subclasses?
IgG
- four subclasses - IgG1, IgG2, IgG3 and IgG4 (abundancy decreases in order of serum e.g. 1>2)
IgA
- two subclasses - IgA1 > IgA2
IgG and IgA antibodies also have polymorphic (genetic) variants, referred to as allotypes
What is the basic structure of an IgG molecule?
Variable regions containing antigen-binding site
Heavy chain and light chain
Constant regions
Heavy chain
Has a valency of two - two binding sites
What is an epitope?
The area of an antigen that fits into the binding site
Usually just a small number of amino acids
What is Fab?
Fragment antigen binding
Four domains above the hinge region
What is Fc?
Fragment crystallisable
Terminal portions of heavy chain - below hinge
What is the function of Fc?
Can be bound by receptors on surfaces of cells
What is VL and CL?
Variable light
Constant light
Two domains of the light chain above the hinge region
What is VH, Cgamma1, Cgamma2 and Cgamma3?
Variable heavy
Constant gamma 1 etc
Domains of heavy chain - VH and Cgamma1 are above the hinge the other 2 are below
How are IgG molecules flexible?
Due to the hinge region
Allows the two arms to move
How do antibodies have infinite different specificity?
Antibody molecule is encoded for by multiple gene segments
- Germ line diversity of genes
- Combinatorial diversity (somatic recombination)
- Junctional diversity (somatic recombination)
- Somatic hypermutation (during ongoing immune response) - when affinity changes during response
Results in approx 10^11 different antibody combining sites
Where are the immunoglobulin genes?
Heavy - Chromosome 14 (50 Vh, 25 DH and 6 JH gene segments) (Different CH gene segment for each antibody isotype and subclass)
Kappa light - Chromosome 2 (30 VK, 25 and 5 JH gene segments)
Lambda light - Chromosome 22
What are the basic principles of somatic recombination?
Length of DNA loops to excise a circle and leftover rearranged DNA is read
Each individual B cell undergoes this process during maturation, independently of all other B cells
Recombination only occurs once in the life of a B cell, so each B has a fixed ‘specificity’
Defect in enzymes that cut DNA leads to lack of B lymphocytes as they cannot mature
What is the process of clonal selection of B cells?
Immature B cell
- Proliferation and diversification of progeny cells
Different nonactivated B cells = SPECIFICITY
- Antigen binding to specific B cell (+ antigen)
Activated B cell
- Proliferation and differentiation
Multiple identical B cells = AMPLIFICATION
Somatic hypermutation acts later to select for increased affinity
How does somatic hypermutation (SHM) occur?
Introduces random changes into the antibody binding site
- Activation-induced cytidine deaminase (AID) directs replacement mutations
- Sequences in the antibody binding site have been selected as hot spots for AID
- The antibody binding site accumulates point mutations >1M x faster than other sequences
- Mutations can increase, decrease or not change Ab affinity
- Therefore, a selection mechanism is required to generate higher affinity clones
Where does somatic hypermutation occur?
Germinal centres in lymphoid tissue
What are the different functions of Ig isotypes and subclasses?
Neutralisation - IgG1-4, IgA
Opsonisation - IgG1, IgG3
Sensitisation for killing by NK cells - IgG1, IgG3
Sensitisation of mast cells - IgE
Activates complement system- IgM, IgG3, IgG1
Where are the different classes and subclasses of Ig’s distributed?
Transport across epithelium - IgA (dimer)
Transport across placenta - IgG1, IgG3
Diffusion in extravascular sites - IgG1-4, IgA (monomer)
Mean serum level (h - l) - IgM, IgG1, IgG2, IgA, IgG3, IgG4, IgD, IgE
How can Ig’s change function?
By class (isotype) switching (this does not affect the specificity of the Ab) Occurs due to cytokines IL-4: Inhibits - IgM, IgG3, IgG2a Induces - IgG1, IgE
IL-5:
Augments production - IgA
IFN-gamma:
Inhibits - IgM, IgG1, IgE
Induces - IgG3, IgG2a
TGF-beta:
Inhibits - IgM, IgG3
Induces - IgG2b, IgA
Which Ig’s have a higher valency than 2?
IgM - Pentameric structure (5 molecules connected)
IgA - Dimeric structure (2 molecules and a secretory piece which allows it to pass through epithelium)
Multimeric IgM has increased capacity to interact with complement
Where are different Ig isotypes distributed?
IgG and monomeric IgA - General tissue and heart
IgM - Heart
Dimeric IgA - Mucosal tissues e.g gut, lungs, and mammary glands
IgE - Skin and mucosal surfaces
What do antibodies do in the complement system?
