Type 1 Diabetes topic Flashcards

Question

what is insulitis?

Answer 1

Insulitis is inflammation of the insulin producing cells within the islet. This is where we start to see beta cell injury and the destruction of the insulin producing cells. Start to see the loss of beta cell mass

Answer 2

the cells of the immune system are invading these cells, they are passing all the other cells and just targeting the islet cells and destroying the beta cells. - Once the beta cells are destroyed that inflammation within the islets becomes a mix of cytokines, of cell destruction, other cells of the immune system are drawn into the islets . - Once process starts it is unstoppable, once start to lose the beta cell mass this is the point you trigger type 1 diabetes and lose insulin producing capacity.

Answer 3

1) When things start to go wrong in the insulitis and you see the destruction of beta cells, they don’t have a lot of ability to defend themselves from the damage that’s coming 2) Beta cells controls your blood glucose and they are sensitive to any changes In your blood glucose concentration. This is because they contain a range of glucose transporters, glycolytic enzymes, secretory granule proteins, processing enzymes that no other cell type in your body has. Because they are highly specialised, they have low defence 3) The beta cells are dependent on other cells within the islet. The beta cells forming the core of the individual islet of Langerhans. They communicate with the other cells in the pancreas. They are dependent on the other cells in the islet. 4) They express protein that are triggers of cell death: FasL which is a death receptor protein and the FasR receptor. When you are healthy and the pancreas works normally, you get a process of apoptosis within the pancreas, beta cells die, gets replaced by new beta cells and things like FasL play an important role in that. When you see the destruction of beta cells in insulitis, they release the components of the beta cell. The fact that beta cells have both ligands under receptors for triggering apoptosis makes them vulnerable

Answer 4

-low anti-oxidant defences highly specialised cellular functions dependent on other cells within an islets fasL/fasR expression

Answer 5

``` vaccination viral infections beta cell low defences environment interactions with lymph nodes ```

Answer 6

pre- diabetic: - If you have a healthy pancreas, as you eat your breakfast in the morning, the insulin increases and the decreases. Same applies lunch dinner. - See a loss of insulin production as the beta cells starts to die out. diabetes: will experience signs and symptoms. gets diagnosed with diabetes when the beta cell mass is below 10%. most o the beta cells has been destroyed

Answer 7

- The beta cells will try and produce more beta cells to try and increase the production of insulin - In a normal healthy pancreas, some of the beta cells are dying because they have come to the end of their natural lifespan. They die by apoptosis and the white blood cells digests the products. - Neogenesis- the birth of new beta cells within islets - Patients with T1D will still make beta cells throughout their lifetime

Answer 8

All insulin in UK is 100iu/ml

Answer 9

- Human – genetically engineered in yeast or E.coli. important as you will get a consistent insulin activity and purity also gives consistent insulin activity. Must ensure you get a consistent affect from the insulin injections - Range of activities, different combinations depending on the patients metabolism. And how they respond to carbohydrates.

Answer 10

Once daily injection – mixture of very long and very short acting analogues. Doesn’t give any fine tuning for patients depending on what they eat or what exercise or what age Twice daily injections – most common regimen for patients mix of short and long acting insulins Multiple daily injections (before meals) of short acting insulin – most common regiment for patients. and one daily injection of long acting insulin (Basal Bolus), gold standard what NICE guidelines say what we should give to patients to ensure what gives the most tight glycemic control for the patients and see if they get used to it.

Answer 11

Lispro, Aspart, Novorapid, Glulisine

Answer 12

NPH/isophane (pre-mix of NPH/regular)

Answer 13

Glargine, Detemir, Lantus, Levimir

Answer 14

Need to know the speed of onset of activity of the insulin and the duration of that activity - When you inject a patient with a rapid onset insulin it acts very quickly and a sharp decline. It wouldn’t come down to no insulin effect. - So when you are taking insulin you have to think about how much insulin is onboard already - Intermediate insulin has a slower onset but lasts above 14 hrs - Long insulin has a slow onset of action and can last up to 16 hours. May take several hours to get to its maximum effect. Need to know these to calculate the best combination

Answer 15

- NICE guidelines states try to get all patients on Basal Bolus regime. - This is the regime that has insulin production that looks most like a healthy pancreas - Take an insulin before a meal, so there is a sharp peak of activity and drop of from the rapid insulin. once a day sometimes 2x a day you have your long lasting insulin as there is a slower onset of action across the day - It is a combination of taking a short acting analogue with your meals and a long acting analogue that sits in the background just regulating your blood glucose concentration across the course of 24 hrs. - When you are taking your short acting insulin, you will already have a little bit of insulin on board from your long lasting insulin from the day before. And you will have a little bit if activity from the long lasting insulin that you have just taken. - Need to know how much insulin to inject with the meals so calculating the right amount of insulin to inject with meals. This is a challenge of the Basal Bolus regime. Takes a lot of trial and error and so hard to get control.

Answer 16

- Twice daily injections – mix of short and long acting insulins - Multiple daily injections (before meals) of short acting insulin and one daily injection of long acting insulin (Basal Bolus)

Answer 17

``` Where do you start from: advise to tell patients where to start from when they calculate how much insulin they need to inject. After this trial and error can alter the meaurements for individual patients. 10g of carbohydrate = 1 unit of insulin 10g of carbohydrate = 1 carbohydrate portion (CP) = 1 unit of insulin ```

Answer 18

- Portion/ serving size - Total carbohydrate (not of which sugars) - Raw or cooked - Glycaemic index? The immediate impact of something on your blood glucose level. Brown rice or brown pasta will have a lower glycaemic index than white pasta or rice.

Answer 19

- Genetic risk factors - HLA molecules - Environmental triggers

Answer 20

- HLA antigens are glycoproteins found on the cell surface and present antigens that are involved in the immune process - There are two classes (Class I and Class II) which differ in their structure - Class I molecules are found on ALL nucleated cells

Answer 21

- Class II molecules are ONLY found on antigen-presenting cells – such as macrophages - Class II molecules bind foreign antigen peptides and present them to T-helper lymphocytes - There are three types of class II molecule – DP, DQ and DR. Each Class II sub-type is sub-classified by numbers

Answer 22

N-Nitro Compounds: - Streptozotocin - Nitrosamines/Nitrosamides - Alloxan - Vacor

Answer 23

- Muumps - Rubella - Enteroviruses/Coxsackie B virus - Rotaviruses - Cytomegalovirus - Epstein-Barr virus

Answer 24

Child/parent separation - Difficult adaptation - Stress during pregnancy

Answer 25

- Bovine milk/short breastfeeding - Cereals - High protein content - Vitamin deficiency

Answer 26

- High energy intake and weight gain - Beta-cell stress – “Accelerator hypothesis”- type 1 diabetes accelerates and the beta cell fails - Increased insulin resistance - Puberty

Answer 27

- Mimic autoantigens- proteins from the viral coat are presented that mimic auto antigens. They are presented on the surface of cells that triggers the disease process - Rubella Virus- if you present these proteins in combination with that high risk HLA molecule are they presenting something to the immune system that’s triggering beta cell destruction by looking like an auto antigen

Answer 28

- Autoreactive T-cell activation- may be a trigger to start the insulitis process that becomes amplified in patients who are at risk of T1D - beta cell killing- the beta cells presenting antigens on their surface that causes their own destruction and they’re are presenting them because of viral infection - Enterovirus (Cocksackie B)- they infect beta cells, they cause the presentation on the surface of the beta cells of proteins that cause auto reactive t-cell activation. If it triggers cytotoxic t-cells to kill your b-cells , if you get an amplification of the level of cytotoxins and interleukins-2. Because of the presentation on the surface of beta cells, that triggers the beta cell destructive process to insulitis to beta cell mass loss to diabetes - Rotavirus

Answer 29

- The increase production of cytokine, mainly interferons. - In interferon production we see a loss of:  Inhibition of insulin production  HLA genes and interferon- if you have high HLA molecules, this causes an amplification of the immune system. The loss of beta cell, attack on beta cells into insulitis then T1D.  Cytomegalovirus- stimulate the production of cytokines  Epstein-Barr Virus- stimulate the production of cytokines

Answer 30

predominantly linked to T1D - Coxsackie B virus cultured from pancreas of T1D patient - That virus caused diabetes when injected into mice - 30% of newly diagnosed diabetic patients have IgM antibodies to Coxsackie B virus - 5% of controls have IgM antibodies to - Coxsackie B virus - 27% of newly diagnosed diabetic children have enteroviral RNA in serum at diagnosis. - 5% of controls have enteroviral RNA in serum at diagnosis. - 51% of pre-diabetic children have evidence of enteroviral infection in the 6 months before autoantibody seroconversion - 28% in controls.

