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A Level Biology Unit 8 page 1 Heckmondwike Grammar School Biology Department Edexcel A-Level Biology B Contents Practical Microbiology ........................................................... p3 Growth Curves ...................................................................... p12 Antibiotics ............................................................................... p14 Pathogens and Disease ......................................................... p20 Controlling Malaria................................................................. p28 The Immune System............................................................... p30 Immunisation............................................................................ p38 These notes may be used freely by biology students and teachers. I would be interested to hear of any comments and corrections. Neil C Millar ([email protected]) June 2016 Y12 Unit 1 Biochemistry Unit 2 Cells Unit 3 Reproduction Unit 4 Transport Unit 5 Biodiversity Unit 6 Ecology Y13 HGS Biology A-level notes Unit 7 Metabolism Unit 8 Microbes Unit 9 Control Systems Unit 10 Genetics NCM/6/15 A Level Biology Unit 8 page 2 Biology Unit 8 Specification Microbial techniques The principles and aseptic techniques used in culturing microorganisms. The use of different media: broth cultures, agar and selective media. The different methods of measuring the growth of a bacterial culture as illustrated by cell counts, dilution plating, mass and optical methods (turbidity). The different phases of a bacterial growth curve (lag phase, log phase, stationary phase and death phase). Calculate exponential growth rate constants. Antibiotics The action of bactericidal and bacteriostatic antibiotics, as illustrated by penicillin and tetracycline. The development and spread of antibiotic resistance in bacteria. The methods and difficulties of controlling the spread of antibiotic resistance in bacteria. Pathogens and Diseases The transmission, mode of infection and pathogenic effect of the following: Staphylococcus spp. (exotoxins) Salmonella spp. (endotoxins) Mycobacterium tuberculosis (invading and destroying host tissues) influenza virus the malarial parasite (Plasmodium spp). stem rust fungus on cereal crops (Puccinia graminis on wheat) HGS Biology A-level notes Detailed life cycles are not required. Controlling Malaria The social, economic and ethical implications of different control methods for endemic malaria and the role of the scientific community in validating these methods. Immune System The mode of action of macrophages, neutrophils and lymphocytes. The development of the humoral immune response, including the role of antigenpresenting T cells, T helper cells and cytokines, B cells, clonal selection, plasma cells, antibodies. The development of the cell-mediated immune response including the role of antigenpresenting cells, T helper cells and cytokines, T killer cells. Immunity and immunisation The role of T and B memory cells in the secondary immune response. How immunity can be natural or artificial, and active or passive. How vaccination can be used in the control of disease and the development of herd immunity. The potential issues in populations where a proportion choose not to vaccinate. NCM/6/15 A Level Biology Unit 8 page 3 Practical Microbiology Microbiology experiments require special techniques and precautions, for two good reasons: Health and Safety – to prevent the escape of any experimental microbes to the surrounding environment, where they may cause disease. Validity – to prevent contamination of experiments by microbes from the environment. These other microbes could out-compete the experimental microbes or otherwise invalidate the results. Safety Precautions for Practical Microbiology Microbiology is potentially hazardous and safety precautions must be taken seriously Never eat or drink in a microbiology laboratory. Avoid putting your hands in or near your mouth during a microbiology practical. Ask for a plaster to cover any cuts or scratches on your hands or arms. Never open a Petri dish or culture bottle unless you have been specifically told to by your teacher. Never touch cultures with your fingers, clothing or any part of your body. Practice aseptic technique before attempting any transfer for real. This will reduce the chance of mistakes. If you drop or spill a culture do not touch it or attempt to clean it. Report the accident immediately so the teacher can deal with it correctly. There are particular ways of dealing with microbiology spills. Dispose of glassware and other apparatus in the labelled receptacles provided. Never pour any medium or culture down the sink. Wash your hands with a disinfectant soap before you leave the laboratory. You are responsible for your own safety and the safety of everyone else in the lab. The Mirobiology Workspace The area where the microbiology work is to be done should be tidy and should have a lit Bunsen burner. This is used to sterilise equipment, and also to provide a convection current, which draws air and spores up and away from the experimenter. Soak a laminated work-mat in Virkon for 10 minutes, blot dry and place immediately in front of the Bunsen. Do all your work on this mat. When finished, return the mat to the bottom of VirKon bowl for at least 10 minutes. A Level Biology Unit 8 page 4 Microbiology Terms and Techniques Sterilise To kill or remove all mirobes from somewhere. For example all glassware and surfaces must be sterilised before a practical session, and all cultures must be sterilised at the end of the experiment. Equipment and cultures are best sterilised by using an autoclave. This is a large pressure cooker, which heats up steam under pressure to 121°C for at least 15 min. This kills all microbes and bacterial spores. All cultures are sealed and autoclaved after an experiment before being discarded with normal waste. Surfaces, spillages and discard pots (e.g. for slides and syringes) are best sterilised using a disinfectant such as Virkon, hypochlorite (bleach) or ethanol. This isn't as thorough as sterilisation, but it kills likely pathogens and inhibits the growth of most microbes. Medium (pl. media) The mixture that the microbes are grown (or cultured) on. The medium must contain all the nutrients needed for the microbes to grow (e.g. sugars, minerals, proteins, etc.). Culture media can be made up by mixing together known amounts of specific chemicals (a defined or synthetic medium), or they can be made from a natural source such as boiled meat or yeast extract, which generally contains the nutrients required by most microbes but in unknown quantities (an undefined or complex medium). Selective Medium A synthetic medium with specific composition to allow the growth of only one group of microbes and stop the growth of all others. For example: MacConkey medium is selective for Gram-negative bacteria Mannitol salt medium is selective for Gram-positive bacteria Yeast malt extract (YM) medium has a low pH, suitable for fungi but not bacteria Media with antibiotics are selective for antibiotic-resistant bacteria (unit 10) Nutrient Medium A cheap general-purpose complex medium used for growing bacteria in most school experiments. Broth A liquid medium (i.e. without agar) in a test tube, universal bottle or flask. Bacteria grow throughout the liquid, and broth cultures can be scaled up to grow large quantities of bacteria. Agar Agar is mixed with a liquid medium to make a solid medium. Bacteria only grow on the surface of the solid medium (where they can get oxygen), which is very useful to observe, separate and store bacteria cultures. A Level Biology Unit 8 page 5 A solid medium in a petri dish is known as an agar plate, A solid medium in a Universal bottle is called an agar slope. Agar is actually a polysaccharide extracted from seaweed. It melts at 41°C (so can be incubated at 37°C without melting), is reasonably transparent, and is not broken down by microbes, so it remains solid. Do not use the word agar when you mean medium. Inoculate To add cells to a medium, so that they may grow. Cells must be added using aseptic technique, so that no foreign cells are introduced by accident. The bacteria are usually transferred using a wire or glass inoculating loop, which can carry a tiny volume of liquid culture (10 µl) or a scraping of cells from a solid culture. Larger volumes of liquid are transferred using a sterile cotton bud, syringe or pipette. Incubate To leave a culture to grow under defined conditions. Cultures are usually incubated in an incubator, which is basically an oven with a very good thermostat. Petri