Epithelium & Connective Tissue 1. Describe the defining properties of each of the four basic tissue types; list the main tissues included in each of the four types. Epithelium is a group of tissues that cover all body surfaces, and line body cavities and blood vessels. It also invaginates to form glands. It is made up of an uninterrupted layer of cells that forms a barrier and mediates exchange between compartments. Connective tissue includes the capsules of organs, supporting tissue within organs, fascia, tendons, cartilage, bone, blood, and fat. They are grouped because they all share an abundance of extracellular molecules and relatively low density of cells. With the exception of bone, cartilage, and blood, all other connective tissues are classified as Connective Tissue Proper. Connective tissue as a whole is supportive in the sense that it supplies both blood and nutrients to surrounding tissue. Muscle moves things in the body. It is associated with skeletal muscle and blood vessels. (We will cover muscle more in Musculoskeletal Block). Nerve is the final tissue type. It acts in a sensory manner, stimulates glands to secrete things, and stimulates muscles to contract. 2. Compare and contrast the different types of epithelia in terms of structure and function. Given the type of epithelium at a particular location, predict the functional requirements of that location. Epithelia can be classified as simple, indicating a single layer, or as stratified, meaning multiple layers of cells. The cells themselves can be squamous (generally flat and without defined shape), cuboidal (cube-like shaped cells), or columnar (long column shaped cells; lateral sides are longer than basal or apical). The two exceptions to this are psuedostratified cells lining the respiratory tract, in which the nuclei of the cells do not line up because not all cells reach the surface; and transitional cells, which are a special case of stratified capable of extensive stretching. Squamous cells generally line organs, blood vessels, etc. Cuboidal cells line ducts, tubules, and glands. Columnar cells line the gut, bronchioles in the lung, oviducts, and the uterus. 3. Explain the concept of epithelial cell polarity using examples, and discuss its importance in epithelial function. Epithelial cells are polarized, meaning that the apical, lateral, and basal sides can all be differentiated. The cell is able to maintain a non-random distribution of its organelles based on where they belong in the cell. In addition to organelle distribution, plasma membrane proteins and lipid compositions vary across the three domains of the cells. For example, epithelial cells lining the gut need to be able to absorb nutrients from the apical side, and transport them to the basal lamina membrane. This is only possible if the cell maintains polarity. 4. Describe the structure and function of the main apical, basal and lateral membrane specializations of epithelial cells (see also: Cell Biology ILMs) The basal surface of the cell sits on the basal lamina, a membrane layer of glycoproteins necessary to connect epithelium to the underlying connective tissue. Adherens junctions and hemidesmosomes are critical to maintain attachment to the basal lamina. The lateral surfaces of epithelium face other epithelium. Because of this, they are characterized by tight junctions (preventing things from moving between cells), zonula adherens (attaching cells to actin filaments of the cytoskeleton), and desmosomes (linking cells to one another via intermediate filaments in the cytoskeleton). The apical membrane faces the outside world in most epithelia. All apical membranes of epithelial cells form microvilli. In addition, some can form cilia composed of microtubule doublets in a 9+2 array. 5. Briefly describe the structure and function of the basal lamina (basement membrane). The basal lamina is a membrane underneath the basal surface of epithelium. It is a secreted layer of glycoproteins and various other molecules vital to attachment of the epithelium to the underlying connective tissue. 6. Describe the main components of extracellular matrix, including Types I, III and IV collagens, elastin, fibrillin, glycosaminoglycans, proteoglycans, fibronectin and laminin, and explain what each contributes to the properties/functions of connective tissues. Where possible, predict the clinical consequences of defects in these components. Collagen is a fiber necessary for the tensile strength of the extracellular matrix. Type I collagen forms fibrils, which combine to form fibers, which combine to form even larger bundles. The fibers are packed in a regular staggered array visible in electron microscopy. Defects in the synthesis of Type I collagen are usually associated with scurvy. Vitamin C is required for proper collagen processing. Without it, collagen accumulates in the cells and causes cell death. Type III collagen is often referred to as reticular fibers. They group in small bundles forming a 3D structure supporting loose connective tissue, especially in lamina propria of hollow organs. They also underlie the structure of some solid organs, including the lymph nodes, spleen and liver. Type III collagen is secreted by mesenchymal reticular cells. Defects inType III collagen cause severe instability of organ structures. Ehlers Danlos syndrome (Type IV) is a result of mutations in Type III collagen, causing organs to become prone to rupture. Elastin fibers are responsible for the elasticity of connective tissues. They are secreted extracellularly to form fibers and sheets. Assembly requires the glycoprotein fibrillin. Elastin is composed of short hydrophobic segments crossed to one another. Since they are hydrophobic, the segments will coil on themselves until forced to pull apart. This is what gives elastin its elasticity. Marfan‟s syndrome is a genetic disorder in which there is a defect in the glycoprotein fibrillin. This causes widespread defects throughout the body, the most serious being the aorta, which becomes subject to aneurysm and rupture. Glycosaminoglycans (GAGs) form the ground substance surrounding the cells and fibers of connective tissue. They are made from a long protein core that has long polysaccharide chain modifications. The negative charge repulsion causes the side chains to jut out from the protein core, attracting cations and water, creating a hydrated gel. Type IV collagen is present in the basal lamina. It forms a sheet-like network that binds laminin to other components of the basal lamina, as well as to the underlying extracellular matrix. It is essential for basal lamina integrity. 7. Describe resident and itinerant cells of connective tissues. For each resident cell, include the site of origin, and briefly describe the cell’s function (itinerant cells will be covered in more detail in later sessions). Primitive Mesenchymal Cells – derived from embryonic mesenchyme. They are small inconspicuous cells usually situated alongside small blood vessels. They serve as connective tissue stem cells. Fibroblasts – derived from primitive mesenchymal cells. They synthesize extracellular matrix molecules including collagen fibers, elastic fibers, proteoglycans, and glycosaminoglycans. They are a major player in wound healing and fibrosis. During wound healing, fibroblasts differentiate into myofibroblasts which contract and help shrink the wound. Adipocytes – derived from primitive mesenchymal cells. They are very large cells each containing a single large lipid droplet which displaces the nucleus and other organelles into a thin rim of cytoplasm around the periphery. They store and mobilize lipids based on energy needs of the body. Mast cells – derived from stem cells in the bone marrow, travel to the connective tissue via the blood, and reside alongside small blood vessels. They are filled with secretory granules containing histamine, heparin, and proteolytic enzymes. They degranulate in response to mechanical injury, toxins, and allergens. They function in initiation of inflammatory response by increasing vascular permeability and causing itching. Histamine is an important mediator of anaphylaxis. Plasma cells – differentiate from antigen-stimulated B lymphocytes. They reside in connective tissue, usually near the site where the antigen was encountered by the parent B cell. They secrete antibodies, and occur in increased numbers during chronic inflammation. 8. Describe the pathways of white blood cells traveling from their site of origin in bone marrow to their site of action in connective tissues. White blood cells originate in the bone marrow, and travel through the blood to their site of action in the connective tissue. They leave the blood supply and enter the tissues via post capillary venules. These are the leakiest part of the vascular system. In response to chemotactic factors cause gaps to form between endothelial cells lining the post-capillary venules. Post capillary venules also express surface receptors that bind to white blood cells and promote their transfer across the endothelium into the connective tissue. 9. Compare and contrast epithelium and connective tissue in terms of both structure and function. The primary function of epithelium is to act as a barrier and mediate exchange between two compartments, usually the inside and outside of the body. Connective tissue forms capsules of organs, the supporting connective tissue within organs, as well as fascia, tendons, cartilage, bone, blood, and fat. Epithelia form a highly dense cellular layer with very little extracellular matrix. They are also avascular and rely on connective tissue for their metabolic needs. Connective tissue is the opposite with few cells and an abundance of extracellular molecules. Specialization of Proteins I 1. Define the following terms associated with enzymes: catalysis, substrate, reaction specificity, coenzyme, and prosthetic group. Catalysis – the process by which a reaction is sped up due to lowering of activation energy. Substrate – the molecule that the enzyme will be acting on to generate a desired product. Reaction specificity – relates to the fact that enzymes will only bind a specific substrate when catalyzing a reaction. Coenzyme –they are specific for the enzyme, essential for activity, and predominantly derived from vitamins, they interact at the reaction site of the enzyme. Prosthetic Group – organic molecules covalently attached to the enzyme, takes part in the reaction in some manner. 