Answer 31

Promotes glucose uptake into muscle cells • Inhibits glucose production from the liver • Promotes glucose uptake from adipocytes • Inhibits lipolysis • Promotes glycogen synthesis and storage

Answer 32

glucose low) – blood glucose drops below 3mmol/l → alpha cells in the pancreas start releasingglucagon→ glucagon targets the liver and stimulates it to release glucose into the bloodstream → blood glucose goes up

Answer 33

blood glucose high) – blood glucose goes up 10mmol/l beta cells release insulin from the pancreas → insulin targets all cells, including fat cells → fat cells take up glucose from the bloodstream→ bloodglucose goes down

Answer 34

* HbA1c is a huge marker of glucose concentrations * We all have some glucose that is stuck to our red blood cells * If you don’t have diabetes, you will have a low HbA1c value * If you have diabetes, you will have a high HbA1c value * Red blood cells live for around 3 months * It tells us if your blood glucose has been low or high in the past 3 months

Answer 35

Compared to other cells, beta cells have quite low anti-oxidant defenses • Why do they have this? → because of the highly specialized cellular functions of the beta cells • Beta cells are dependent on other cells within an islet • They express FasL/FasR (FasL → apoptosis function, FasR → receptor function) – when beta cells apoptose, they release their cell content, which are received by FasR (receptors) of the beta cell, which cause a chain reaction of apoptosis

Answer 36

* We start to see the loss of insulin production * We start to see the loss of responsiveness * Increased levels of beta cell death

Answer 37

HLA antigens are glycoproteins found on the cell surface that are involved in the immune process • There are 2 classes (class I and class II) which differ in their structure • Class I molecules are found on ALL nucleated cells • In a normal healthy immune system, MHC class I is found on the outside of the cells and they present antigens to the immune system • But in patients with T1DM, this process goes wrong (something to do with the genetic shape of those molecules triggers T1DM) * Class II molecules are ONLY found on antigen presenting cells – such as macrophages • Class II molecules bind foreign antigen peptides and present them to T-helper lymphocytes * There are 3 types of class II molecule – DP, DQ and DR * Each class II sub-type is sub-classified by numbers * In a normal healthy person, your MHC class II molecules present antigens to CD4+ T helper cells * These helper cells are activated and release IL-2, which starts to stimulate the immune system * CD8+ cytotoxic T cells start to get activated, which will help fight infection * In T1DM, this goes wrong → something is being presented to the immune system that starts to stimulate and recruit other cells from the immune system into the pancreas, into the islets, where we start to see insulitis * This leads to beta cell death, which accelerates the recruitment of immune cells even more which leads to even more beta cell death – it is now out of control and we cannot stop it

Answer 38

a chronic disorder characterised by high level of blood glucose that result from wither inadequate insulin production or resistance of the bodys cells to the action of insulin type 1 diabetes is where insulin is destroyed by the beta producing cells type 2 diabetes is where there is insulin resistance and gradual insulin deficiency.

Answer 39

starts with the food that we intake and that gets broken down by our digestive system into glucose. following a meal the glucose concentration in our blood rises. beta cells of the pancreas islets start secreting insulin which is a peptide hormone that binds to the insulin receptor and stimulates glucose uptake by our cells. under the influence of insulin, liver and skeletal muscles store absorbed glucose in the form of glycogen. many other cells quickly break down absorbed glucose to make ATP

Answer 40

the alpha cells of the pancreatic islets release a different peptide hormone that is glucagon which has opposite effect of insulin. for example when it acts on the liver, it causes a breakdown stored glycogen into glucose which is then released into the blood stream.

Answer 41

insulin can be reproduced by recombinant DNA technology using bacteria or yeast. the amino acid sequence of human insulin can be altered to produce insulin analogs with different onset and duration of action. because insulin is a polypeptide, it is susceptible to degradation in the GI tract. therefore in order for it to be effective it is typically administered by subcutaneous injection insulin preparations are generally divided into 3 major categories depending on how quick and how long they work

Answer 42

preparations that fall into this category are insulin lispro insulin, aspart and insulin glulisine which are rapid acting producing peak effects in as quickly as 30 mins and duration of action of up to 5 hrs another analog belonging to this group is regular insulin which is considered as short acting with peak effect as quickly as 2 hrs and duration of action less that 8 hours the insulin molecules naturally stick together forming hexamines that is 6 insulin molecules bound together. theses hexamines are too large to cross from the subcutaneous tissue into the bloodstream therefore they must be separated into single molecules before absorption can occur it was done by altering the amino acid sequence of insulin molecules to make them less likely to aggregate which results in analogs with faster absorption and more rapid action

Answer 43

hypoglycaemia | lipodystrophy- can develop at the site of repeated insulin injection

Answer 44

preparation that falls into this category is NPH insulin also known as isophane insulin. NPH has a little slower onset of action, it produces its peak effects around 6 hrs mark and lasts for 18 hrs. these longer lasting effects are achieved by the addition of zinc and protamine to regulate insulin which results in a complex that is less soluble. the final outcome is delayed absorption and therefore longer duration of action

Answer 45

preparations that fall into this category is detemir with a peak effect between 6-8 hours and a duration of approx 24 hrs glargine doesnt produce peak effect due to its steady delivery of insulin over a period of approx 24hr degludec doesnt produce peak effect due to steady release and has a duration greater than 24 hrs all these long lasting effects are due to the modification of the insulin molecule. in the case of detemir, the fatty acid chain was added to the insulin molecule which allows it to bind to albumin and thus slows down its release into the blood stream glargine is modified to have low solubility at neutral pH which causes it to precipitate in the subcutaneous tissue that slowly releases insulin into the bloodstream degludec was designed from a long chain of hexamines in subcutaneous tissue that serves as a depot from which insulin is continuously and slowly released

Answer 46

pancreatic cells not only secrete insulin but also another peptide hormone called amylin. amylin job is to delay the gastric emptying to suppress postprandial glucagon secretion and to promote satiety, the only amylin mimetic thats currently in the market is PRAMLINTIDE. allows insulin to be reduced however the risk of hypoglycaemia is still there and other side effects includes nausea and weight loss

Answer 47

mechanism of glucose dependent insulin secretion from pancreatic beta cells the available glucose enters beta cella through glucose transporter 2 once inside the cell glucose gets metabolised to create a bunch of ATP. the rising levels of ATP lead to inhibition of ATP-sensitive potassium channels thus blocking the inflow of potassium ions this leads to depolarisation of the cell membrane which triggers activated voltage gated calcium channels and then an influx of calcium increased levels of calcium mediate fusion of insulin containing vesicles with the membrane leading to insulin release

Answer 48

they bind to and inhibit the activity of ATP-sensitive potassium channels. this just like incoming glucose triggers membrane depolarisation, calcium influx and ultimately secretion of insulin other actions includes increased sensitivity to beta cells to glucose and reduced hepatic glucose production. examples includes glimepiride, glyburide, and glipizide side effects includes hypoglycaemia and weight gain because they are protein bound and most are metabolised in the liver cytochrome p450 enzymes and they often tend to react with other drugs.