dishes are incubated upside down, so that condensation doesn’t drip onto the agar and mix up the culture. Broth cultures may be incubated in a water bath, preferably with constant shaking. In schools, cultures must be incubated at a maximum of 30ºC to discourage the growth of human pathogens. Culture A growth of microbes in a medium. The culture can be pure (one species of microbe) or mixed (many species). Mixed cultures can be obtained from natural sources such as soil, milk, food, saliva, faeces, etc., and pure cultures may be obtained from microbiological suppliers, or from a single colony on a streak plate (see below). Colony A visible circular growth of bacteria on an agar plate containing many millions of cells. The key point is that each colony grows from a single original cell, so is a pure culture of geneticallyidentical cloned cells. Colonies are usually circular because they grow outwards in all directions from the original cell. Streak Plate A method of inoculating an agar plate with bacteria so that the bacteria are gradually diluted. The aim is to separate individual cells, which can grow into visible colonies. This is used to separate pure cultures of bacteria from a mixed starting culture, such as soil, food or bodily fluids. Bacterial Lawn A smooth even layer of bacteria covering the surface of an agar plate. This is useful to test the effectiveness of antimicrobial substances such as antiseptics or antibiotics, using a disk diffusion test. A Level Biology Unit 8 page 6 Blank Page A Level Biology Unit 8 page 7 Measuring the Growth of Microbes Microbes are mostly unicellular, so their “bodies” don’t get any bigger as they grow, like an animal or plant. So measuring the growth of microbes in a culture generally means just counting the number of cells. There are various techniques for doing this, and some give total cell counts, which include both living and dead cells, while others give viable cell counts, which only include living cells. Serial Dilution Microbial cultures can have a very large concentration of cells in them – 109 cell mL-1 for example, which is far too many to count. So you often start by making a serial dilution of your culture to give you a large range of concentrations. One of these will be suitable for counting. We will look at four techniques for measuring microbial growth: 1. Cell counting with a haemocytometer 2. Turbidometry with a colorimeter 3. Dilution plating 4. Dry mass for measuring fungal growth A Level Biology Unit 8 page 8 1. Cell Counter (Haemocytometer) This counts the total cells in a broth culture by simply observing the individual cells under the microscope. This is reasonably easy for large cells like yeast, but is more difficult for bacterial cells, since they are so small. The cell counter (or haemocytometer) is a large microscope slide with a very accurate grid drawn in the centre. The grid marks out squares with 1 mm, 0.2 mm and 0.05 mm sides. There is an accurate gap of 0.1 mm between the grid and the thick coverslip, so the volume of liquid above the grid is known. The number of cells in a known small volume can thus be counted, and so scaled up. The units are cells mL-1. The haemocytometer grid has a range of different-sized squares, from 5mm to 0.05mm, so you can choose the most appropriate sized square to use depending on the size and density of cells. The most commonly-used size is the 0.2m “triple-lined” square. For example a 0.2 mm square has an average of 80 cells in a 1000x dilution Volume above square = 0.2 x 0.2 x 0.1 = 0.004 mm³ So there are 80 cells in 0.004 mm³ in the diluted suspension Which is 20 000 cells per mm³ in the diluted suspension which is 20 000 x 1000 = 2 x 107 cells per mm³ of undiluted suspension or 2 x 1010 cells cm-3 (cells mL-1) What do you do about cells that cross the line between two squares? They are partly in both squares, but you don’t want to count them twice. The convention is to count cells that cross the line on the north and west sides of the square, but not the on the east or south sides. This way every cell will be counted once and only once. In the triple-lined squares the middle line is the actual boundary. Counting Viable Cells Normally the haemocytometer counts total cells, since you can’t tell by looking if a cell is alive or dead. However, by using a vital stains (like trypan blue) that selectively stain only dead cells and not living ones, you can distinguish between living and dead cells under the microscope. This allows both total and viable cell counts to be made in a haemocytometer. A Level Biology Unit 8 page 9 2. Turbidometry This technique measures the density of cells indirectly. A sample of the liquid culture is placed in a cuvette in a colorimeter, and the absorbance of light is measured. The greater the concentration of the cells, the more cloudy or turbid the liquid is, so the more light it scatters, so the less light is transmitted to the detector, so the higher the absorbance reading. A wavelength of 600 nm is normally used. Although the absorbance scale of the colorimeter is used, technically light is not actually absorbed by the cells (as it is by pigment molecules), but scattered. Since the cells are in suspension, they will settle over time, so it is important to shake the culture flask before collecting a sample so that the mixture is homogenous. For the same reason the turbidity reading should be taken quickly before the cells settle to the bottom of the cuvette. If the absorbance reading is too high, the original culture will have to be diluted using serial dilution to obtain a reading below 0.5. If a range of culture samples are counted in a haemocytometer and also in a colorimeter, then a calibration curve can be plotted. Generally, if the absorbance is below 0.5, the calibration curve is a straight line. From this calibration curve the concentration of cells in a new sample can be read off for any absorbance, without having to use a haemocytometer. A Level Biology Unit 8 page 10 3. Dilution Plating This technique counts viable cells. A small sample is taken from a broth culture using aseptic technique and a sterile pipette. This sample is then spread evenly onto an agar plate using a sterile glass or plastic spreader. The agar plate is incubated at 30ºC for 24h. Each viable cell in the sample will multiply to form a colony. The number of colonies on the agar plate is counted. Since each colony arose from a single living cell, this is the number of viable cells in the sample. For most cultures there will be too many colonies to count, so a serial dilution is used to get a range of cell concentrations, and a sample from each dilution is spread on an agar plate. For most of the agar plates there will be too many colonies to count, and in some agar plates there will be too few, so any count would be unreliable. But in one of the dilutions there will be a good number (20-200) of individual colonies. From this we can calculate the concentration of viable cells in the original culture. For example: suppose there were 83 colonies in the x10 000 dilution agar plate. This means there were 83 viable cells in the 0.1 cm³ sample of the x10 000 dilution So there were 83 cells ×10 000 0.1 cm3 = 8.3 × 106 cells cm-3 in the original culture This method is very accurate, but tedious. It is the only good way to count viable cells, because only those cells that grow into living colonies are counted. A Level Biology Unit 8 page 11 4. Measuring Fungal Growth Fungi can be grown in solid or liquid culture, just like bacteria. Unicellular fungi (yeasts) can be treated just like bacteria and any of the cell counting methods above can be used. But multicellular fungi (moulds) form a large mass called a mycelium, make of thread-like hyphae (see unit 6). Mould growth can be measured in two ways: Solid Cultures. Moulds growing on a solid agar plate tend to form fairly flat circular mycelia, so the area of the mycelium is an easy measure of growth. If the mycelium is not circular it can be treated as an ellipse, and the area calculated using: area = x r1 x r2, where r1 and r2 are the longest and shortest radii. Liquid Cultures. For moulds growing in liquid cultures the best measure of growth is their dry mass. A sample of the culture is taken and filtered or centrifuged to separate the fungi from the solution. The mould is then dried fully by heating in an oven at 100ºC to evaporate the water and then weighed. The cycle of heating and weighing is repeated until the mass is constant, which means that all the water has been driven off. This gives the dry mass (i.e. fungal cell mass) in the original sample volume. The dry mass of bacterial cells growing in liquid culture can also be measured using this dry mass technique. Comparison of Counting Methods Viable or total count Gives exact number of cells Speed Used for Haemocytometer Turbidity Spread Plate Dry Mass total total viable total Slow (hour) Fast (minutes) Very slow (24 h) Slow (hours) bacteria bacteria bacteria Moulds (or viable with vital stain) A Level Biology Unit 8 page 12 Bacterial Growth Curves By starting a new culture with a small number of cells, and using one of the counting methods to measure the number of cells over a period if time, a growth curve can be obtained. This growth curve is typical of unicellular microbes such as bacteria and yeast that increase their numbers by binary fission. The curve is called a sigmoid growth curve, and can be plotted on a linear or logarithmic scale. It has four phases: 1. Lag phase, while cell division and cell death rates are both low, so the population does not change much. The microbes are adjusting to their new conditions by absorbing water and nutrients from the medium, switching on genes for biochemical pathways, transcribing mRNA and synthesising proteins and enzymes. 