Create the antibody-antigen complex which stimulates the classical pathway
What is the function of Fc?
Binds to certain receptors in the immune system to induce certain actions e.g. mast cells to release granules or macrophages and beginning phagocytosis
What is needed to turn a resting B cell into an Ig secreting plasma cell?
Microenvironment, follicular dendritic cells, chemokines, APCs, CD4+ T cells, cytokines
Which T cell is required to help B cells?
CD4 + ‘helper’ cells
Where do T cells come from?
Derived from bone marrow-derived haematopoietic stem cells that migrate to the thymus
98% of T cells die in the thymus by positive and negative selection
Only T cells with TCR able to recognise foreign peptides bound by MHC escape to the periphery
What is the structure of a TCR?
Very similar to antibody
alpha and beta chain
Single antigen-binding site
Transmembrane region
How do T and B cells recognise different forms of antigen?
B cells:
Use Ab to recognise ‘conformational epitopes’ or tertiary structure
Conformational epitopes can be made up from discontinuous stretches of amino acids
T cells:
Use TCR to recognise ‘linear epitopes’ enzymatically digested out of antigens (antigen processing) by phagocytic cells
How does the structure of MHC I differ from MHC II?
MHC class I
- single heavy chain
- 3 domains connected to beta 2 microglobulin
- peptide binding site
MHC II
- one alpha one beta chain
- peptide binding site
Are MHC molecules polymorphic?
Yes, this can lead to certain people being more susceptible to certain diseases
What is the function of MHC class I?
Expressed on all nucleated cells
Carry peptides generated in the cytosol or ER
Present peptides to CD8+ T cells
Surveillance for virus infection and altered self
What is the function of MHC class II?
Expressed on specialised APCs (normally)
Carry peptides generated in the endosomal compartments
Present peptides to CD4+ T cells
Surveillance for exogenous pathogens
What 4 ways do T cells differ?
Phenotype
MHC restriction
Function
Cytokine production
How may T cells differ by phenotype?
- Assessed by identifying molecules expressed on the surface or inside the cell
- May help distinguish origin, differentiation , activation or exhaustion states and/or function
- Many molecules used for phenotyping are called cluster of differentiation (CD) antigens e.g. CD3 is a molecule associated with the TCR so marks T cells
- T cells subsets develop unde the control of dfferent transcription factors (e.g. Tbet, Gata3, RoRγ, Fox P3, EOMES)
How do T cells differ by MHC restriction?
- Used to denote whether the TCR recognises peptides presented by MHCI or MHCII molecules
- Defines what type of antigen expressing cells they can recognise:
CD4+ T cells are MHCII restricted and see antigens on specialised APC
CD8+ T cells are MHCI restricted and see antigen on any nucleated cell
How do T cells differ by function?
- Help formation of germinal centres and B cell antibody affinity maturation: CD4 + Tfh (T follicular helper) cells
- Activation of phagocytes for enhanced killing of intracellular pathogens: CD4+ Th1 (T helper 1) cells and CD8+ T cells
- Activation of responses targeting extracellular pathogens (e.g. worms): CD4+ Th2 (T helper 2) cells
- Activation of neutrophils/anti-fungal responses/autoimmunity: CD4+ Th17 (T helper 17) cells
- Direct killing of infected ‘target’ cells or cancer cells: CD8+ CTL (cytotoxic T lymphocytes)
- Inhibit the function of other T cells: CD4+ Treg (T regulatory) cells
How do T cells differ by cytokine production?
IL-2: CD4+ IFNγ: Th1 and CD8+ IL-4: Th2 IL-17: Th17 IL-21: Tfh
How does MHCI-restricted antigen presentation to CD8+ T cells happen?
Virus infects cell
Viral proteins synthesised in cytoplasm
Peptide fragments of viral proteins bound by MHC class I in ER
Bound peptides transported by MHC class I to the cell surface
Cytotoxic T cell recognises complex of viral peptide with MHC class I and kills infected cell
How does MHCII-restricted antigen presentation to CD4+ T cells happen?
Th1 response
- Macrophage engulfs and degrades bacterium, producing peptides
- Bacterial peptides bound by MHC class II in vesicles
- Bacterial peptides transported by MHC class II to the cell surface
- Th1 cell recognises complex of peptide antigen with MHC class II and activates macrophage
Th2 response
- Cell-surface immunoglobulin of B cell binds bacteria and engulfs and degrades them, producing peptides
- Bacterial peptides bound by MHC class II in vesicles
- Bound peptides transported by MHC class II to the cell surface
- Th2 cell recognises complex of peptide antigen with MHC class II and activates B cell
What are the three signals required to support T cell activation and differentiation?