Answer 49

also stimulate insulin secretion from pancreatic beta cells however they achieve this by binding to ATP sensitive potassium channels at different site and different kinetics they have a faster onset of action and shorter duration. good for patients who have postprandial hyperglycaemia examples includes Nateglinide and Repaglinide side effects includes hypoglycaemia and weight gain

Answer 50

MoA is not understood but the main blood glucose lowering activity appears primarily through reduction of hepatic glucose production they appear to slow intestinal absorption of glucose and increase insulin sensitivity which enhances peripheral glucose uptake examples include metformin side effects include nausea, vomiting loss of appetite and weight loss because metformin increases hepatic uptake of lactate it may increase the risk of lactic acidosis especially in patients who have organ dysfunction such as heart failure/ renal impairment

Answer 51

works by selectively activating nuclear receptor called PRAP- gamma.when activated this receptor binds to DNA triggering expression and repression of specific gene encoding proteins that regulate glucose and lipid metabolites as well as insulin signalling transducers also promotes fatty acid uptake and utilisation of adipocytes. this decreases in fatty acid concentration which leads to increased uptake of glucose. examples includes piolitazone and rosiglitazone side effects includes lowering blood glucose levels and also increase HDL

Answer 52

inhibits glucose transporters located in the proximal convoluted tubules of the kidney that is responsible for glucose reabsorption. inhibition of sodium glucose co-transporter leads to increased urinary glucose secretion and thus reduced levels of blood glucose this increased level in glucose and sodium in the urine generates mild osmotic diuresis which may contribute to small reduction in blood pressure and weight loss. drugs includes conagliflozin and dapagliflozine side effects includes thirst, increased urination and increased risk of UTI

Answer 53

alpha glucosidase is an enzyme that is located in the intestinal brush border that is responsible for breaking down carbohydrates into simple sugars such as glucose when alpha-glucosidase inhibitors blocks this enzyme, the absorption of glucose is delayed resulting in lower postprandial glucose levels example include acarbose and miglitol side effects includes GI effects

Answer 54

are a group of metabolic hormones that are secreted in from the gut in response to food ingestion and their job is to stimulate the pancreas to produce more insulin. the two primary incretin hormones are glucagon like peptide-1 and GIP by increasing the concentration of these hormones would benefit the patient who have diabetes however their actions are limited and are rapidly inactivated by the enzyme DPP-4. to counter this drugs such as exanatide and liraglutide that are resitant to DPP-4 degradation. in addition to stimulating GLP-1 mimetics also slow down gastric emptying and promotes satiety so can experience wight loss, GI problems and nausea

Answer 55

another way to enhance incretin hormones is to simply inhibit DPP-4 enzyme which is responsible for the activation of GLP-1 and GIP by romoting the activity of GLP-1 and GIP. this increases insulin secretion decreases gastric emptying and reduce glucagon release. examples of drugs in this class is Alogliptin, Linogliptin, saxagliptin and sitagliptin

Answer 56

Rapid onset of thirst, polyuria, unexplained weight loss AND Finger-prick blood glucose level > 11 mmol/L

Answer 57

multiple daily injection basal-bolus insulin regimens which comprises of 2 components: 1) rapid acting bolus therapy which is injected BEFORE meals. it serves the purpose as being bolus or mealtime insulin replacement. discourage use after meals 2)long acting basal insulin therapy- nice 1st line is insulin detemir twice daily in the morning and evening. this allows for constant levels of insulin to meet background metabolic demands and not causing nocturnal hypoglycaemia

Answer 58

- NICE guidelines states try to get all patients on Basal Bolus regime. - This is the regime that has insulin production that looks most like a healthy pancreas - Take an insulin before a meal, so there is a sharp peak of activity and drop of from the rapid insulin. once a day sometimes 2x a day you have your long lasting insulin as there is a slower onset of action across the day - It is a combination of taking a short acting analogue with your meals and a long acting analogue that sits in the background just regulating your blood glucose concentration across the course of 24 hrs. - When you are taking your short acting insulin, you will already have a little bit of insulin on board from your long lasting insulin from the day before. And you will have a little bit if activity from the long lasting insulin that you have just taken. - Need to know how much insulin to inject with the meals so calculating the right amount of insulin to inject with meals. This is a challenge of the Basal Bolus regime. Takes a lot of trial and error and so hard to get control.

Answer 59

- Twice daily injections – mix of short and long acting insulins - Multiple daily injections (before meals) of short acting insulin and one daily injection of long acting insulin (Basal Bolus)

Answer 60

The pancreas is responsible for the maintenance of BG levels. Once food is digested, the pancreas responds to reduce blood glucose (BG) levels.

Answer 61

When BG is low (< 3 mmol/L), alpha -cells release glucagon and the liver releases glucose into the blood.

Answer 62

When BG is high (> 10 mmol/L), ß-cells release insulin and fat cells take in glucose from the blood.

Answer 63

Ideal BG level = 4-6 mmol/L.

Answer 64

ß-cells, alpha-cells, delta -cells and pp-cells.

Answer 65

ß-cells are extremely sensitive to changes in BG, secreting active (fully-processed) insulin in response to elevated BG. Insulin is a peptide hormone produced by ß-cells. Its effect is to decrease BG levels by taking glucose into cells for storage or use. It also uptakes fats and amino acids. It blocks synthesis of glycogen, glycogenolysis and gluconeogenesis. storage or use. It also uptakes fats and amino acids. It blocks synthesis of glycogen, glycogenolysis and gluconeogenesis. T1-diabetics do not produce endogenous insulin.

Answer 66

Glucose enters the cell via GLUT-2 transporter. It is metabolised by glucose kinase (rate limiting step) which raises intracellular ATP levels. ATP-sensitive K channels close, causing the depolarisation of the membrane and voltage-gated Ca channels to open. A wave of calcium releases insulin into the bloodstream.

Answer 67

In Type 1 DM, there are no ß cells in the islet; immune cells (seen as black dots in the picture below) have destroyed ß-cells, but alpha cells, delta cells, lymph nodes are unaffected. With T1DM, there is no remission; it is a constant immune attack. Insulin-dependent diabetes mellitus (IDDM) has an early/juvenile onset and accounts for 5-10% of all diabetes cases. It is important to note that in Type 2 DM, ß cells are still present, but in smaller concentrations & structure of islet is disrupted.

Answer 68

- hereditary - auto antibodies/HLA molecule - environmental triggers

Answer 69

If your father has T1DM, there is an 8% risk of developing it; if your mother has T1DM, there is a 2% chance of developing it. If both parents have T1DM, there is a 30% chance of developing T1DM.

Answer 70

HLA antigens are responsible for presenting foreign bodies to the immune system. Class I molecules are found on ALL nucleated cells. Class II molecules are ONLY found on antigen-presenting cells – such as macrophages. There are 3 types of Class II molecules (DP, DQ, DR) which bind foreign antigen peptides and present them to the T-helper lymphocytes. In T1MD, there is a breakdown of immunological self-tolerance to ß cells, with early involvement of APCs. These recruit T cells (CD4+) which leads to targeted ß-cell destruction and autoimmunity, by the release of inflammatory mediators such as IL-2. Recent studies have confirmed strong associations between T1D and HLA genes. However, there are at least 47 non-HLA genetic factors are believed to contribute to the development of T1D; these genes include the INS gene, responsible for insulin production and metabolism), genes responsible for ß-cell apoptosis protection & many other genes related to immunity.

Answer 71

ß cells have low antioxidant protection, so N-nitro compounds (those containing ≥ 1 NO2 groups). • Nitrosamines (found in smoked meats) may act as triggers to T1DM. • Streptozotocin, nitrosamines, alloxan & vacor directly cause ß cell death, leading to further apoptosis/necrosis.

Answer 72

Some viruses and bacteria cause an autoimmune response by a variety of mechanisms. • The entero-& rotaviruses cause autoreactive T-cell activation, leading to ß cell death; 51% of pre-diabetic children have evidence of enteroviral infection in the 6 months before autoantibody seroconversion. • The Mumps virus moves through the pancreas and causes cytolysis of ß-cells, the contents of which can be released to APCs, inducing autoimmunity by the recruitment of T cells. • The Rubella virus presents foreign bodies on the surface of the body’s cells, prompting an immune response & killing the cell. Maternal rubella (where the mother contracts rubella during pregnancy) increase risk of child. • Cytomegalovirus and Epstein-Barr Virus (EBV) inhibit insulin production and a protein from the EBV (EBNA2) binds to multiple locations along the human genome that are associated with these autoimmune disorders, promoting interferon (IFN-α) production. • Streptomyces in the diet may contribute to the cause of type 1 diabetes.

Answer 73

Child/parent separation, difficult adaptation and stress during pregnancy can lead to autoimmunity.

Answer 74

* A high energy intake (and associated weight gain) can overwork ß cells; this is the accelerator hypothesis where the increasingly 'obesogenic' environment promotes insulin resistance – this mechanism is more closely attributed with T2DM * If one is celiac and eats gluten, they have an increased T1D risk. * Vitamin D deficiency may contribute; intake is generally decreasing in populations.