2. Discuss the following concepts associated with enzymes: Vmax, Kcat, substrate affinity, and Km. Vmax – the rate of the reaction when all enzymes are saturated with substrate. Kcat – the reaction efficiency (how fast each individual enzyme turns reactant to product. Substrate affinity – how well the substrate is able to interact with the enzyme active site. Km – the concentration of substrate required to achieve half of the maximal velocity (Vmax). 3. Describe the steps and relevant cofactors in the synthesis and processing of collagen including the post-translational modifications of amino acid residues during its maturation (i.e., proteolysis, hydroxylation, glycosylation and oxidation). Collagen is first synthesized as preprocollagen in the ER. The „pre‟ signal sequence is cleaved by a signal peptidase as the protein is synthesized. Prolyl hydroxylase hydroxylates proline residues to hydroxyproline on each alpha chain. Lysine residues are also hydroxylated, by lysyl hydroxylase. Both hydroxylase enzymes require iron and ascorbic acid (vitamin C). Hydroxylysines are subsequently glycosylated. However, the hydroxylation and glycosylation of lysine is not well regulated, and is dependent on the time procollagen spends in the ER. The three alpha chains are aligned by disulfide bonds, helping them to achieve proper register to form the triple helix. The procollagen triple helix is then sent to the Golgi, where it is packaged and excreted from the cell. After secretion into the extracellular matrix, the collagen is modified by removal of the C and N terminal domains. This decreases solubility and causing the collagen to spontaneously form into fibrils. Fibrils are then stabilized through crosslinking by lysyl oxidase. Lysyl oxidase requires copper and vitamin B6 as cofactors. It oxidizes hydrolysines to allysines which react with other fibers, forming the crosslinking. 4. Describe the causes and consequences of Osteogenesis Imperfecta, Scurvy, and Ehlers Danlos Syndrome Types V, VI and VIIB. Osteogenesis Imperfecta is caused by mutations in the collagen genes. These defects affect Type I collagen, found in the skin, tendons, arterial walls, and bones. If even one of the genes encoding collagen is mutated, the triple helix is unable to form properly. The cell views this misfolded protein as abnormal and destroys it. Scurvy is caused by vitamin C deficiency. Vitamin C is essential for both prolyl and lysyl hydroxylase function. Without it, proper collagen processing is unable to occur. This causes weakening of the blood vessels, and characteristic bleeding of the gums. Ehlers Danlos Syndrome is a family of collagen processing diseases. EDS types V and VI are caused by defects in lysyl hydroxylase and lysyl oxidase, respectively. EDS V is characterized by thin skin and cardiac valves, as well as hernias. EDS VI is characterized by hyperextendible joints and eye defects. EDS type VIIB is caused by a defect in the N- terminal protease required to cleave the collagen in the extracellular matrix; it is characterized by chronic hip dislocations. 5. Describe how Advanced Glycosylation End-products (AGEs) are formed, and their clinicopathological significance. When blood sugar reaches high levels, the aldehydes on glucose spontaneously react, glycosylating molecules in a non-enzymatic fashion. One common target of these glycosylations is collagen. Collagen has a natural turnover in both adults and children; however, these glycosylations inhibit that turnover. Short term glycosylation is reversible, but long term uncontrolled glucose levels can lead to glucose rearrangement and protein cross-links. These crosslinks are non-reversible, and accumulated in the tissues. AGE formation in connective tissue proteins may be related to complications of diabetes. Muscle & Peripheral Neural Tissue 1. Compare and contrast skeletal, cardiac, and smooth muscle with respect to their major structural and functional properties. Skeletal muscle is formed of fibers of the same relative size. The nucleus in each is pushed to the side in each cell. Skeletal muscle is responsible for strong, voluntary movements. Cardiac muscle is of varying size, with the cell‟s nucleus in the center. Each cell is stuck to the one next to it via intercalated disc. Unlike skeletal muscle, cardiac muscle fibers can be branched. Cardiac muscle is responsible for strong involuntary contractions. Smooth muscle is so called because of the lack of striations in its cross sections. Smooth muscle nuclei are located in the center of the cell, which tapers off on each end. Cells lay together, in an overlapping fashion, making cross sections appear to have different sized cells. 2. Name and describe the 3 levels of connective tissue coverings in skeletal muscle. Skeletal muscle has three levels of connective tissue, the epimysium, the perimysium, and the endomysium. The epimysium is made of dense collagen surrounding the entire muscle. The perimysium is thinner than the epimysium, and surrounds the muscle fascicle (composed of multiple muscle fibers). The endomysium is the thinnest and surrounds each individual muscle fiber 3. Describe the location and functions of satellite cells in muscle. Satellite cells are small, flat cells sitting on the membrane of the muscle fiber‟s basal lamina. They are metabolically inactive until the muscle is damaged. Damaged muscle will cause them to grow and fuse with muscle fibers or each other in order to produce proteins to repair the damage. 4. Describe the location, the structural components, and the functions of intercalated discs. Intercalated discs are found throughout cardiac muscle at the junction where two muscle fibers meet end to end. They anchor sarcomeres, provide intercellular adhesion, and allow rapid communication of electrical and chemical signals between muscle fibers. It is composed of a fascia adherens, desmosomes, and gap junctions, each performing the respective duty of the aforementioned function. 5. Describe the locations and functions of Schwann cells. Schwann cells are located in peripheral nerves, supporting their associated neuron axon. Schwann cells wrap the axon of some nerves with myelin. Myelin is an extension of the Schwann cell that acts to increase conduction velocity down the axon. 6. Describe the structure and summarize the function of a node of Ranvier. A node of Ranvier is the gap between myelin segments. Nodes of Ranvier are critical to conduction of action potentials. 7. Describe the structure and functions of peripheral autonomic and sensory ganglia. Peripheral nerves are the nerves extending out into the periphery of the body. Peripheral nerves have three layers of connective tissue, the epineurium, the perineurium, and the endoneurium. They are used to transmit signals to target cells, as well as other neurons. Organ Architecture I & II 1. Define the following terms, and describe the components and function of each: mucosa; lamina propria; muscularis; serosa; adventitia; Mucosa – the innermost layer next to the lumen, consisting of the epithelium and its lamina propria. Lamina propria – the layer of loose connective tissue supporting the epithelium. Muscularis – the layer of smooth muscle deep to the mucosa or submucosa. It usually consists of a circular and longitundal layer of muscle allowing for peristalsis. Serosa – part of the outer layer. If the organ lies within a body cavity, it is covered by an outer layer consisting of connective tissue covered by a simple squamous mesothelium, together making up the serosa. Adventitia – part of the outer layer. If the organ does not lie in a body cavity, the outer layer is composed of connective tissue attaching the organ to adjacent structures, this is called the adventitia. 2. Compare and contrast the architecture of a medium artery or vein with hollow organs such as the digestive tube. Blood vessels have much of the same structure as hollow organs. They are composed of three layers, the tunica intima (mucosa), the tunica media (muscularis), and the tunica adventitia (outer layer). Unlike hollow organs in the digestive tract, blood vessels do not have multiple layers of mucosa, i.e. the mucosa and submucosa. Also, the tunica intima is flat more like the esophagus, and not invaginated or evaginated like the small and large intestines. 3. Compare and contrast bronchi, bronchioles and alveoli in lung in terms of structure and function. Bronchi are the largest of the airways in the lungs. They are lines with a pseudostratified ciliated epithelium with goblet cells that enable the secretion of mucus and the movement of it up the bronchi, trachea, and into the throat to be swallowed. Bronchi are lined with a muscularis of smooth muscle, and an adventitia with hyaline cartilage to hold the airways open. Bronchioles are smaller and lack goblet cells, seromucous glands, and cartilage. Mucous secretion would be undesirable since bronchioles are close to the alveoli where gas exchange occurs. Alveoli are small sacs lined with squamous epithelial cells separated by a thin basal lamina from the capillaries in the alveolar septa. 4. Describe the basic organization of simple and compound exocrine glands. Simple exocrine glands are formed by unbranched invaginations of an epithelial surface. Cells lining the invagination differentiate into secretory cells. They can be rounded or tubular; tubular can be straight or coiled. Compound glands arise from multiple branches of an invaginated epithelium. The branching ends in secretory acini which are connected by a duct system. The acini and their ducts are surrounded by supporting connective tissue. 