Signal 1 - TCR engagement: is the antigen present (from antigen processing)? (TCR-MHCII & CD4)
Signal 2 - Costimulation: is the antigen meaningful (is it pathogen derived)? (CD80/CD28)
Signal 3 - Cytokine: what type of T cell should I become (to respond correctly)?
What is the normal sequence of intracellular signalling following TCR stimulation?
- Signalling via the T cell receptor induces AP-1 activation and increases intracellular Ca2+ concentration
- Raised intracellular Ca2+ activates calcineurin, a phosphatase that activates NFATc family members
- Activated NFATc family members migrate to the nucleus and bind to AP-1 and other partner proteins to form active transcription factors
- Activation of specific genes including the IL-2 gene (leading to clonal expansion of the activated T cell
What is the sequence of intracellular signalling following TCR stimulation but in the presence of cyclosporin A and tacrolimus?
- The immunosuppressive drugs cyclosporin A (CsA) and tacrolimus act in the cytoplasm
- CsA and tacrolimus bind to distinct targets, the intracellular proteins cyclophilin (CyP) and FK-binding protein (FKBP)
- Both CsA:CyP and the tacrolimus:FKBP complex bind to calcineurin, preventing its activation by calcium and blocking activation of NFATc
- No activation of transcription
What are the immunological effects of cyclosporin A and tacrolimus?
T lymphocyte:
Reduced expression of IL-2, IL-3, IL-4, GM-CSF, TNF-α
Reduced proliferation following decreased IL-2 production
Reduced Ca2+-dependent exocytosis of granule-associated serine esterases
Inhibition of antigen-driven apoptosis
B lymphocyte:
Inhibition of proliferation secondary to reduced cytokine production by T lymphocytes
Inhibition of proliferation following ligation of surface immunoglobulin
Induction of apoptosis following B-cell activation
Granulocyte:
Reduced Ca2+-dependent exocytosis of granule-associated serine esterases
What is the role of IL-2?
Helps maintain T-cell population - key to T cell survival
In higher levels causes proliferation - key to clonal expansion
What are memory consequences of clonal expansion?
1 - Naive T cell sees antigen - naive T cells
2 - Quiescent memory cells may derive from activated naive T cells - activated effector cells and ‘memory’ cells
3 - EITHER - long term memory or death
- Other memory cells express CCR7 and remain in lymphoid tissue
- Some memory cells become effectors and migrate to tissues
What are effector consequences of clonal expansion?
1 - Naive T cell sees antigen - LN normal
2 - Effector T cells differentiate, secrete cytokines and express cytokine receptors - LN enlargement
3 - EITHER - LN normal
- Some effector cells may become quiescent memory cells
- Most effector cells die after a few days
What is the basis of acquired or adaptive immunity?
Memory to previous exposure
Memory T cells remain at higher frequency after immunisation and react quicker
Secondary antibody response far quicker and bigger than primary
What are the dynamics of the immune response in a lymph node
APC or bacteria in afferent lymphatic vessel enters lymph node
Pathogens can be picked up by phagocytes in T cell area and have their antigens displayed for T cells to survey them
T cells may recognise and expand clonally and may also interact with B cells to provide help and eventually produce antibodies
All of these products move out of the efferent lymph
What is the role of T follicular helper cells?
Helps B cells in the follicle area with affinity maturation
How do Th1 and Th2 cytokines effect macrophage function?
Th1 - IFN-γ, TNF, IL-1
- Microbial killing, tissue damage, DTH
- Classical activation
Th2 - IL-4/IL-13, IL-10, GM-CSF
- Helminth responses, fibrosis and repair
- Alternate activation
What is the role of Tregs?
Inhibit immune response via multiple pathways
- Consume IL-2 leading to loss of effector T cells
- Produces TGF-β, IL-10, IL-35 to suppress effector T cells
- Suppress APC via CTLA-4
What happens when T cells stop working?
Senescent T cells
- Cells that enter a terminal differentiation state owing to excessive cell replication
- This state is associated with irreversible cell cycle arrest and telomere shortening
Anergic T cells
- An unresponsive state that is induced by suboptimal stimulation (that is, signal 1 without signal 2/3) at the time of priming by antigen
Exhausted T cells
- A state characterised by an overstimulation via signal 1 and 3 leading to hierarchical loss of effector functions and memory T cell properties, and by the expression of multiple inhibitory receptors
What are the potential uses of T cell exhaustion for immunotherapy?