Answer 75

* A higher maternal age (older mother) * Gestational infections (e.g. maternal rubella) * Heavier birth weight * Birth order (i.e. being first born)

Answer 76

ß-cells have low anti-oxidant defences, so once destruction begins, they are very vulnerable. ß-cells are dependent on other cells within the islet, so when some start to die, others trigger apoptosis. In addition, necrosis (attack by immune cells) depletes ß-cell numbers. The Fas/ Fas-ligand (FasL) interaction is a common effector mechanism of β-cell apoptosis and islet injury under inflammatory conditions; FasL is an apoptosis-inducing surface molecule that triggers cell death by binding to Fas. Active autoimmunity is associated with increased ß-cell replication & increased Reg gene/protein expression because ß-cell proliferation is caused by: • Adaptive response to damage or loss of ß cells – because other ß-cells are destroyed, more are created. • Response to increased metabolic demand • Non-specific effect of stress

Answer 77

There is a genetic predisposition to T1DM, however environmental modifiers must be present to trigger a reduction in ß-cell function and initiate ß-cell pathology. The pathology stage is where ß-cells are attacked by autoantibodies to insulin: GAD65 and IA-2. This is known as insulitis. The pre-diabetes stage, where over half of ß cells are lost, is where the first phase of insulin response; IV glucose can be given and there is no insulin response seen. The progression to diabetes is seen when there is a loss of oral glucose tolerance.

Answer 78

test is where no food or drink is consumed for 8-12 hours before the test.

Answer 79

is where no food or drink for 8-12 hours, then a drink containing 75g of glucose is ingested; the test is taken after 2 hours. Random plasma glucose (RPG) test result is when a test is performed with no dietary modifications, and the glucose concentration ranges are the same as OGTT.

Answer 80

measures average blood glucose levels over a period of 2-3 months, indicating the development and management of diabetes. It allows HCPs to change regimes if treatment is not effective.

Answer 81

RPG level of 11.1 mmol/L is indicative of diabetes

Answer 82

* The body is unable to utilise glucose or store glucose as fat, so hunger signals are continually sent out; polyphagia (increased hunger), unexplained weight loss and fatigue are be seen. * Polydipsia (increased thirst) is experienced, in response to highly elevated BG levels; osmotic pressure drives the body to dilute the blood. * Polyuria (frequent urination) is experienced as a result of increased water intake, but this allows high levels of glucose excretion. * Osmotic changes in the lens of the eye cause blurred vision in one or both eyes * High BG conc causes BP to drop, which may cause dizziness * A high BG conc creates the ideal environment for yeast infections, so genital itching may be reported * Slow wound healing is experienced as a result of nerve damage, caused by high BG levels. the patient may not notice an abrasion so re-infection is likely.

Answer 83

Poor glycaemic control (swings in BG levels) leads to a number of complications. Complications are seen more frequently in poorly-controlled T1DM. All mechanisms act via mitochondrial overproduction of superoxide (ROS), as a result of excess glucose metabolism: • Increased glucose flux through polyol pathways causes vascular & nerve damage • Increased formation of advanced glycation end products (AGEs), where glucose-derived peptides cross-link, alters the structure of proteins • Activation of PKC isoforms leads to increased membrane permeability, synthesis of basement membrane and raised levels of vasoactive hormones which raises the blood pressure. • Over-activity of the hexosamine pathway, where PAI-1 (a protein involved in inappropriate blood clotting) is activated leading to CV events * T1DM sees higher prevalence of liver, pancreas, colon, rectum, breast and bladder cancers; treatment with insulin is also associated with an increased risk whereas metformin is associated with a lower risk. * Being diabetic causes difficulties in conceiving and increases the risk of miscarriage. During pregnancy, there is an increased risk of congenital malformations by 6-10-fold. * Living with a chronic disease increases likelihood of depression; in T1DM, the risk is doubled. Depression decreases compliance to treatment & management of the condition; patients are less likely to have a healthy lifestyle or monitor their blood glucose as often as necessary. There is an association between depression and a spiralling drop-off in glycaemic control.

Answer 84

Complications can be divided in macro- and micro-vascular sub-categories. As the name suggests, macrovascular relates to large blood vessels and microvascular relates to smaller blood vessels.

Answer 85

Diabetes is also the leading cause of blindness. Eye tests should be offered to patients annually. Background retinopathy is the first stage of retinopathy, characterised by microaneurysms. 1/3 of patients will develop sight-threatening retinopathy, which is asymptomatic and can only be spotted at diabetes clinic. In retinopathy, venous irregularities, blot haemorrhages and cotton-wool spots are seen. The progression of retinopathy can be delayed, but not reversed, by tight glycaemic control & BP control. Diabetes also increase the rate of age-related cataract formation, and the prevalence of glaucoma is increased. Hyperglycaemia alters the osmotic pressure within the lens leading to refractory defects and increases the risk of infection.

Answer 86

Diabetes can lead to renal failure and as a result, 20-45% of all kidney transplants are due to diabetes. There are 4 stages of nephropathy (microalbuminuria, persistent proteinuria, renal impairment, and stage 4 chronic kidney disease) characterised by the increase of albuminuria, serum creatinine, BP & decrease in renal function (eGFR) decreases as it progresses. Nephropathy should be monitored by assessing serum creatinine & eGFR annually. Nephropathy is tested for using a urine dipstick to assess proteinuria. Abnormal levels of protein may indicate kidney damage.

Answer 87

Neuropathy is damage to the nerves. This may be peripheral (where damage to long nerve axons causes sensory loss) or autonomic (gastrointestinal, cardiovascular, genitourinary, musculoskeletal, retinal) neuropathy. Peripheral neuropathy is the most common form. Paralysis of the bladder is a common symptom of this type of neuropathy. When this happens, the nerves of the bladder no longer respond normally to pressure as the bladder fills with urine. As a result, urine stays in the bladder, leading to urinary tract infections. 50-60% of men with long-standing diabetes are affected with erectile dysfunction, as a result of autonomic neuropathy. Improving glycaemic control, reducing alcohol and drug intake can reduce erectile dysfunction. Diarrhoea and constipation can occur when the nerves that control the small intestine are damaged. Sometimes, gastric motility is affected; the ability to move food through the digestive system is impaired, causing vomiting and bloating. Charcot foot is where the foot’s structural integrity is lost and the shape/ posture of the foot is altered. Pain may be experienced in peripheral neuropathy. It can be managed by Amitriptyline (10-75mg daily) or Duloxetine (60-120mg daily). If these are unsuccessful, pregabalin (150-500mg daily) or tramadol (200mg daily) should be used following referral to

Answer 88

Retinopathy Nephropathy Neuropathy

Answer 89

cardiovascular foot problems Hyperosmolar hyperglycaemic state (HHS)

Answer 90

Diabetes is the leading cause of heart disease, conferring a 2-4-fold increase risk of MI & stroke in men); 10-fold in women. Patients can reduce their risk of CV events by maintaining tight glycaemic control and reducing cholesterol levels. Lifestyle modifications include stopping smoking, exercising and eating a balanced diet. If patients have hypertension, an ACE inhibitor/ ARB can be given; renal function and electrolyte levels should be monitored.

Answer 91

15% of people with diabetes develop foot ulcers. Of these, 5-15% require amputation of the foot. Diabetic foot ulcers are caused by peripheral neuropathy (lack of sensation), motor neuropathy (changes to posture), autonomic neuropathy (reduced sweating leading to dry, cracked feet) and peripheral vascular disease (reduced blood & oxygen supply). If the skin is then penetrated, the high glucose conc invites infective agents. To reduce risk of diabetic foot, patients should be advised to wash & check their feet daily, wear comfortable shoes, see a chiropodist regularly and seek urgent treatment if problems, not use hot water bottles, as neuropathy increases likelihood of injury and not attempt to self-treat any problems – see a doctor! Where injury has occurred, there are a small number of novel treatments; growth factors (e.g. platelet-derived growth factors) and bioengineered skin substitutes aim to replace the skin which has been damaged. Negative pressure wound therapies decrease swelling by removing pro-inflammatory cytokines and optimising blood flow.

Answer 92

This is a complication of diabetes mellitus in which high blood sugar results in high osmolarity without significant ketoacidosis. This causes water to be pulled from the tissues into the blood, leading to excessive urination (more specifically an osmotic diuresis), which, in turn, leads to volume depletion and haemoconcentration that causes a further increase in blood glucose level. Symptoms include signs of dehydration, weakness, legs cramps, vision problems, and an altered level of consciousness.

Answer 93

DKA is a metabolic complication of diabetes. It occurs as a result of hyperglycaemia. A series of metabolic pathways fails and the body’s attempt to correct itself can lead to shock, coma and death. Incidence is 5-8 per 1000 and mortality is < 1% (mainly due to corrective healthcare; if no healthcare is sought, there is a high risk of death). Glucagon production is not affected in T1-diabetics, so it is able to exert its effects: gluconeogenesis (increasing blood glucose levels) and lipolysis (breaking down fats into fatty acids). The free fatty acids produced from lipolysis are taken into the liver and oxidised to acidic ketone bodies, which are released into the bloodstream and are taken into cells for energy. However, because there is a lack of endogenous insulin, an excess of free fatty acids (ketones) causes the pH of the blood to drop.