5. Describe the histological organization of the pancreas. The pancreas can be divided into two organizations, the exocrine portion and the endocrine portion. The exocrine portion has an organization of a compound multicellular gland. The pancreatic acini secrete their product into intercalated ducts, all joining into eventually one large duct that drains into the duodenum. Among the acini of the exocrine pancreas, there are small islands of endocrine tissue. This tissue consists of epithelial cells that are secretory but do not connect via ducts with any surface epithelium. These islands are so called the Islets of Langerhans. 6. In the liver, describe blood flow through portal tracts, sinusoids and central veins, and contrast with bile flow. Explain the importance of albumin to maintaining colloid osmotic pressure of blood and as a carrier protein. Blood from the hepatic portal vein flows into the liver and is dispersed via portal tracts. Portal tracts carry blood to a sheet of hepatic tissue where it trickles through the sinusoids, where it is cleared of toxins, before it reaches the central vein where it is dumped into the hepatic vein to return to the heart. Bile ducts (canaliculi) run in between hepatocytes, draining into a larger system of bile ducts which eventually drain into the gall bladder. Albumin is the most abundant protein synthesized by the liver. If the liver fails to produce enough albumin, the osmotic pressure of the blood drops, and fluid leaks into surrounding tissues, resulting in edema. 7. In the kidney, distinguish glomeruli, kidney tubules, blood vessels and interstitium in terms of structure and function. Glomeruli are the beginning of filtration in the kidneys. They are the capillary tuft forced up against the Bowman‟s capsule. The pressure in the glomerulus will force a substantial portion of blood, minus the formed elements and large proteins, into the lumen of the Bowman‟s capsule. Epithelial cells lining the nephron then reabsorb most of the ultrafiltrate and return it to the blood via capillaries in the interstitium (connective tissue). 8. Describe basic functions of digestive tract, lung, airways, pancreas, liver, and kidney, and relate structure to function. The primary function of the digestive tract is to break down food and absorb nutrients. Because of this, both the small and large intestines have extended epithelial surface with microvilli to increase absorption. The primary function of the lung is to facilitate gas exchange with the blood stream. Thus, the lung has large airways that are ciliated and mucus lined to trap particles that would inhibit gas exchange, and remove them from the lung. The small airways of the lung are very thin with easy access to the bloodstream to better facilitate the exchange. The primary function of the pancreas is to secrete both endocrine and exocrine products. Therefore, the pancreas is composed of clusters of secretory cells and their ducts, as well as secretory cells that secrete to the surrounding connective tissue. The primary function of the liver is to remove toxins from the blood. Because of this, all blood supply from the GI tract, including the spleen, goes to the liver before recirculating. The liver filters it through portal tracts and sinusoids before it returns to central veins, and ultimately the hepatic vein. It is also the producer of bile, whose canaliculi ducts run between hepatocytes collecting bile for storage in the gall bladder. The primary function of the kidney is to filter blood, removing wastes, as well as regulate blood pressure, electrolyte concentration, and pH. To do so, blood in glomeruli capillaries is forced into the Bowman‟s capsule, which dumps into tubules lined with epithelial cells that selectively reabsorb vital nutrients for reentry into the blood supply. Blood & Lymphatic Vessels 1. Name the three layers in the walls of blood vessels and briefly describe the cells, key molecules, and function(s) of each one. Tunica Intima – the innermost layer, contains endothelium and underlying basal lamina and connective tissue. Internal elastic lamina is considered to be part of tunica intima. Tunica Media – layers of smooth muscle, also contains interspersed elastic sheets in large/medium arteries. Large arteries usually have external elastic lamina as the outer boundary of the tunica media. Tunica Adventitia – outermost layer consisting of connective tissue, collagen, and reticular fibers. It attaches vessel to adjacent tissues. In large arteries/veins, the tunica adventitia contains blood vessels and peripheral nerves which supply outer portions of the vessel wall with nutrients and oxygen. 2. Compare and contrast the structure and function of small, medium, and large arteries and veins, capillaries, and lymphatics. Arteries‟ primary function is to carry blood away from the heart. Large arteries have a tunica media with dozens of interspersed smooth muscle cells. The elastic layers smooth out pulses by storing energy during systole and releasing it during diastole. Medium arteries have several layers of smooth muscle in the tunica media, but not as many as large arteries. Has clearly visible internal elastic lamina. Arterioles (small arteries) have the fewest number of smooth muscle layers. They don‟t usually have an internal elastic lamina, unlike medium and large arteries. Their primary function is to deliver blood to the capillary bed. Capillaries are the smallest blood vessel where exchange of gases, nutrients, and wastes takes place. Capillaries can be continuous, fenestrated or sinusoidal. Continuous capillaries are sealed and have no holes (fenestrae). Materials are transported across the endothelium via transcytosis. Fenestrated capillaries have small holes that allow for faster exchange between the blood and tissue. Sinusoidal capillaries have the largest lumen of the capillaries, which slows blood flow even more. The endothelial cells have large fenestrae allowing plasma direct access to the surrounding tissue, making for the greatest possible exchange of materials. Veins‟ primary function is to collect blood and return it to the heart. Large veins have a tunica media with some smooth muscle, but much less than a comparable artery. Large veins located below the heart have longitudinal smooth muscle to help move blood up towards the heart. In medium veins, the tunica media contains a couple of layers of smooth muscle, and is smaller than the tunica adventitia. Venules are large diameter endothelial tubes with little or no smooth muscle. They are the primary site for white blood cells to leave the blood and enter the tissue. Veins located below the heart are equipped with valves that prevent backflow. Valves are thin flaps of endothelium that extend into the lumen of the vein. Lymphatic vessels collect and transport extracellular fluid and white blood cells from connective tissue spaces, through lymph nodes, and back into the blood. Lymphatic vessels have extremely thin walls, which are highly permeable, and low internal pressure, which allows for easy entry of fluids and white blood cells. Lymphatic vessels, like some veins, are equipped with valves to prevent backflow of lymph. 3. Explain the function of vasa vasorum and nervi vasorum. Vasa vasorum and nervi vasorum are the vessels of the vessel and the nerves of the vessel, respectively. They are the blood supply and innervation of the outer walls of larger blood vessels. Vasa vasorum are small arteries located in the tunica adventitia, and branching into the tunica media to supply oxygen and nutrients to the outer layers. (Coronary arteries of the heart are vasa vasorum.) Nervi vasorum innervates the outer layers of the blood vessels. The nerve terminals are in the tunica adventitia, but can act on the tunica media through diffusion. Release of norepinephrine causes the smooth muscle to contract, while release of acetylcholine will cause them to relax. 4. Compare and contrast the structures and functions of the 3 types of capillaries. Capillaries can be continuous, fenestrated or sinusoidal. Continuous capillaries are sealed and have no holes (fenestrae). Materials are transported across the endothelium via transcytosis. Fenestrated capillaries have small holes that allow for faster exchange between the blood and tissue. Sinusoidal capillaries have the largest lumen of the capillaries, which slows blood flow even more. The endothelial cells have large fenestrae allowing plasma direct access to the surrounding tissue, making for the greatest possible exchange of materials. 5. Explain the function of lymphatic vessels and explain how the structure of lymphatic vessels supports their function. Lymphatic vessels collect and transport extracellular fluid and white blood cells from connective tissue spaces, through lymph nodes, and back into the blood. Lymphatic vessels have extremely thin walls, which are highly permeable, and low internal pressure, which allows for easy entry of fluids and white blood cells. Lymphatic vessels, like some veins, are equipped with valves to prevent backflow of lymph. Introduction to Blood I & II 1. Describe the components of blood and their basic function(s). Blood consists of formed (cellular) elements, and nonformed elements. The cellular components include red blood cells (RBCs), white blood cells (WBCs), and platelets. Red blood cells transport oxygen from the lungs to the tissue, and carbon dioxide from the tissue to the lung. White blood cells are responsible for defending the body against infection and foreign material. Platelets are responsible for aiding in the clotting cascade. The nonformed elements are everything in the plasma, including water, sugars, lipids, vitamins, minerals, electrolytes, and proteins. 