2. Exponential phase, or log phase, while the cells divide without any external limiting factors (i.e. there is plenty of space and nutrients). Bacteria divide by binary fission, so the population doubles in a set time known as the generation time. This generation time can be as short as 20min for bacteria under optimal conditions. Growth with a constant doubling time is called exponential growth, and if the growth curve is plotted on a log scale the exponential phase is linear, which is why this phase is also called the log phase. In practice exponential growth only lasts a short time until some factor limits growth rate. 3. Stationary phase, when cell division slows down and cell death increases until the two rates are approximately equal, so the population of viable cells is roughly constant. The growth rate is limited by lack of nutrients, lack of oxygen, lack of space, change in pH, or accumulation of toxic metabolic waste products. A total cell count may still increase during the stationary phase, as dead cells are included. 4. Death phase, when cell death rate is greater than cell division rate, so number of viable cells decreases. The total cell count may also decrease as dead cells lyse. A Level Biology Unit 8 page 13 Measuring the Exponential Growth Rate Exponential growth is common in everyday life (e.g. population growth, chain reactions, compound interest), and is described by the equation: 𝑁𝑡 = 𝑁0 × 𝑒 𝑘𝑡 Where Nt = N0 = e= k= t= the population at time t the population at time zero the mathematical constant known as Euler’s number, roughly 2.71828. the exponential rate constant, with units of time-1 (h-1, day-1, etc.) time The exponential rate constant k can most easily be calculated from a growth curve by re-writing the equation in its logarithmic form: 𝐿𝑛(𝑁𝑡 ) = 𝐿𝑛(𝑁0 ) + 𝑘𝑡, where Ln means natural (base e) log. This equation can be re-arranged to: 𝑘= 𝐿𝑛(𝑁𝑡 ) − 𝐿𝑛(𝑁0 ) 𝑡 In other words k is the gradient of the growth curve when plotted on a natural log scale. If base 10 logs are used instead, then a conversion factor of log(e), 0.434 is needed: 𝑘= 𝐿𝑜𝑔(𝑁𝑡 )−𝐿𝑜𝑔(𝑁0 ) 0.434× 𝑡 A different rate constant, the generation (or doubling) rate constant is given by a different conversion factor of log(2), 0.301: 𝜈= 𝐿𝑜𝑔(𝑁𝑡 )−𝐿𝑜𝑔(𝑁0 ) 0.301× 𝑡 The advantage of is that it is the reciprocal of the generation or doubling time T, i.e. 𝜈 = 1⁄𝑇. The generation time is the time taken for one generation, or for the population to double in size. A feature of exponential growth is that it has a constant doubling time. So for example if T = 20min then 𝜈 = 1⁄20 =0.05 min-1. Sometimes, just to be confusing, is called the exponential growth rate constant and given the symbol k! However, you don’t need to learn any of these equations, or even understand them. You will be given an equation and you just have to plug in the correct numbers. A Level Biology Unit 8 page 14 Antibiotics Antibiotics are antimicrobial chemicals produced naturally by other microbes (usually fungi or bacteria). The first antibiotic was discovered in 1896 by Ernest Duchesne and "rediscovered" by Alexander Fleming in 1928 from the filamentous fungus Penicilium notatum. Neither investigator appreciated the importance of what he had found, and the antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war. The discovery of antibiotics was probably the greatest medical advance of the 20th century, saving many millions of lives. Today there are hundreds of different antibiotics, amongst many other antimicrobial drugs: antimicrobial drug antibiotic drug antifungal drug antiviral drug kills or inhibits any microbe kills or inhibits bacteria (also called antibacterial) kills or inhibits fungi (also called fungicides) kills or inhibits viruses Action of Antibiotics Many chemicals kill bacteria. But a therapeutically useful antibiotic must be selectively toxic i.e. it must kill bacteria growing in human tissue, without also killing the host human cells. Antibiotics do this by inhibiting enzymes that are unique to prokaryotic cells, such those involved in synthesising the bacterial cell wall or 70S ribosomes. For example: Penicillin (and related antibiotics ampicillin, amoxicillin and methicillin) inhibits the enzyme transpeptidase, which cross-links peptidoglycan chains in the bacterial cell wall. Without these crosslinks the cell wall fall apart and the cell is left without a cell wall. This means the cell can’t withstand the turgor pressure due to osmosis and the cell bursts by osmotic lysis. Since this kills bacteria, penicillin is called a bactericidal antibiotic. Penicillin is most effective against Gram positive bacteria, because they rely on a thick peptidoglycan cell wall. Gram negative bacteria are not affected by penicillin because the antibiotic can’t easily cross the outer lipopolysaccharide membrane of Gram negative cell walls to reach their thin peptidoglycan cell wall. Since penicillin only affects some bacteria it is called a narrow-spectrum antibiotic. A Level Biology Unit 8 page 15 Tetracycline (and streptomycin and erythromycin) binds to 70S bacterial ribosomes, blocking the attachment of tRNA, so stopping protein synthesis. This stops further growth and cell division, but doesn’t kill the bacterium. Tetracycline is therefore called a bacteriostatic antibiotic. Bacteriostatic antibiotics are still useful medically, because if the growth of an pathogen is stopped, the body's immune system will usually be able to kill it. Because all bacteria have 70S ribosomes, tetracycline is effective against almost all bacteria and so is a broad-spectrum antibiotic. These graphs show how bactericidal and bacteriostatic antibiotics affect bacterial growth curves: Testing Antibiotics The effectiveness of different antibiotics against different bacteria can be tested using a disk diffusion test. A bacterial lawn is made on an agar plate using a pure sample of bacteria or a sample from a patient, and different antibiotics are then applied to the agar on filter paper disks. Special disks, called multidisks or mast rings, can be used to test 8 different antibiotics at once: The antibiotic diffuses out from the filter paper through the agar, killing or preventing growth of the bacteria as it goes and leaving a clear inhibition zone. In general, the larger the inhibition zone around the disk, the more effective the antibiotic is against this particular bacterium, but what exactly does “more effective” mean? A Level Biology Unit 8 page 16 As the antibiotic diffuses out, its concentration decreases, as shown on the graph on the right. So a large inhibition zone means the antibiotic kills bacteria at a low concentration and a small zone means that a large concentration of the antibiotic is needed. So “more effective” means kills at a lower critical concentration. This graph also shows that the size of the inhibition zone also depends on the concentration of antibiotic on the filter paper. The disk diffusion test does not distinguish between bactericidal and bacteriostatic antibiotics. Resistance to Antibiotics When antibiotics were first introduced after the Second World War, they were seen as "miracle drugs" because they cured all bacterial diseases. However, within a few years, some antibiotics stopped working as bacteria became resistant to them (note that bacteria can’t be immune). Development of Antibiotic Resistance How do bacteria become resistant to antibiotics? Resistance first develops due to a mutation. Bacteria reproduce asexually, so all the offspring should