Answer 94

undiagnosed diabetes Body’s demand for insulin increases: e.g in cases of illnesses or use of corticosteroids or thiazide diuretics Reduced appetite- body goes into starvation mode & digestion of fats Not taking insulin as scheduled

Answer 95

Blood glucose >11.0mmol.L Ketonaemia ≥ 3.0 mmol/L or ketonuria (++ on urine sticks) Significant acidosis (venous bicarbonate < 15 mmol/L or venous pH < 7.3)

Answer 96

Insulin is regarded as the key that allows for influx of glucose from the blood into our body cells. Cells then use the glucose as fuel for energy! But in DKA, insulin is virtually not present!So there is no way for the glucose to enter the cells and be used as a fuel source. Thus, glucose accumulates in the blood resulting in hyperglycaemia!

Answer 97

The liver recognises okay..the body cells seem to not have a source of energy! The pancreas then secretes glucagon. Glucagon then causes the liver to engage in glycogenolysis whereby the stored glycogen gets converted to glucose which is released into the blood!

Answer 98

However, the body really begins to need a decent source of fuel to keep the cells functioning. As a result it resorts to the break down of fat since glucose cannot be utilised as a fuel source with no insulin around! Breakdown of fats results in weight loss and the production of ketones. Ketones have a fruity smell thus corresponding to the fruity breath seen in some patients. Ketones are also acidic which then results in metabolic acidosis, lowering of blood pH and causing a kissmoul breathing & ketonuria.

Answer 99

Recall how the kidney serves a purpose in reabsorbing glucose via the renal tubules. However, with hyperglycaemia due to no insulin and glycogenolysis occurring, the kidneys can really only reabsorb so much glucose!! The amount of glucose in the blood ends up exceeding the kidney’s capacities. Thus, glucose begins to be excreted in the urine. This results in glucosuria, polyuria (water goes where glucose goes due to osmotic diuresis). Patient also gets dehydrated, thirsty (polydipsia) and hypotensive due to this loss of water! Electrolyte imbalances also occur!

Answer 100

fluid replacement aims: ketone clearance, restore circulatory volume & correct electrolyte imbalance! Administer IV fluids: Sodium Chloride infusion, Potassium Chloride also included to prevent hypokalaemia Increase insulin levels & inhibit gluconeogenesis by- administer regular soluble insulin IV via continuous infusion

Answer 101

Hyperglycaemia Weight loss Fruity breath Kissmoul breathing Ketonuria Glucosuria Polyuria Dehydration Polydipsia Hypotension Abdominal pain- children Breathlessness Confusion Leg cramps Nausea

Answer 102

give 500mls 0.9% sodium chloride solution over 15 mins via IV pump. fix electrolyte levels (k+) give iv short acting insulin infusion-50 units made up to 50ml using 0.9% NaCL solution monitor blood glucose blood ketones respiratory rate ECG urea and electrolytes

Answer 103

reassess the pt and monitor for vital signs blood glucose hourly rate ketones pH, bicarbonate potassium continue fluid replacement via infusion pump as follows with caution with 18-25, elderly, pregnancy, heart or renal failure 0.9% NaCl 1L with potassium chloride over next 2 hrs add 10% glucose 125ml/L if blood glucose falls below 14mmol/L assess response to treatment insulin infusion rate may need review if ketones is not falling by >0.5mmol/l/hr bicarbonate is not raising by >3mmol/l/hr glucose is not falling by 3mmol/l/hr if equipment is working but reposes is inadequate then increase the insulin infusion rate bu one unit per hr increment hourly until targets achieved

Answer 104

Convert to rapid acting SC insulin regime when ketones < 0.3 mmol/L and blood pH > 7.3) and the patient is able to eat but intravenous insulin must not be discontinued until 30 minutes after SC insulin has been given.

Answer 105

Stage of disease Therapy -Triggering – virus/toxins N/A -Breakdown of tolerance Preventative -Autoimmune attack Immunotherapy -ß-cell destruction ß-cell deference/repair & growth promoters -Diabetes Replacement Insulin therapy

Answer 106

The equation below shows how excess acid drives the equation to the right. The oblongata (the respiratory centre in medulla) tries to correct the excess CO2 by causing hyperventilation. Acidosis causes vasodilation and hypotension which results in the under-perfusion of organs (insufficient oxygen supply); the body goes into shock.

Answer 107

Most patients use flash (finger prick) glucose monitor. A small sample of blood is taken, which enzymatically produces a new advancement is a continuous glucose monitor (CGM), which is attached to the abdomen. Limits can be set so the patient can visualise their blood glucose levels, to avoid hyperglycaemia and hypoglycaemia – there is also an alarm which alerts patients to hypo events. Both use a lancet with a capillary needle and test strip. Blood glucose testing can help to control diabetes by: • Informing food choices and portion quantities • Assisting medication dosing decisions • Identifying periods of high or low blood glucose levels

Answer 108

A novel treatment (not yet licensed in the UK) provides insulin to enter the lung as a dry powder. As the powder travels through the bronchial corridor, they absorb moisture. Some particles dissolve, but in the alveolar sacs, air speed lessens, and insulin particles penetrate the alveoli – on average, one EXUBERA particle enters a single alveolus. The thin cells provide a large surface area for absorption, where they enter tight junctions between cells before entering the blood stream. Reaching the site absorption may not occur, because of a variety of defence mechanisms. Oropharyngeal & bronchial filters & mucociliary clearance can prevent insulin from reaching the site of absorption. The lungs have a rich supply of antigen-presenting cells including the lung epithelium, pulmonary dendritic cells and alveolar macrophages.

Answer 109

Insulins are characterised by onset, peak and duration of insulin and their route of delivery. Insulin is given by SC injection, as a suspension. The drug enters capillaries by diffusion/filtration, and the rate of absorption is affected by the blood supply; the closer a good blood supply is to the injection site, the more rapid the onset of action. In addition, exercise increases blood flow, so if exercising, insulin should be injected into the abdomen.

Answer 110

Calculating the dose is difficult, because everyone’s metabolism is different. Insulin injections come as preparations where the concentration is 100 international units per ml (100 iu/mL). 1 unit = 10g of carbohydrate ingested. ``` Carbohydrate foods that are broken down quickly by your body and cause a rapid increase in blood glucose have a high GI rating. High GI foods include: • sugar and sugary foods • sugary soft drinks • white bread • potatoes • white rice ``` Carbohydrate foods that are broken down more slowly and cause a gradual rise in blood sugar levels over time have a low/medium GI rating. They include: • some fruit and vegetables • pulses • wholegrain foods, such as porridge oats Other foods impact carbohydrate metabolism. Protein is broken down into glucose less efficiently than carbohydrate and, as a result, any effects of protein on blood glucose levels tend to occur anywhere between a few hours and several hours after eating. Fat impairs insulin sensitivity and increase glucose production. The way that a meal is prepared/cooked changes the glycaemic index. shows how quickly each food affects your blood sugar (glucose) level when that food is eaten on its own.

Answer 111

* Once daily – mix of very long and very short acting analogues; this does not give tight glycaemic control so is rarely regimented. * Twice daily, biphasic – mix of short and long acting insulins. A twice daily regimen is suitable in people who have a consistent day to day routine. * Basal Bolus – mix of short and long acting insulins. Long acting insulin is taken once daily and short acting insulin is taken at each meal. This is the gold standard for tightest glycaemic control; it offers more flexibility over when meals are taken and also allows doses to be varied in response to different carbohydrate quantities in meals.