2. Describe how cells of the blood develop from multipotent progenitor stem cells in the bone marrow. A pluripotent stem cell gives rise to two progenitor multipotent lines, the common lymphoid lineage, and the common myeloid lineage. The myeloid stem cell gives rise to cells that differentiate into either granulocytes/monocytes, megakaryocytes, or erythrocytes. The lymphoid lineage gives rise to precursors of T-cells, B-cells, and natural killer cells. 3. Know approximate time it takes for development in the marrow of the various blood cells (RBCs, WBCs and platelets) and how long the mature cell lasts in the periphery. Granulocytes – take 10-14 days to develop, neutrophils last ~1 day in circulation, and 1-2 days in tissue. Monocytes – take 2-3 days to develop, circulate for ~3 days, and may last for up to 3 months in tissue. Erythrocytes – takes 7 days to mature, last 120 days in circulation under normal circumstances. Megakaryocytes (platelet producers) – takes 5-10 days to produce platelets, survive for 10 days in the circulation. 4. Describe the morphologic features of the white blood cells in the peripheral blood and be able to identify them. Granulocytes – Basophils, Neutrophils, and Eosinophils (granules in cytoplasm) Basophils have a bilobed nucleus usually partial obscured by large dark purple granules. Neutrophils have a multilobed nucleus (3-5 lobes) and have 3 types of granules. Eosinophils have a multilobed nucleus (2-3 lobes) and are filled with large red- orange granules containing histaminase and collagenase. Lymphocytes – round nucleus with pale blue cytoplasm. Monocyte – lobated nucleus (not segmented), with abundant blue-gray cytoplasm with small blue granules. 5. Describe the function of the different WBCs. Basophils are functionally related to mast cells. They release vasoactive agents upon stimulation. Neutrophils‟ primary function is in acute inflammatory response to tissue injury (secrete enzymes to degrade dead tissue, ingest/destroy damaged tissue, digest foreign substances) Eosinophils are associated with allergic reactions, parasitic infections, and chronic inflammation. Lymphocytes are the main cells of the immune system; functionality will depend on the type of lymphocyte. Monocytes are precursors of mononuclear phagocytes. Once differentiated, they will digest bacteria, tissue debris, other cells, and present antigens to T-cells. 6. Relate major causes of leukocytosis and leukopenia to functions of white blood cells. Leukopenia is a decreased leukocyte count, usually due to a reduction in the number of neutrophils. The causes can either be reduced production, indicating a vitamin deficiency, a bone marrow condition, or myelodysplasia; or increased destruction due to drugs, autoimmune conditions, or infection. Leukocytosis is an increase in the white blood cell count. There are three main causes, increased release from the marrow (infection), decreased margination and extravasation (leaving the blood stream to go to the tissues), and increased number of marrow precursors (infection, inflammation, neoplasms). Leukocytosis can be further classified according to which white blood cell is increased. Neutrophilia – due to acute bacterial infections, non-infectious inflammation Eosinophilia – due to allergic disorders, parasitic infections, drug reactions, some malignancies. Basophilia – rare, chronic myelogenous leukemia. Monocytosis – chronic infections, lupus, inflammatory bowel disease, monocytic leukemia. Lymphocytosis – viral infections, TB, neoplasms. 7. Describe the significance of a left-shift of granulocytes in the blood A left-shift of granulocytes means that there are precursor cells in the blood. This indicates that the blood is undergoing inflammation, infection, bone marrow disease, or some other stressor causing release of earlier precursors into the blood. 8. Define anemia, and outline the classification of anemia. Anemia is a reduction in the number of red blood cells or hemoglobin content of the blood. It is classified according to the underlying mechanism or alterations that take place in red blood cell morphology. The underlying mechanisms include impaired production of RBCs, increased blood loss, or increased destruction of RBCs. Changes in morphology are either in shape (microcytic, macrocytic, or normocytic) or in color (hypochromic or normochromic). 9. Describe the main functions and components of the hemostasis system. Hemostasis consists of a highly regulated set of events with 2 main functions – rapid formation of a hemostatic plug to stop bleeding, and prevention of out of control clot formation. It is composed of the vessel wall and platelets, forming the primary hemostasis, coagulation cascade (secondary hemostasis), and the counter-regulatory mechanisms. 10. Compare and contrast bleeding defects in terms of type of bleeding and onset of bleeding. Bleeding defects can be due to platelet abnormalities, vessel wall abnormalities, or coagulation factor deficiencies. Platelet abnormalities can be due to reduced platelet number, or inability of platelets to bind properly. Vessel wall abnormalities can cause leakage of blood into surrounding tissues. Coagulation factor deficiencies affect the ability to stabilize the clot for long-term healing. Platelet abnormalities result in petechae (pinpoint hemorrhages). Vessel wall abnormalities result in purpura (3-10 mm hemorrhages). Coagulation factor deficiencies result in ecchymosis (bruises), and deep tissue bleeding. 11. Describe the two main causes of thrombocytopenia. Thrombocytopenia (reduced platelet count) can be caused by either decreased production by the marrow, due to drugs, bone marrow disorders, vitamin deficiencies, congenital disorders, or infection; or by accelerated destruction due to either immune mediated or non-immune mediated/mechanical processes. 12. Compare and contrast von Willebrand disease and Hemophilia A in terms of type of bleeding, defect, lab features and treatment. Von Willebrand disease is a platelet adhesion disorder that results in spontaneous mucocutaneous bleeding. The defect is in the von Willebrand Factor, which acts as a stabilizer of coagulant factor VIII. Lab results will show a normal platelet count, but decreased vWF protein or function. It is treated with vasopressin (causes the release of vWF and FVIII from endothelial cells) or vWF concentrates. Hemophilia A is a coagulation factor deficiency, specifically in factor VIII, that causes easy bruising, hemorrhage after trauma, and spontaneous hemorrhage into joints. Laboratory tests will show a prolonged prothrombin time and a low factor VIII level. Hemophilia A is treated with factor VIII replacement. Specialization of Proteins II 1. Differentiate between reticulocytes and erythrocytes, and discuss how reticulocyte and erythrocyte count may be used diagnostically. Reticulocytes are an immature form of erythrocytes. They have no nucleus, but contain some ribosomes. Because red blood cells lack nuclei, they are unable to replace proteins that are degraded. Erythrocytes comprise 35% of the volume of the blood, whereas reticulocytes make up only 1% of all red blood cells. Since reticulocytes are precursors to erythrocytes, destruction of erythrocytes in anemias, or infection can lead to an increase in the reticulocyte count. 2. Describe the role carbohydrates, band 3 protein (anion exchange protein) and cytoskeletal proteins associated with the red blood cell membrane. Carbohydrates covering the surface of red blood cells denote which blood type it is (A, AB, B or O). This is critical during blood transfusions in which the immune system will attack and destroy blood with a different surface antigen. Band 3 protein is a transmembrane glycoprotein that forms a dimeric channel. The opening of this channel allows the cell to exchange chloride ion with the cell bicarbonate, which in the long run facilitates the exhalation of CO2. Spectrin, ankyrin, and actin are the main cytoskeletal proteins of the erythrocyte. Spectrin is comprised of a pair of heterodimers (α and β) that bind band 4.1 protein, ankyrin, and actin. Ankyrin binds to band 3 protein, and anchors spectrin to the plasma membrane. The overall structure of this network of proteins is what provides the shape and flexibility of red blood cells which allows them to squeeze through capillaries. 3. Describe similarities and differences between Myoglobin and Hemoglobin. Myoglobin and hemoglobin are both globin proteins whose primary function is transportation. They are responsible for the transport and storage of oxygen in the body. This is done via oxygen binding to a heme prosthetic group nestled in their active site. The globins are primarily composed of α-helices. Myoglobin is a monomeric protein, whereas hemoglobin is a tetrameric protein. Despite this, the monomers of hemoglobin are structurally similar to myoglobin, essentially making hemoglobin the same protein, just with the ability to carry 4x as much oxygen. Hemoglobin is comprised of a dimer of dimers, meaning that the individual subunits are α and β types, which associate to form αβ dimers that then form the hemoglobin tetramer. The largest difference between the two is affinity for oxygen. Hemoglobin exhibits cooperativity in its binding, whereas myoglobin does not. This is one of the factors that add to myoglobin having a higher affinity for oxygen than hemoglobin does. 4. Describe the beta and alpha globin gene clusters and identify the different globin chain compositions that comprise HbA1, HbA2 and HbF. The α and β subunits of hemoglobin are encoded by two different gene clusters. The two families exist as clusters on our chromosomes. The α cluster has δ and two α genes (which are duplicates). The β cluster is more complicated and has ε, two γ‟s, a δ, and then the β gene. HbA1 – adult hemoglobin (95%), α2β2 HbA2 – adult hemoglobin (5%), α2δ2 HbF – fetal hemoglobin, α2γ2 5. Describe the molecular