be the same, but sometimes, at random, mutations occur when DNA is replicated. These mutations may have any effect (and most will be fatal), but just occasionally a mutation occurs that makes that bacterium resistant to an antibiotic. For example: A mutation could slightly alter an enzyme, changing its substrate specificity so that its active site will now bind penicillin. Some bacteria now have penicillinase enzymes that modify or break down penicillin, rendering the antibiotic useless. Antibiotics are often similar to normal bacterial metabolites, and a small mutation in an existing enzyme can modify its active site to fit an antibiotic. A mutation could modify a transport protein so that the antibiotic is transported out of the bacterial cell, or not transported into the bacterial cell. A mutation could slightly alter a ribosome so that antibiotics like tetracycline and streptomycin can no longer bind and inhibit protein synthesis. Such mutations are very rare, but bacteria reproduce so rapidly, and there are so many bacterial cells, that new resistance mutations do crop up at a significant rate (a few times per year somewhere on the planet). Remember that development of antibiotic resistance is a random event, and is not caused by the presence of the antibiotic. It is certainly not an adaptation that bacteria acquire. A Level Biology Unit 8 page 17 Spread of Antibiotic Resistance Resistance spreads from one mutated bacterial cell to another by vertical and horizontal gene transfer. Vertical Gene Transfer Imagine a community of different bacterial species living in your gut, and one particular cell has just mutated to become resistant to penicillin. What happens next? It will reproduce by binary fission and pass on its resistance gene to all its offspring, forming a new strain of bacteria in your gut. If there is no antibiotic present in your gut (most likely) this mutated strain may well die out due to competition with all the other bacteria, and the mutation will be lost again. However, if you are taking penicillin, then penicillin will be present in the bacteria's environment, and these mutated cells are now at a selective advantage: the antibiotic kills all the normal bacterial cells, leaving only the mutant cells alive. These cells can then reproduce rapidly without competition and will colonise the whole environment. This is a good example of natural selection at work. The mutant cells have been selected by the environment and so the frequency of the resistance allele in the population has increased. Horizontal Gene Transfer Bacteria have a trick that no other organisms can do: they can transfer genes between each other by conjugation. This is the transfer of DNA between bacterial cells via a conjugation tube or pilus. From time to time two bacterial cells can join together (conjugate), and DNA passes from one cell (the donor) to the other (the recipient). The transferred DNA is usually a plasmid but can be part of the chromosome. Conjugation means a resistance gene can spread from the bacterium in which it arose to other, perhaps more dangerous, species. It is also the cause of multiple resistance. It is highly unlikely that a single strain will mutate twice to develop resistance to antibiotics, but it is perfectly likely that it could receive genes for resistance to different antibiotics by horizontal gene transfer. This has led to strains of bacteria that are resistant to many (or even all) antibiotics. A Level Biology Unit 8 page 18 Multi-Resistant Bacteria Antibiotic-resistant bacteria can spread to other people by any of the normal methods of spreading an infection: through faeces, water, food, sneezing, infected instruments, etc. A common source of antibioticresistant bacteria (and especially multiple-resistant bacteria) is hospitals. This is partly because hospitals have a high concentration of people with bacterial infections, but also because the use of antibiotics means any antibiotic-resistant strains can multiply in the absence of competition. A good example is Staphylococcus aureus, a bacterium responsible for a variety of diseases from staphylococcal food poisoning to toxic shock syndrome. This species has been resistant to penicillin for years, due to the possession of the penicillinase enzyme. Methicillin is unaffected by penicillinase and so was effective against S. aureus. However, within a year of the introduction of methicillin, methicillin-resistant strains of S. aureus (MRSA) were found in hospitals where methicillin was in regular use. Infections by MRSA were very difficult to treat, responding only to the antibiotic vancomycin. In 1997 vanocmycinresitant, methicillin-resistant Staphylococcus aureus appeared in Japan. These bacteria are effectively untreatable at present. Mycobacterium tuberculosis, the bacterium that causes TB (see pxx), has developed resistance to streptomycin. An extreme drug-resistant TB strain (XDR-TB) has been found in South Africa that kills 98% of those infected within two weeks, and is resistant to most antibiotics. Resistant strains of M. tuberculosis can only be treated using a cocktail of four different antibiotics over a prolonged period of months, which is expensive and unlikely to be achieved in developing countries. Controlling the Spread of Antibiotic Resistance How can the spread of antibiotic resistance be controlled? There are many different measures that are being taken, but none is effective on its own, and they all have difficulties. 1. Use a wider range of antibiotics. There is a constant industrial search for new antibiotics for which there is no resistance. However, it is becoming more and more expensive to find truly novel drugs and, sooner or later the bacteria will develop resistance, so this is only a short-term solution. 2. Prescribe fewer antibiotics. This will reduced the selection pressure in favour of resistant strains. Most resistant strains are inferior to the "wild type" strains in other respects (perhaps they reproduce more slowly), so in an antibiotic-free environment the mutants are generally out-competed and will die out. The antibiotic resistance genes will therefore disappear from the gene pool of that population. Unfortunately we are now in an "antibiotic culture" where many doctors prescribe antibiotics routinely for common ailments such as the flu (even though they have no effect), simply to keep the patient happy. And in some countries antibiotics can be bought over the counter without a doctor’s prescription, so are impossible to control. A Level Biology Unit 8 page 19 3. Stop using antibiotics in animal feed. Many farmers routinely feed their livestock small concentrations of antibiotics to promote growth and as a prophylactic against possible infection. This agricultural use accounts for an astonishing 90% of all antibiotic use worldwide, and provides very strong selection for antibiotic-resistant bacteria. The practice was made illegal in the EU in 2006, but this has had little effect and anyway few other countries have passed any laws, partly because the agriculture industry is a very powerful lobby. 4. Improve hospital hygiene. Transfer of bacteria between patients in hospitals is a major cause of spread of antibiotic resistance genes. Hand washing with antibacterial wash, using disposable gloves and aprons when handling patients, deep cleaning wards and all soft furnishings periodically all help to reduce spread. But the measures are expensive and rely on compliance of all staff and visitors. 5. Screening and isolating patients. New patients can be tested for the presence of MRSA and other resistant “superbugs” and then isolated in wards separate from uninfected patients. However, screening takes time (24h typically) and many hospitals don’t have space for isolation wards. If we cannot control the spread of antibiotic resistance, then most antibiotics will become useless, and we will revert to the pre-antibiotic age where bacterial infections are effectively untreatable. A Level Biology Unit 8 page 20 Pathogens and Disease To most people “disease” means an infectious disease, and these are the diseases you can "catch". Infectious diseases are caused by a variety of pathogens, including viruses, bacteria, fungi and protoctists. A few of the common pathogens are shown in this table: Viral Diseases Bacterial Diseases Fungal Diseases Protoctist Diseases Disease common cold influenza measles mumps chickenpox AIDS tuberculosis typhoid food poisoning cholera tetanus whooping cough pneumonia thrush athletes foot ringworm malaria amoebic dysentery sleeping sickness Pathogen Rhinovirus Myovirus Paramyxovirus paramyxovirus Varicella zoster virus HIV Mycobacterium tuberculosis Salmonella typhi Salmonella enterica Vibrio cholerae Clostridium tetani Bordetella pertussis Streptococcus pneumoniae Candida albicans Tinea pedis Tinea capititis Plasmodium vivax Entamoeba histolytica Trypanosoma spp. Some pathogens are more harmful than others; in other words they are have a greater pathogenicity or virulence. For a pathogen to cause a disease these steps must take place: 1. Transmission. The pathogen must be transmitted to the human host. Pathogens can be transmitted through drinking water, eating food, breathing aerosol droplets, animal bites, or direct contact. 