Lecture Notes on Epithelial Tissue PDF

1 1 Lecture The Covering of Epithelium ILOs By the end of this lecture, students will be able to 1. Interpret the significance of general features of epithelium in relevance to its functional requirements. 2. Correlate structural adaptation of each type of epithe...

1 1 Lecture The Covering of Epithelium ILOs By the end of this lecture, students will be able to 1. Interpret the significance of general features of epithelium in relevance to its functional requirements. 2. Correlate structural adaptation of each type of epithelium to its function. 3. Recognize significance of epithelial in view of its structure & function. Epithelial tissue Epithelial tissue is present in two forms: (1) as sheets of contiguous cells (epithelia) that cover the body on its external surface and line the hollow organs on the internal surface, and (2) as glands, which originate from invaginated epithelial cells. Epithelial tissues have numerous functions Protection of underlying tissues of the body from abrasion and injury. Transcellular transport of molecules across epithelial layers. Secretion of mucus, hormones, enzymes, and so forth, from various glands. Absorption of material from a lumen (e.g., intestinal tract or certain kidney tubules). Control of movement of materials between body compartments via selective permeability of intercellular junctions between epithelial cells. Detection of sensations via taste buds, retina of the eye, and specialized hair cells in the ear. General features of epithelium 1. It is formed of closely packed cells with limited intercellular spaces. 2. The cells are related by intercellular junctions. 3. They are separated from the underlying connective tissue by an extracellular matrix, the basal lamina. 4. It has a high regenerative capacity through stem cells division. Epithelium possess a unipotent stem cell that is capable of division and differentiation to one type of epithelium only. 5. It is a non-vascular tissue but richly innervated. 6. The adjacent supporting connective tissue through its capillary beds supplies nourishment and oxygen via diffusion through the basal lamina. 1  2 Classification of Epithelial Membranes Cell arrangement and morphology are the bases of classification of epithelium Simple; formed of a single cell layer. Stratified; formed of several cell layers. Types Shape of cell Site Function I- Simple 1. Squamous Flat cells forming thin Lining: pulmonary Limiting membrane, sheets alveoli, parietal layer of fluid transport, Bowman capsule, blood gaseous exchange. and lymphatic vessels. 2. Cuboidal Cuboidal Thyroid gland follicles, Secretion, absorption, covering of ovary, protection kidney tubules. 3.Columanr Columnar Lining: oviducts, uterus, Transportation, much of digestive tract absorption, secretion, (stomach, small & large protection intestine). 4.Pseudostratified All cells rest on basal lamina Lining: most of trachea, Secretion, absorption columnar but not all reach epithelial primary bronchi. lubrication, protection, surface; surface cells are transportation. columnar Figure 1. Types of covering epithelium 2  3 Types Shape of cell Site Function II- Stratified 1. Non-keratinized Flat cells forming the most Lining: oral cavity, Form a physical Squamous superficial cell layer oesophagus & vagina. barrier for protection. 2. keratinized Flat cells form the most Skin. Protection Squamous superficial cell layer & covered by a keratin layer 3.Cuboidal Cuboidal cells form the Ducts of sweat glands Protection & secretion most superficial cell layer 4.Columnar Surface cells are columnar Conjunctiva of eye. Secretion & protection 5.Transitional Surface cells are dome Lining the ureters & Protection, distensible shaped urinary bladder Transitional Epithelium ⮚ It is structurally adapted to volume changes. ⮚ It has wide intercellular spaces allowing cells to glide and reduce number of cell layers in response to pressure by full bladder. 3  4 ⮚ Dome shaped cells form a barrier between urine and the blood vessels in the underlying connective tissue. 4  2 Lecture Other forms of epithelium & epithelial cells polarity ILOs By the end of this lecture, students will be able to 1. Interpret structural adaptation of special epithelium to its relative function. 2. Correlate cell membrane specialization to its function. 3. Interpret some clinical disorders related to structural abnormalities of cell membrane specialization. Specialized forms of covering epithelium These are types of epithelium that have an additional special function. 1. Germinal epithelium; cells that differentiate into sperms in male or ovum in females. It is present in the seminiferous tubules of testis in male and in ovaries in female. 2. Sensory epithelium; cells have a sensory nerve supply and function as sensory receptors, such as taste buds, hair cells of the ear and retina of the eye. Epithelial cell polarity Most epithelial cells demonstrate structural changes in one or more of its cell membranes that represent cell adaptation to perform a special function. Subsequently, cell membranes with special structure have a polarity; directed towards a certain surface in relation to function. Figure 1. Types of cell surface specializations & polarity 1 Types of polarity I- Apical domain ⮚ The apical domain, the region of the epithelial cell facing the lumen. ⮚ Functional specialization; It functions in transport of ions, molecules and water. ⮚ It is rich in ion channels, carrier proteins, H+-ATPase (adenosine triphosphatase), glycoproteins, and hydrolytic enzymes, as well as aquaporins, channel-forming proteins that function in regulation of water balance. Structural modification in the apical domain: These include microvilli with associated glycocalyx and, in some cases, stereocilia, cilia, and flagella in sperms. a. Microvilli ⮚ Microvilli are small finger-like cytoplasmic projections, covered by the cell membrane, emanating from the free surface of the cell into the lumen. They increase surface area of upper cell membrane involved in high rate of absorption. ⮚ They contain a bundle of microfilaments in the core to maintain rigidity. (Fig. 2) ⮚ Microvilli represent the striated border of the intestinal absorptive cells and the brush border of the kidney proximal tubule cells observed by light microscopy. Figure 2. Structure of microvilli 2  Scanning EM b. Stereocilia (not be confused with cilia) are long microvilli found only in the epididymis and on the sensory hair cells of the cochlea (inner ear). ⮚ These nonmotile structures are unusually rigid because of their core of actin filaments. ⮚ In the epididymis, they probably function in increasing the surface area; in the hair cells of the ear, they function in signal generation. Figure 3. Structure of stereocilia c. Cilia; are long motile apical cell processes that have a regular rhythmic bending movement. ⮚ The core of the cilium contains a complex of uniformly arranged microtubules called the axoneme. The axoneme is composed of a constant number of longitudinal microtubules arranged in a consistent 9 + 2 organization (two central single microtubules and outer nine doublets). ⮚ Each of the nine doublets is composed of two subunits A and B. 3  ⮚ The microtubule-associated protein dynein, also active in flagella, which has ATPase activity to release energy, radiates from subunit A of one doublet toward subunit B of the neighbouring doublet. ⮚ In the ciliated epithelia of the respiratory system (e.g., trachea and bronchi) and in the oviduct, there may be hundreds of cilia in orderly arrays on the luminal surface of the cells. Figure 4. Structure of a cilium Scanning EM Clinical Hint: Kartagener's syndrome results from hereditary defects in the ciliary dynein that would normally provide the energy for ciliary bending. Thus, ciliated cells without functional dynein are prohibited from functioning. Persons having this syndrome are susceptible to lung infections since their ciliated respiratory cells fail to clear the tract of debris and bacteria. Additionally, males with this syndrome are sterile since their sperm are immotile. II- Basolateral domain ⮚ The basolateral domain may be subdivided into two regions: the lateral plasma membrane and the basal plasma membrane. Each region possesses its own junctional specializations and receptors for hormones and neurotransmitters. In addition, these regions are rich in Na+,K+- ATPase and ion channels and are sites for constitutive secretion. The basal domain specializations Three important features mark the basal surface of epithelia: the basal lamina, plasma membrane enfoldings, and hemidesmosomes, which anchor the basal plasma membrane to the basal lamina. a. Basal enfoldings 4 The basal surface of some epithelia, especially those involved in ion transport, possesses multiple finger- like enfoldings of the basal plasma membranes that increase the surface area of the plasmalemma and partition the mitochondria-rich basal cytoplasm. b. Hemidesmosomes ⮚ They resemble half desmosomes and serve to attach the basal cell membrane to the basal lamina. Keratin tonofilaments insert into the plaques, unlike those in the desmosome, where the filaments enter the plaque and then make a sharp turn to exit it. ⮚ The cytoplasmic aspects of transmembrane linker proteins are attached to the plaque, whereas their extracellular moieties bind to laminin and type IV collagen of the basal lamina. ⮚ The transmembrane linker proteins of hemidesmosomes are integrins, a family of extracellular matrix receptors, whereas those of desmosomes belong to the cadherin family of cell-to-cell adhesion proteins. Figure 5. Structure of hemidesmosome c. Basal lamina ⮚ It is located at the plane of junction between the epithelium & the underlying C.T. 5 ⮚ It is formed of adhesive glycoproteins as laminin and collagen type IV. Clinical Hint Metaplasia; is transformation of one type of epithelium into another with loss of function, for example; Pseudostratified ciliated columnar epithelium of the bronchi of heavy smokers may undergo squamous metaplasia. Tumors that arise from epithelial cells may be benign (nonmalignant) or malignant. Malignant tumors arising from epithelia are called carcinomas. If the malignant cells cross the basal lamina, the tumour classification is upgraded to a more dangerous level with a worse outcome. Figure. Basal domain specializations Basal enfoldin gs b. Basal lamina Glandular epithelium ⮚ It is the type of epithelium found in different types of glands. ⮚ Glands are classified according to mode of secretion into exocrine (have a duct that opens into a cavity) or endocrine that secrete hormones directly into the circulation. ⮚ Epithelium form the secretory units of glands, as well as, lining the ducts of exocrine glands. 