genetic basis of the various thalassemias, the genotype- phenotype relationships in these diseases and the aberrant hemoglobins that are produced in α-thalassemias. The thalassemias are a class of diseases in which there is a mutation in the gene for one of the subunits. This results in inefficient production of globin, which leads to anemia. The severity of the thalassemia is dependent on which gene and how many are mutated. In the α cluster, there are two copies of each of the genes, but no replacements. Thus, one mutation of the four is a silent carrier, two has minor anemia, three results in replacement of the α subunits with β chains, and four results in fetal death. In the case of mutations at three loci, hemoglobin is synthesized as HbH (β4) and Hb Barts (γ4) in utero due to the lack of functional α subunits. The β thalassemias are simpler, due to the lack of gene duplications, but more survivable due to the larger gene family. One allelic mutation/deletion is generally asymptomatic, two mutations range between asymptomatic and anemia, two deletions causes severe anemia, usually resulting in death by the age of twenty. Specialization of Proteins III 1. Discuss how the R(elaxed) and T(ense) configurations relate to the positioning of the heme iron, the role of the proximal histidine, and the binding or releasing of oxygen. In the tense state, the proximal histidine pulls the iron out of the plane of the heme. Binding of oxygen is coordinated with the distal histidine, allowing oxygen to pull iron back into the plane of the ring, changing the configuration from tense state to relaxed state. The release of oxygen terminates the pull on the iron, allowing the proximal histidine to pull it back out of the plane of the heme. 2. Describe how cooperative binding of oxygen by hemoglobin improves its effectiveness as an oxygen transporter. Hemoglobin is tetrameric, binding of oxygen in one subunit affects the binding of oxygen in the other subunits. For example, binding of oxygen in subunit one will pull that subunit into the R state, which will force the other three subunits into the R state. While in the R state, they are more effective at binding oxygen. 3. Discuss the physiological significance of protons (Bohr Effect), carbon dioxide, BPG (2,3-bisphosphoglycerate) and the presence of the γ- subunit rather than the β-subunit in HbF on the affinity of Hb for oxygen and under what circumstances these effects are important. Protons are at high concentrations in the tissue when there are high rates of metabolism. Free protons are able to bind a free histidine, giving it a positive charge, which results in a salt bridge favored by the T state. This causes hemoglobin to have less affinity for oxygen in the tissues, which is desirable under high metabolic conditions. Carbon dioxide is also a byproduct of metabolism. It is able to bind to hemoglobin at the N-terminus, forming a carbamino group. This favors a salt bridge that stabilizes the T state, lowering affinity for oxygen in the tissues. This also helps to favor oxygen dissociation in tissues. BPG is produced in red blood cells undergoing the metabolism of glucose in low oxygen content. It is highly negative charged, and can insert itself in the cleft present in the T state. There it is able to interact favorable with positively charged side chains, stabilizing the T state. (BPG is integral to expression of cooperativity). Fetal hemoglobin has a γ subunit instead of a β subunit. The γ subunit decreases the affinity for BPG relative to adult hemoglobin because it disrupts the salt bridges present in adult hemoglobin; which in turn increases the affinity of fetal hemoglobin for oxygen. This is essential in the placenta, where oxygen must be transferred from the mother‟s hemoglobin to the fetal hemoglobin. 4. Discuss how carbon monoxide (CO) competes with oxygen for binding to Hb and how its greater affinity relative to O2 is reduced by the distal histidine. Carbon monoxide binds both hemoglobin and myoglobin at the same site as oxygen, but with a 200x greater affinity. Its affinity would be increased if it weren‟t for the steric hindrance caused by the distal histidine used to coordinate oxygen when binding. This hindrance causes carbon monoxide to bind at an angle instead of straight on, decreasing its affinity. 5. Describe the relationship between hemoglobin and acid-base homeostasis. Carbon dioxide and protons are produced as endproducts of metabolism. Hydration of carbon dioxide gives bicarbonate and a proton. This equilibrium is used to maintain acid- base homeostasis. Hemoglobin is able to bind protons at histidine residues, and bind carbon dioxide at the N-terminus, enabling it to buffer against acidosis. Introduction to the Immune System I and II 1. Describe the general features of innate versus adaptive immunity. Innate immunity exists prior to exposure to a foreign pathogen and is not improved by repeated exposure. It is not antigen specific and lacks a memory component. Adaptive immunity continually develops in response to antigen exposures. It is improved by repeated exposures, antigen specific, and has immunological memory. 2. Describe the mechanisms of innate immunity. Innate immunity can be split into physical components, biochemical components, and cells. Physical components include all epithelial surfaces, which act as barriers to the outside with anti-microbial properties. This also includes mucus, tears, urine, as well as coughing and sneezing. The biochemical components include the complement system, which is capable of lysis, opsonization, and chemotaxis, lysozymes, low pH, and interferons. The cells of innate immunity are phagocytes, natural killer cells, and others all capable of killing microbes and initiating an inflammatory response. 3. Describe the roles of complement components in the innate immune response. Complements are proteins in circulation that function to induce inflammation, opsonize pathogens, and induce lysis. Complement can be activated by 3 general pathways, the alternative pathway (first to act), the lectin pathway (second to act) and the classical pathway (third to act). 4. Explain how the innate immune system recognizes “non-self”. Innate immunity cells are equipped with Toll-Like Receptors. These receptors are pattern recognition receptors that bind molecules unique to microbes. They bind peptidoglycans lipoproteins, dsRNA, ssRNA, flagellin, etc. This binding activates a cell to increase microbe killing capacity, enhance ability to active adaptive immunity, increased cytokine secretion, and induction of interferons. 5. Explain the clonal selection model. Every lymphocyte in the body has a distinct antigen receptor which specifies the antigen it will bind to, prior to exposure to the antigen. On each lymphocyte, there are only receptors for a single antigen on the cell surface. Antigens bind to the lymphocyte with a receptor specific for that antigen. Binding stimulates the lymphocyte to proliferate. B cells differentiate into plasma cells and secrete antibodies. T cells differentiate into effector cells. Some of each of these is saved as memory cells. Any cell capable of bind self-antigens is destroyed or rendered unresponsive. 6. Explain how antigen receptor diversity is generated for B and T cells. Antibody diversity is generated through recombination of multiple gene segments encoding the variable region. One segment of each type is randomly combined to create a functional gene. There is pairing of heavy chains with light chains to further increase diversity. Imprecise joining of segments allows for additional diversity. A similar mechanism exists for T cell receptors. 7. Describe the effector functions of the various T lymphocyte subsets. T lymphocytes have three subsets: T helper, T cytotoxic, and T regulatory cells. T helper cells secrete cytokines, active dendritic cells and “help” B cells produce antibodies. T cytotoxic cells destroy virus infected cells and tumors. T regulatory cells regulate immune responses by controlling the activities of other immune cells. 8. Describe the kinetics of the antibody response. The antibody response has four phases: lag phase, exponential phase, steady-state phase, and declining phase. The lag phase is the time between the initial exposure and detection of antibodies in the body (4-7 days). The exponential phase is the rapid increase in antibodies due to the increased number and activity of plasma cells. The steady-state phase is when the rate of antibody synthesis becomes equal to the rate of degradation. The declining phase is when antibody synthesis wanes as pathogen is eliminated. 9. Explain the differences between primary and secondary immune responses in the case of antibody production. The secondary response has a shorter lag period due to the memory cells. There is also an extended plateau with higher levels of antibodies, followed by a slower decline of antibody levels. IgG is the major antibody class in the secondary response in comparison to IgM in the primary response. The binding affinity of the antibody is higher in the second response than in the first, this is known as “affinity maturation”. 