2. Entry. The pathogen must gain entry inside the human body. The human body is protected by a tough layer of endodermis (skin), but pathogens can enter via breaks in the skin (e.g. malaria); or through the epithelium exchange interfaces, such as in the digestive system (e.g. cholera) or lungs (e.g. tuberculosis). 3. Evasion. The pathogen must evade the defences of the host. Humans have a range of defences, such as stomach acid, lysozyme enzymes and the immune system, and these defences are usually very effective at preventing disease. But it only takes a few pathogen cells resisting the defences to multiply and cause a disease. 4. Invasion and Reproduction. The pathogen must reproduce. Viruses, bacteria and protoctists are all smaller than human cells and so can colonise human cells and reproduce inside them. This reproduction digests and uses the host cell’s contents and so prevents the host cell from carrying out its normal reactions. The microbes then usually burst out of the host cell, rupturing the cell membrane and killing the cell in the process. A Level Biology Unit 8 page 21 5. Harm. To be pathogenic, the pathogen must harm the host. As we’ve just seen, reproducing inside cells harms the host, but many pathogens also cause harm by producing toxins – chemicals that interfere with the body's reactions. These chemicals may inhibit enzymes, bind to receptors, bind to DNA causing mutations, interfere with synapses and so on. There are two kinds of toxins: Exotoxins are proteins secreted by bacteria while they are growing. Each different exotoxin has a specific effect. For example the tetanus bacterium colonises nerve cells and secretes a toxin that inhibits motor neurones, causing muscle paralysis; while the cholera bacterium colonises the intestine and secretes a toxin that causes violent inflammation of the intestine and diarrhoea. Viruses can also produce toxins. For example the HIV protein “tat”, which helps to transcribe the HIV’s DNA, is a toxin because it prevents T helper cells from working. Some bacterial exotoxins (e.g. cholera) are actually proteins made by viruses that are infecting the bacteria that are infecting the human! Endotoxins are lipopolysaccharides released from the cell wall of Gram negative bacteria when they are killed and digested by the host. They all cause the same general effects, which include fever, weakness and aching. We will look at six diseases and their pathogens in detail: 1. Staphylococcal infections 2. Salmonellosis 3. Tuberculosis 4. Influenza 5. Malaria 6. Stem Rust of wheat A Level Biology Unit 8 page 22 Disease Staphylococcal (Staph) infections Pathogen Various species of the genus Staphylococcus, Gram-positive bacteria, including MRSA (methicillin-resistant Staphylococcus aureus). Transmission Most people have commensal Staphylococcus bacteria growing harmlessly on their skin and on their nasal mucosa. The bacteria become pathogens when they enter the body through a break in the skin, such as a cut, burn or insect bite. Mode of Infection Once in the body, Staphylococcus release various exotoxins, including haemolysins – polypeptides that form pores in the host cell membranes; and superantigens – proteins that stimulate a massive primary immune response (see p xx), which can cause toxic shock and death. Signs and Symptoms Staphylococcal infections can be put into two groups: Skin infections include boils (red, painful lumps on the skin); impetigo (sores, blisters and crusts on the skin); cellulitis (an infection of the deep layers of the skin, which become red, painful, swollen and hot); and abscesses (a painful lump under the skin containing pus). Invasive infections include septic arthritis (a joint infection with pain, swelling and tenderness); osteomyelitis (a bone infection causing pain, restricted movement and swelling); pneumonia (an infection of the lungs with persistent coughing, breathing difficulties and chest pain); sepsis (an infection of the blood with a fever, rapid heartbeat and rapid breathing); and toxic shock syndrome (where bacteria release toxins into the blood causing fever, vomiting, diarrhoea, fainting, dizziness, confusion and a rash). Treatment Skin infections don't usually need any treatment and will get better on their own within a few days or weeks, but some may require antibiotic tablets or creams. Invasive infections usually require treatment with antibiotic injections for several days in hospital. Hospitalacquired infections with MRSA need to be identified and treated with vancomycin, one of the few effective antibiotics. Prevention Staphylococcal infections can be reduced by hygiene. A Level Biology Unit 8 page 23 Disease Salmonellosis (Salmonella Food Poisoning) Pathogen Various strains of Salmonella enterica, a Gram-negative bacterium Transmission Through food, especially poultry and eggs, especially when uncooked (e.g. mayonnaise and ice cream), or lightly-cooked (e.g. meringue and soft-boiled eggs); or through unpasteurised milk. Other sources are contamination of foods and water by human or household pet faeces. Mode of Infection The bacteria colonise the lumen of the small intestine and remain there. Dead Salmonella cells release endotoxins, which stimulate an inflammatory response in the epithelial cells. This response reduces the absorption of water, resulting in diarrhoea. Signs and Symptoms Sudden signs 1-2 days after eating infected food. Symptoms include moderate diarrhoea, Treatment Treatment usually by fluid replacement. Antibiotics are unnecessary since the symptoms vomiting, abdominal pain, mild fever and headache. Recovery usually in 2-3 days. are usually so mild. Prevention Prevention is mainly by hygiene in food preparation; thorough cooking of poultry and eggs; and pasteurisation of milk. Chickens are vaccinated and less crowded poultry farming methods also reduce infection rates in hens. Eggs in UK are now always pasteurised. A Level Biology Unit 8 page 24 Disease Tuberculosis (TB) Pathogen Mycobacterium tuberculosis, a bacterium Transmission Through aerosol droplets from coughs and sneezes of infected persons. Mode of Infection The disease has two stages: 1. Latent tuberculosis starts when bacterial cells invade the epithelial cells of the alveoli and multiply to form lumps called tubercles, in which the bacteria remain alive but dormant. The tubercles stimulate an inflammatory response by the white blood cells of the immune system, resulting in the formation of fibrous scar tissue. 2. Active tuberculosis starts after a delay of months to years, when the bacteria emerge from the tubercles and start reproducing inside the lung epithelial cells, lysing and killing them. The TB bacteria can also spread through the bloodstream to other organs, like the kidney, bone and nervous tissue. Again the bacteria invade the cells in these tissues and digest them. This causes weakness as the body wastes away and the bacteria appear to “consume” the body – hence the old name for TB: consumption. Signs and Symptoms Typical symptoms of TB include a persistent long-lasting cough that may bring up bloody Treatment Since it is a bacterial disease, TB can be treated by antibiotics. However, many strains of phlegm; weight loss; night sweats; fever; tiredness; loss of appetite. Mycobacterium tuberculosis are resistance to streptomycin and there are extreme drugresistant M. tuberculosis strains (XDR-TB) resistant to most antibiotics. Prevention The BCG vaccine can provide effective protection against TB and is given to at-risk groups. A Level Biology Unit 8 page 25 Disease Influenza Pathogen Human Influenza virus (type A, B or C). Transmission Through air-borne droplet infection from the coughs and sneezes of infected individuals. Infected people are infectious from a day before symptoms show themselves until a week afterwards. Mode of Infection The virus invades the epithelial cells lining the upper respiratory tract (nose, mouth, throat, trachea and bronchi) and reproduces inside them, using up their resources and killing them by lysis. The death of the ciliated epithelial