6  3 Absorption Across the Epithelium ILOs By the end of this lecture, students will be able to 1. Outline the model of Victorian transport across varied epithelial tissue 2. Interpret how properties of substances affect the transfer pattern 3. Appraise the relevance of its manipulation to achievement of therapeutic merits TRANSPORT of SUBSTANCES ACROSS the PHOSPHOLIPID BILAYER of a CELL MEMBRANE was reviewed in the previous block VICTORIAN TRANSPORT ACROSS THE EPITHELIUM At many body sites, any substance must be transported all the way through the whole cellular sheet instead of simply through the cell membrane. Transport of this type is termed Victorian Transport. It exists through the (1) intestinal epithelium, (2) epithelium of the renal tubules, (3) epithelium of all exocrine glands, (4) epithelium of the gallbladder, and (5) membrane of the choroid plexus of the brain (6) the placenta and other membranes. The basic mechanism for such Victorian Transport is that (1) active transport through the cell membrane occurs on one side of the transporting cells / then (2) either simple diffusion or facilitated diffusion occur through the membrane on the opposite side of transporting cell. This means that the UNIDIRECTIONAL TRANSPORT of substances TRANSCELLULAR is accomplished by presence of non-uniform distribution of transporters on opposing face of a cell. In (Figure 1); Solute carrier (SLC) superfamily; are mostly influx transporters involved in secondary active or facilitated transport. ATP-binding cassette (ABC) superfamily; are efflux transporters involved in active transport. Figure 1: Illustrate different examples of Victorian Transport. SLC; Solute carrier – ABC; ATP-binding cassette Thus, entry of any substance (or drug) into a cell by secondary active transport is achieved by the SLCs and its exit out of the cell is by facilitated diffusion. Both forces are driven by energy from Na+ gradient + that keeps and maintains intracellular concentration of Na+ low by the pumping action of the ABCs. VARIABLE FACTORS THAT INFLUENCE ABSORPTION ACROSS THE EPITHELIUM In Link to the Absorption Site: ➔ Physiological versus pathological considerations:  a. Temperature: The higher the temperature, the faster the rate of diffusion. Thus, the absorption process can occur more rapidly in a person with a fever. b. Surface area: The larger the membrane surface area available for diffusion, the faster is the absorption. For example, lung air sacs have a large surface area for diffusion of oxygen from air to blood. In emphysema, there is a reduction in that surface area that slows the rate of oxygen diffusion and makes breathing more difficult. c. Diffusion distance: The greater the distance that must be crossed the longer time it takes for absorption. For example, it takes only a fraction of a second for absorption of O across alveolar 2 epithelium but in pneumonia, fluid collects and increases the diffusion distance. This prolongs its absorption time as O must move through both the built-up fluid and the membrane to reach 2 the bloodstream. ➔ Pharmacological considerations: a. Stomach acidity can affect acid-labile drugs altering their property and impairing their absorption by intestine. So, these drugs must be given as enteric-coated tablets. b. GIT Motility changes: - A delay in gastric emptying time (as during food intake) will decrease the rate of intestinal absorption of some drugs, so are better taken on empty stomach. - An increase in intestinal motility (extensive diarrhea) can decrease contact time needed for proper absorption of some drugs by the intestine; to decrease their absorption. c. Presence of efflux transporters; as P-glycoprotein (P-Gp) that extrude back some drugs to the intestinal lumen; to decrease their absorption. This is taken in consideration during: - Dosage calculation when initially administering such drugs - Dosage readjustment, when concomitant inhibitors of P-Gp are used with them that will impair their back efflux, enhancing more absorption with liability to develop toxicity. d. Presence of variable food or other drugs in intestinal absorption milieu; for instant; caffeine and Vitamin C can accelerate absorption of acetaminophen & iron, respectively. In Link to The Absorbed Substance Itself: a. Its size and molecular weight; the smaller is the size and mass of a diffusing substance the more it is readily absorbed while if larger, it will be slower and less absorbed. b. Its degree of ionization; if that substance is a DRUG, then it can exist either in an ionized (polarized) water soluble or a non-ionized (non-polarized) lipid soluble form. When in a non-ionized form (uncharged / lipophilic); the drug will pass and diffuse readily across cell membranes. When in an ionized form (weak acid or base / hydrophilic) as most drugs are, then their degree of ionization will be dependent on their pKa (i.e., the pH at which their molecule is 50% ionized and 50% nonionized state) and the pH of the site it exists and is dissolved in (whether gastric secretion, intestinal secretion, urine, bile, …. etc.). Thus, drugs can pass, get absorbed and transported when they become poorly ionized. On the contrary, they tend to accumulate in the fluid compartment they exist in, when they are most highly ionized. This simply means that: Weak acids are more absorbed in fluid with lower pH and tend to accumulate in that with higher pH, as acetyl salicylic acid, …etc. Weak bases are more absorbed in fluid with higher pH and tend to accumulate in that with lower pH as atropine, …etc. IMPACT of MANIPULATION of VICTORIAN TRANSPORT by DRUGS  REGARDING THERAPEUTIC USES The use of Ezetimibe to inhibit the selective-cholesterol transporter in enterocyte. This anti- dyslipidemic agent will block exogenous cholesterol intake to help in treatment of hypercholesterolemia, Figure 2.  Figure 2: Cholesterol transporter as a target of action for ezetimibe. The use of the Gliflozins; the Sodium-Glucose Co-transporter-2 Inhibitors to block glucose reabsorption in proximal convoluted tubules back to the plasma i.e., lowering blood glucose level. This antidiabetic drugs are used in treatment of diabetes and control of heart failure manifestations, Figure 3. Figure 3: Sodium-Glucose transporter-2 (SGLT-2) as a target of action for Dapagliflozin  REGARDING DRUG INTERACTIONS [CAUSING ADVERSE EFFECTS]: The cardiac glycoside: digoxin that is of limited use to control advanced manifestations of heart failure; can develop some adverse effects when other drugs are used with it at the same time during its intake. This is due to development of interaction occurring at the level of P-glycoprotein (P-Gp) superfamily of efflux transporters. Normally Digoxin is partially extruded after absorption back to intestinal lumen and after reabsorption back to be eliminated through renal tubules. But the concomitant use of ➔ Macrolide antibiotics; as erythromycin, that inhibits the P-Gp efflux transporters will: - Increase digoxin absorption from intestine. - Suppress its elimination in urine. Yield digoxin toxicity ➔ Some anti-epileptics; as carbamazepine, that activates the P-Gp efflux transporters will: - Suppress digoxin absorption from intestine. - Enhance its elimination in urine. Decrease digoxin efficacy. __________  4 Drugs Bioavailability and Distribution ILOs By the end of this lecture, students will be able to 1. Interpret the importance of bioavailability when deciding oral versus parenteral doses of drugs, considering first-pass metabolism. 2. Appraise the importance of bioequivalence between similar drug formulations. 3. Co-relate the volume of distribution of different drugs to their dose calculation or management of toxicity. 4. Distinguish the concept of free versus bound drug fractions in relevance to its clinical implication. For any drug to exert its action, it must first enter the bloodstream and be bioavailable in the systemic circulation (unless it is locally acting at the site of its application) to be distributed to its site of action. Therefore, to get an effect, the drug should undergo two important processes:  Absorption which means the transport of drug form the site of its administration to the blood.  Distribution which means the delivery of the drug from the blood to the tissues. The changes of the drug concentration with time after oral administration are shown in figure 1, where after absorption and distribution the drug will be eliminated form the body by metabolism and excretion, and the time course of the drug concentration in the body is represented by the area under the curve (AUC). The study of all these processes is called pharmacokinetics. Because absorption entails the Fig. 1: Plot of plasma concentration versus time after oral administration, AUC: Area under the curve. transport of drugs across cellular membranes, the extent and rate of absorption will be dependent on the effect of the different factors governing transport. (Refer to factors governing absorption and transport across membranes). This means that unless the drug is administered directly into the blood by an intravenous (IV) administration, not all the amount of the administered drug can reach the blood stream to be bioavailable for further distribution. The comparison of the impact of the routes of drug administration especially the oral route on drug concentration versus, IV administration leads to the important concept of drug bioavailability. Oral bioavailability As discussed before, many factors can affect the absorption of a drug after its oral administration, which may not allow the whole administered dose to reach the blood. Not only that, but the absorbed content from the intestine passes directly through the portal blood to the liver first, before it reaches the systemic circulation. In the liver cells, many drugs are further bio-transformed (metabolized) to Page 1 of 4 metabolite(s) that is/are different from the administered drug. This, in-turn, will further decrease the fraction of the parent drug that reaches the systemic circulation to be available for distribution into the tissue to exert its action. This kind of drug metabolism or biotransformation is thus termed pre-systemic elimination. Some degree of drug metabolism may also occur in the gut wall before reaching the liver as shown in Fig 2. In contrast, drugs given by IV administration, are delivered directly in full amount (100% of the administered dose) to the systemic circulation which means that they escape the effect of both absorption and pre-systemic elimination. N.B., Pre-systemic elimination is also known as Fig 2: Factors affecting drug bioavailability. hepatic first pass metabolism, differentiating it from the metabolism that can occur further after distribution of the drug to all tissues including the liver again. This further metabolism can occur for drugs given by the oral or IV routes (all routes for systemic drug administration) and play an important role in drug elimination as will be further explained in next Blocks). Therefore, Bioavailability can be defined as: The fraction of the administered drug reaching the systemic circulation as intact (unchanged) drug. Accordingly, intravenously administered drug will exhibit a bioavailability of 1, since the entire dose reaches the systemic circulation as intact drug. However, for other routes of administration, it may be less than 1. The oral bioavailability (designated as F) is estimated from comparing the AUC of the of the drug concentration-time relationship (Figure 3) corrected for the dose and is calculated as 𝑫𝒐𝒔𝒆 𝑰𝑽. 