BIO 102 Lab 04, The Immune System and Blood Cellstrnn PDF

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BIO 102 Lab 04: The Immune System and Blood Cells To submit, print or edit this document (use a different font color for your answers), complete all lab activities, and answer the review questions. Scan (using the free phone app AdobeScan) or save your lab pages, and upload your Word or PDF file to Canvas. If you have a disability that makes it difficult to complete this lab, please contact your instructor. Please provide your instructor a copy of the Memorandum of Accommodation (MOA) from NVCC Disability Support Services. OBJECTIVES:   

Become familiar with the different types of blood cells and their functions Understand antigen-antibody binding and its importance in transplants Learn about the effects of vaccination on a population and the protection gained from herd immunity

INTRODUCTION Our immune system functions to keep us safe by destroying and removing things that can make us sick. This system is not composed of individual organs, but several structures in our body work as part of the immune system to produce, house, or circulate immune components, including the bone marrow, spleen, and lymphatic vessels (Figure 4.1). The immune system is composed of physical, chemical, and cellular parts that work together to destroy foreign particles that enter our bodies. Foreign particles can include dangerous pathogens (e.g., bacteria, viruses, parasites, fungi), toxins, benign molecules such as pet dander or pollen, and even beneficial bacteria or fungi. One part of our immune system, the innate system we are born with fully intact and it naturally produces components to fight things we might face in our everyday lives. The second part of our immune system, the adaptive system, we generate through lifetime exposure and we produce specialized components that are trained during our development and growth. Innate Immunity The innate immune system—called that because you and everyone around you is born with it—acts in a non-specific way against foreign particles, meaning this “pre-made” innate immune system will attack particles in the same way regardless of the nature of a particle (i.e., virus vs grass pollen). This system makes no memory about previous encounters or whether its defense to a previous invader was effective or ineffective. The nonspecific immune system's primary function is to prevent us from getting sick from common potentially harmful things in our everyday environment. Major structures of the innate system include our skin, mucus membranes, ear wax, stomach acid, sweat, tears, vaginal secretions, all which help prevent or block entry of harmful particles. If harmful particle is able to enter our bodies, then cells of the innate system called leukocytes or white blood cells (WBCs) can destroy it or can produce antimicrobial proteins that destroy the invaders. Leukocytes can be distinguished from erythrocytes (red blood cells) that function in oxygen distribution and platelets that function in clotting and tissue repair because they are larger, have a clear cytoplasm (thus white blood cell), and they have large BIO 102 Lab 04: The Immune System and Blood Cells

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nuclei with distinctive shapes that stain purple when dyed for microscopy. Leukocytes that function in innate immunity, include macrophages, dendritic cells, neutrophils, basophils, eosinophils, and each of these cells has a specific phenotype (characteristic look) as well as particular functions (Table 4.1).

The innate immune system is powerful and is fundamental to keeping us safe during everyday encounters. Sometimes the response from the innate immune system—and all its physical, chemical, and cellular components—is insufficient to fight of a pathogen, such as a virus. This stimulates the second (more targeted) portion of our immune system; the adaptive immune system. Adapted Immunity The adaptive immune system—called that because the components are “adapted” to the environment each individual has experienced—acts in a unique way against every foreign particle your body encounters during its lifetime. Our adaptive immune system specializes in clearing (destroying) viruses and other particles that our innate immune system is not able to clear. This is because the cells of our adaptive immune system, called T cells and B cells (Table 4.1), are made in the presence of the invading particle and therefore are specific to that particle; it is a very targeted attack. However, because the body has to build a defense from scratch, so to speak, the initial formation of an immune BIO 102 Lab 04: The Immune System and Blood Cells

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defense can take up to 2 weeks. This is called the primary immune response and it is a dangerous time for us because if some pathogens replicate very fast, then they can cause a lot of harm in our bodies. Some people are not able to build a primary immune response fast enough to fight of a pathogen and they perish. But if the adaptive response is able to control a pathogen, a small subset of the components used to destroy that pathogen are maintained in the body. This is referred to as memory because your body will “remember” that encounter. The next time your body sees that same intruder, a faster and much more efficient defense, called the secondary immune response) can be built. The memory components from the adaptive response are memory T-cells (a specialized subset of killer T cells) and antibodies, produced by B cells. Antibodies are Y-shaped proteins that can specifically recognize cell surface molecules of foreign origin (Figure 4.2). T cells and antibodies are able to recognize and target foreign particles because all the cells in our bodies have molecules on their surfaces. These surface molecules are important for cell communication, transportation across the membrane, and many other cellular activities. Many cell surface molecules are unique to individuals, and so during development our T cells and B cells are trained to recognize the molecules on our cells, referred to as recognition of self. When foreign particles such as viruses, toxins, or poisons enter our bodies, immune components recognize the different surface molecules as non-self and they can attack and destroy them. A molecule that stimulates an antibody response is called an antigen (antibody generating chemical). As an example, the ABO blood group are carbohydrate molecules that appear on the surface of red blood cells. A common cause of rejection in blood transfusions is an antibody response to different surface antigens. This is why it is it is vital that blood type between donor and recipients match. Non-matching surface proteins on blood cells could induce a massive adaptive immune response that could kill the recipient. Memory in adaptive immunity Antibodies will bind to their corresponding antigen molecule, and they coat or “tag” the entire particle for destruction by other immune cells (such as T cells). The tagged cells or particles are then destroyed. Memory T cells and B cells remain and circulate in your blood or take up residence in different parts of your body (such as the spleen). When a virus that our adaptive immune system has already “seen” enters again, memory T cells and B cells in circulation will detect it, proliferate (divide rapidly), and control the infection before we even realize we came into contact with that pathogen again. Interestingly, people can share their antibodies with others! This is called passive immunity. For example, when you are bitten by a venomous snake, you would not survive a bite if you had to wait 2 weeks until your body made T cells and B cells that would fight off that venom. Instead, you receive anti-venom—a solution that is full of antibodies which have been grown in laboratory animals and have been extracted from their blood to create that medicine for you. In this way, you get the benefits of adaptive immune components without having to wait to build them yourself! Unfortunately, because you did not build your own antibodies against the snake venom, you did not make memory B cells that can produce antibodies in the future. The memory from an adaptive immune response is why the first time you eat something you are allergic to, you may only get mild discomfort, but the second or third time you eat it, you can go into BIO 102 Lab 04: The Immune System and Blood Cells