cells stops the mucus being removed, so encourages infection by airborne bacteria. These dead cells increase the amount and thickness of mucus produced during an infection, which irritates the throat, causing coughing. Signs and Symptoms The onset is sudden after an incubation period of 1-4 days, and the symptoms include fever, shivering, headache, muscular pain, coughing of excess mucus and a loss of appetite. Recovery normally takes about 4 days, unless there are secondary infections, which can be fatal if untreated. The influenza pandemic of 1918 killed over 20 million people world-wide, making it the greatest killer disease ever. Treatment Treatment is by bed rest with plenty of fluids and analgesic drugs like asprin or paracetamol. Since this is a viral infection, antibiotics are useless. Prevention Vaccination is difficult because of genetic changes in the influenza virus (antigenic variability), but vaccinations based on a variety of antigens are now used to protect atrisk groups (babies and elderly). Prevention would require the isolation of flu victims, which is not practical. A Level Biology Unit 8 page 26 Disease Malaria Pathogen Various species of the genus Plasmodium, a protoctist. Transmission Via the Anopheles mosquito vector. Anopheles has a long sharp proboscis for feeding, which can penetrate human skin and reach blood vessels. Plasmodium cells are sucked into female mosquitoes when they take a blood meal from an infected human, and are passed on to the next human she drinks from. Mode of Infection The plasmodium is carried through the host’s bloodstream to the liver, where it reproduces asexually producing thousands of clones. These clones invade red blood cells and start a multiplication cycle. The Plasmodium reproduce asexually over 2-3 days, producing 8-24 cells, which then burst out of their host cell, lysing it, and infect more red blood cells. Eventually the lack of red blood cells leads to anaemia and death. a mosquito sucking human blood Signs and Symptoms plasmodium inside red blood cells The symptoms of malaria typically begin 1-4 weeks after infection, and at first are similar to flu symptoms: headache, fever, shivering, muscle pain and vomiting. The classic symptom of malaria is paroxysm – a cycle of coldness followed by fever, occurring every two days. Treatment Malaria can be treated with anti-malaria drugs such as quinine, chloroquine and artemisinin. These drugs are only effective if they are taken very soon after (or even before) infection, and Plasmodium is becoming resistant to many of them. Prevention There is no vaccine for malaria and prevention is based on eliminating the Anopheles mosquito with insecticides, or preventing it biting with mosquito nets. A Level Biology Unit 8 page 27 Disease Stem Rust of wheat Pathogen Puccinia graminis, a fungus commonly known as stem rust fungus Transmission By wind-dispersed spores of Puccinia, spread from infected plants. Mode of Infection The Puccinia spores on the surface of leaves grow hyphae that enter the leaves through stomata. Inside the leaves the hyphae branch to form a mycelium that fills the leaf. The fungus secretes digestive enzymes, including cellulase, to digest the plant tissue and absorb the products. This cell death reduces photosynthesis so the cereal plant can’t grow. The hyphae also absorb water and minerals from the xylem vessels and digest the xylem cell walls so that the stems are weakened and fall over. Signs and Symptoms The fungus forms brick-red blisters on the surface of the leaves and stem, giving the Treatment Puccinia can be killed with fungicide (antifungal) chemicals, but this treatment is not infected plant its “rusty” appearance. economic, since the cost of fungicide is usually more than the crop is worth. Prevention Preventative measures include: removing Berberis plants close to cereal crops, since Puccinia spends part of its life cycle on this plant; reducing the use of fertilisers, which encourage rust growth; changing the season of the crops so they no longer match the season of the fungus; and breeding resistant strains of wheat. A Level Biology Unit 8 page 28 Controlling Malaria Malaria is endemic to many tropical parts of the world. An endemic disease is one that is always present in a population in a given area or country (for example chicken pox and flu are endemic in the UK). This map shows that today malaria is endemic to tropical regions, but it used to be much more widespread. During the 20th century, intensive international eradication programs reduced malaria distribution around the world by half, from 53% of the Earth’s land surface to 27%. The campaign involved pesticides, antimalarial drugs and destruction of mosquito breeding sites. Today, malaria remains endemic in tropical areas, but it appears to be very difficult to eradicate completely. So how is malaria controlled? Controlling Plasmodium The obvious strategy is to kill or control the pathogen. But Plasmodium is one of the most complex of human pathogens, and it is has proved impossible to control Plasmodium in the way many bacterial and fungal diseases can be controlled. Pasmodium is a eukaryotic protoctist, so has few unique targets for a selectively-toxic drug that don’t also harm the human host. Plasmodium has a complex life cycle involving stages in humans and mosquitoes. The cells of each stage have different enzymes and antigens, making it difficult to find a feature for a drug to target. In humans, Plasmodium is always found inside liver or blood cells, so shielding it from the immune system. Plasmodium has high antigenic variability, so vaccines don’t work. A Level Biology Unit 8 page 29 Controlling Anopheles If Plasmodium can’t be controlled, an alternative is to control the Anopheles mosquito vector instead. Mosquito control measures include: Insecticides. These have been very successful in some areas, and the insecticide DDT has successfully wiped out Anopheles from the Mediterranean, where it used to be endemic. However, DDT has side effects on other species, so its use for crop protection is now banned in many countries, though its use is still allowed for disease control. Spraying insecticides onto the walls of houses is an effective local measure. Biological control. Predators of mosquito larvae, including fish and dragonflies, can be introduced to reduce mosquito populations with some success. Attempts have also been made to control mosquitoes by releasing thousands of sterile male mosquitoes so that reproduction is unsuccessful. Mosquito nets. The female Anopheles mosquitoes only feed at night, so using insecticide-treated bed nets is a simple but very effective way of preventing biting. Although cheap, nets are still too expensive for many communities and charities always want to provide more nets. In 2015, 68% of African children were using mosquito nets, leaving many millions unprotected. Nets protect humans, but don’t kill mosquitoes, and mosquitoes that feed during the day are now starting emerge. Remove standing water. Mosquitoes breed on standing water, so removing any potential puddles from areas around homes can reduce the prevalence of mosquitoes locally. Large-scale drainage or flowing-water schemes are used in the USA, but are too expensive for developing countries. Some of these schemes can be successful on a small scale, but most require considerable expense and large-scale organisation, which is not available in many of the countries where malaria is most prevalent. In many areas eradicating malaria is not as high a priority as malnutrition, cholera or fighting wars. The Role of Science Scientists are actively involved in all stages of the war on malaria by: Studying the biology and ecology of Plasmodium and Anopheles, in order to understand their life cycles and so derive new treatments to control them. Evaluating the success of existing treatments in the lab and in the field. This can be very difficult to do, since there are so many confounding variables, and appropriate collection of unbiased data together with statistical analyses are essential. Advising governments and local hospitals on the best treatments for each situation Training local health-workers in the most appropriate techniques. A Level Biology Unit 8 page 30 The Immune System The Immune System is the body’s defence system against disease. It is made up of white blood cells (or leukocytes), which are found in the blood, lymph, tissue fluid and body cavities (such as alveoli). In unit 4 we listed the blood cells by their appearance, but it is more useful now to sort the leukocytes by their function: As this diagram shows there are two branches to the immune system: the non-specific immune system and the specific immune system. We’ll look at each in turn. A Level Biology Unit 8 page 31 The Non-Specific Immune System The non-specific immune system is a collection of general methods of destroying foreign bodies that have entered the body. The main methods are phagocytosis and inflammation. Phagocytosis Phagocytosis is the digestion of microbes and other foreign bodies by phagocytes: neutrophils, monocytes and macrophages. Phagocytes are large, irregularly-shaped white blood cells with a complex cytoskeleton that allows them to move and change shape. 