𝑨𝑼𝑪 𝒐𝒓𝒂𝒍 F= 𝑫𝒐𝒔𝒆 𝑶𝒓𝒂𝒍. 𝑨𝑼𝑪 𝑰𝑽 N.B., bioavailability of other routes of administration can also be determined using the same equation but replacing the oral with the specific route utilized. Clinical relevance of bioavailability: Knowing the bioavailability of drugs is important for drug dosing when changing their administration from one route to another especially form IV to oral or vice versa as explained below:  Drugs with low bioavailability will have their oral Fig 3: Drug Bioavailability. AUC: Area under the curve dose much higher than the IV dose, e.g., Propranolol, where F≈0.2.  Drugs with high oral bioavailability like for e.g., Phenytoin (an anti-epileptic drug), whose F≈1, both the oral and IV dose would be almost equal.  Drugs with very low bioavailability, are most probably not suitable for oral administration and are given only by IV or other routes. e.g., the antibiotic vancomycin. Page 2 of 4  Sometimes, changing the drug formulations may change their absorptive properties and hence, their bioavailability. E.g., new formulations of oral cyclosporine have better bioavailability than the older ones. To compare formulations of the same drug produced by different companies, it is important not only to have the same bioavailability, but also, they should be bioequivalent. Bioequivalence means that the time- concentration curves of the two formulations are identical. This means that they not only should have the same AUC, but also the same maximal concentration (Cmax), and the same time to reach maximal concentration (Tmax). Bioequivalence is commonly calculated in the generic drug industry to determine that the generic formulation (e.g., a tablet) is bioequivalent to the original formulation (e.g., another tablet). (What is a generic drug and what is a drug generic name?) Clinical significance of bioequivalence: Variation between different formulations may be of practical significance for drugs with low safety margin (digoxin) or where dosage needs precise control (e.g., oral hypoglycemics, oral anticoagulants or anti-epileptic drugs). It may also be responsible for success or failure of an antimicrobial regimen. Distribution Once the drug is present in the systemic circulation following absorption and 1st pass metabolism, it will be bioavailable to the tissue through the process of distribution. Distribution simply means the movement of a drug from the bloodstream to the various tissues of the body including their target site of action. Since the distribution of drugs to the tissues involves crossing of cellular membranes, the extent of distribution or the compartments in which the drug is distributed will, again, depend upon the factors governing transport across membranes, studied before (Refer to factors governing absorption and transport across membranes) including: Factors related to the tissue itself, and here they can be in the form of 1. The local tissue blood flow, where organs of high blood flow (e.g., liver and the kidney) usually receive larger amount of drug relative to organs with low blood flow (e.g., the skin), where very high doses are required to achieve an effective concentration. Therefore, most of the skin diseases are primarily treated by topical preparations. 2. The presence of special barriers like the blood brain barrier, the blood placental barrier and the blood testicular barrier, which limit the passage of drugs into these tissues unless they have favorable molecular characteristics or a specific transport mechanism. Factors related to the drug itself including: 1. The molecular characteristics of the drug. (How can it affect drug distribution?) 2. The drug affinity to plasma protein: These are large molecular weight protein molecules that carry substances including hormones and other chemicals in the blood. They are of several types including mainly albumin that binds to acidic drugs like aspirin, and alpha acid glycoproteins which bind basic drugs, e.g., propranolol. Drugs bind plasma protein with an equilibrium between bound and free molecules and this binding has the following clinical implications:  It limits their distribution to the tissues as only the free drug fraction (unbound fraction) can cross membranes to tissues and exert a pharmacological action.  It acts as a drug reservoir limiting their elimination as only free fraction can be metabolized or excreted.  It may be a mechanism of drug interaction, where competition between two drugs (or drug and a body chemical) for the plasma protein may result into displacement of one of them transiently increasing its free fraction and thus may increase the risk of adverse effects. This Page 3 of 4  could be of significance with the highly protein bound drugs (>90 %) like the oral antidiabetic drugs belonging to the sulphonylurea (SU).  The total drug concentration may be changed in case of diseases affecting the synthesis of plasma protein as in liver disease or causing loss of plasma protein as in renal disease. 7n Therefore, for monitoring drugs of high risk of toxicity, it is important to measure the free fraction rather than the total drug concentration. The sum of all these factors will finally cause each drug to be distributed into a volume of fluid (compartment) that is different from that of other drugs even if given with the same dose, e.g.  If a drug is of high molecular weight e.g., Heparin, or is highly bound to plasma protein e.g., warfarin, it will be retained into the vascular compartment which is only about 3 L (the plasma volume)  If the drug is polar, but of small molecular weight, it may escape the endothelial lining of the blood vessel and reach the interstitial space, in addition to the plasma, where it will be distributed into the extracellular fluid of about 15 L. e.g., Gentamicin.  If a drug is highly lipid soluble e.g., Propranolol, or is of very small molecular weight e.g., alcohol can cross all cell membranes and pass intracellular, and it can be distributed to the whole-body water which is about 42 L. This difference in distribution of drugs is quantified roughly by what is known as the volume of distribution. The Volume of distribution (Vd) simply means the volume into which a drug appears to be distributed with a concentration equal to that of plasma. In such a case, this volume can be calculated form the ratio of the total dose to the plasma concentration. 𝐷𝑟𝑢𝑔 𝑑𝑜𝑠𝑒 (𝑚𝑔) Vd (L) =𝐷𝑟𝑢𝑔 𝑝𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑧𝑒𝑟𝑜 𝑡𝑖𝑚𝑒 (𝑚𝑔/𝐿) The Vd is a specific property for each drug, and either is expressed as the total volume of distribution per whole body weight or more accurately is expressed as volume /kg body weight. Accordingly, drugs with very high Vd, e.g., digoxin (500 L) are expected to be concentrated mainly intracellular, while those with low volume of distribution, e.g., heparin (5L), are limited to blood. (How can the Vd be larger than the volume of the body water?) N.B., Toxicities of drugs with high Vd are less likely to be treated by hemodialysis. (Why?) Since the Vd of all drugs is a known value as revealed form their pharmacokinetic studies, it can be used for calculation of what is called the loading dose, which is the dose needed to load all the expected tissues within the known Vd to achieve the target plasma concentration as soon as possible. Accordingly, The loading dose = Vd X required plasma concentration. After distribution to the tissues, the drug can exert its pharmacological action, and also can undergo elimination by distribution to the liver and kidney as will be discussed in the further blocks. Page 4 of 4 5 Connective tissue Cells & immune cells ILOs By the end of this lecture, students will be able to 1. Value the significance of stem cell origin of CT cells. 2. Correlate the structure of each cell to its function. 3. Interpret the response of CT cells to physiological & pathological conditions (immune response, hypersensitivity reaction, tissue repair, inflammation) 4. Differentiate types of white blood cells on basis of their origin and histological features. 5. Interpret structural adaptation of immune cells in different medical conditions 6. Interpret stages of lymphocyte maturation & the expression of their receptors What is connective tissue (CT)? Connective tissue, as the name implies, forms a continuum with epithelial tissue, muscle, and nervous tissue, as well as, with other components of connective tissues to maintain a functionally integrated body. ⮚ Origin; Most connective tissues originate from mesoderm, the middle germ layer of the embryonic tissue that contains mesenchymal stem cells (multipotent cells). (Fig. 1) ⮚ Components of the connective tissue; Connective tissue is composed of cells and extracellular matrix consisting of ground substance and fibers. ⮚ Functions of the CT; differ according to the type of CT and its components. Generally, functions include: Providing structural support Serving as a medium for exchange Aiding in the defense and protection of the body Forming a site for storage of fat Classification of CT cells: 1. Fixed cells are a resident population of cells that have developed and remain in place within the connective tissue, where they perform their  functions. The fixed cells are a stable and long-lived population that includes fibroblasts& adipose cells (fat cells). 2. Transient cells (free or wandering cells) originate mainly in the bone marrow and circulate in the bloodstream, where they reach the CT in response to specific signals. These include mast cells and macrophages (the 2 cells may become resident in the CT), plasma cells, Lymphocytes, Neutrophils, Eosinophils, Basophils, monocytes. Structure & function of CT cells: 1. Fibroblasts ⮚ Fibroblasts, the most abundant. ⮚ Function; responsible for synthesis and secretion of extracellular components (fibers & ground substance molecules). ⮚ Functional forms: it may be active fibroblasts with protein synthesis machine, especially when the cell is actively manufacturing matrix, as in wound healing. (Fig 2a) ⮚ Inactive fibroblasts (fibrocytes) are smaller and more ovoid with less organelles. ⮚ Myofibroblasts represent transitional modifications of fibroblasts responding to signalling molecules within a regional intercellular matrix, as in areas undergoing wound healing. They contain actin and myosin filaments thus resembling features of a smooth muscle. Role of myofibroblasts in health and disease Early in process of wound healing, myofibroblasts produce a contractile action to reduce the size of the wound. If the injurious stimulus persists, continuous stimulation of myofibroblasts results in excessive collagen deposition by myofibroblasts with subsequent fibrosis, for example in chronic hepatitis (liver inflammation) and complicated lung infection. 