LIST OF SUBJECTS - IMMUNOLOGY EXAM 1. Immunity and the immune system: definition, cells, molecules, phases (stages) I M M U N I T Y property of the body that defines – defense against the structures considered FOREIGN, both external (microorganisms from environment) and internal (tumor cells). IMMUNE is considered an organism that, naturally or following some treatments, is protected against a certain disease. IMMUNE SYSTEM is specialized directly and exclusively in defense against all diseases, but especially against microbial, parasitic and neoplastic diseases (cancer). The immune recognition – extremely specific, the immune system being capable to recognize minimal difference between foreign structures. The immune system develops responses only against foreign structures”immune response”. In the same time, the system “knows” to distinguish between foreign (non self) and its own (self) molecules. - Own (self) - structures, cells and molecules of the organism; - Foreign (non self) - any cellular or molecular structure that has a different chemical composition compared to the own components or structures of the organism; IMMUNE SYSTEM consist of organs; tissues;cells; molecules – that are participating to defense through immune mechanisms. IMMUNE SYSTEMS consists of ORGANS – two categories: - central (primary): bone marrow, thymus, fetal liver and bursa of Fabricius; - peripheral (secondary): spleen, lymph nodes, mucosa-associated lymphoid tissues, Harderian gland). CELLS participating in the immune response are: Mononuclear phagocyte system – monocytes and macrophages; LYMPHOCYTES (T AND B);Accessory cells (dendritic, interdigitating and FOLLICULAR); polymorphonuclear cells; Killer Cells. MOLECULES are classified in two categories: - molecules present on the cells’ surface that are: receptor molecules (TCR and BCR); cellular markers (CD molecules); major histocompatibility complex (MHC) molecules; -free molecules (circulating) that are: immunoglobulins; complement system; cytokines; acute phase proteins. MAIN PHASES OF THE IMMUNE RESPONSE ARE: l RECOGNITION (cognitive) PHASE – the elements of the immune system take note about the entrance of an intruder (non self) in the organism; l ACTIVATION PHASE - characterized by the entrance in the cell cycle of the cell that are participating to the immune response, followed by the proliferation and synthesis of some characteristic proteins and glycoprotein (especially antibodies and cytokines); l EFFECTOR PHASE – cells and their molecules react with the intruders. 2. Natural immunity IMMUNE SYSTEM – is a biological and open system that receives information from the environment (exterior) and assimilates them through recognition of foreign information( pathogens). IMMUNE RESPONSE – is accomplished by the cooperation of two categories of complex biological mechanisms: innate immunity (non specific, natural);acquired immunity (specific, adaptive). NATURAL IMMUNITY (INNATE, NON SPECIFIC) is represented by the totality of mechanisms of born defense,that are the first mechanisms and which act regardless if the “attackers” are viruses, bacteria or parasites.This type of immunity, characteristic to a species, defines the resistance of all the individuals of the respective species to some microorganisms, which for other species can be pathogenic.Ex: horses are resistant to swine fewer; The FACTORS that ensure the natural immunity are classified in: passive factors; active factors. PASSIVE FACTORS OF NATURAL IMMUNITY represented by various anatomical structures, included in 4 barriers : 1.PHYSICAL BARRIERS or primary barrier through which the body is opposing to the entrance of the microorganisms in tissues and body fluids ex: skin and mucous membranes. SKIN obstructs the invasion of infectious agents, due to a compact cellular structure on the level of epidermis and by sebum secretion, it resists to the entrance of microorganisms, through the following mechanisms: - mechanical resistance (the infections occurs only in alteration of the continuous structure of the skin like wounds); - acidity;- antagonistic action of the endemic microbial flora;- continuous peeling;secretion of annex glands. MUCOUS MEMBRANES - are limiting the access of microorganisms to subjacent tissues due to the special structure that confers them integrity, as well as due to their secretions (mucus). Mucus - secreted by the specialized epithelial cells, integrate the foreign microorganisms. 2.MECHANICAL BARRIERS also found in skin and mucous membranes: - peristaltic movements; - cilia of some cells of respiratory mucosa (microorganisms are eliminated cilia movements together with the mucus is which are fixed). 3.CHEMICAL BARRIERS represented by various chemical factors: - low pH of some glands’ secretion in skin; - low gastric pH; - other chemical factors (lactoferrin, lactoperoxidase), and also various enzymes and blood. 4. BIOLOGICAL BARRIERS represented by bacteria and protozoa, especially in the digestive tract, especially in oral cavity, but also at the level of respiratory and genital mucosa. ACTIVE FACTORS OF THE NATURAL IMMUNITY Are represented by: - molecular elements – present at humoral level – so called “humoral factors”; - cellular elements – with certain function, so called “cellular factors”. THE MAIN CHARACTERISTICS OF THE NATURAL IMMUNE RESPONSE ARE -The natural immunity is non specific, no matter the nature of the etiological agent; -natural immunity is instant, spontaneous and doesn’t need any kind of preparation in time; -natural immunity is without memory and doesn’t amplify due to a first aggression; -has a genetic character and is hereditary transmitted. 