Answer 112

``` Insulin lispro (Humalog) Insulin aspart (Novorapid) Insulin glulisine (Apidra) onset -15 min peak- 15mins 1hr duration- 2-5 hrs ```

Answer 113

``` Regular or neutral insulin (Actrapid) onset -30-60 min peak- 1-3 hrs duration approx 8 hrs ```

Answer 114

Isophane insulin (NPH) onset approx 2 hrs peak 2-12 hrs duration 18-24 hrs

Answer 115

``` Insulin glargine (Lantus) Insulin detemir (Levemir) onset-1 hr peak none up to 12 hrs up to 24hrs ```

Answer 116

Rapid and short acting insulins are given as a bolus to mimic the insulin secreted by the pancreas in response to post-prandial increase in glucose levels, providing 10 – 20% of total daily insulin. Rapid acting insulins have a lower tendency to form hexamers; existing as monomers enables them to have a more rapid absorption & faster onset of action. They are relatively resistant to degradation at high temperatures and in prolonged storage (≥ 10 days) but manufacturers still stress that insulin exposed to temperatures above 37 °C should be discarded.

Answer 117

Intermediate and long-acting insulins are basal; they supress hepatic glucose between meals/overnight, providing 50% of total daily insulin. ``` Insulin detemir (long-acting) has a fatty acid (myristic acid) tail attached to the 29th position of the B-chain. This gives high binding affinity for albumin. > 98% of insulin detemir being protein-bound, which forms a depot from and slowly dissociates, releasing insulin over a long amount of time. It is less potent than endogenous insulin, because the structural changes affect the affinity for the insulin receptor. The duration of action of is dose-dependent, and ranges from 8-22 hours at higher doses but it’s peak action comes between 6-8 hours after injection. ``` With insulin glargine, asparagine is replaced by glycine. This provides stability and the addition of 2 arginines at C-terminus of the B chain alters the iso-electric point and so decreases solubility at pH 7. Therefore, it can easily self-aggregates and precipitates to form microcrystals, which act as an insulin depot preparation. The depot breaks up into hexamers, then dimers, then monomers.

Answer 118

On one end is a small needle, which is disposable, on the other is a plunger that you press to deliver the insulin under the skin. It is convenient as pens can be prepared at home and a more accurate dose can be delivered whenever it is needed. It is easier to use those with visual or fine motor skills impairments although, not for patients with joint conditions. They are slightly more expensive and are restrictive in only being able to give one or half-unit dosing.

Answer 119

This device releases a fine spray of insulin at such a fast speed that the insulin passes directly through the skin; no injection is required. Jet injectors are great for people who don't like to use needles, but they can be costly, may be painful and can cause bruising, especially in thin people.

Answer 120

Continuous subcutaneous insulin infusion (CSII) devices provide a replacement to the basal insulin regime and mealtime-high blood sugar insulin. The pump has a reservoir, which usually holds about two to three days’ supply of insulin. This insulin is delivered through an infusion set (a very fine tube that runs from the pump to a cannula), which is inserted under the skin. The cannula can be left in for two to three days before needing to be replaced and repositioned somewhere else on the body. The pump is battery operated and indicates if power is low. They are the best choice for patients with a fear of needles, have difficulty managing high/ low BG levels, if their HbA1c level is outside of target range and seeking more flexibility in everyday life. It allows for tighter glycaemic control with fewer injections and a possible reduction in total dose of insulin. However, BG must be tested more frequently (every 3-4 hours), with the amount of insulin given adjusted accordingly. There is an increased infection risk at insertion site and scarring may occur. There may also be interactions between the pump and insulin. Insulin may adsorb onto hydrophobic surfaces (e.g. glass) causing a loss of activity and it may aggregate (mis-fold and attach to one another, forming large fibrils) due to elevated temperatures and agitation causing a loss of activity and blockage of infusion set. Insulin pumps must offer optimal basal and postprandial glycaemic control with no risk of hypoglycaemia. There must be a buffered environment, to avoid fibrillation and risk of catheter occlusion, with protective compartmentalization of the insulin from direct sunlight and reduced exposure which could allow adsorption to hydrophobic interfaces. It must be free of immunogenic and microbial compounds. It is also important that appropriate education for CSII users is available in terms of the practical aspects related to correct insertion of infusion cannula, the need to change the infusion systems at a frequency recommended by the manufacturers, and what to do in the event of catheter occlusion.

Answer 121

A novel treatment (not yet licensed in the UK) provides insulin to enter the lung as a dry powder. As the powder travels through the bronchial corridor, they absorb moisture. Some particles dissolve, but in the alveolar sacs, air speed lessens, and insulin particles penetrate the alveoli – on average, one EXUBERA particle enters a single alveolus. The thin cells provide a large surface area for absorption, where they enter tight junctions between cells before entering the blood stream. Reaching the site absorption may not occur, because of a variety of defence mechanisms. Oropharyngeal & bronchial filters & mucociliary clearance can prevent insulin from reaching the site of absorption. The lungs have a rich supply of antigen-presenting cells including the lung epithelium, pulmonary dendritic cells and alveolar macrophages.

Answer 122

This does not involve actual transplantation of a pancreas but is an extension of the cover that an insulin pump may give. It is an autonomous system for tight glycaemic control. A. Insulin pump acts as others (see above) B. Cannula delivers insulin from the reservoir in the pump SC to the abdomen C. Glucose sensor indicates the BG levels, requiring the patient to input the information into the insulin pump D. Small transmitter sends information to a receiver New advances aim to remove the patient input step; a control algorithm receives information from the continuous glucose monitor and performs a series of mathematical calculations. Based on these calculations, the controller sends dosing instructions to the infusion pump. Complex algorithms must predict BG levels to ensure effective coverage; after eating, there is usually a 15/ 20-minute lag where BG levels are very high. Some novel treatments also release glucagon to counterbalance the effects of insulin, to further avoid hypoglycaemic events/ insulin shock. The artificial pancreas is extremely expensive, but with more clinical evidence to gives tighter glycaemic control, it will become cheaper. It must be as safe, or safer than what is already available for treatment.

Answer 123

Replacing ß-cells provides a huge challenge; the aim is to replace ß-cells in a person whose immune system actively destroys ß-cells. Transplantation gives freedom from insulin injections, (reduces anxiety of a night due to) hypoglycaemic events, however immunosuppressants are needed for life, and they are limited to severe cases of poorly controlled T1D due to a shortage in donated pancreases.

Answer 124

Transplantation of a pancreas is used to replace ß-cells in patients with T1D. The donated pancreas is implanted onto the small intestine. Pancreas transplantation is not a first line treatment; 90% of pancreas transplants are due for a kidney transplant simultaneously, as a result of uncontrolled diabetes.

Answer 125

* cancer that has spread in the last 12 months * a severe or untreated infection * Creutzfeldt-Jakob Disease (CJD) – a rare condition that affects the nervous system and causes brain damage

Answer 126

Only the ß-cells in a diabetic pancreas are affected, so there have been efforts to only transplant the islets of Langerhans. To obtain the islets, a donated pancreas is digested and purified. The islets are injected to the hepatic portal vein, where they lodged in the liver. The islets act in the exact same way as they would in a non-diabetic pancreas: sensing BG concentrations and giving a natural insulin response. The more cells that are transplanted, the longer they will last for, but eventually they will stop producing insulin and therefore insulin replacement may be needed (after 5-10 years). The lifespan of transplanted cells is also affected by immunosuppressant use; the immune system will begin to destroy new, transplanted cells if immunosuppressants are not given. Safely transplanting insulin-producing clusters is a challenge because the mechanism of immunology is still unknown.

Answer 127

Encaptra ® is a capsular device which is implanted into the abdomen, using purified human islets. There is a semi-permeable membrane so immune cells don’t stick to it, or attack it, but glucose can enter, and insulin can exit. Lifelong immunosuppression is not needed. PEC-01 cells are a mixture of islets with stem cells, giving it a longer lifespan; when ß-cells die, they are replaced by stem cells.

Answer 128

Pancreas transplantation requires lifelong immunosuppressant therapy (Cyclosporine/ Tacrolimus/ Sirolimus/ Azathioprine/ Mycophenolate mofetil) and steroids. Corticosteroids are damaging for the pancreas, ß-cell function & insulin sensitivity. They Islet transplantation still requires lifelong immunosuppressant therapy (Tacrolimus/Sirolimus) but is steroid free. The development of monoclonal antibodies is extremely important. * Alemtuzumab targets CD52 (cell-surface antigen on lymphocytes) and is the best monoclonal antibody for immunosuppressive therapy due to its specificity. It prevents the interaction of T cells and expansion of clones. * Sirolimus is an intracellular cell-signalling inhibitor, which targets mTOR. mTOR is a key signalling molecule involved in the insulin signalling cascade. * Tacrolimus is an intracellular cell-signalling inhibitor, which targets calcineurinA, preventing T-cell function of the cells which attack insulin-producing cells.