- T cell exhaustion prevents optimal control of infections and tumours
- Exhausted T cells are a distinct lineage of differentiated T cells
- Altered usage of transcription factors is a key feature of T cell exhaustion
- Exhaustion of CD4+ T cells is distinct from that of CD8+ T cells
- Modulating inhibitory pathways that are over-expressed in exhaustion can reverse this state and reinvigorate immune responses
Can exhaustion be reversed?
Basis of antibody-based immunotherapy
Inhibitory immune receptors are often called ‘immune checkpoints’ and immunotherapeutic antibodies are called ‘ immune checkpoint inhibitors’
How can T cells be engineered for immunotherapy?
CAR-T cell therapy
Chimeric antigen receptor
Cancer-specific mAb, placed on TCR/co-stim molecule complex as variable region to create a specific CTL
Shares advantages of mAbs and CTLs
What is the difference between primary, secondary and relative polcythaemias?
Primary
- Genetic problems in RBC
Primary familial and congenital polycythaemia - due to enhanced responsiveness to EPO due to mutations in the EPOR
Polycythaemia rubra vera (PV) - Most commonly associated with JAK2V617F mutation; increased RBC, white blood cells and platelet production; may lead to myelofibrosis and acute leukaemia
Secondary
- Due to conditions that promote RBC development
Hypoxia - e.g. due to COPD, CHF, kidney transplant (major source of EPO)
EPO secreting tumours - e.g. Renal cell carcinoma, hepatocellular carcinoma
Neonatal polycythaemia - seen in 1-5% of neonates, due to maternal RBC transfusion after delivery, intrauterine hypoxia
Relative
- RBCs normal but reduced plasma volume, due to e.g. dehydration, severe vomiting
What are the clinical signs of essential thrombocythaemia?
Frequently asymptomatic
Haemorrhage or thrombosis
Occasional progression to myelofibrosis and leukaemia
What are the clinical signs of idiopathic myelofibrosis?
Leukoerythroblastic blood picture Splenomegaly and bone marrow fibrosis Anaemia Thrombocythaemia or thrombocytopenia and variable white cell counts Usual progression to acute leukaemia
Describe the JAK2 gene.
Member of a family of four janus kinases 1, 2, and 3 and tyrosine kinase 2
These are non-receptor kinases - binds to a receptor and aids in signal transduction
One of the receptors it binds to is EPOR
Describe the structure of the JAK2 gene.
4 domais
(N) FERM, Sh2, JH2, JH1 (C)
FERM - Interaction with cytokine receptor
SH2 - Role in conformational conservation
JH2 - TK like domain - auto-inhibitory role
JH1 - Tyrosine kinase (TK) activity
V617F mutation is in the JH2 region
How does the V617F mutation affect the function of JAK2?
The mutation is in the JH2 domain of the gene
Mutation of this auto-inhibitory domain makes JAK2 constitutively active in the absence of ligand binding
How may the outcome of the V617F mutation be modified by other factors?
Some factors may cause the patient to be more likely to have thrombocythaemia:
- Depleted iron stores
- Genetic modifiers
- EPO level
Other factors may cause the patient to be more likely to have polycythaemia:
- Sex
- genetic modifiers
- V617F homozygosity
How many types of hypersensitivity are there?
4 Type I - Immediate (Allergy) Type II - Cell-bound antigen Type III - Immune complex Type IV - Delayed type hypersensitivity
What is type I hypersensitivity?
Allergy and atopy
Immediate
Immune reactant - IgE
Antigen - Soluble antigen
Effector mechanism - Mast-cell activation
Example - Allergic rhinitis, asthma, systemic anaphylaxis
Mast cells have FcεRi receptors for Fc region of IgE
IgE binds to this (preloaded) and when antigen binds to IgE mast cells degranulate
IgE is low in serum as it binds to mast cells
What are the consequences of mast-cell activation and granule release in type I hypersensitivity?
Gastrointestinal tract
- Increased fluid secretion, increased peristalsis
Expulsion of gastrointestinal tract contents (diarrhoea, vomiting)
Airways
- Decreased diameter, increased mucous secretion
Expulsion of airway contents (phlegm, coughing)
Blood vessels
- Increased blood flow, increased permeability
Oedema, inflammation, increased lymph flow and carriage of antigen to lymph nodes
What is type II hypersensitivity?
Anitbody-mediated
Hours to days
Immune reactant - IgG or IgM
Depends on antigen type
Cell- or matrix- associated antigen
Effector mechanism - complement, FcR+ cells (phagocytes, NK cells)
Example - some drug allergies (eg. penicillin)
Cell-surface receptor
Effector mechanism - antibody alters signalling
Example - chronic urticaria (antibody to FαRIα)
What is type III hypersensitivity?