10. Describe the properties and functions of each antibody isotype. IgM – primarily exists as a pentamer, which confines it to the bloodstream. It is expressed on the surface of naïve B cells as an antigen receptor. Does not cross the placenta, predominant during first week of infection, strong activator of complement. IgG – principle immunoglobulin of serum with the longest half-life, has four subclasses, is able to cross the placenta and provide immunity for up to a year after birth, activator of complement, can neutralize toxins and viruses, and has opsonic activity. IgA – most abundant immunoglobulin in the body (including mucosal surfaces), can exist as a monomer or dimer, protects exposed mucosal regions, predominant form of Ig in sero-mucous secretions, transferred to newborns from breast milk, functions primarily as a neutralizing antibody. IgE – low concentration in serum, important for allergic reactions, induced in response to parasitic infections. Introduction to the Immune System III 1. Describe the cell types and steps involved in T cell activation. T cells are activated by contact with peptides displayed on antigen presenting cells. Antigen presenting cells include dendritic cells, macrophages, and B lymphocytes. Antigen presenting cells interact with the T cell receptor, and are co-stimulated by the CD4/CD8 proteins. The type of response depends on the type of MHC class the antigen is presented on. MHC Class I will only interact with CD8 T cells, and Class II will only interact with CD4 T cells. T cell activation requires that the antigen presenting cell has already digested the antigen, loaded part of it onto the respective MHC, and transported it to the cell surface to be displayed. 2. Explain the structural and functional differences between MHC Class I and MHC Class II. MHC Class I interacts with CD8 T cells (and NK cells), whereas Class II interacts with CD4 cells. Class I binds 8-10-mer peptides, and Class II will bind anything over 13. Class I is expressed in all nucleated cells; Class II is only expressed in professional antigen presenting cells. Class I binds primarily in the ER; Class II binds in endo-lysosomes. Class I primarily binds peptides from endogenous proteins; Class II primarily binds from exogenous proteins. Both proteins are composed of four subunits (which vary between the two classes). Class I is only anchored to the membrane with one subunit; Class II is anchored with two subunits. 3. Describe how MHC Class I and Class II peptides are generated. MHC is a large locus in the genome, containing roughly 400 genes. MCH Class I is encoded by multiple genes for either the heavy chain (which binds antigen) or the light chain. Class II is also encoded by multiple genes for two chains of equal size that together bind the peptide. Binding peptides are generated as a result of ingestion and presentation of the antigen. Class I presenting cells are infected by a virus, begin reproduction of its genetic material, and bind peptide fragments in the ER, which are then transported to the surface of the cell. Class II presenting cells present fragments left over from degradation by the lysosome. This can either be from the pathogen itself, or from antigens bound to surface antibodies. 4. Explain the basic, unique genetic features of MHC molecules. MHC genes have a high rate of polymorphisms, meaning that in a given population there is a high frequency of alternate alleles. This broadens the pool of peptides that will be able to bind the MHC, increasing the likelihood the immune system will “see” a pathogen. 5. Describe the effector functions of Cytotoxic, Helper, and Regulatory T cells. T helper cells react with antigenic peptides presented by MHC Class II (exogenous). They help B cells make antibodies, and macrophages become active. They also secrete factors that influence immune response. T cytotoxic cells react with antigens presented by MHC Class I (endogenous). They have cytolytic activity towards virus infected cells and tumor cells. T regulatory cells regulate immune responses, which help prevents autoimmunity. They are only able to interact with MHC Class II. 6. Explain the characteristics of active immunization and how they differ from passive immunization. Passive immunization is transfer of antibodies/serum from an immunized individual to a non-immunized individual. Active immunization is exposure to a foreign antigen. Active immunization can be natural (arising from infection) or artificial (vaccination). Vaccinations can be live attenuated organisms, killed organisms, inactivated bacterial product, bacterial components, peptide vaccines, or DNA vaccines. 7. Describe some of the undesirable effects of the immune response. Hypersensitivity is exaggerated reactions to innocuous substances, i.e. allergies. Autoimmunity is when your adaptive immune system targets self-antigens. Transplant rejection occurs when organs are not accepted between genetically distant individuals. This is largely due to differences in MHC molecules. Introduction to the Immune System IV and V 1. Outline the path of lymph flow, including the 4 major lymph vessels and the regions they drain. Explain a major clinical implication of the asymmetric pattern of lymph drainage. Lymph flows from the periphery through multiple lymph nodes, draining into major lymph vessels, which eventually drain into the venous system. The four major lymph vessels are the left jugular trunk (which drains the head), the right jugular trunk (which also drains the head), the right lymphatic duct (which drains the right side of the chest as well as the right arm), and the thoracic duct (which drains the rest of the body, including the left jugular trunk). This results in ~75% of the body draining to the left side of the neck. Asymmetric lymph drainage will result in more pathogens, tumors, infections, etc. in nodes along the thoracic duct. 2. Describe the pathways that lymphocytes use to travel between blood and tissues, and compare to pathways used by WBCs (re-circulation vs. one-way street). Lymphocytes are made in the bone marrow and either mature there or in the thymus before entering circulation. They leave the blood into the connective tissues via post capillary venules, exactly as white blood cells do. However, white blood cells act and die in the tissues, whereas lymphocytes have the option of recirculating to a new tissue. To do so, they enter lymphatic capillaries in the connective tissue, circulate through the lymphatic system until they are dumped back into the blood circulation. 3. Explain the difference between primary and secondary lymphoid organs. Primary lymphoid organs are so called because they are the sites of production of naïve lymphocytes; the bone marrow and thymus gland are primary lymphoid organs. Secondary lymphoid organs are where cells of the immune system interact with antigens and generate an immune response. Mucosa associated lymphoid tissues of the gut, respiratory, and urogenital tracts, as well as lymph nodes in the white pulp of the spleen are all secondary lymphoid organs/tissues. 4. Describe the histology of bone marrow and thymus. Relate structure to function. The thymus is divided into lobules by connective tissue septa. Each lobule is a network of epithelial reticular cells infiltrated by pre-T lymphocytes. The epithelial reticular cells are connected via desmosomes and support the parenchyma of the thymus. Each lobule has a cortex and a medulla. Lymphocytes are predominant in the cortex, whereas epithelial cells predominate in the medulla. As part of the maturation process, pre-T lymphocytes are positively selected if they are able to recognize MHC proteins presented to them, and negatively selected if their receptors bind any self-antigens. Self-antigen binding cells are triggered to undergo apoptosis and are removed by macrophages. The thymus is also riddled with Hassall‟s corpuscles. They are collections of involuted epithelial cells of unknown function (characteristic of the medullary regions). The thymus has a blood thymus barrier composed of 4 layers that act to prevent pathogens from entering the thymus. Pre-T lymphocytes that recognize antigen are assumed to be recognizing self, and are destroyed; if a pathogen got into the thymus, any pre-t lymphocytes recognizing it would be destroyed. The bone marrow is encased by a cortical bone, and traversed by trabecular bone. It is a highly organized meshwork of thin-walled capillary-venous sinuses with surrounding extracellular matrix, fat, and hematopoietic compartment. The capillaries are necessary because all cells in the bone marrow develop from a progenitor cell, and eventually leave through the capillary sinusoids. 5. Describe the structure and function of lymphoid follicles. Distinguish lymphoid follicles from thymic lobules, both structurally and functionally. A lymphoid follicle (which is synonymous with a lymphoid nodule, but NOT with a lymph node) is composed of two areas. The inner circle of a follicle is the germinal center. It is surrounded by a mantle. B lymphocytes are concentrated in the germinal center with some in the inner region of the mantle. T lymphocytes are in the outer mantle. The follicle itself is supported by a connective tissue network of mesenchymal reticular cells and the fibers they secrete (note that these are not the same as the reticular cells in the thymus). Germinal centers are the location of affinity maturation in B lymphocytes. A B lymphocyte with antigen bound undergoes rapid proliferation with hypermutation in order to create a diverse population of B cells with antigen bind sites of varying affinities for the antigen. Whichever site binds with the highest affinity is selected to undergo further proliferation, differentiate into pre-plasma cells, and leave the germinal center to secrete antibodies. Germinal centers are also the site of class switching in B lymphocyte antigen receptor types. Thymic lobules have two layers, like follicles, but are called cortex and medulla, not mantle and germinal center. Although both have an underlying network of reticular cells, the thymus has epithelial reticular cells, and the follicles have mesenchymal reticular cells. Follicles have High Endothelial Venules, which allow easy access to the follicle, whereas thymic lobules have thymus blood barriers that prevent direct access to the thymus. The thymus is the site of maturation of T cells, where the follicles are more the site of proliferation of B cells. 