Answer 129

Ideal replacement ß-cells must be capable of expansion to large numbers which can secrete useful amounts of insulin which responds to BG levels in the blood. There also must be an ability to control the proliferative capacity so they do not continue to grow and evade immune destruction, like cancers.

Answer 130

ESCs originate from donated embryos where they are cultured and expanded in a lab. The inner cell mass is isolated and are further proliferated. These ESC cultures are grown in Leukaemia inhibitory factor (LIF), preventing differentiation into other cells. Once LIF is removed, they form embryoid bodies and communicate with other ESCs. They are able to develop into any cell type and act as normal cells. ß-cells can be created at this stage; PDX1 is a transcription factor protein which is necessary for β-cells maturation and regulation of pancreas development. It can be switched on so that stem cells differentiate to pancreatic precursor cells and then ß-cells. Engineered ESC grown in normal culture environments (on a surface – 2D) and do not communicate with other cells. This means if a single cell was stimulated to express PDX1, only 5-10% develop into ß-cells and so cannot be transplanted. A technological breakthrough – microgravity bioreactors – allow the development of 3D culture systems. Cells do not touch any surfaces, instead growing in floating space. This allowed the development of an extracellular matrix, which promotes cell communication. This means if a single cell was stimulated to express PDX1, the signal spreads across the entire cluster, forming something very similar to an islet. Once ß-cells have been made, they must be pure populations of fully functional ß-cells, secreting insulin in response to BG, and stopping secretion if hypoglycaemia occurs. Their growth must be controllable, so they are safe and transplantable.

Answer 131

Due to ethical issues around using embryonic stem cells, ß-cells can be generated using adult stem cells. Adult cells can be transdifferentiated; the cell changes into a different cell type and function effectively. Adult stem cells can be obtained from bone marrow, cord blood and liver cells. For ß-cells, pancreatic stem cells are used. Pancreatic ductal cells produce most new ß-cells and can differentiate into ß-cells.

Answer 132

DNA stores the instructions/signals/packages needed for a cell to reproduce. RNA in synthesized by transcription, and the genetic code within RNA is translated in the ribosomes. This produces a string of amino acids that fold together and form a bioactive protein. Cloning is the process of creating an exact copy of a biological unit. A gene is a stretch of DNA that codes for a type of protein that has a specific function in an organism. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring.

Answer 133

* A promotor sequence acts as the on switch, starting the transcription into messenger RNA. * Untranslated DNA are not transcribed into mRNA but can regulate production. * The actual DNA sequence is an interspersion of exons and introns. Exons make the protein and introns intervene sequences, regulating expression. * The Poly A sequence protects messenger RNA when it is transported out of the nucleus and into the ribosomes. * A terminator sequence acts as the off switch, stopping transcription.

Answer 134

1. Isolate your gene from DNA using restriction enzymes or the Polymerase Chain Reaction (PCR) & purify 2. Insert the gene into a suitable cloning vector to create a chimaeric vector (e.g. a plasmid) 3. Introduce the vector carrying the gene into prokaryotic cell (such as E. Coli; cells that take up vector is transformed) so it can copy the gene into mRNA and manufacture the protein this gene codes for. 4. Screen transformed cells to ensure correct gene 5. Scale-up production of recombinant protein, purify, formulate and characterise for therapeutic use

Answer 135

In cells, the gene is transcribed into “pre-messenger-RNA”. This codes for introns and exons. Cellular factors cut-out the introns and splice the exons together. mRNA only encodes the proteins; it does not express them. To produce a recombinant protein the messenger RNA is isolated. Using an enzyme called reverse transcriptase (found in a group of viruses), mRNA can be converted back to cloned DNA (cDNA). cDNA contains the gene in question as well as all the other active genes, so the cDNA must be modified to isolate the single gene in question. This can be done in two ways – see below.

Answer 136

When we wish to locate a new gene, PCR is used; only very small quantities of DNA (can be impure) are needed. By using a pair of primers, the desired DNA sequence can be defined. If the sequence is known, a primer can be designed to delineate where to start and end. Primers also allow the addition of restriction endonuclease recognition sites. These assist subsequent cloning steps as they can be detected by a monoclonal antibody to purify a protein. 1) During the denaturation stage, the two strands of DNA come apart. 2) During the annealing stage, primers stick to the stands. 3) During the elongation stage, DNA polymerase recruits nucleotides giving new copies of DNA. This process is repeated 20-30 times using a thermocycler.

Answer 137

E. Coli have an enzymatic immune system which recognises and destroys foreign DNA; it also modifies native DNA to prevent self-destruction. These DNA-cutting enzymes are called restriction endonucleases (RE). Where genes are flanked by specific DNA sequences, Res can recognise & cleave DNA at specific sites, and then clone them. This is particularly useful if the gene is already in a plasmid and you wish to sub-clone it into another vector. There are four types, but only Type II are used to manipulate DNA. Restriction endonuclease recognition sites (RERS) consist of specific sequences of 4 to 8 base pairs – GAATTC for E. Coli restriction endonuclease #1, Eco R1. They are recognised in a forward and reserve direction. Eco RI makes staggered cuts in both DNA strands, creating single stranded overhangs known as sticky ends. If these ends find each other, the complementary bases will anneal back together and allow cloning. Some restriction enzymes cuts are not staggered and give rise to blunt ends. These can be manipulated but it is more difficult. A major disadvantage of restriction endonucleases is if the same RERS are in the middle of the gene, or is absent, this technique will not work. When we wish to locate a new gene, PCR is therefore used.

Answer 138

All DNA required for cloning must be part of a larger, stable structure that is capable of being copied; normal cellular DNA in living organisms is organised into chromosomes, which are very stable and can be copied. Chromosomes are rarely used for cloning, so cloning vectors are used. Cloning vectors include plasmids and viruses, amongst other. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. Plasmids are found in the cytoplasm of bacteria and some yeasts. They are small, double-stranded, self-replicating circular DNA molecules, distinct from the normal bacterial chromosome (extrachromosomal – away from the chromosome). Plasmids often carry genes that may benefit the survival of the organism and can be transmitted from one bacterium to another, for example antibiotic resistance. Most cloning plasmids now used are artificial constructs made from naturally occurring plasmids.

Answer 139

* DNA replication origin – for the vector to replicate inside the host organism * Multiple cloning site, containing multiple RERS – to serve as sites where foreign DNA may be introduced * Selectable genetic marker gene – used to enable the survival of cells that have taken up vector sequences * Tag gene – used to screen for cells containing the foreign DNA

Answer 140

To insert a gene into a vector both the gene and vector must be cut at the same restriction site that is cleaved by a restriction endonuclease, producing matching ‘sticky ends’ in the gene & vector. The gene & vector are added together, and complementary base pairing allows association, by hydrogen bonding. PCR DNA can be directly cloned without using RERS. TA cloning is the most common method; it is easier and quicker than traditional sub-cloning. TA cloning relies on the ability of adenine (A) and thymine (T) (complementary base pairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together. This is quick and cheap (15 minutes/£2 per ligation). Another method of TA cloning uses TOPO vectors. DNA topoisomerase I can cleave & re-join supercoiled DNA ends. TOP vectors contain a DNA sequence, at each free end, to which topoisomerase enzyme is bound. When the PCR DNA is added, the enzyme links the vector to the insert, without requiring Ligase. This is the method of choice for PCR DNA, but it is expensive (£25 per ligation).

Answer 141

Transformation is used to introduce recombinant DNA into a prokaryotic cell (e.g. E. Coli). The cell is made temporarily permeable, so it takes up plasmid DNA, then permeability is reversed so the cell’s contents don’t leak out.

Answer 142

Chemical transformation/Heat shock This is a cheap and easy method. The prokaryotic is suspended in a CaCl2 solution before the chimaeric plasmid is added. The temperature is raised to 42ºC, then reduced to 0ºC and the bacterial membrane becomes temporarily permeable to plasmid DNA. Electro-transformation/electroporation This method requires expensive equipment, and half of the cells can be killed. Electrocompetent E. Coli cells are placed in a cuvette and subjected to a voltage spike of 200-2000 Volts for a few milliseconds. The bacterial membrane becomes temporarily permeable to plasmid DNA. Needs expensive equipment and can kill half of the cells Lipofection This method can be very expensive (£25/ reaction) due to the reagents used. The DNA is encased, using liposomes, and fused with mammalian/ insect cells. The bacterial membrane becomes temporarily permeable to plasmid DNA.

Answer 143

Not all of the bacteria will take up the plasmid, so selectable markers must be used; transformed cells are grown on agar containing a type of selection agent (usually an antibiotic). Only the bacteria that have taken up the plasmid will grow. A single colony is selected then scaled up into a culture.

Answer 144