2. Adipose cells ⮚ Adipose cells are fully differentiated and do not undergo cell division. ⮚ They function in the synthesis and storage of triglycerides. ⮚ Types; unilocular fat cells: Cells with a single, large lipid droplet and form white adipose tissue, and multilocular fat cells with multiple, small lipid droplets and form brown adipose tissue. (Fig 2b & c) ⮚ Unilocular fat cells continuously store fat in the form of a single droplet, which enlarges so much that the cytoplasm and nucleus are displaced peripherally against the plasma membrane, thus giving these cells a "signet ring" profile when viewed by light microscopy.  ⮚ Multilocular fat cells are smaller and more polygonal than white fat cells. Multilocular fat cells contain many more mitochondria than unilocular fat cells. 3. Mast cells ⮚ Mast cells are ovoid and possess a centrally placed, spherical nucleus. ⮚ They are characterized by abundant secretory granules that contain different chemical mediators, such as, heparin (Anticoagulant), histamine & Leukotrienes (vasodilators and smooth muscle contractors of bronchi, interleukins (IL-4, IL-5, IL- 6), and tumour necrosis factor-alpha (TNF-α) (enhances the inflammatory response). (Fig 2d) ⮚ Function: They function in the immune system by initiating an inflammatory response known as the immediate hypersensitivity reaction (whose systemic form, known as an anaphylactic reaction, may have lethal consequences). This response commonly is induced by foreign proteins (antigens) such as bee venom, pollen, and certain drugs. (It will be discussed in detail in the following module). Clinical hint Victims of hay fever attacks suffer from the effects of histamine being released by the mast cells of the nasal mucosa, results in feeling "stuffed up" and hinders breathing. (Why?)  Victims of asthma attacks suffer from extra-release of leukotrienes in the lungs and difficulty in breathing (why?). Degranulation of mast cells usually is a localized phenomenon; the typical inflammatory response is mild and site-specific. However, a risk also exists for hyperallergic persons who may experience a systemic and severe immediate hypersensitivity reaction (systemic anaphylaxis) following a secondary exposure to an allergen (e.g., insect stings, antibiotics). (What are the expected signs? and Why?) 4. Macrophages ⮚ Macrophages arise from circulating monocytes, and they are the most abundant phagocytic cells in connective tissue. ⮚ They function in removing cellular debris and in protecting the body against foreign invaders. ⮚ Structure: large cells with irregular surface that show blunt projections or filopodia reflecting cell motility and abundance of lysosomes reflecting active phagocytosis. (Fig 3a) 5. Plasma cells ⮚ Plasma cells are derived from B lymphocytes and manufacture antibodies. ⮚ Their cytoplasm is intensely basophilic (Why?) except in the prenuclear area which appear pale and referred to as a negative Golgi image. (Fig 3 b& c). IMMUNE CELLS Leukocytes (white blood cells) All leukocytes originate in the bone marrow. Leukocytes do not function within the bloodstream but use it as a means of traveling from one region of the body to another. they leave the blood stream by migrating between the endothelial cells of the blood vessels (diapedesis), enter the connective tissue spaces and perform their specific function. (Fig.4) Classification of white blood cells I- Granulocytes, which have both specific granules and non-specific granules (lysosomes) in their cytoplasm 1. Neutrophils 2. Eosinophils 3. Basophils II- Non-granulocytes; which lack specific granules and contain only non-specific granules (lysosomes) 1. Lymphocytes 2. Monocytes. Note: the suffix “ilia” when add to any blood cells means increase in the number of this cells Ex; Neutrophilia means increase in the number of Neutrophils 1. Neutrophils Polymorphonuclear leukocytes (polys, neutrophils) are the most numerous of the white blood cells, constituting 60% to 70% of the total leukocyte population. It is recognized in a blood film by its multilobed nucleus, where lobes are interconnected by chromatin. It has a pale cytoplasm. In females, the nucleus presents a characteristic small appendage. the "drumstick," which contains the condensed, inactive second X chromosome, called the Barr body, which is used for sex identification. (Fig. 5A) Functions They are first line of defence against bacterial infection. They phagocytose and destroy bacteria by the content of their granules. Neutrophils can also migrate through blood capillary wall to reach connective tissue sites where there is bacterial infection to act in phagocytosis. After performing their function, cells die and form pus cells. 2. Eosinophils They are characterized by a bilobed nucleus and eosinophilic granules in cytoplasm. (Fig 5B) Function: Eosinophils phagocytose antigen-antibody complexes and kill parasitic invaders Clinical hint: Connective tissue cells in the vicinity of antigen-antibody complexes release the pharmacological agents histamine and IL-5, causing increased formation and release of eosinophils from the bone marrow. In contrast, elevation of blood corticosteroid levels depresses the number of eosinophils in circulation. 3. Basophils They are characterized by S-shaped nucleus and abundant basophilic granules in cytoplasm. (Fig 5C) Function: They mediate allergic and inflammatory reaction through release of granules content, such as histamine and heparin. Clinical hint In certain hyperallergic individuals, a second exposure to the same allergen may result in an intense generalized response. A large number of basophils (and mast cells) degranulate, resulting in widespread vasodilation and sweeping reduction in blood volume (because of vessel leakiness). Thus, the person goes into circulatory shock. The smooth muscles of the bronchial tree constrict, causing respiratory insufficiency. The combined effect is a life-threatening condition known as anaphylactic shock. 4. Monocytes They are the largest white blood cells in a blood smear and recognized by a large, eccentric and kidney shaped nucleus. It has irregular surface with projection from the cell membrane(filopodia). They possess abundant lysosomes. Monocytes migrate into the connective tissue and become named macrophages. They belong to the mononuclear phagocytic system. They phagocytose and destroy dead and defunct cells (such as senescent erythrocytes) as well as antigens and foreign particulate matter (such as bacteria). The destruction occurs within the phagosomes. Functions Macrophages phagocytose unwanted particular matter. Produce cytokines that are required for the inflammatory and immune responses. Present epitopes (processed foreign antigens) to T lymphocytes, thus acting as antigen presenting cells to elicit an immune response. 5. Lymphocytes They are rounded cells that have a slightly indented, round nucleus that occupies most of the cell. Lymphocytes are subdivided into three functional categories: 1. B lymphocytes (B cells) 2. T lymphocytes (T cells) 3. Null cells The three types are similar morphologically. They are differentiated on bases of their surface receptors. Approximately 80% of the circulating lymphocytes are T cells, about 15% are B cells, and the remainder are null cells. Their life spans also  differ widely: some T cells may live for years, whereas some B cells may die in a few months. Lymphocytes have no function in the bloodstream, but in the connective tissue these cells are responsible for the proper functioning of the immune system To be immunologically competent, they migrate to specific body compartments to mature and to express specific surface markers and receptors. B- cells mature leave the bone marrow as mature cells, while T-cells migrate to the thymus where they become mature and immunocompetent. Once they have become immunologically competent, lymphocytes leave their respective sites of maturation, enter the lymphoid system, and undergo mitosis, forming a group of identical cells, known as a clone. All members of a particular clone can recognize and respond to the same antigen. After stimulation by a specific antigen, both B and T cells proliferate and differentiate into two subpopulations: Memory cells do not participate in the immune response but remain as part of the clone with an "immunological memory," ready to undergo cell division and mount a response against a subsequent exposure to a particular antigen or foreign substance. Effector cells are classified as B cells and T cells (and their subtypes).  6 Lecture Classification of C.T proper & Embryonic ILOs By the end of this lecture, students will be able to 1. Deduce significance of functional role of CT components in its distribution in the human body. 2. Correlate structural components of each type of CT to its function. 3. Interpret some medical problems in relevance to CT features. 4. Value the role of embryonic CT as a potential source of mesenchymal stem cells. Classification of connective tissue I- Embryonic CT that includes: a) Mesenchymal CT b) Mucous CT II- Connective tissue proper that includes: a) Loose areolar b) Dense CT; regular & irregular c) Reticular d) Elastic III- Special types: a) Cartilage b) Bone c) Blood I- Embryonic CT a- Mesenchymal CT ⮚ Mesenchymal connective tissue is present only in the embryo and consists of mesenchymal cells in a gel-like, amorphous ground substance containing scattered reticular fibers. (Fig 1a) ⮚ Mitotic figures frequently are observed in mesenchymal cells. (Why?) ⮚ In adults it is present in the pulp of teeth. b- Mucous tissue is a loose, amorphous connective tissue exhibiting a jelly-like matrix primarily composed of hyaluronic acid and sparsely populated with type I and type III collagen fibers and fibroblasts. This tissue, also known as Wharton's jelly, is found in the umbilical cord. (Fig 1b)  Figure 1. Embryonic CT Mesenchymal b. Mucous type type II- Connective Tissue Proper a) Loose areolar connective tissue ⮚ It is formed of a loose arrangement of fibers and dispersed cells embedded in a gel-like ground substance. (Fig 1a) ⮚ Sites; Under the epithelium, where it is called lamina propria in mucous membranes such as in the alimentary tract or dermis in the skin. It also surrounds the blood vessels. (Fig 1b) ⮚ Components; Loose connective tissue is characterized by abundant ground substance and tissue fluid (extracellular fluid) housing the fixed connective tissue cells and all types of fibers. ⮚ Structural correlation to site and function; this tissue lies immediately beneath the thin epithelia of the digestive and respiratory tracts, this is where the body first attacks antigens, bacteria, and other foreign invaders. Therefore, loose connective tissue contains many transient cells responsible for inflammation, allergic reactions, and the immune response. (refer to CT cells) ⮚ Clinical Hint: Under normal circumstances, extracellular fluid returns to the blood capillaries or enters lymph vessels to be returned to the blood. A potent and prolonged inflammatory response, however, causes accumulation of excess tissue fluid within loose connective tissue beyond what can be returned via the capillaries and lymph vessels, causing a clinical condition called oedema.  