6. Describe the structure and function of lymph nodes and spleen. Explain the special anatomic features that reflect the function of lymph nodes as filters of lymph, and spleen as filter of blood. Based on this information, discuss causes of lymphadenopathy and splenomegaly. Lymph nodes are supported by an outer capsule with trabeculae that extend into the node. The parenchyma of the node is supported by a matrix of reticular fibers secreted by mesenchymal reticular cells. Lymph nodes are organized into a cortex and a medulla, with follicles present in the outer part of the cortex containing mainly B lymphocytes in both the germinal center and the mantle. T lymphocytes predominate in the inner cortex, but are not organized into follicles. Cells in the medulla are organized into cords, which are cross-linked to one another and supported by the framework of mesenchymal reticular cells. Medullary cords are composed of T and B lymphocytes, macrophages, plasma cells, and mesenchymal reticular cells and the fibers they secrete. Lymph is brought from upstream of the node and forces to percolate through the fibers, causing turbulence that allows macrophages to trap particles in the lymph and travel to the cortex and present the antigen so an immune response may be triggered. Blood flows through the lymph node into capillaries, HEV in the follicles, and exit the lymph node at the hilus. The spleen performs the same functions as a lymph node with the addition of blood filtration. Because of this, the spleen is split into red pulp and white pulp. The white pulp is responsible for generating an immune response against blood borne antigens. As blood spills out of the arterioles into the parenchyma, macrophages ingest particular matter and present it to the lymphocytes in the white pulp. The red pulp is composed of all the components of the blood, in association with splenic cords. After the blood has spilled into the parenchyma, it must reenter the splenic sinusoids to continue to circulate. To do so, the red blood cells must pass through the network of fibers, and the porous walls of the sinusoids. This requires that the cell be very flexible; old or defective cells will get stuck in the spleen and will be destroyed by macrophages. Lymphadenopathy is a disease of the lymph nodes. This would be caused by the fact that lymph nodes are intentionally placed “in harm‟s way”. They are the situated such that they will catch any tumors, infections, pathogens, etc. trying to travel the blood through the lymph. This makes them prone to infection. Splenomegaly can be caused by a number of things, including infection, liver diseases, anemia, blood cancers, blood clot, etc. The things that make splenomegaly different than lymphadenopathy are those causes associated with blood. Since the spleen filters the blood, disorders affecting the blood will also cause problems with the spleens ability to filter the blood. Cell Injury and Cell Death I and II 1. Explain why an elevated blood level of cardiac troponin is used as an indicator of a myocardial infarct. Cardiac troponin is an intracellular enzyme specific to the cardiac muscles of the heart. Upon cell death, intracellular enzymes are released and gain access to the bloodstream. Elevated levels of such enzymes are indicative of cell death in their respective origins. Therefore, presence of cardiac troponin is an indicator of myocardial infarction. 2. Differentiate the types of necrosis by their etiology and morphologic features Coagulation necrosis – occurs due to hypoxic death of cells. Predominant feature is protein denaturation. Infarcted tissue remains firm, but microscopically, the cells are eosinophilic and have nuclear changes (karyolysis). Liquefactive necrosis – occurs with focal infection by bacteria/fungi due to accumulation of neutrophils. Predominant feature is tissue digestion. Tissue appears as an abscess (collection of pus), but microscopically the tissue is digested and inflammatory cells and debris are present. Fat necrosis – necrosis affecting adipose tissue, occurs when acute pancreatitis or trauma causes the digestion of fat cell membranes and the triglycerides stored in fat cells. Grossly, the tissue will have a chalky white appearance due to saponification; microscopically, vague outlines of fat cells will be present, with basophilic calcium deposits. Caseous necrosis – necrosis whose appearance is akin to that of soft cheese. It is seen in the central areas of certain granulomas, representing the necrotic cell debris. Microscopically, the cells no longer have their outlines, and appear as granular eosinophilic debris. Fibrinoid necrosis – necrosis in injured blood vessels. Microscopically, the tissue has a bright pink, granular appearance resembling fibrin, which is actually composed of fibrin, plasma proteins and complement. Gangrenous necrosis – ischemic necrosis usually in the lower extremities. Coagulative necrosis pattern will dominate if bacterial and inflammatory cells are less involved. Liquefactive necrosis dominates if bacteria and/or inflammatory cells are recruited to the tissue. 3. Summarize the molecular changes occurring in cells exposed to hypoxia and relate these changes to the microscopic appearance of reversibly and irreversibly injured cells. Hypoxia is decreased oxygen delivery to the cells. The first thing to happen is decreased oxidative phosphorylation in the mitochondria, which decreases ATP production. This causes Na+ pumps to shut down, allowing Na+, Ca++ and water to enter the cell, while K+ leaves. This cascade causes cell swelling. ATP is then produced via anaerobic glycolysis. This causes an accumulation of lactic acid, which lowers the cell pH, causes chromatin to clump. ATP is required for ribosomal attachment. Lower levels of ATP cause ribosomes to detach, which compromises protein synthesis. Loss of protein synthesis renders the cell unable to export lipids, which causes lipid accumulation. 4. Describe the mechanism by which cyanide injures cells. Cyanide injures cells by binding to cytochrome oxidase in the mitochondria. This binding inhibits ATP production by oxidative phosphorylation. 5. Explain why N-acetylcysteine is used to treat acetaminophen poisoning. Metabolism of acetaminophen cases the generation of a free radical. Glutathione is a chemical in the body that scavenges the free radical. However, after glutathione levels are depleted, the free radicals can cause damage to proteins, DNA, and lipids. N- acetylcysteine can be taken up by the liver (unlike glutathione supplements) and be used to supply the cysteine required for glutathione synthesis. 6. Describe the function of Bcl-2 and p53 in pathways to apoptosis. Bcl-2 was the first oncogene to be discovered to inhibit cancer cell death. It prevents apoptosis by inhibiting the release of pro-apoptotic proteins from the mitochondria. It has been found to be overexpressed in ~85% of follicular lymphomas dues to translocation. p53 normally functions to mediate growth arrest, repair in response to DNA damage, and induce apoptosis when DNA cannot be repaired. It is found to be frequently mutated in cancers, leading to resistance to apoptosis. 7. Explain the process underlying the accumulation of various substances in tissues: steatosis, atherosclerotic plaques; neuritic plaques, and anthracotic pigment. Endogenous substances being produced faster than metabolism can handle them can cause steatosis. Steatosis is caused by toxic levels of alcohol or accumulation of cholesterol in atherosclerotic plaques. Abnormal endogenous substances can accumulate due to defects in protein folding, transport, or degradation. Neuritic plaques are caused by such accumulations. Normal endogenous substances can accumulate because of defects in enzymes needed for their metabolism. Exogenous substances that cannot be metabolized or removed from the tissues will also lead to accumulation; such is the case in anthracotic pigment. 8. Describe the etiology and features of dystrophic calcification versus metastatic calcification. Dystrophic calcification occurs in damaged or dying tissues, but serum calcium levels and metabolism are normal. Metastatic calcification occurs in normal tissues because of elevated serum calcium levels and/or abnormal calcium metabolism. H & E will stain calcium deposits a dark blue to purple. Thrombosis, Thromboembolism and Infarction 1. Define thrombosis and summarize the 3 major predisposing factors. Thrombosis is formation of a blood clot (thrombus) in a blood vessel that has been damaged. The Virchow‟s triad is the three predisposing factors for thrombosis, endothelial injury, altered blood flow, and increased coagulability of blood. Endothelial injury can cause a thrombus to form over the exposed subendothelium, which leads to adhesion of platelets. Altered blood flow can contribute to thrombosis by damaging endothelium and creating local pockets of stasis. Increased coagulability of the blood is an alteration of coagulation pathways, can be acquired or inherited. 2. Describe the features that differentiate a true thrombus from a postmortem clot. True thrombi tend to be attached to the vessel, have tissue like consistency, and have characteristic lines of Zahn. Lines of Zahn are produced by alternating pale layers of platelets mixed with fibrin, and dark layers of red blood cells. A postmortem clot will be more gelatinous in appearance. 3. Describe the potential fates of a thrombus and their clinical significance. A thrombus can be dissolved, emoblized, propagated, or organized and recanalized. Dissolution occurs when thrombi are cleared by the fibrinolytic system. Embolization occurs when portions of the thrombus dislodge and travel to other areas in the body. They inevitably lodge in small vessels and cause partial or complete occlusion. Propagation occurs when the thrombus grows and accumulates more platelets and fibrin. Organization and recanalization occur when the thrombus undergoes fibrosis (organization) and may eventually be recanalized (reestablish blood flow). 