Topic 6 INFECTION, IMMUNITY AND INFECTION Fingerprint methods are used to identify a dead body with no identification papers

on them. Fingerprints are small ridges caused by folds in the epidermis of the skin. Sweat and oil leave impressions on surfaces we touch. Oils are secreted from sebaceous glands (non on palms/fingers) are and transferred to fingers when touching other parts of our body. There are 4 types of finger prints: Arch, Tented arch, whorl, loop. This is known as the Henry Classification. Fingerprints are made visible by using fine aluminium, iron or carbon powders; using superglue; using ninhydrin; magnets and iron flakes are sometimes used. The police have a national database of biomedical information called IDENT1. Everyone who is arrested has their prints taken. Dental records are used if someone has no fingerprints on file or their body has been burnt. This is because teeth and fillings are more resistant to burning and decay slowly. DNA profiling relies on the fact that everyones DNA is unique (apart from identical twins). The non-coding blocks on DNA are called introns and the coding blocks are called extrons. In introns there are sequences of repeated bases known as short tandem repeats (STRs) or satellites. The same STRs occur at the same place (locus) on both chromosomes of a homologous pair. The number of repeats on each homologous pair can be different which causes variation in individuals. E.G. A DNA profile is made by cutting up DNA, separating the fragments and then comparing it to some reference (e.g. a suspect, a relative of the corpse). To obtain a DNA sample take any tissue sample (blood, cheek cells, semen, bone). Physically break it down in a buffer solution that includes salt and a detergent to disrupt the cell membranes. The DNA is separated from the rest of the cell debris by filtering or centrifuging. Protease enzymes remove proteins, and then cold ethanol is added to precipitate out the DNA. Washing with buffer solution then follows.

Restriction enzymes (restriction endonucleases) will only cut DNA at specific base sequences. The short tandem repeats remain intact but it will be cut away from the rest of the genome. They are found in bacteria; they protect themselves by changing the bases in their own sequences that are targeted by their own restriction enzymes. Polymerase Chain Reaction allows scientists to use tiny amounts of DNA, which is copied numerous times. It uses DNA Primers which are short DNA sequences complementary to the DNA adjacent to the STR. The DNA primers are marked with a fluorescent tag. The forensic sample is placed in a reaction tube with DNA polymerase, DNA primers and nucleotides .Once in the PCR machine, the tube undergoes a cycle of temperature changes. The first separates the double stranded DNA. The second temperature optimises prime binding to the target DNA sequence in the sample. The polymerase attaches and replication occurs. The final temperature is the optimum temperature for the heat stable DNA polymerase.

DNA fragments can be separated by gel electrophoresis according to their size. The gel (agarose or polyacrylamide) is submerged in a buffer solution and connected to electrodes. The negatively charged DNA fragments move across the solution with the small fragments moving further and faster (closer to the positive electrode). Southern blotting is used to transfer the fragments to a more resilient nylon or nitrocellulose. The membrane is placed on top with a wad of dry absorbent paper on top, which draws up the buffer solution carrying the DNA fragments onto the membrane. The membrane is placed in a bag with DNA probe. Single-stranded DNA

probe binds to fragments with a complementary sequence. If the DNA probe is radioactive, X-ray film is used to detect the fragments. If the DNA probe is fluorescent it is viewed using UV light. The STR is inherited like genes so are used for identification purposes.

Determining Time of Death

The temperature of the body, the degree of rigor mortis and the state of decomposition can be used to estimate time of death. In addition, entomological (insect) evidence can provide further clues about when the person died. Body Temperature: Core temperature in a human is usually 36.2 to 37.6oc, but when a person dies the body cools down due to no heat producing reactions taking place. Temperature is measured through the rectum or an abdominal stab wound, with a long thermometer (normal ones are too short and have a lower temperature range). However, environmental conditions must be noted as they can change the rate at which the body cools. The cooling of a body follows a sigmoid curve which shows that normal body temperature lasts for 30-60 mins after death. But if the person had a fever or had hypothermia then the body temperature would not be normal. Many factors will effect post-mortem cooling including; body size, body position, clothing, air movement, humidity, surrounding temperature. For example if a body is in water it will cool much quicker than on land as water is a better conductor of heat than air. Rigor Mortis: After death muscles totally relax and then stiffen again, this is rigor mortis (stiffness of death). After a period of time the muscles become relaxed again. The sequence in which this happens is 1) after death, muscle cells become starved of oxygen, and oxygendependant reactions stop. 2) Respiration in the cells becomes anaerobic and produces lactic acid. 3) The pH of cells fall, inhibiting enzymes and thus inhibiting anaerobic respiration. 4) The ATP needed for muscle contraction is no longer produced. AS a result, bonds between the muscle proteins become fixed.