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anaphylactic shock and even die. This is because your T cell and B cell response is so massive after subsequent exposure that they can damage your organs, and your body shuts down. However, we can use the memory response to our benefit by stimulating an immune response to deadly pathogens before we actually encounter them. This is called immunization.

Immunity and vaccination Immunization refers to the formation of memory to specific pathogens. This occurs through direct exposure and survival or through vaccination. Vaccines work when the antigenic portions of a pathogen, or the inactivated pathogen (with its antigenic portion intact), are used to stimulate a primary immune response. The vaccination will stimulate an adaptive immune response and build memory without actually causing disease, which could be deadly. After vaccination, if you are ever exposed to the real pathogen in the future, you will now have circulating memory T cells and B cells that can respond and control the pathogen immediately. The length of memory you get depends on the type of pathogen you get infected with, the quantity of antibodies you create, and the type of antibody that is made in the response. For example, vaccinations against the Clostridium tetani bacteria, which causes tetanus, builds immunity for about 10 years. After that time, the memory B cells have mostly died off and disappeared, and so you need a booster shot about every 10 years to prevent infections. But antibodies against the measles virus remain in circulation in your body for the rest of your life, so you only need to be vaccinated once. When the majority of the population is immunized (either through exposure or vaccines), the high level of immunization in the population becomes a barrier to disease transmission. This is because some pathogens such as viruses require infecting individuals for survival, but if most people are immunized, a virus may not find enough hosts to keep the infection going, and the virus will die off. Immunization is protective for the few individuals in a population that are susceptible to an infection due to immune deficiencies or other ailments. This is referred to as herd immunity. Just as a wild herd of animals protect their weakest by moving them towards the inside of the heard (and away from danger), immunization protects susceptible individuals from infection by “hiding” them away from transmission possibilities. Medical and scientific use of immune components Blood cell counts The proportion of different blood cells in blood (Figure 4.3) can be indicative of various diseased conditions and so medical professionals will often do blood cell counts to detect abnormalities. Blood levels that are too high or too low may indicate health problems. For example, specific leukocytes will replicate in response to infections, disease, or injury. High WBC counts with neutrophils could indicate infections with bacteria or other pathogens. Sometimes, the immune system will recognize benign substances, such as pollen, dirt, or pet dander, as foreign and dangerous. These substances will stimulate an immune response from basophils, eosinophils, and other immune components. That is the cause of allergic reactions. Sometimes an individuals' immune components can recognize molecules on self-tissues as foreign or dangerous and will attack those tissues. This is how autoimmune diseases such as asthma or diabetes BIO 102 Lab 04: The Immune System and Blood Cells

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can arise. Low WBC counts may also indicate disease. For example, autoimmune disorders such as leukemia (cancer of the blood and bone marrow) can result in WBC loss as the cancer destroys blood cells. Infectious pathogens such the Human Immunodeficiency Virus (HIV) specifically targets certain WBCs as hosts for replication. The virus then destroys the host cell as it leaves and once WBC counts drop below 200 cells/mm3 in blood, an infected person is diagnosed with AIDS (acquired immune deficiency syndrome). This means that they have so few white blood cells present in their blood that they are no longer able to fight off any pathogens, even very mild ones. ELISA (enzyme-linked immunosorbent assay) The ELISA (enzyme-linked immunosorbent assay) can be used to detect the presence of antibodies in solution (as well as proteins, or other molecules). This is because antibodies will only bind to their specific antigen, and so you can test for the presence of specific antibodies if their corresponding antigen is present in a solution. If the antigen is not present, no binding will occur. There are several variations of ELISAs, but two of the most commonly used are direct ELISA and indirect ELISA. In a direct ELISA, antibodies that are conjugated (bound) to an enzyme reporter molecule are used to detect antigen (Figure 2). Indirect ELISA is similar to direct ELISA, but it requires a second and different type of antibody to anchor antigen molecules to the bottom of the test well. In both types of ELISA, the enzyme reporter molecule functions when a substrate (a molecule that the enzyme works on) is added to the solution that has antigen-conjugated antibody binding. The enzyme reaction causes a noticeable color change (Figure 2, step 4). This will only happen if the enzyme-conjugated antibody has been able to bind its antigen. The deeper the intensity of the color change, the more antigen-antibody interactions occurred. No color change indicates no antigen-antibody interaction. The procedure is completed on a small plastic plate that has many wells so numerous samples, at different dilutions, can be run simultaneously.