4. Describe the mechanism whereby factor V Leiden and the prothrombin gene mutation lead to hypercoagulability. Factor V Leiden is a genetic point mutation that renders Factor V resistant to inactivation by activated protein C. This results in loss of one of the major coagulation system inhibitors, which can lead to hypercoagulability. The prothrombin gene mutation is also a point mutation that results in increased levels of prothrombin. This will drive the coagulation cascade to increase fibrin formation, making the blood hypercoagulable. 5. Describe the symptoms of deep leg venous thrombosis and pulmonary embolism. Deep vein thrombosis (DVT) common occurs in the deep veins of the leg. It can cause pain and swelling, but roughly half of patients are symptomatic because collateral circulation bypasses the obstruction. DVT‟s are dangerous because than can embolize. Pulmonary embolisms are caused by a thrombus that embolizes and lodges in the lung. 95% of cases of PE are a result of DVT. 6. Describe the potential outcomes of pulmonary embolism. Most pulmonary embolisms are small and silent. A few result in sudden death, due to right heart failure or cardiovascular collapse. Obstruction of medium arteries results in pulmonary hemorrhage. Small emboli can lodge in end-arteriolar small branches. Multiple episodes of small thromboemboli can result in pulmonary hypertension. 7. Define infarction and describe the clinical significance. An infarct is an area of ischemic necrosis due to occlusion of either the artery supply or the venous drainage. More than half of all deaths in the US are caused by infarction, usually myocardial or cerebral infarction. 8. Describe gross and microscopic features of the two types of infarcts and explain why they occur in different organs. Infarcts can either be white or red. White infarcts occur with arterial occlusions in organs with end-arterial circulation (no collaterals or dual blood supply), i.e. the heart, kidneys, spleen. Red infarcts occur with venous occlusion, in tissues with dual circulation (lung, small intestine), or in loose tissues (lung), that allow blood to collect in the infarcted zone. Infarcts are usually wedge shaped, with the occluded vessel at the apex. As time passes, the infarct will become more defined. Infarcts in organs without a dual blood supply become progressively paler (white infarcts). Extravasated red blood cells in hemorrhagic infarcts are phagocytosed and converted to hemosiderin. Microscopically, the basic outline of cells is preserved, but the nuclei are lost. An inflammatory response will develop along the border within a few hours. There is gradual clearly of the cellular debris by neutrophils and macrophages; most infarcts will be replaced by scar tissue. 9. Summarize the factors that influence the development of an infarct. Development of an infarct is influenced by four factors: nature of vascular supply, rate of development of occlusion, vulnerability to hypoxia, and oxygen content of the blood. If there is an alternate blood supply, the area will be relatively insensitive to small occlusions. Slowly developed occlusions are less likely to cause infarcts because there is time for collateral circulation to develop. Tissues that are particularly sensitive to hypoxia are more likely to infarct. Anemia and pulmonary diseases are high risk factors for partial flow obstructions to lead to infarcts. Acute Inflammation 1. Explain the three major components of acute inflammation. 2. Describe four common types of stimuli that trigger acute inflammation. 3. Describe the various types of extracellular fluid collections, including transudate, exudate, edema and pus. 4. Understand the two major results of the vascular response in acute inflammation to be vasodilation and increased vascular permeability, and describe the forces favoring fluid movement out of vessels. 5. Name the chemical mediator commonly implicated in the primary, transient vascular permeability response and identify the type of blood vessel in which increased permeability primarily occurs. 6. Know the sequence of events in leukocyte extravasation, and explain how adhesion molecules are involved at most steps. 7. Explain the three main steps of phagocytosis, and name two oxygen dependent processes that are important in the third step (killing and degradation step). 8. Name two diseases that occur due to defects in leukocyte function. 9. Know the two main types of vasoactive amines, their source and explain their main effects. 10. Know the two main pathways of the arachadonic acid cascade and understand the role of anti-inflammatory medications in inhibiting the production of prostaglandins and leukotrienes. 11. Explain how nitric oxide acts as an endogenous regulator of inflammation. 12. Know the two main cytokines involved in inflammation and describe their major effects. 13. Recognize the complement, kinin and clotting systems as enzyme cascades that can act to modify the inflammatory response. 14. Explain the four common morphologic patterns of acute inflammation in tissues, and be able to give an example of each pattern. 15. Describe the three main outcomes of acute inflammation and explain factors favoring each outcome. Explain why some types of ongoing or repeated injury responses may display a mixed (both acute and chronic) inflammatory picture Chronic Inflammation I 1. Define chronic inflammation. 2. Describe three major causes of chronic inflammation, and be able to give an example of each. 3. Describe the morphologic features of chronic inflammation, including the predominant cell types. 4. Describe the main functions of macrophages in chronic inflammation. 5. Understand that unregulated inflammation can cause tissue damage and identify two types of substances macrophages secrete that can injure host tissues. 6. Compare and contrast tissue regeneration with healing by scar formation 7. Explain the differences between embryonic and adult stem cells. 8. Know the main growth factors involved in tissue regeneration and repair. 9. Know the main components of granulation tissue and be able to recognize its appearance in tissue. 10. Explain two separate mechanisms of angiogenesis. 11. Explain the three main processes in scar formation. 12. Contrast wound healing by first intention to healing by second intention. 13. Recognize the main systemic and local factors that influence wound healing. 14. Describe three complications of wound healing. 15. Explain the main way in which fibrosis associated with chronic inflammatory conditions differs from healing of a cutaneous wound. Chronic Inflammation II 1. Define a granuloma conceptually and histologically. 2. Describe the two main situations in which granulomas occur (non-immune and immune). 3. List the common causative agents of granuloma formation. 4. Diagram the dynamic cellular events and mediators in granuloma formation. 5. Understand the relationship between blood monocyte, macrophages, epithelioid cells and giant cells. 6. Discuss the possible sequelae of granulomas. 7. Compare and contrast granulomatous with nongranulomatous inflammation. 8. Describe the difference between granulomas and granulation tissue. 9. Give a brief summary of sarcoidosis as a clinicopathologic disease. Principles of Infectious Disease I 1. Discuss the structure and replication requirements of viruses, bacteria, fungi, and parasites. 2. Recognize the structural, metabolic, and genetic components of microbes (bacteria, fungi, parasites, and viruses) and how these impact the pathogenesis of disease. 3. Understand the structural, structural, metabolic, and genetic components, features which separate viruses, bacteria, fungi, and parasites 4. Categorize bacteria by cell shape and Gram stain. 5. Describe the role of the clinical microbiologist, the clinical laboratory, and the epidemiologist in diagnosis and management and prevention of infectious diseases. 6. Understand the epidemiologic basis for the acquisition and spread of viral, bacterial, fungal, and parasitic infections, and the role of epidemiology in disease diagnosis and infection prevention. Principles of Infectious Disease II 1. Summarize human-microbe interactions and describe differences between true pathogens and commensal organisms 2. List the factors that may affect the composition of normal flora, focus on antibiotics 3. Understand the pathogenic steps, which must exist for an infection to occur colonization, adhesion, etc 4. Describe the concept of virulence 5. Discuss genetics of pathogenicity 6. Discuss bacterial strategies of pathogenicity like adherence, invasion, toxin production Immunopathology of Hypersensitivity Reactions I 1. Define immunopathology. 2. Diagram the mechanism involved in IgE- mediated immediate hypersensitivity. 3. Contrast Type I and Type II hypersensitivity in terms of cells involved and diseases resulting from each 4. List the cell/tissue targets for two examples of Type II hypersensitivity. 5. Contrast Type I and III types of hypersensitivity. 6. Outline the immunologic steps in the development of post-Streptococcal glomrulonephritis as an example of Type III hypersensitivity. 7. List three other diseases (other than post-Streptococcal glomerulonephritis) mediated by Type III hypersensitivity. 8. Contrast the elements of Type III-mediated hypersensitivity from those of Type IV delayed hypersensitivity. 9. List the series of cellular/ tissue events which lead to the formation of a granuloma. Immunopathology II 1. Give the three defining properties of an autoimmune disease. 2. Contrast clonal anergy with clonal deletion. 3. Describe six mechanisms by which immunologic tolerance may be lost. 4. Explain how superantigens can break tolerance. 5. Know two common laboratory tests for SLE. 6. Describe the abnormalities of facial skin and renal glomeruli in SLE. 7. List three non-genetic factors which may act as triggering events for clinically apparent SLE. 8. Describe the paradox of anti-cardiolipin antibody results in vitro and in vivo.
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