5) The proteins can no longer move over one another to shorten the muscle, fixing the muscles and joints. Smaller joints stiffen before larger ones. Rigor mortis passes off as muscle tissue starts to break down, in the same order in which it developed. Decomposition: After death, tissues start to break down due to the action of enzymes. Autolysis occurs first, which is where the bodys own enzymes from the digestive track, break down cells. First sign is a greenish colour (discolouration) of the skin, in the lower abdomen. Due to the formation of sulphaemoglobin in the blood. Due to action of bacteria, gases including hydrogen sulphide, methane, carbon dioxide, ammonia, hydrogen form in the intestines and tissues. This causes the body to smell and become bloated, which deflates as further tissue decomposes and gas is released. Decomposition varies but in average conditions, discolouration occurs 36-72 hours after death. Gas formation about 1 week. Low temperatures means low decomposition rate. Warm temperatures speed up decomposition. Injuries to the body allow the entry of bacteria that aid decomposition. Forensic Entomology The presence of insects allows forensic entomologists to make an estimate of how much time has elapsed since death. The temperature of the air, ground, body and maggot mass are measured in order that the rate of maggot development can be determined. For the commonest bluebottle species found on bodies, Calliphora Vicina, a graph is seen to determine age. (only used if temperature conditions have stayed constant). Fly lifecycle... egg: 1 day. Lava: 9 days. Pupa: 6-12 days. Other factors e.g. toxins in the body will affect the results cocaine would accelerate development. There is succession on a dead body. Normally eggs are laid in wounds or at openings to the body (mouth or nose) . The season, weather conditions, size and location of the body will all influence the type and number of species present.

Cause of Death A post-mortem may be done. This is an internal and external examination of the body to help determine cause of death.

Viruses consist of a strand of nucleic acid (RNA or DNA) enclosed in a protein coat. Some viruses can have an outer envelope taken from the host cells surface membrane. They have glycoproteins from the virus itself. These are antigens, molecules recognised by the hosts immune system as not being its own self. Viruses lack some internal structures required for growth and reproduction. This means that they have to enter the hosts cells and use the hosts metabolic systems to make new viruses. After reproducing inside the hosts cells, new virus particles may bud from the cell surface or burst out of the cell splitting it open. This splitting kills the cell and is called lysis.

Transmission of TB bacterium It is carried in the droplets of mucus and saliva released into the air when an infected person talks, coughs or sneezes. This is known as a droplet infection. The droplets can stay in the air for several hours and as dust for several weeks making bedclothes potentially dangerous. Close contact, poor health, poor diet and overcrowding living conditions increase the risk of developing the disease. Transmission of HIV It cant survive outside the body. It can be passes on by bodily fluids but not saliva or urine. For infection to occur, the body fluids have to be transferred directly into the body of the nest host (sharing needles while taking drugs, unprotected sex, blood to blood transfer through cuts and grazes, maternal transfer from mother to child or in breast milk). The Bodys Response to Infection Non-specific responses help to destroy any invading pathogens, whereas specific immunity is always directed at a specific pathogen. If dust lands in your eye, tears appear. These contain lysosome that kills bacteria by breaking down their cell walls. The same enzyme is found in

saliva and nasal secretions. An injury enables microbes to enter the body. The inflammatory response destroys these foreign organisms. Damaged white blood cells and mast cells releases special chemicals such as histamines, causing the arterioles to dilate, increasing blood flow to the area. Plasma fluid, white blood cells and antibodies leak from the blood causing oedema (swelling) the microbes can now be attacked by intact white blood cells. Phagocytosis Phagocytes are white blood cells that engulf bacteria and other foreign pathogens. They include both neutrophils and macrophages. Neutrophils = 70% of WBC leave blood capillaries by squeezing between the cells of capillary walls. Monocytes = Circulate in the blood for a day or two before they move into the tissue by squeezing between the cells of the capillary walls. Here they become macrophages and engulf bacteria, foreign matter and cell debris. Neutrophils are first to arrive (5-20 bacteria) followed by macrophages (up to 100). The ingested material is enclosed within a vacuole. Lysosomes containing digestive enzymes fuse with the vacuole the enzymes are released and destroyed. Sometimes bacteria get carried away. The lymphatic system tries to stop this.

Interferon provides non-specific defence against viruses. Lymphocytes are white blood cells that help to defend the body against specific diseases. They circulate in the blood and lymph and gather at the site of infection. B and T cells (Lymphocytes) This is the specific immune response. B cells = secrete antibodies to antigens. Antibodies are known as immunoglobulins (acts as labels). B cells are specific. Produced in bone marrow.

T cells + produced in bone marrow but mature in the thymus gland. They are specific. There are 2 types of T cell. T helper cells (stimulate B cells to divide, also enhance phagocyte activity). T killer cells = destroy anything with an antigen on its surface membrane (could include transplanted tissue). When foreign material is engulfed by a macrophage, an antigen is added to the cell surface membrane. Macrophages displaying non-self peptides are antigen-presenting cells (APCs). A T helper cell with a complementary shape (called a CD4 receptor) binds to the antigen. This binding activates T helper cells to divide and produce more T helper cells and a clone of T memory cells, which stays in the body for years in case the pathogen enters the body again.

Cloning of B cells Antigen-presenting B cells bind to a T helper cell which release a chemical called cytokines.