Immune complex
Hours to days
Immune reactant - IgG
Antigen - soluble antigen
Effector mechanism - Complement, phagocytes
Examples - Serum sickness, Arthus reaction
What are the consequences of type III hypersensitivity depending on route of administration of antigen?
Route
Resulting disease - site of immune-complex deposition
Intravenous (high dose)
Vasculitis - blood vessel walls
Nephritis - renal glomeruli
Arthritis - joint spaces
Subcutaneous
Arthus reaction - perivascular area
Inhaled
Farmer’s lung - alveolar/capillary interface
What is a type IV hypersensitivity reaction?
Cell-mediated
Delayed (days)
Depends on immune reactant:
Th1 cells:
Antigen - soluble antigen
Effector mechanism - macrophage activation
Example - contact dermatitis, tuberculin reaction
Th2 cells:
Antigen - soluble antigen
Effector mechanism - eosinophil activation
Example - chronic asthma, chronic allergic rhinitis
CTL:
Antigen - cell-associated antigen
Effector mechanism - cytotoxicity
Example - contact dermatitis
Why is there a delay in type IV hypersensitivity?
Reflects the need to recruit lymphocytes to the challenge site - 24-72 hours
- Antigen is introduced into subcutaneous tissue and processed by local local antigen-presenting cells
- A Th1 effector cell recognises antigen and releases cytokines which act on vascular endothelium
- Recruitment of T cells. phagocytes, fluid, and protein to site of antigen injection causes visible lesion
What is an example of a clinically significant type IV hypersensitivity?
Granulomatous inflammation in schistosomiasis
Granulomas form around schistosome eggs
Can lead to ascites, due to portal hypertension from granuloma blocking ducts in liver
What are the two types of immunodeficiency?
Primary (Genetic cause)
- e.g. chronic granulomatous disease
Secondary (Unrelated cause)
- HIV/AIDS
What is chronic granulomatous disease?
Genetic disorder affecting how phagocytes kill bacteria, fungi and parasites
4 major forms due to mutation in the different proteins that comprise the phagocyte oxidase system (gp91-phox, p22-phox, p47-phox, p67-phox)
Recessive sex-linkes (X-) CGD is due to mutations of gp91-phox (three forms depending on whether protein is absent, low, or nonfunctional: X91º X91- X91+)
What are the clinical signs and histopathology of chronic granulomatous disease?
Clinical signs
- Impetigo
- Skin and rectal abscesses
- Chronic lymphadenopathy
- Recurrent pneumonia
Histopathology
- Presence of granulomas (as you can’t kill the pathogen)
What is HIV/AIDS?
A retroviral infection leading to loss of CD4+ T cells
HIV infection - diagnosis of HIV infection, regardless of the stage of disease at diagnosis (i.e., HIV infection Stage 1, 2, 3 (AIDS), or unknown)
AIDS - diagnosed with Stage 3 HIV infection (AIDS), based on case definitions for adults, adolescents of children
What is the impact of secondary immunodeficiency on T cell regulation?
Reduced number of naive CD4 T cells results in deficiency in CD4+ T cells and their function
- Th17 - Extracellular bacteria, fungi, autoimmunity
- Th1 - Intracellular pathogens, autoimmunity
- Th2 - Extracellular parasites, allergy and asthma
- iTreg - Immune tolerance, lymphocyte homeostasis, regulation of immune responses
What is the impact of HIV associated immunodeficiency?
Normal CD4 count = 500-1500/mm3
Above 500 CD4 cells - no unusual infections likely to appear
200-500 - increased risk for shingles, thrush, skin infections, bacterial sinus and lung infections, and TB
<200 - increased risk for PCP (pneumonia)
<100 - increased risk of MAC and toxoplasmosis
<50 - increased risk for cytomegalovirus
How can cancers arise due to the extremes of B and T cell production?
Too much - Acute lymphoblastic leukaemia of B or T cell (B-ALL/T-ALL)
Too little - Insufficient T or B cell response to recogniser and kill the tumour cells
What is the result of the too much immune activity?
Autoinflammation - prolonged, unwanted innate immunity
Autoimmunity - loss/failure of self/non-self recognition
What is autoinflammation and some examples of it?
Prolonged, unwanted innate immunity
Familial mediterranean fever
- Most common in Jewish, Armenian, Turkish and Arab populations
- Mutation in FMF gene encoding pyrin
- Recurrent fever and peritonitis, chest pain and pleurisy
Neonatal onset multisystem inflammatory disease (NOMID)
- Enhanced IL-1 production
- Rash, fever, meningitis, joint damage, vision and hearing loss, and mental retardation, poor survival to adulthood
Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS)
Deficiency of the Interleukin-1 receptor antagonist (DIRA)
Behçet’s Disease
How are autoimmune diseases classified
By target organ, mechanism of destruction, mode of induction
How may autoimmunity be induced by infection?