1. Phagocytes crawl through the blood and tissue fluid in response to chemicals released by microbes or other white blood cells. 2. When they reach the microbe they surround and engulf it through the process of phagocytosis. The microbe is now trapped in a membrane sac called a phagosome. 3. The phagosome then fuses with lysosomes small vesicles containing lysozymes, which are released into the phagosome. These lysozymes are digestive enzymes (proteases, carbohydrases and lipases) that hydrolyse the proteins, carbohydrates and lipids that make up the microbe, so killing it. Different phagocyte cells work in different locations: neutrophils circulate in the blood, while macrophages and monocytes are found in lymph, tissue fluid, lungs and other spaces, where they kill microbes before they enter the blood. Macrophages are also important as antigen-presenting cells (see p33). Inflammation Inflammation is a localised response to an injury or infection, driven by granulocyte cells (eosinophils and basophils). Granulocytes release chemicals, including histamines and prostaglandins, which stimulate: Vasodilation to increase the flow of blood to the area, so the area turns red. Capillary leakage so that phagocytes and granulocytes can enter the local tissue fluid. The area swells and the dead pathogens and phagocytes, together with excess tissue fluid, are released as pus. Sensory neurone impulses, so the area is tender or painful. Blood clotting to seal a wound, so a scab is formed. Fever to raise the body temperature, which is more harmful to pathogens than humans. Tissue repair by depositing collagen and stimulating the growth of new cells, called scar tissue. A Level Biology Unit 8 page 32 The Specific Immune System The specific immune system is a more complex and sophisticated collection of reactions that not only kills invading pathogens, but also leaves a “memory” of the pathogen so that it can be killed quickly on subsequent infections. While all animals have a non-specific immune system, only vertebrates have a specific immune system, so it must be a later evolutionary advance. The specific immune system involves the lymphocyte cells, which are made in the bone marrow. There are two kinds of lymphocyte – Blymphocytes (or just B-cells) and T-lymphocytes (or just T-cells). The key feature of the specific immune system is that it is capable of recognising foreign cells as distinct from its own cells, an ability called self/nonself recognition. It does this by making use of antigens. Antigens Antigens are large molecules that bind to the lymphocyte cells and so trigger a specific immune response (the name is short for “antibody generator”). All living cells and viruses have molecules (usually glycoproteins) on their surface that can act as antigens. Antigens are genetically controlled, so close relative have more similar antigens than unrelated individuals or species. The “A/B” proteins on red blood cells that cause blood groups are an example of antigens, but all cells have them. B-Lymphocytes and Antibodies B-lymphocytes make antibodies. An antibody (also called an immunoglobulin) is a protein molecule that can bind specifically to an antigen. Antibodies all have a similar structure composed of 4 polypeptide chains (2 heavy chains and 2 light chains) joined together by strong disulphide bonds to form a Y-shaped structure. The stem of the Y is called the constant region because in all immunoglobulins it has the same amino acid sequence, and therefore same structure. The ends of the arms of the Y are called the variable regions of the molecule because different immunoglobulin molecules have a different amino acid sequence and therefore different structures. These variable regions are where the antigens bind to form a highly specific antigen-antibody complex, much like an enzyme-substrate complex. variable region constant region disulphide bridges antigen-binding site stylised diagram Rasmol molecular model Each B-cell has around 105 membrane-bound antibody molecules on its surface and can also secrete soluble antibodies into its surroundings. A Level Biology Unit 8 page 33 T-Lymphocytes and Receptors T-lymphocytes have receptor proteins on their surfaces. Receptor proteins are very similar to antibodies, but receptor proteins have only one binding site, and are only found on the surface of T-cells, never free in solution. Receptors proteins bind specifically to antigens to form antigen-receptor complexes. Each T-cell has around 105 receptor proteins. T-cells do not secrete soluble proteins. B-lymphocyte T-lymphocyte Self Tolerance Every human has around 108 different types of B and T cell, each making antibodies or receptor proteins with slightly different binding sites. Between them, these antibodies and receptor proteins can therefore bind specifically to 108 different antigens, so there will be an antibody or receptor proteins to match almost every conceivable antigen that might enter the body. At birth we have less than 100 copies of each type of B or T lymphocyte. The B and T cells with receptors for "self" antigens are quickly destroyed early in life. From then on self antigens are ignored, but any non-self antigens are recognised and stimulate an immune response as described below. Occasionally this self-tolerance fails, leading to autoimmune diseases, such as type I diabetes. We’ll now look at each stage of the actions of the specific immune system in detail. 1. Macrophages and Antigen Presentation Infection is started when cells with non-self antigens enter the blood or tissue fluid. The antigens can be from a variety of sources: e.g. a virus; a bacterial cell; a toxin released from a bacterium; on a cell infected with a virus so that it has viral proteins on its surface; on a transplanted cell; on a cancerous cell. These foreign antigens need to be presented on antigen-presenting cells in order to initiate the specific immune response. Many body cells can act as antigenpresenting cells, but the most important are the macrophages, because they are the most numerous phagocytes. As we’ve seen, macrophages ingest A Level Biology Unit 8 page 34 pathogens by phagocytosis, digest the pathogen but keep the antigens intact. The macrophages then “present” these foreign antigens on their surface, bound to special membrane proteins. The macrophage is now an antigen-presenting cell. This method of presenting antigens amplifies the number of foreign antigens in the blood without increasing the number of pathogens. The macrophage also secretes cytokine chemicals to stimulate the next stage of the immune response – clonal selection. 2. Helper T-cells and Clonal Selection The antigen-presenting cells interact with the Thelper cells in the blood. Sooner or later the antigen-presenting cell will encounter a T-helper cell with a matching receptor molecule, so the two cells bind tightly to each other. It's a bit like Prince Charming trying to fit the glass slipper (the antigen) onto all the girls in the kingdom (the receptors on the different T-cells) until eventually he finds Cinderella, who is an exact fit. As soon as a match is found, the tight binding of the antigen-presenting cell to the T-helper cell stimulates the T-helper cell to release signalling molecules called cytokines (or lymphokines, since they are released by lymphocytes). These cytokines stimulate immature T and B lymphocyte cells to activate, proliferate and differentiate. When activated, the lymphocyte cells divide repeatedly by mitosis, making a clone army of about 106 genetically-identical cloned lymphocyte cells. This is called clonal selection, because only the selected cells are cloned. There is now a clone army of B and T lymphocytes with identical binding sites on their cell-surface proteins – binding sites that specifically complement the foreign antigen. The clone army can now destroy the infecting microbe, as described below. 