ELISAs are used by medical professionals to test for the presence of antibodies in serum (fluid portion of blood) that were generated by infections from certain pathogens (adaptive immunity). For example, you might hear in the news that many clinics are currently running antibody testing to see what BIO 102 Lab 04: The Immune System and Blood Cells

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proportion of the population has been infected with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) which causes COVID-19 (coronavirus disease 2019). The tests that are being performed are indirect ELISAs. In this case, the antigen that is bound to the wells comes from SARSCoV-2, and the antibodies come from the serum of individuals that may have previously been infected with the virus. In this way, we can figure out if we may have reached herd immunity levels in the population. You may also hear that people that test positive for the presence of antibodies can donate their blood. That is because their blood contains circulating antibodies, and therefore, individuals that have survived COVID-19 can donate their antibodies (passive immunity!) to people sick with severe COVID-19 in the hopes of reducing the severity of the viral infection. PROCEDURE: For this lab, you will be completing 3 activities. In Activity A you will identify leukocytes by studying blood smears. In Activity B you will learn how to analyze ELISA results and interpret their meaning. In Activity C you will examine transmission simulations to understand immunity. ACTIVITY A: Leukocyte identification and analysis of blood smears 1. Circulating RBCs and a variety of WBCs. Using information from the introduction of this lab and your textbook, identify the different cells in the blood smear image by their phenotypes and state their primary function in the table below.

Letter A B C

Leukocyte name

Primary function

Lymphocyte

Part of the white blood cells, they split into two different types, B cells and T cells. B cells are like antibodies which attack bacteria and viruses. They release an inflammatory in the immune system called cytokines.

Neutrophil Eosinophil

Helps to protect against the types of parasite infections.

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2. Analyze the following pictures of blood samples from two different patients, Patient A and Patient B. Use information in the introduction to answer the questions below. Assume these slides are representative of the rest of the blood in the patient.

a. Is blood sample A normal/healthy? Explain why or why not and hypothesize on the health status of Patient A. What might be the cause for any abnormalities (be sure to name any leukocytes present).

. Patient A looks as though they are not in a healthy condition. The leukocytes present are the lymphocyte, neutrophil, and monocyte. There for , i believe there is an infection or maybe inflammation in the body.

b. Is blood sample B normal/healthy? Explain why or why not and hypothesize on the health status of Patient B. What might be the cause for any abnormalities.

Patient B looks healthy and/or normal. There is no evidence of leukocytes present and I do not see any cause for abnormalities, looks to be functioning properly and with no negative reaction.

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ACTIVITY B: ELISA assay for detecting the presence of antigen 3. Measles is caused by a virus that is easily transmitted between individuals through the air and it is highly contagious. After a small gathering of 9 people, the host began showing symptoms consistent with measles infection, and then tested positive for the virus. At the hospital, medical personnel wanted to test everyone that attended the party to determine who had been infected with the virus and had generated an immune response (remember ELISAs test for the presence of antibodies against specific antigen). By using an ELISA test, measles infections can be confirmed despite the absence of symptoms (after people have cleared the virus). a. In your own words, briefly explain how the antibody-antigen portion of the ELISA technique works.

The ELISA technique is used to find substances such any peptides or proteins in the body b. What causes changes of color in the wells?

The color changes when a chemical reaction occurs with the antibodies of the enzymes. c. What does a color change from a sample indicate for the individual?

The color change indicates that the antibodies are positive for the thing that it is trying to get rid of. Below are the color results from an ELISA completed on the 8 party guests. Please note the following:  The first and second rows are the positive (+) and (-) controls for the test.  The reporter enzyme turned the solution blue in the presence of corresponding antigenantibody binding.  The serum samples from each individual were diluted up to 5 times (2-fold each time) to get an idea of antibody concentration (how much antibody is present in the original sample).  The undiluted row would have the highest concentration of antibodies for each individual. As such, the higher the dilution, the lower the concentration of antibody present in the well.  These results can be further processed using a device (photometer) that can read specific color intensity, but you will only be doing interpretation of initial pos/neg results.

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d. Row 1 (+) and row 2 (–) indicate the positive and negative controls. What could be used as positive and negative controls and why are controls necessary in this experiment?

An antigen could be used as a positive control and the negative control would consist of nothing. The controls are necessary as they help us understand what it is to be on the lookout for in the color changing and further producing scientific results.

e. The rows of wells in the ELISA plate contain serum at 2 -fold dilutions. What does this mean in terms of the concentration of antibodies in the undilu...