Molecular mimicry
- A pathogen that expresses a molecule very similar to a self-molecule resulting in T and B cells not being able to tell the difference between the two and attacking both
Protein changes, cryptic antigens
- During infection some antigens or cell contents released that aren’t usually present which the immune system becomes polarised against
Superantigens
- Non-specifically bypass signals required for activation to activate T cells
Bystander activation
- Self-reactive cells are activated just as they are in the area
What are some therapeutic interventions in auto-inflammatory and autoimmune diseases?
Cytokine therapies
- anti-TNF
- anti-IL-1
Reducing lymphocyte activation
- Interfering with signalling pathways e.g. steroids
- Blocking cell surface receptors
- Active induction of ‘tolerance’
Killing the cells causing disease
- Anti-B cell antibodies (Rituximab)
How long do hypersensitivity reactions take to appear?
Type I - Minutes (degranulation of mast cells)
Type II - Minutes to hours (antibody induced cytotoxicity)
Type III - Hours (antibody binding to soluble antigen)
Type IV - Days (T cell mediated damage)
What are allergens?
Antigens which cause allergy are called allergens
Small proteins
No clear feature shared by all allergens but many are a specific type of enzyme called a protease
Exposure generally through inhalation, ingestion or administered as drugs
Exposure is too low dose of allergen
Most allergens promote a Th2 immune response
Why does an allergy occur?
It is a result of repeated exposure to a allergen which generates an IgE mediated immune response
What is atopy?
The term used to describe a predisposition for an IgE mediated immune response
What is the first step of forming an allergy?
Sensitisation - the first encounter with an allergen
An APC may pick up this allergen and present it on its surface to a T cell
This activates the T cell and causes it to become a Th2 cell which then produces type 2 cytokines (IL-4 and IL-13)
This will drive B cells to class switch to IgE
A memory pool of Th2 cells specific for this allergen will then form
What are the two key components to an allergic reaction?
IgE and mast cells
IgE binds to mast cells via the IgE receptor (FcεR1)
IgE not the most abundant antibody, but preformed and loaded onto mast cells makes this a really potent response
- Pre loaded
- Increases the half-life of IgE
What are some common allergens for systemic anaphylaxis?
Drugs, serum, venoms, food (e.g. peanuts)
Intravenous route (either directly or following oral absorption into the blood)
Response - Oedema, increased vascular permeability, laryngeal oedema, circulatory collapse, death
What are some common allergens for acute urticaria (wheal-and-flare)?
Animal hair, insect bites, allergy testing
Route of entry is either through skin or systemic
Response is local increase in blood flow and vascular permeability
What are some common allergens for seasonal rhinoconjunctivitis (hay fever)?
Pollen (ragweed, trees, grasses), dust-mite faeces
Route of entry is inhalation
Response is oedema of nasal mucosa and sneezing
What are common allergens in asthma?
Danders (cats), pollens, dust-mite faeces
Route of entry is inhalation
Response is bronchial constriction, increased mucous production, airway inflammation
What are common allergens in food allergy?
Tree nuts, shellfish, peanuts, milk, eggs, fish soy, wheat
Route of entry is oral
Response is vomiting, diarrhoea, pruritis (itching), urticaria (hives), anaphylaxis (rarely)
What are the two types of reaction in an allergic response?
Immediate and late reaction
What characterises an immediate reaction in an allergic response?
Characterised by release of mast cell mediators:
Histamine
- smooth muscle contraction of lungs, gut and blood vessels promoting oedema
- acts as a chemoattractant to other WBC - particularly neutrophils and eosinophils
- causes skin itching
Cytokines
- pro-inflammatory induce inflammation e.g. TNFα
Enzymes (e.g. Tryptase)
- activate complement
Th2 cytokines
- IL-4 to activate Th2 cells and IL-3 and IL-5 to induce eosinophil activation
Leukotrienes
- bronchial and gut contraction and chemotaxis of eosinophils and neutrophils
Prostaglandins
- vasodilation, increased vascular permeability and constriction of gut and bronchial smooth muscle
What is a late response in an allergic reaction?
Occurs hours after exposure
- Migration of leukocytes especially eosinophils
Eosinophils release peroxidase and other mediators which cause further tissue damage
What are the effects of mast cell degranulation?