Figure 2. Structure of loose CT a b b) Dense CT ⮚ Dense connective tissue contains the same components of loose CT but with more fibers and fewer cells than loose connective tissue. ⮚ Dense connective tissue is less flexible and far more resistant to stress than the loose type. ⮚ According to orientation and arrangement of collagen fibers, dense CT is further subdivided to: 1- Regular type with regularly arranged collagen bundles that have regular orientation. It includes dense regular & elastic types. In the dense regular type, collagen bundles are densely packed and oriented into parallel cylinders or sheets that resist tensile forces. (Fig 2a) Sites; It is present in tendons of muscle and ligaments of joints. Thin, sheet-like fibroblasts are located between bundles of collagen with their long axes parallel to the bundles. In the elastic CT Type, coarse branching elastic fibers with only a few collagen fibers forming networks. The elastic fibers are arranged parallel to one another and form either thin sheets or fenestrated membranes. Sites; It is present in the wall of large arteries and some ligaments of the vertebral column. 2- Dense Irregular type with randomly oriented collagen bundles, such as in dermis of skin and in capsule covering solid organs as the kidney and lymph nodes. (Fig 2b) It has a limited amount of ground substance.  Fibroblasts are the most abundant cells. Figure 3. Dense CT b. Irregular Regular c) Reticular CT Type III collagen is the major fiber component of reticular tissue. The collagen fibers form mesh-like networks interspersed with fibroblasts and macrophages. Reticular tissue forms the architectural framework (fine stroma) of adipose tissue, bone marrow, lymph nodes, and spleen. Figure 4. Elastic & Reticular CT  Elastic CT b. Reticular CT 7 Structure & Chemistry of C.T. Ground Substance: ILOs By the end of this lecture, students will be able to 1. Interpret general features of CT in relevance to its components 2. Describe glycosaminoglycans and proteoglycans of importance in ground substance function 3. Correlate the structure of the components of ground substance to their function 4. Deduce the effects of abnormal degradation of glycosaminoglycans The 'ground substance' is an amorphous gelatinous material. It is transparent and fills the spaces between fibres and cells. It actually consists of large molecules called glycosoaminoglycans (GAGs) which link together to form even larger molecules called proteoglycans. ❖ What are Glycosaminoglycans? Glycosaminoglycans (GAG) are large complexes of negatively charged heteropolysaccharide chains. They are generally associated with a small amount of protein (core protein), forming proteoglycans, which typically consist of up to 95% carbohydrate. GAGs have the special ability to bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body’s ground substance. The hydrated GAG serve as a flexible support for the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of GAG, which led to the original naming of these compounds as mucopolysaccharides. ❖ What is their structure? GAG are long, unbranched, heteropolysaccharide chains composed of a repeating disaccharide unit [acidic sugar– amino sugar]n. The acidic sugar as D-glucuronic acid. While in amino sugar is either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated. The amino sugar may also be sulfated. The source of those sulfates is 3´- phosphoadenosyl-5´-phosphosulfate [PAPS]. Figure 1: Structure of GAGs The sulfate groups together with the presence of the acidic sugar, give GAG their strongly negative nature. ❖ What is its Structure–function relationship? Because of the high concentration of negative charges, these heteropolysaccharide chains tend to be extended in solution. They repel each other and are surrounded by a shell of water molecules. When brought together, they slide past each other, much as two magnets with the same polarity seem to slide past each other. This produces the slippery consistency of mucous secretions and synovial fluid. Page 1 of 3  When a solution of GAG is compressed, the water is squeezed out, and the GAG are forced to occupy a smaller volume. When the compression is released, the GAG spring back to their original, hydrated volume because of the repulsion of their negative charges. This property contributes to the resilience of synovial fluid and the vitreous humor of the eye. The specific GAGs of physiological significance include: o Hyaluronic acid (also called hyaluronan)(HA): - It is unique among the GAGs in that it does not contain any sulfate and is not found covalently attached to proteins forming a proteoglycan. It is, however, a component of non-covalently formed complexes with proteoglycans in the ECM. (see proteoglycans below) - Hyaluronic acid polymers are the largest polysaccharides produced by humans and can displace a large volume of water. HA is ubiquitous in body tissues and is best-known for its capability of attracting water molecules. HA is capable of binding 10000 times its own weight in water. - Due to this characteristic, it plays a key role in lubrication of synovial joints and wound healing processes. HA is also used exogenously by clinicians for promotion of tissue regeneration and skin repair. It is used in a variety of cosmetic products and shows promising efficacy in promoting skin tightness, elasticity, and improving aesthetic scores. o Chondroitin sulfate: - It is the most abundant GAG in the body, principally associated with protein to form proteoglycans. The sulfation of chondroitin sulfates occurs on several positions. - They are found in cartilage, tendons, ligaments and aorta. - One of the leading causes of Osteoarthritis relates to loss of chondroitin sulfate from articular cartilage in joints, leading to inflammation and catabolism of cartilage. o Heparin: - Unlike other GAGs that are extracellular compounds, heparin is intracellular component of mast cells, lining the arteries of the lungs, liver and skin. - It is highly sulfated; clinically useful as an injectable anticoagulant. Heparin does not break down clots directly, but enhances the body’s natural clot lysis mechanisms. o Keratan sulfate: - Present in Cornea, bone, cartilage aggregated with chondroitin sulfates, usually associated with protein forming proteoglycans. - The cornea comprises the richest known source of keratan sulfate in the body. Its role in the cornea includes regulation of collagen fibril spacing that is essential for optical clarity, as well as optimization of corneal hydration based on its interaction with water molecules. - Keratan sulfate has also been shown to play an important regulatory role in the development of neural tissue and might have key roles in promotion of axonal repair following injury. Page 2 of 3  ❖ Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. A proteoglycan monomer consists of a core protein to which up to 100 linear chains of GAG are covalently attached. These chains, which may each be composed of up to 200 disaccharide units, extend out from the core protein and remain separated from each other because of charge repulsion. The resulting structure resembles a bottle brush. Figure 2: Proteoglycan structure In cartilage proteoglycans, the species of GAG include chondroitin sulfate and keratan sulfate. GAG–protein linkage: This covalent linkage is most commonly through a (galactose-galactose-xylose) and a serine residue in the protein. An O-glycosidic bond is formed between the xylose and the hydroxyl group of the serine. Aggregate formation: Many proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent and occurs primarily through ionic interactions between the Figure 3: GAG protein linkage core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins. These aggregates are the major structural component of the ECM of the cartilage providing the cartilage with unique gel-like property and resistance to deformation through water absorption. ❖ Degradation: GAGs are degraded in lysosomes, which contain hydrolytic enzymes that are most active at a pH of ~5. Therefore, as a group, these enzymes are called acid hydrolases. The low pH optimum is a protective mechanism that prevents the enzymes from destroying the cell in the cytosol where the pH is neutral. Mucopolysaccharidoses: - The mucopolysaccharidoses are hereditary diseases caused by a deficiency of any one of the lysosomal hydrolases. They are progressive disorders characterized by lysosomal accumulation of GAG in various tissues, causing a range of symptoms, such as skeletal and ECM deformities, and intellectual disability. - Incomplete lysosomal degradation of GAG results in the presence of oligosaccharides in the urine. These fragments can be used to diagnose mucopolysaccharidosis. Diagnosis is confirmed by measuring the patient’s cellular level of the lysosomal hydrolases. - There currently is no cure. Bone marrow and cord blood transplants, in which transplanted macrophages produce the enzymes that degrade GAG, have been used to treat mucopolysaccharidoses , with limited success. Page 3 of 3 8 Structure & Chemistry of C.T. Fibers ILOs By the end of this lecture, students will be able to 1. Describe distribution and function of CT fibers. 2. Explain synthesis and degradation of collagen. 3. Deduce the structure-function relationship of Collagen and Elastin 4. Interpret how biochemical defects in collagen and elastin can impair morphology. Types of CT fibers: Collagen and elastic fibers, the two major fibrous proteins of connective tissue, have distinctive biochemical and mechanical properties as a consequence of their structural characteristics. I- Collagen (Structure and Function) Collagen is the most abundant protein in the human body representing about 25% of the protein in the body. It is found in tissues with tensile strength and rigidity, where collagen resists tensile forces (stretching or pulling forces) thus providing rigidity to the connective tissue. It is a long triple helix of peptide chains, known as α-chains. Each individual collagen polypeptide is an α-chain of about 1400 residues. Each α-chain is coded by a separate messenger ribonucleic acid (mRNA). Every third amino acid is glycine (-Gly-X-Y-) with a very high proportion of proline and lysine in the other two positions. Proline and glycine, are both important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each α chain because its ring structure causes “kinks” in the peptide chain. Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces Figure 1: Structure of Collagen where the three chains of the helix come together. Many proline and lysine residues are hydroxylated to hydroxyproline and hydroxylysine after synthesis of the α-chain (post-translational modification). The extent of lysine hydroxylation of collagen is highly variable in comparison with proline hydroxylation. Hydroxyproline residues are crucial for collagen folding and stability. Approximately 50% of proline residues in collagen are hydroxylated in various types of collagen in different tissues, and it does not significantly change under physiological conditions. Page 1 of 5  The extent of lysine hydroxylation, however, can vary from 15 to 90% depending on the collagen types and, even within the same type, it varies significantly from tissue to tissue and under the physiological/pathological condition of the tissue. Synthesis and Degradation: (Figure5) - Collagen will spontaneously assemble into fibrils. To avoid premature assembly of fibers within the cell, precursor forms are first synthesized. - The α-chain (preprocollagen) is first targeted to the endoplasmic reticulum (ER) with a signal sequence that is immediately removed in the ER. - Selected proline and lysine residues are then hydroxylated in the ER to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, ferrous iron (Fe2+), and the reducing agent vitamin C (ascorbic acid), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function. - The pro-α-chains spontaneously assemble into procollagen triple helices within the ER. The resulting molecule has propeptide extensions on both ends (carboxy and amino ends), still preventing spontaneous assembly into collagen fibrils. - The procollagen is translocated from the ER into the Golgi apparatus and packaged in secretory vesicles. It is then secreted into the extracellular matrix by exocytosis (fusion with the plasma membrane), and procollagen peptidases remove the propeptide ends - Procollagen then forms units called tropocollagen, which spontaneously assemble into collagen fibrils. Figure 5: Collagen Synthesis Page 2 of 5 - Collagen fibrils are packed regularly to form bundles that appear white glistening in the fresh state. When stained with hematoxylin and eosin, they appear as long, wavy, pink fiber bundles. (Fig 6) - The collagen fibrils are strengthened further by crosslinking between adjacent lysine side chains by the enzyme lysyl oxidase. This is a slow, continuous process throughout an individual’s life. Figure 6: Collagen H&E stained - Collagen can be remodeled by with metalloproteinases. - The action of these digestive enzymes degrade collagen and is balanced by a tissue inhibitor of metalloproteinases (TIMP). - Cross-linking permits scar tissue to strengthen long after a wound has healed, but it also contributes to the decline in collagen quality with aging causing collagen to stiffen, contributing to the decline in vascular elasticity, weakening of the cartilage and the visible signs on the skin, which becomes less firm and supple with age. Clinical Implications: - In the case of ascorbic acid deficiency (and, therefore, a lack of proline and lysine hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked, greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses (bruise-like discolorations) on the limbs as a result of subcutaneous extravasation (leakage) of blood due to capillary fragility - Collagenopathies: Defects in any one of the many steps in collagen fiber synthesis 1-Ehlers-Danlos syndrome:(EDS) is a connective tissue disorder caused by a deficiency of collagen- processing enzymes (as lysyl hydroxylase).The classic form of EDS, is characterized by skin extensibility and joint hypermobility. The vascular form, is the most serious form because it is associated with potentially lethal arterial rupture. 2- Osteogenesis imperfecta: This syndrome, known as “brittle bone disease,” is a genetic disorder characterized by bones that fracture easily, with minor or no trauma. The most common mutations cause the replacement of glycine by amino acids with bulky side chains. The resultant structurally abnormal α chains prevent the formation of the required triple-helical conformation. Phenotypic severity ranges from mild to lethal. Elastic fibers In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue fibrous protein with rubber-like properties. Elastic fibers provide elasticity to the connective tissue, thus most abundant in areas of the body subjected to volume or pressure changes as lungs, the walls of large arteries, and elastic ligaments. They are highly accommodating and may be stretched one and a half Page 3 of 5  times their resting length without breaking. When the force is released, elastic fibers return to their resting length. It is rich in proline and lysine but contains scant hydroxyproline and hydroxylysine. Structural organization; These fibers are usually slender, long, and branching in loose connective tissue (Fig 7A) or form coarser bundles in ligaments and fenestrated sheets (Fig 7B) (discussed later in types of CT). The core of elastic fibers is composed of elastin and is surrounded by a sheath of microfibrils; each microfibril is about 10 nm in diameter and is composed of the glycoprotein fibrillin. During the formation of elastic fibers, the microfibrils are elaborated first, and the elastin is then deposited in the space surrounded by the microfibrils. Elastin is an extremely durable and does not turn over appreciably in healthy tissue. It is estimated to have a half-life of about 70 years. However our neutrophils secrete elastase enzyme a powerful protease that is released into the extracellular space and degrades elastin of alveolar walls as well as other structural proteins in a variety of tissues. To counteract this enzyme the liver secretes α1-antitrypsin (AAT), which inhibits a number of proteolytic enzymes including elastase Patients with inherited defects in α1-antitrypsin are at serious risk for emphysema; intravenous administration of α1-antitrypsin is an effective treatment. Smoking also can cause emphysema, since a methionine residue in α1-antitrypsin essential for binding to elastase is vulnerable to oxidization by cigarette smoke. Clinical hint: The integrity of elastic fibers depends on the presence of microfibrils. Patients with Marfan syndrome have a defect in the gene on chromosome 15 that codes for fibrillin; therefore, their elastic fibers do not develop normally. Typically, patients present with tall stature and aortic root dilatation. People who are severely affected with this condition are predisposed to fatal rupture of the aorta. Figure 7. Elastic fibers A B Page 4 of 5 Page 5 of 5 L9 Adipose CT and energy storage ILOs By the end of this lecture, students will be able to 1. Compare between white & brown adipose CT structurally & functionally. 2. Interpret ultra-structural adaptation of unilocular & multilocular adipocyte. 3. Describe process of fat storage in adipose tissue 4. Interpret adipose CT changes in normal and disturbed lipid metabolism. ⮚ Adipose tissue is classified into two types according to whether it is composed of unilocular or multilocular adipocytes. I- Unilocular (white) adipose CT Components: Unilocular adipocytes; contain a single large lipid droplet that occupy most of the cytoplasm. (Fig 1) Ultra-structurally, contain well developed smooth endoplasmic reticulum and Golgi complex (Why?). The plasma membranes of the unilocular adipose cells contain receptors for several substances, including insulin, growth hormone, norepinephrine, and glucocorticoids, that facilitate the uptake and release of free fatty acids and glycerol. Reticular fibers that support the cells. Limited amount of ground substance. It is a highly vascular tissue (Why?). Fig.1 White adipose tissue  Fig 2. Sites of white adipose CT Subcutaneous layer Sites In the deep layer under the skin (subcutaneous). Distribution of adipose tissue is variable between males and females. In males, mainly abdominal, while in females more in breasts and buttocks. Around vital organs such as the heart and kidney to act as shock absorbent. Multilocular(brown) adipose CT Components Brown adipose tissue (brown fat) is composed of multilocular fat cells, which store fat in multiple droplets. This tissue may appear tan to reddish brown because of its extensive vascularity and the cytochromes present in its abundant mitochondria. Unmyelinated nerve fibers enter the tissue, with the axons ending on the blood vessels as well as on fat cells, whereas in white fat tissue, the neurons end only on the blood vessels. Functions 1. Brown adipose tissue is associated with production of body heat because of the large number of mitochondria in the multilocular adipocytes composing this tissue. These cells can oxidize fatty acids at up to 20 times the rate of white fat, increasing body heat production three-fold in cold environments 2. Sensory receptors in the skin send signals to the temperature-regulating center of the brain, resulting in the relaying of sympathetic nerve impulses directly to the brown fat cells. The neurotransmitter  norepinephrine activates the enzyme that cleaves triglycerides into fatty acids and glycerol, initiating heat production by oxidation of fatty acids in the mitochondria. Fig 3. Brown adipose CT Sites: It is the only type in the in the new born, where it is located in the neck region and in the interscapular region. Brown adipose tissue transforms into white adipose tissue with age advancement. Clinical correlation: Since brown adipocytes contain an extremely high number of mitochondria (where the respiratory chain complexes are anchored). Therefore, when heat is required (exposure to the cold, for example), norepinephrine released by sympathetic nerves rapidly activates brown adipocytes resulting in fatty acid oxidation. Due to the presence of uncoupler protein in the brown adipose tissue the oxidation energy produced is lost as heat (thermogenesis) instead of fat synthesis. -In adults, obesity develops in two ways. Hypertrophic obesity results from the accumulation and storage of fat in unilocular fat cells, which may increase their size by as much as four times. Hypercellular obesity, as the name implies, results from an overabundance of adipocytes. This type of obesity usually is severe. There also appears to be a genetic basis in some cases of obesity. Mutations in the gene responsible for the coding for leptin produces an inactive