3. T-Cells and Cellular Immunity The activated T-lymphocytes differentiate into T-killer cells (or cytotoxic T-cells). These Tkiller cells bind to antigens on infected human cells or pathogen cells and kill them by secreting porin proteins that embed in the pathogen cell membrane. These make pores that allow water to diffuse in so that the cell lyses (bursts). This is called cellular immunity because the foreign cells are killed directly by the lymphocyte cells. A Level Biology Unit 8 page 35 4. B-Cells and Humoral Immunity The activated B-lymphocytes differentiate into plasma cells. Plasma cells contain large amounts of rough endoplasmic reticulum and are protein factories, synthesising and secreting large numbers of soluble antibodies. A single B-cell can divide to form 106 plasma cells, each of which can release 103 antibodies each second for 4 days. These antibodies are carried around the blood, lymph and tissue fluid binding to any antigens they come into contact with and forming antibody-antigen complexes. This is called humoral immunity because the foreign cells are killed by soluble antibodies dissolved in the blood plasma (body fluids were called humours in old terminology). The binding of antibodies to antigens kills pathogens in various ways: 1. By binding to antigens on viruses and bacteria they prevent the viruses or bacteria attaching to cells and so infecting them. 2. By binding to free toxin proteins they change the shape of the active region so that these proteins can no longer take part in the reactions that caused disease. 3. By stimulating phagocytosis and the non-specific immune response (e.g. inflammation). This process is called opsonisation. 4. By linking cells together. Each antibody molecule has two antigen-binding sites (one on each arm of the Y), so can potentially bind to two different pathogens. Some antibodies are joined in groups of 5 or more by their constant regions, and both of these features mean that antibodies can stick cells together into large clumps. This process, called agglutination, immobilises viruses and cells, and precipitates soluble toxins so that they can easily be destroyed by phagocytes or T-killer-cells. 5. Memory Cells and Immunological Memory The clone army of B and T cells only lasts for a few days, after which the cells are destroyed and recycled by phagocytes. However, some of the activated B and T cells differentiate into memory cells. These memory cells remain in the blood for many years after the infection and the memory B cells continue to secrete antibodies in small quantities. If the same antigen is encountered later in a subsequent infection these memory cells will quickly divide to form a new clone army, without having to go through the clonal selection step. This is called the secondary immune response (see below). A Level Biology Unit 8 The five stages of the specific immune system are summarised in this diagram: page 36 A Level Biology Unit 8 page 37 Primary and Secondary Immune Responses The first time a new antigen is encountered there are only a few lymphocyte cells of each kind (<100) for the antigen to encounter, so it can take several days for clonal selection to take place and the clone army to be assembled. Furthermore the clone army tends to be fairly small. This slow and weak response to a first infection is called the primary immune response. It is during this period that the symptoms of the disease are shown, partly due to toxins and cell death due to the pathogen, and partly due to the immune response itself (e.g. fever, inflammation). After a primary response memory cells (both T and B lymphocytes) remain in the blood. This means that after a subsequent infection by the same antigen the clonal selection stage can be by-passed and the specific immune response is much faster and much greater (i.e. more clone B and T lymphocytes and antibodies are produced). This is called the secondary immune response, and is so fast that the pathogen is destroyed before it reproduces enough to cause disease. In other words the individual is immune to that disease. Note that the non-specific immune response is the same in all infections. Antigenic Variability Some pathogens have antigens that remain constant, so we remain immune to them, and can only catch them once (e.g. chicken pox, measles or mumps). Other pathogens develop new strains every few years, with different antigens (e.g. the common cold, flu, malaria). The body does not have memory cells against the new antigens, so infection by a new strain of the microbe causes a new primary response, with all the trappings of the accompanying disease. These pathogens with many different strains show antigenic variability. It is caused by mutations during replication of the pathogen. A Level Biology Unit 8 page 38 Immunisation We have been able to make use of the immune system's memory to artificially make people immune to certain diseases even without ever having caught them. The trick is to inject with an antigen that will promote the primary immune response, but has been modified so that it is non-virulent (or nonpathogenic), i.e. will not cause the disease. The immune system is thus fooled into making memory cells so that if the person is ever infected with the real virulent pathogen, the more powerful secondary immune response is triggered and the pathogen is killed before it can cause the disease. This technique is called vaccination and is commonly used to provide artificial immunity to a number of potentially-fatal diseases. In the UK children are commonly vaccinated against diphtheria, tetanus, whooping cough, polio, measles, mumps, rubella and TB. Any process that promotes a primary immune response and leaves memory cells is called active immunity (e.g. catching a disease or vaccination). Passive Immunity It is also possible to inject antibodies against certain pathogens into the blood. This is called passive immunity and is used when someone has already been infected (or is likely to become infected) with a pathogen. The antibodies in it assist the body's normal immune response and help it deal with serious diseases. Antibodies are either prepared from the blood serum of an infected human (or rarely animal), called an antiserum, or are made by genetic engineering. Passive immunisation is not very common, but can be used for rabies, tetanus, measles and hepatitis B, and is being tried to combat AIDS. Passive immunity also occurs naturally when a mother passes antibodies to her child. Antibodies can pass across the placenta to the foetus and are also found in colostrum, the milk produced in the first few days after birth. Since the baby's digestive system does not function at this stage, the immunoglobulin proteins can be absorbed intact. This passive immunity helps the new-born baby survive in a world full of pathogens, and is one reason why breast feeding is so important. The different kinds of active and passive immunity are summarised in the table. Active Immunity (antigens received) Passive Immunity (antibodies received) Natural Achieved through the passing of antibodies Achieved through the primary immune from mother to child through the placenta response following an infection and milk. Artificial Achieved through the primary immune Achieved through injection of antibodies in response following injection of modified an antiserum. antigens in a vaccination. A Level Biology Unit 8 page 39 Herd Immunity If enough people are vaccinated in a population (typically 85-95%), then even the few that are not, or cannot be, vaccinated are protected by herd immunity, since there are not enough hosts for the pathogen to survive and reproduce. Some individuals cannot be safely vaccinated (e.g. new-born infants, those with immunosuppressant diseases or undergoing chemotherapy and some with particular genetic conditions) and they rely on herd immunity to protect them. Unfortunately, some people choose to not vaccinate themselves or their families and rely on herd immunity to protect them. These so-called free-riders put themselves and others at risk because the threshold required for herd immunity is very high, and too many free-riders may compromise the herd immunity. One recent example of the problems of free-riding is the MMR hoax that started with a paper published by Dr Andrew Wakefield in 1989, linking autism to the MMR vaccine. The paper was later shown to be fraudulent and was withdrawn, while Wakefield was struck off the UK medical register for unethical conduct. However the story was widely reported in the press and vaccination rates fell from 92% in 1996 to 84% in 2002, with the result that the incidence of both measles and mumps increased over the next ten years to 37 times their previous level. Measles and mumps are now both endemic in the UK, having been almost eradicated, and several children have died.