Once scaled up into a culture, the gene must be expressed to produce the desired protein. A therapeutic antibody must have full biological functionality to be effective. For this to occur, the expression system must perform all the post-translational modifications, so the recombinant protein resembles the native protein as closely as possible. These can include cleavage of precursor proteins, amino acid modification (e.g. glycosylation – addition of carbohydrate chains to form glycoproteins). Glycosylation can modify protein folding, cellular localisation and protein activity. Prokaryotes do not glycosylate their proteins, so glycoproteins must be made in eukaryotic expression systems.

Answer 145

1. Expression host (e.g. CHO cells) 2. Expression vector 3. Transfection reagent/device, which gets the vector into the host.

Answer 146

An expression host provides the cellular machinery required for DNA replication, transcription and translation. Hosts can be prokaryotic such as bacteria (particularly E. Coli) or eukaryotic, such as yeast cells, mammalian or insect cell lines. E. Coli expression (prokaryotic) system produce short-acting insulin (humulin) & human growth hormone. Yeast (eukaryotic) expression systems produce short-acting insulin (alternative to Humulin) & Hep B vaccine. They are a good compromise between E. Coli and CHO cells. They are easy, cheap and quick, and able to perform some post-translational modifications; however, the product is usually not secreted and may show aberrant glycosylation. Mammalian cell expression systems tend to use Chinese Hamster Ovary (CHO) cells, and they produce interferons, interleukin-2, monoclonal antibodies.

Answer 147

Pharming refers to the use of genetic engineering to make therapeutics.

Answer 148

* Good quality of raw materials ensures quality of product * Grading & isolating the product to ensure suitability * Compliance to Product Licence to ensure purity * Effective sterilisation to ensure safety (septic filtration, gamma radiation, ethylene oxide used to circumvent thermal damage to proteins if using heat treatment)

Answer 149

Stage 1: Upstream processing • The medium must be formulated and optimised for host organisms, to result in optimum product yield at minimum cost • The bioreactor must be sterilised and the air purified, to avoid unwanted microbial growth • There must be an adequate supply of producer cells o Cell banking is where a culture of newly-constructed producer cells are stored in a master bank and working cell bank at – 170ºC. The master cell bank must be extensively validated before use

Answer 150

• A vial from the working cell bank is removed from storage and starter cultures are generated • Production of cell cultures is scaled up. Small bioreactors contain several litres of medium; production scale bioreactors contain several thousands of litres of medium • Stirred tank bioreactors have the following features o High grade stainless steel, which allows for full sterility o Temperature jacketed improves control o Impeller ensure even distribution of nutrients & cells o Sampling points enable monitoring for pH, temperature & concentration of critical metabolite o Sparger gives forced aeration, providing O2

Answer 151

Stage 3: Downstream processing (clean-up) Sterility is essential during this stage. Clean rooms with positive pressure and highly filtered air, are required because of the bacterial load. Equipment and personnel must be segregated between virus-present and sterile environments. 1. Cell harvesting and recovery of crude product. If the protein is expressed intracellularly, producer cells are recovered by centrifugation/filtration. Cells are disrupted using surfactants or enzymatic treatment, which lyses the cell, then cellular debris (insolubles) is removed by centrifugation/filtration. If the protein is secreted, cells are removed from media by centrifugation/filtration. 2. Purification & removal of viruses/ pyrogens by chromatic purification 3. Final formulation. Adjustment of potency and addition of excipients (e.g. serum albumin which is used to disguise a protein, amino acids, polyols, surfactants) stabilises and/ or enhances the product.

Answer 152

A range of potential impurities present in pharmaceutical products may have medical consequences and requires testing: * Microorganisms – risk of microbial infection, septicaemia * Viral particles – risk of viral infections * Pyrogenic substances (LPS) – fever response * DNA – possible immune reaction * Contaminating proteins – immune reaction or unwanted biological activity

Answer 153

The ultimate goal is to achieve a high degree of quality assurance. There is, however, a risk of variability in the consistency of biological material; uniformity from batch to batch varies which have detrimental knock-on effects if given as a medicine.

Answer 154

* Non-inert growth medium * Lyophilisation * Potency (proteins may denature over time if they are exposed to heat or humidity) * Preserving globular integrity and subsequent efficacy * Isomeric forms

Answer 155

From 1992-2001, there has been an 800% increase in the number of biopharmaceutical patents. The world’s largest biotechnology company (Amgen, owned by Pfizer) has a market value of £53 billion. There are risks associated with any type of reduced testing/ restricted process monitoring, which is inferred by some biotechnic practices. The FDA regulates because European Medicines Agency (EMEA)-regulated states trade with the US. The EMEA may grant Market Authorisation to medicinal products from biotechnology and other higher-technology processes. The safety of medicines is monitored: guidelines on quality, safety and efficacy requirements. A special office works for - small and medium-sized enterprises (SMEs).

Answer 156

* Just-in-time: nothing is made until it’s needed to reduce storage requirements * Kanban: based on need of customer * Kaizen: cyclical approach of learning from mistakes * Value stream mapping: only produce what will make the most profits * Total production maintenance: ensuring full use of a facility The Qualified person must follow directives & guidelines as specified by GMP, GCP, Product License, as well as ensuring validation and production processes to gain approval from regulatory bodies. Shewhart’s cycle of validation can be applied: Plan  Do  Check  Act

Answer 157

Biosimilars are biological drugs or therapeutics that are produced by recombinant DNA technologies (MAb’s) and are found (via clinical trials) to be “similar” to a reference [first-generation] product already authorized in the EU. The regulator grants a marketing authorisation to a compound of this type when it has been compared to, and matches with, the reference medicine in terms of quality, safety and efficacy. The process of gaining a MA takes ~ 12 years.

Answer 158

Recombinant insulin is cheap to manufacture and has 98% purity. A 40m3 bioreactor provides ~ 100g of pure insulin (equivalent to 1% of the annual global demand). Polyribosomes bound to the ER of insulin-producing cells encode a precursor protein, prerpoinsulin. Preproinsulin is cleaved, giving a second precursor, proinsulin. Proinsulin is converted to insulin by proteolysis. Insulin is an amino acid heterodimer (two chains: A – 21  and B – 34 ) linked by disulphide bonds; these post-translational modifications are required when making insulin. The heterodimers form into a trimer (6 molecules). Regular insulin forms hexamers.

Answer 159

Chain recombinant bacterial Insulin (CRB), used an E. Coli expression system. It gave the first recombinant therapeutic protein was Humulin, approved for use in 1982. Unfortunately, but the method gave low yields and the cross-linking of strands was inefficient. Proinsulin recombinant bacterial (Insulin PRB) produced proinsulin and cleaved the central peptide enzymatically. It gave higher yields, with more efficient strand cross-linking. Insulin PRB was the first generation of recombinant insulins.

Answer 160

For production in yeasts, modification of the proinsulin gene construct was required; fusion of native A-chain to B-chain lacking the C-terminal B30 threonine. This made it easier to purify so less was lost and a higher yield could be obtained. Codon-optimization refers to the improvement of the sequence of three nucleotides which together form a unit of genetic code in a DNA or RNA, based on various criteria, without altering the amino acid sequence. E. Coli / yeast. • Rapid-acting (Analogue) o Aspart o Lispro o Glulisine • Long-acting (Analogue) o Detemir o Glargine To convert native insulin into analogues, genetic engineering is utilised. • Single/ small numbers of nucleotides can be changed by o Site-directed mutagenesis, which involves the specific substitution of one DNA base for another. Unlike the nonspecific mutations described above, site-directed mutagenesis allows precise control of the number, placement, and base substitution of mutants. The two classes of site-directed mutagenesis include methods that use double-stranded DNA cassettes and those that use single-stranded oligonucleotide primers o PCR induces mutations on one of the primers, in order to produce a modified PCR product. This is very easy when the changes are introduced at the end of a sequence • If several changes or codon-optimisation is required, the new gene can be synthesised

Answer 161

• Single/ small numbers of nucleotides can be changed by o Site-directed mutagenesis, which involves the specific substitution of one DNA base for another. Unlike the nonspecific mutations described above, site-directed mutagenesis allows precise control of the number, placement, and base substitution of mutants. The two classes of site-directed mutagenesis include methods that use double-stranded DNA cassettes and those that use single-stranded oligonucleotide primers o PCR induces mutations on one of the primers, in order to produce a modified PCR product. This is very easy when the changes are introduced at the end of a sequence • If several changes or codon-optimisation is required, the new gene can be synthesised

Answer 162

Tobacco, lettuce, Arabidopsis hold potential for easy production of future insulin analogues. They are cost effective, give a high quality of protein processing, are absent of human pathogens and have eukaryotic machinery for posttranslational modifications.