form of that hormone. ❖ How are fatty acids mobilized from adipose tissue? When the energy supply from dietary carbohydrates is limited, the body responds to this deficiency by mobilization of stored fat (Lipolysis), this requires the hydrolytic release of FFA and glycerol from their TAG form. It is initiated by adipose triglyceride lipase (ATGL), which generates a diacylglycerol that is the preferred substrate for hormone sensitive lipase (HSL). The monoacylglycerol (MAG) product of HSL is acted upon by MAG lipase. These reactions result in 3 free fatty acids and glycerol  The free fatty acids (FFA) produced by lipolysis move through the plasma membranes of the adipose cells and endothelial cells of blood capillaries by simple diffusion and bind to albumin in the blood plasma, which are transported to peripheral tissues where it might undergo oxidation to produce energy (Discussed later). The glycerol produced is taken up by liver, phosphorylated and oxidized to dihydroxyacetone phosphate, which is isomerised to glyceraldehydes-3-phosphate, an intermediate of both glycolysis and gluconeogenesis. Therefore, the glycerol is either converted to glucose (gluconeogenesis) or to pyruvate (glycolysis). Regulation of lipolysis: In the fed state: Insulin inhibits lipolysis by converting HSL to its inactive dephosphorylated form. In the fasting state: Primarily epinephrine and to a little extent glucagon hormones activate lipolysis by converting HSL to its active phosphorylated form. ❖ The Biosynthesis of Fatty Acids Apart from diet fatty acids can be synthesized in the body. De novo fatty acid synthesis occurs primarily in the liver and lactating mammary glands and, to a lesser extent, in adipose tissue. It is a cytosolic process that takes place by the action of a single multifunctional enzyme complex called “fatty acid synthase” that contains 7 enzyme activities. It adds two carbons by two to the elongating chain. Its function stops upon formation of palmitate (C16). The carbons incorporated to the growing fatty acid chain are from acetyl Co A (source of carbon), for this process to occur it uses ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH) (source of Hydrogen) The Acetyl CoA is produced in the mitochondria by PDH while the FA synthesis occurs in the cytoplasm, Citrate shuttle is responsible for transport of acetyl COA from mitochondria to the cytoplasm Acetyl-CoA needs to be activated to malonyl- CoA to be used as the elongating unit during fatty acid synthesis. This occurs by CO2 fixation, catalyzed by the enzyme Acetyl-CoA carboxylase, this is the committed step for FA synthesis.  The free palmitate, the end product of denovo synthesis of fatty acids, must be activated to palmitoyl CoA before it can proceed through any metabolic pathway. Its usual fate is esterification to form TAG. Elongation (after formation of palmitate) requires a system of separate enzymes rather than a multifunctional enzyme. Malonyl CoA is the two carbon donor, and NADPH supplies the electrons. The brain has additional elongation capabilities, allowing it to produce the very-long-chain fatty acids ([VLCFA] over 22 carbons) that are required for synthesis of brain lipids. REGULATION OF FATTY ACID SYNTHESIS: - Through the regulation of acetyl CoA carboxylase. o Hormonal Regulation: Insulin: (in fed state) - Activates acetyl CoA carboxylase by Dephosphorylation. - It induce the synthesis of acetyl COA carboxylase Glucagon and catecholamines: (During fasting) - Inhibit acetyl CoA carboxylase by phosphorylation. - It repress the synthesis of acetyl COA carboxylase o Allosteric regulation: - Acyl CoA (palmitoyl CoA): Is an allosteric inhibitor of acetyl CoA carboxylase. It also inhibits the transport of citrate from mitochondria to the cytosol. - Citrate is an allosteric activator of the enzyme Biosynthesis of Triacylglycerols The starting point is glycerol-3-(P) which is formed either by reduction of dihydroxy acetone phosphate particularly in adipose tissue or by glycerol kinase occurs in liver, intestine, kidney and lactating mammary gland  10 ANTIGENS AND ANTIBODIES ILOs By the end of this lecture, students will be able to 1. Differentiate between super-antigens and classical antigens. 2. Analyse the impact of super-antigens and classical antigens on the immune system. 3. Interpret the relationship between the biochemical structure and the function of each immunoglobulin class. 4. Depict the role of antibody mediated mechanisms in host defence. 5. Relate the monoclonal antibody production to their diagnostic &therapeutic uses. ANTIGENS (Ags) Antigens are immunogens that react with specific receptors on B cells or T cells and stimulates a specific immune response. For a substance to be immunogenic, it must be recognized by the body as being foreign or non-self. The most potent immunogens are proteins with high molecular weight. Polysaccharides, lipopolysaccharide, lipoproteins, nucleoproteins are antigenic. Haptens: hapten is a molecule that is not immunogenic by itself. Haptens can be small molecules, nucleic acids, lipids or drugs (e.g., penicillin) Although haptens cannot stimulate a primary adaptive response by themselves, they can do so when bound to a carrier protein. Many haptens bind to our normal proteins to which we are tolerant and modify these proteins. The hapten protein combination now becomes immunogenic. This strategy is used when designing conjugate vaccine in which weak immunogen is conjugated to a strong peptide antigen. Superantigens: Super antigens are viral and bacterial proteins that cross link the variable ß-domain of a T cell receptor to the major histocompatibility complex «MHC» class II of antigen-presenting cells outside the normal peptide binding groove. This cross-linkage provides an activating signal that induces T-cell activation and proliferation, in the absence of antigen specific recognition of peptides. Because superantigens binds outside of the antigen binding cleft, they activate any clones of T cells expressing a particular variable ß sequence and thus cause polyclonal activation of T cells resulting in the overproduction of proinflammatory cytokines. Excessive amounts of these cytokines induce systemic toxicity. Page 1 of 5 Molecules produced during the infectious process and known to act as superantigens include staphylococcal enterotoxins, toxic shock syndrome toxin, exfoliative dermatitis toxin and streptococcal pyrogenic exotoxins. (Figure 1) Figure 1: Differences between Antigen and Superantigen. ANTIBODIES Antibodies or immunoglobulins are glycoproteins expressed as: - Soluble molecules present in serum and tissue fluids function to protect against microbes - Membrane bound antibodies (IgM, IgD ) on the surface of B cells function as antigen receptor Antibodies provide protection against extracellular pathogens in the blood, mucosal surfaces and tissues. Immunoglobulin Classes: Immunoglobulin G (IgG) the major class of immunoglobulin present in serum. Representing approximately 75% of serum antibodies in humans. It consists of two L chains and two H chains (H2L2) held by disulphide bridges. The L and H chains of an Ig molecule are subdivided into variable regions and constant regions. An L chain is composed of one variable domain (VL) and one constant domain (CL) whereas most H chains have one variable domain (VH) and three or more constant domains (CH). Each domain is approximately 110 amino acids in length. The variable regions of the Ig molecule are involved in antigen binding, whereas the constant regions are responsible for the biologic functions. IgG exists in four subclasses IgG1, IgG2, IgG3 and IgG4. Ø The protective role of IgG is carried out by: Page 2 of 5  Activates complement leading to cell lysis. Acts as an opsonin, enhancing phagocytosis Neutralises pathogens (e.g., block viral binding sites) and toxins. Mediates antibody dependent cell mediated cytotoxicity: The lytic destruction of infected or tumour cells is mediated by a killer cell (NK, macrophage, neutrophil) that binds to the Fc portion of the bound antibody. (Figure 2) Figure 2: Antibody dependent cell mediated cytotoxicity IgG is the only antibody cross the placenta and plays a crucial role in the protection of new-borns. IgM comprises about 7% of Igs in normal human serum. It consists of 5 units, joined together by the J-chain, each unit is similar in structure to one IgG (2 L and 2 H chains). So, IgM is formed of 10 L and 10 H chains. It has the highest molecular weight of Igs (pentamer). IgM is the Ig which appears early in the specific immune response. It can neutralize pathogens and it is the most effective antibody at immobilizing antigen (agglutination) and in activating the classical pathway of complement. IgA is the major immunoglobulin responsible for mucosal immunity. The level of IgA in the serum are low, consisting of only 10-15% of total serum immunoglobulins. In contrast, IgA is the predominate class of immunoglobulin found in mucous membranes and in body secretions such as, mucus,saliva, and tears, and in breast milk,other secretions of the respiratory, intestinal, and genital tracts. In serum, IgA is secreted as a monomer. In mucous secretions, IgA is a dimer and is referred to as secretory IgA. This secretory IgA consists of two monomers that contain two additional polypeptides: the J chain that stabilizes the molecule and a secretory component that is incorporated into the secretory IgA when it is transported through an epithelial cell. The main function of secretory IgA is probably to prevent the attachment of microbial pathogens to mucosal surfaces. Page 3 of 5 IgD: Found in a small concentration in the serum. IgD is found mainly on the surface of B- lymphocytes. These B cells contain IgD and IgM at a ratio of 3 to1.The function of IgD is unclear. IgE: Is also called cytotropic Ab. It is found in trace amounts in normal serum. IgE binds directly to Fc Receptors present on mast cells ,eosinophils and is involved in elicitation of protective immune responses against parasites and allergens. (Figure 3) Figure 3: Structure of Antibodies Immunoglobulin class switching Initially, all B cells bound to an antigen carry IgM specific for that antigen and produce IgM in response to this antigen. Later, gene rearrangements generate antibodies of the same antigen specificity but of different immunoglobulin classes, so that the immunoglobulin produced later (IgG, IgA or IgE) has the same specificity as the original IgM but with different biologic characteristics. Class switching is dependent on cytokines released from T cells. Monoclonal Antibodies These are highly specific antibodies produced against a single epitope. They are obtained by fusion of a myeloma cell (malignant plasma cell) with a B cell producing antibody against a single epitope (derived from the spleen of mice immunized with this epitope). The resulting fused cell is called hybridoma cell, this cell has the ability to produce unlimited quantities of highly specific monoclonal antibody. This is the murine type. Another humanized one is produced by genetic engineering (recombinant DNA technology). (Figure 4) The monoclonal antibodies have several diagnostic and therapeutic uses in the medical field Diagnostic as in lymphocyte subsets determination, HLA typing (by flow cytometry) and serological tests as ELISA, chemiluminescence and immunofluorescence. Therapeutic as anti-tumour therapy, antiallergic, and immunosuppressive therapy to prevent graft rejection and in the treatment of viral infections and autoimmune diseases. Page 4 of 5 Figure 4: Production of monoclonal antibodies Page 5 of 5  11 The Complement system, major histocompatibility complex & Cytokines ILOs By the end of this lecture, students will be able to 1. Interpret the role of the complement as an effector mechanism of both innate and adaptive immunity. 