These stimulate division and differentiation of B cells into 2 types. B effector cells differentiate into plasma cells (release antibodies into the blood and lymph). B memory cells long lives, enable body to respond more quickly to the same antigen in the future. The process of B cell division is called Clonal Selection. It takes about 10-17 days to produce sufficient antibodies called the primary immune response. T killer cells bind to the anti. It divides to form a clone (stimulated by cytokines). T killer cells release enzymes that create pores in the membrane of the infected cell which allows water and ions into it. It swells and burst and the pathogens in the cell are released so they can be labelled by antibodies for destruction by macrophages.

The secondary immune response Involves memory cells. B memory cells produce plasma cells immediately and release antibodies. There is a greater production of antibodies and it lasts longer. Membrane proteins on the surface of our cells act as bar codes and mark the cell as self. Any lymphocytes with self membrane proteins are destroyed by apoptosis (programmed cell death) only lymphocytes with receptors for foreign antigens remain. Tuberculosis TB is caused by the bacterium Mycobacterium tuberculosis. Infection may occur when the bacteria are inhaled and lodge in the lungs. There are 2 phases of the disease Primary Infection: The immune system responds by an inflammatory response. In a healthy person, macrophages engulf the bacteria. A mass of tissue known as a granuloma forms. These are anaerobic

and have dead bacteria and macrophages in the middle. They are called tubercules. After 3-8 weeks it is controlled and the infected area heels over. Invading the immune system M.tuberculosis can survive inside macrophages, resisting killing mechanisms by having a thick waxy cell wall which makes them difficult to break down and also hides their antigens from other macrophages. They also reproduce inside the macrophage. They can be dormant for years and also target immune system cells. TB bacterium can suppress T cells, reducing antibody production and attach killer cells. Active Tuberculosis (2nd phase) If patients immune systems cant contain the diseases when it first arrives or an old infection may break out if the immune system isnt working properly. Activity of immune system is reduced in old and very young ages, also by malnutrition and poor living conditions. Most significant factor is AIDS. HIV the virus that causes AIDS directly targets white blood cells and reduces patients ability to fight infection. Bacteria in lungs destroy tissues creating holes and cavities. The lung damage will eventually kill the sufferer. Symptoms of active TB Coughing (blood sometimes) Shortness of breath Loss of appetite and weight Fever and extreme fatigue Role of Fever Fever occurs because of inflammatory response causes fever-causing chemicals to be released from neutrophils and macrophages. The chemicals affect the hypothalamus and alter core body temperature by the use of effectors. Raised temperature is thought to increase immune function and phagocytosis while also making it harder for bacteria to reproduce. Glandular TB

TB can move to other parts of the body. Main sites are the bones, lymph glands and central nervous system. enlarged lymph glands are signs of glandular TB (neck or armpits) Diagnosis A history is taken from the patient of their symptoms. Skin and blood tests: Tuberculin (extracts from several species of Mycobacteria) is injected under the skin. A positive result shows inflamed skin showing antibodies are already present. This can be wrong so a blood test is done to find specific T cells. Identification: a sample of sputum is coughed up and cultured. Using staining techniques different bacteria can be identified. X rays can also be used on the lungs to discovered extent of the disease. Treatment is usually antibiotics and a better lifestyle. The bodys responses to HIV and AIDS AIDS (acquired immune deficiency syndrome is caused by infection with the human immunodeficiency virus, HIV. A syndrome is a collection of symptoms related to the same cause. HIV is an example of an enveloped virus, with the envelope coming from the hosts cell membrane. HIV invades T helper cells Particular glycoprotein molecules called gp120 (located on the virus surface) bind to the CD4 receptors on the T helper cells. They fuse with another gp120 receptor which enables the envelope surrounding the virus to fuse with the T helper cell membrane, allowing the viral RNA to enter the cell. (can also happen with macrophages). HIV hijacks the cells protein synthesis Once inside the T helper cell the virus needs to make viral components. This means making DNA from the virus RNA. They do this by using an enzyme called reverse transcriptase. Viruses that contain RNA and use reverse transcriptase in this way are known as retroviruses. Once the HIV DNA strand is produced, it is integrated into the hosts DNA by another enzyme, integrase. Once the HIV genome is integrated into the hosts cell genome it can be transcribed and translated to produce new viral proteins. Protein Synthesis

DNA in the nucleus unwinds its double helix to form a sense strand and an antisense strand. Free nucleotides in the nucleus line up on the antisense strand, due to complementary base pairing, making a mRNA strand. It then travels out of a nuclear pore to the ribosomes. This is transcription. Once on a ribosome, the mRNA is read in codons (3 base pairs make a codon). For each codon there is a complementary anticodon on a tRNA molecule. Each anticodon codes for a specific amino acid which joins together in the ribosome by a peptide bond. mRNA splicing Between transcription and translation mRNA s often edited with the introns (non-coding sections of DNA) being removed. This means that several proteins can formed from one length of mRNA if it is spliced in different ways. The killing of T helper cells The new virus is assembled and bud out of the T cell taking some of the cell surface membrane with it and killing the cell as it leaves. Infected T helper cells will be destroyed by T killer cells. As the number of T helper cells decrease, macrophages, B cells and T killer cells are not successfully activated an the immune system therefore doesnt work properly.