Skin sensitivity - reddened and inflamed area resulting in itching
Airway sensitivity - results in sneezing and rhinitis or wheezing and asthma
Gastrointestinal tract - increased fluid secretion, increased peristalsis
- Expulsion of gastrointestinal tract contents (diarrhoea, vomiting)
Eyes, nasal passages, and airways - decreased diameter, increased mucous secretion
- Congestion and blockage of airways (wheezing, coughing, phlegm)
- Swelling and mucous secretion in nasal passages
Blood vessels - increased blood flow, increased permeability
- Increased fluid in tissues causing increased flow of lymph to lymph nodes, increased cells and protein in tissues, increased effector response in tissues, hypotension potentially leading to anaphylactic shock
What are the two types of allergic rhinitis (hay fever)?
Seasonal
- grass pollen is the most common allergen (May to July), but tree (February to June) and weed (June to September) pollens can also cause allergic reaction we know as hay fever
Perennial allergic rhinitis
- symptoms continue all year round and usually relate to indoor allergens, such as house dust mites, pets, including birds, or moulds
How can allergic rhinitis be treated?
Reduced exposure
Intranasal corticosteroids
Anti-histamines
Some instances - immunotherapy
What is the pathology of asthma?
Normal airway - open bronchiole with a thin wall
Asthmatic airway - thickened bronchiole wall and narrowed lumen
During an asthmatic attack, airway undergoes further restriction
Does histamine has as much of a role in asthma as other allergic diseases?
No
What is the treatment for asthma?
Reliever inhalers - Salbutamol (β2 agonist) Preventative inhalers - Corticosteroids Leukotriene receptor antagonists Theophyllines (inhibit leukotriene synthesis)
What is allergic eczema?
Itchy, dry, red and cracked skin
Genetic link - barrier dysfunction - protein called Filaggrin
Thymic stromal lymphopoietin (TSLP) - epithelial derived cytokine (perpetuates scratch response)
What are the treatments of allergic eczema?
Reducing scratching
Emollients (moisturising)
Topical corticosteroids
What is urticaria?
Raised red itchy bumps or wheals
Response to histamine release within the skin
What is the treatment for urticaria?
Trigger avoidance Anti-histamines Corticosteroids Leukotriene receptor antagonists Ciclosporin Omalizumab (anti-IgE therapy)
What is anaphylaxis?
Systemic response to an allergen via any exposure route
Rapid synthesis of prostaglandins and leukotriene
- Systemic vasodilation and increased vascular permeability
- Fluid enters extravascular space
- Fall in blood pressure
- Severe bronchiole constriction, oedema, and shock
Similar to systemic inflammation
What are the symptoms of anaphylaxis?
Swollen eyes, lips, hands and feet
Feeling lightheaded or faint
Narrowing of the airways, which can cause wheezing and breathing difficulties
Abdominal pain, nausea and vomiting
Collapsing and becoming unconscious
Anaphylaxis should always be treated as a medical emergency
What are some examples of type II hypersensitivity diseases?
Haemolytic disease of the foetus and newborn (HDFN)/Rhesus disease Drug induced haemolytic anaemia Goodpasture syndrome Myasthenia gravis Graves disease
What is drug induced haemolytic anaemia?
Binding of drugs to cells can induce an immune response
Antibodies recognise drug in combination with cells
Drug acts as an immunological hapten (binding creates a new antigen)
What is Goodpasture syndrome?
IgG antibodies recognise collagen within the kidney basement membrane
IgG binding to basement membrane results in complement activation
What is the treatment of Goodpasture syndrome?
Oral immunosuppressants
Plasmapheresis
What is myasthenia gravis?
Antibodies block the acetylcholine receptor at neuromuscular junctions preventing nerve impulse transmission
Presents as muscle weakness especially of the eyes and face but all muscles can be affected
What is the treatment of myasthenia gravis?
Pyridostigmine - blocks acetylcholinesterase which suppresses ACh Corticosteroids Immunosuppressants Thymectomy Biologics - B cell depleting therapy
What is Graves disease?
Antibodies bind to the thyroid hormone receptor causing activation of the receptor and increased thyroid hormone production
What is the treatment for Graves disease?
Thionamides
Radioactive iodine therapy
Surgery
What are the mechanisms of type II hypersensitivity and the associated immunoglobulins?
Antagonism - IgG4, IgG1
Signalling - IgG
CDC - IgG1, IgG2, IgG3, IgM
ADCC - IgG1, IgG3
How do immune complexes cause type III hypersensitivity?
Antibody-antigen complexes if not removed deposit in tissues resulting in tissue damage by complement or phagocytes
IgG mediated
What causes the immune complexes in type III hypersensitivity?