2. Correlate the biological functions of complement activation with its components. 3. Compare between MHC Class I and MHC class II antigens regarding its types and role in adaptive immunity. 4. Interpret the role of MHC polymorphism in paternity testing, disease association and organ transplantation. 5. Interpret the importance of various cytokines in the immune and inflammatory responses. 6. Explain the biologic effects of interferons to control viral infection. 7. Relate the clinical uses of cytokines as antimicrobial, immunomodulatory & antiproliferative factors. The complement system The complement consists of approximately 30 proteins that are present in normal human serum. The term complement refers to the ability of these proteins to complement or assist the effects of other components of the immune system. Activation of Complement: There are three pathways of complement activation: the classical the alternative and lectin pathways. (Figure 1) o The classical pathway (the pathway of acquired immunity) is activated by antigen-antibody complexes; thus, it is triggered after the generation of specific antibody to a particular Ag. Both IgG and IgM can activate the system by this pathway. o The alternative pathway can be activated by microbial cell surface substances. Bacterial polysaccharides and lipopolysaccharides of the cell envelop of gram-negative bacteria both serve as potent initiating stimuli. o The lectin binding or mannose binding pathway (MBP) is activated when mannose binding lectin binds to carbohydrates on the pathogen. TheThe lectin and the alternative alternative pathway andpathway are pathway the lectin considered arepart of the innate considered part ofimmune system innate immunity itysystem The activation of complement components involves sequential proteolytic cleavage of complement proteins, leading to the generation of effector molecules that participate in eliminating microbes in different ways. Activated complement proteins become covalently attached to the microbial cell surfaces where the activation occurs. Page 1 of 8  Many components are pro-enzymes which must be cleaved to form active enzymes. On cleavage of a complement component, the large fragment is given the suffix “b” e.g. C5b and the smaller fragment is given the suffix “a” e.g. C5a.. Complement pathways lead to the formation of a complex enzyme (C3 convertase) capable of binding and cleaving a key protein C3, common to all three pathways. Ø C3 activation is the common central event. After that, the three pathways proceed in the same fashion together through binding of the late-acting components to form a membrane-attack complex (MAC) which is C5b,6,7,8,9 which becomes inserted in the lipid bilayers of foreign membranes, ultimately causing cell lysis. Biologic Functions of the Complement System: 1. Cell lysis: Insertion of the membrane attack complex (C5b,6,7,8,9) into the cell membrane leads to lysis of many types of cells, including erythrocytes, bacteria and tumour cells. 2. Opsonization of pathogens: Microbes such as bacteria and viruses are phagocytized much more efficiently in the presence of C3b bound on their surfaces, because of the presence of C3b receptors on the surface of many phagocytes. 3. Inflammatory function: a. Chemotaxis: C5a attracts phagocytic cells, mainly polymorphs to the site of inflammation and increases their activity. b. Anaphylatoxins: C3a, C4a and C5a can produce degranulation of mast cells with release of mediators which cause increased capillary dilatation. 4. Enhancement of antibody production: The binding of C3b derivatives to its receptor on the surface of B lymphocytes enhances antibody production. Page 2 of 8  Figure 1: The classical, alternative & lectin pathways of the Complement system MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) The MHC is a collection of highly polymorphic genes on the short arm of chromosome 6 in the human. There are 2 major classes of cell bound MHC gene products: I and II. MHC gene products are also called Human Leucocytic Antigens (HLA). Figure 2 Figure 2: The major histocompatibility gene complex and the corresponding MHC class I and II molecules or antigens on the cell surface o Class I MHC–antigens: are glycoproteins and include HLA-A, HLA-B, and HLA-C. They are expressed on all nucleated cells in the body. They are expressed in a codominant fashion, meaning that each cell expresses 2 A ,2B,2C products (one from each parent). Class I MHC enables cytotoxic T cells (CD8) to recognize foreign antigen on the surface of graft cells, tumour cells or virus infected cells and kill these cells. In other words, cytotoxic T cells are triggered only when they recognize both antigen and class I MHC molecules in close association on the surface of cells. This is known as MHC-restriction (Figure 3). Page 3 of 8  o Class II MHC-antigens: or HLA-D antigens are glycoproteins, and include HLA- DP, HLA-DQ and HLA-DR. They are expressed on antigen presenting cells of the body such as macrophages, dendritic cells and B lymphocytes. Helper T cells (CD4) will recognize foreign antigen on the surface of APC only if they are associated with class II MHC molecules (MHC restriction). (Figure 3) - Both classes could be typed by: Flow cytometry or molecular techniques such as PCR. Figure 3: Role of MHC in the immune response Ø Importance of MHC: Organ transplantation: the likelihood that a transplanted organ is accepted by the recipient’s immune system depends on the compatibility of the MHC genes of the donor and the recipient. MHC restricted antigen presentation: The ability of T cells to recognize antigen is dependent on the association of the antigen with either class I or class II proteins. o Tc cells recognize the Ag in association with MHC I. o Th cells recognize the Ag in association with MHC II. Disease association: It is found that the presence of certain HLA antigens is often associated with a particular disease: o HLA-B27 with ankylosing spondylitis. o B8 with myasthenia gravis. o DR2 with multiple sclerosis. o DR4 with rheumatoid arthritis. Paternity testing and forensic investigations (Each person have two sets of these genes -one on the paternal and the other on the maternal chromosome 6). Page 4 of 8 CYTOKINES Cytokines are glycoproteins of low molecular weight consisting of a large group of molecules involved in signalling between cells during the immune response. Mode of action: Cytokines are secreted by many cell types in response to specific stimuli. A cytokine acts on a target cell through a high-affinity cytokine receptor triggering subsequent signalling inside the cell. Cytokines mediate and regulate immune and inflammatory reactions. Most cytokines act in a paracrine or autocrine manner, whereas only a small part of the cytokines may function at the systemic level, by the endocrine way, like hormones (Figure 1) Figure 1: Mechanism of action of cytokines Classification of cytokines: Classifications based upon structural and biochemical properties of cytokines are complex. Classical nomenclature of cytokines includes: 1. Interleukins (ILs) 2. Colony-stimulating factors (CSFs) 3. Tumor necrosis factors (TNFs) 4. Transforming growth factors (TGFs) 5. Interferons (IFNs) 6. Chemokines and a variety of other proteins 1. Interleukins: This is a large group of cytokines which are produced mainly by T-cells, but also by mononuclear phagocytes or by tissue cells. Most are involved in directing other cells to divide and differentiate or their activation during an immune response. Each interleukin acts on a specific limited group of cells which express the correct receptors for that interleukin E.g.IL 2: The most powerful activator and growth factor for T-cells. 2. Colony stimulating factors (CSFs): are cytokines primarily involved in directing the division and differentiation of bone marrow stem cells. The CSFs direct immature bone marrow stem cells to develop into red blood cells (RBCs), platelets, and the various types of white blood cells. The balance of different CSFs is partially responsible for the proportion of different immune cell types produced. Some CSFs also promote further differentiation of cells outside the bone marrow, e.g., macrophage CSF (M-CSF) promotes the development of monocytes in blood and macrophages in tissues. Page 5 of 8 3. Tumour necrosis factors: TNFα acts synergistically with TNFβ, IL1, IL6, and IFNγ. It is a potent pro- inflammatory cytokine and even endogenous toxin, an endogenous pyrogen, and a regulator of adaptive immune responses. Historically, TNFα was discovered and named in such manner as could lyse particular tumor cell lines. TNFβ (“lymphotoxin”) is secreted by lymphocytes. Its effects are similar to TNFα’s effects, but TNFβ is more critical for the development of lymphoid tissue. 4. Transforming growth factors (TGFs): Transforming growth factors (TGFs) are a subset of a larger family of protein hormones that induce growth regardless of target cell anchorage and can play a role in embryological development, wound healing and tissue repair. TGF-β acts as a potent immunosuppressor, inhibiting proliferation in T and B cells and downregulating T-cell activity. 5. Interferons (IFN):-IFNα is produced by leukocytes and other cell types and takes part in the innate immunity as a potent antiviral agent to promote the cytostasis of target cells. IFNα is an anti- inflammatory cytokine and a stimulator of NK cell and macrophage activity. It belongs to type I of the IFN subfamily. IFNβ is secreted by fibroblasts and other cell types. It exerts the same effects as IFNα, in particular, in defense against viral infections. It also belongs to type I of the IFN subfamily. IFNγ is produced by lymphocytes upon their activation, belongs to the Th1 profile of cytokines, and exhibits wide immunoregulatory qualities in the immune processes. IFNγ is related to type II of the IFN subfamily. -Type I can be induced i