Infectious Ags (e.g. Streptococcal or Hep B infection) Environmental Ags (e.g. Fungal spores - Farmer's lung) Autoantigens (e.g. DNA)
Normally removed by complement
Constant activation of complement results in tissue damage
IC disease prevented by avoidance of trigger antigen however this is not possible in the case of autoantigens
Where might immune complexes deposit?
Kidneys - glomerulonephritis
Blood vessels - vasculitis
Synovial joints - arthritis
What are some examples of type II hypersensitivity reaction diseases?
Systemic lupus erythematosus
Arthus reaction
Serum sickness
What is systemic lupus erythematosus?
Autoantigen is DNA
Failure of clearance of apoptotic cells
Anti-DNA antibodies result in systemic damage to tissues - kidney, skin, heart, joints
What is the treatment of systemic lupus erythematosus?
Alkylating agents - e.g. cyclophosphamide suppress DNA synthesis
Immunosuppressants
Biologic therapies - B cell depleting therapy Rituximab and an antibody which prevents B cell activation - Belimumab - both aim to reduce amount of antibody in blood
What is Arthus reaction?
Complication of vaccination e.g. tetanus if delivered in a bolus (intradermal vs intramuscular administration)
What is serum sickness?
Non-blanchable, painful purpuric patches
Response to anti-venom to treat snake bite
Antibodies within the circulation bind to antibodies present within the anti-venom
IC formation in response to injection of foreign protein or serum
Self resolving but future prevention - avoidance
What immune response characterises type IV hypersensitivity?
Th1 immune response - doesn’t involve IgE
Present as allergic reactions but delayed - removal of trigger, symptoms alleviated
Autoimmune disease - autoantigen exposure results in chronic disease
What are some examples of type IV hypersensitivity?
Delayed-type hypersensitivity:
Antigen
- Proteins - insect venom, mycobacterial proteins (tuberculin, lepromin)
Consequence
- Local skin swelling - erythema, induration, cellular infiltrate, dermatitis
Contact hypersensitivity:
Antigen
- Haptens - pentadecacatechol (poison ivy), DNFB
- Small metal ions - nickel chromate
Consequence
- Local epidermal reaction - erythema, cellular infiltrate, vesicles, intraepidermal abscesses
Gluten-sensitive enteropathy (celiac disease):
Antigen
- Gliadin
Consequence
- Villous atrophy in small bowel, malabsorption
Where does contact hypersensitivity take place?
Superficial skin
Hapten binds to Langerhans’ cells in skin which stimulates Th1 response (IFNγ)
What are the exposure stages of the delayed-type hypersensitivity?
Primary exposure - sensitisation
- DC induce memory cell production - Th1 type cells
Secondary exposure - delayed type hypersensitivity response
- Memory Th1 cells become activated to produce
IFN-γ and TNFα - tissue destruction and inflammation
Il-2 - activate T helper and T cytotoxic cells
Chemokines - cell recruitment esp. macrophages
Il-3 and GM-CSF - production of monocytes and macrophages
What is contact sensitisation?
Delayed response - cells must migrate to the area Compounds include - Nickel - Poison oak/poison ivy Treatment - trigger avoidance
What is the tuberculin test?
Clinically important test to detect infection with Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB)
Antigen is injected into subcutaneous tissue - positive result causes visible lesion
What is rheumatoid arthritis?
Most common autoimmune disease - 1% of the population - spectrum of diseases
Dysregulation in cytokine production - TNFα and IL-6
Immune cell mediated joint tissue destruction
Autoantibodies - rheumatoid factor which recognise IgG and anti-citrullinated peptide antigen (ACPA) antibodies which target proteins modified by citrullination
What is the treatment of rheumatoid arthritis?
NSAIDs
Corticosteroids
Biologic therapies (e.g. anti-cytokine therapies)
What is multiple sclerosis?
Affects 1 in 1000 people in Northern Europe and central North America
Multiple forms of disease
Immune cell mediated destruction of myelin resulting in paralysis
Diagnosis is complex - neurological examination - physical and visual changes, MRI for changes in brain tissue, analysis of CSF for presence of autoantibodies
What is the treatment for multiple sclerosis?
Immunosuppression
Biologic therapies - anti-cytokine and cell function modifying mAbs
What is inflammation?
The body’s immune systems response to an irritant
Acute - occurs quickly but is resolved
Chronic - an illness persisting for a long time or constantly recurring
What is an NSAID?
Non-steroidal anti-inflammatory drug
Widely used to relieve pain, reduce inflammation and bring down a high temperature
What is a metabolite?
A substance essential to the metabolism of a particular metabolic process
What are the features of inflammation?
Redness Swelling Heat Pain Loss of function