3. Adaptive (aquired) immunity IMMUNE SYSTEM – is a biological and open system that receives information from the environment (exterior) and assimilates them through recognition of foreign information, represented by various pathogens. IMMUNE RESPONSE – is accomplished by the cooperation of two categories of complex biological mechanisms: adaptive immunity (specific, aquired); innate immunity (non specific, natural); ADAPTIVE IMMUNITY (ACQUIRED, SPECIFIC, ARTIFICIAL) is a system able to RECOGNIZE SPECIFICALLY and to ELIMINATE SELECTIVELY the foreign macromolecules and microorganisms.The ADAPTIVE IMMUNE RESPONSE is SPECIFIC to each etiologic agent meaning the defense mechanisms act differently, with very high precision. This type of immunity expresses on maximum intensity in adult animals and is partially deficitary,in young and old animals.The specific mechanisms of adaptive immunity amplify after the first contact with an etiological agent, so that on the subsequent contacts (second, third etc.) the mechanisms are more efficient. This is due to the fact that, following the first contact, there are formed cells with “immunological memory”.To accomplish this type of immune response, specialized immune cells participates – T and B lymphocytes, NK cells, and antigen presenting cells (APC). On this level there can be described macromolecular structures implicated in: -recognition – receptors of B and T cells and MHC macromolecules; -activation – different markers of surface and adhesion molecules; -elimination of antigens – immunoglobulins, perforins, granzymes. The main features of the adaptive immune response are the following: -The specificity : only against a certain antigen. -The diversity : the organism is capable to respond against an extraordinary multitude of etiological agents. -The capacity of differentiating between foreign and own structures: lymphocytes react only against the foreign molecules (non self), which are recognized as non self by them and against which elaborate an immune response. This capacity of recognition of non self is due to a process of lymphocytes selection during their maturation and in primary lymphoid organs. In the process of lymphocytes’ maturation, there are eliminated almost all T and B cells clones capable to react with the biochemical (self) struct. -The self-limitation is a property of the immune system through which the intensity and duration of the immune response is limited because of some regulatory mechanisms through the contribution of foreign antigens, some cells and some molecules of the immune system. -The memory following the first immune response against a certain “non self”, results T and B cells that memorize the foreign agent, being thus prepared for a second contact with the same etiologic agent. CLASSIFICATION OF THE ACQUIRED IMMUNE RESPONSE - I) active immunity - II) passive immunity 4. Active and passive immunity 1.ACTIVE IMMUNITY It is induced by the direct conflict of the organism and the antigenic components.Accomplished: in natural conditions (post-infectious immunity) as a result of the organism passing through disease ; in artificial conditions(postvaccination immunity) by vaccination . According to the immune effectors involved, the active immunity can be: of humoral type or humoral immunity; of cellular type or cellular immunity. - NATURAL ACTIVE IMMUNITY (after infections): Occurs when the body is exposed to a live pathogen, develops the disease, and becomes immune as a result of the primary immune response OR An active immunity acquired by experiencing and having recovered from a disease. After the conflict between the body and the microorganism, the body can win a strong specific resistance, sometimes for the entire life. - ARTIFICIAL ACTIVE IMMUNITY(after vaccination): It is obtained following the administration of vaccines, in 7-14 days after the contact with the innoculated antigen and ensure body protection during 3-6 months up to 1 year, rarely more. VACCINATION OF SICK ANIMALS IS PROHIBITED. 2.PASSIVE IMMUNITY It is the immunity obtained due to the transfer of antibodies from an immune organism to a non immune one; it always has a humoral character (humoral immunity); it is called passive because the immunized organism receives the immune effectors produced by another organism. There are 2 known types of passive immunity: naturally acquired - maternal immunity; artificially acquired – transfer of the antibodies (serum) from an immune organism to a non immune one. - NATURAL/MATERNAL IMMUNITY It is accomplished in natural conditions by the transfer of maternal antibodies to the product of conception. Maternal antibodies can be transferred through: placenta; colostrum; egg yolk. TRANSFER THROUGH PLACENTA:The placenta represents the morpho-physiological link between mother and fetus, respectively between the chorial villosities and the uterine mucosa.The transfer of maternal-fetal antibodies depends on the: type of de immunoglobulin; structure of the placenta. Types of placenta: 1. HEMOCHORIAL PLACENTA – in primates (including humans) and rodents; the most permeable;the maternal blood is in direct contact with the trophoblast. 2. ENDOTHELIOCHORIAL PLACENTA – in dogs and cats; medium permeability; chorionic epithelium is in contact with the endothelium of the maternal capilaries. 3. SYSNDESMOCHORIAL PLACENTA – in cow, sheep, goat; low permeability; chorionic epithelium is in contact with the uterine tissue. 4. EPITHELIOCHORIAL PLACENTA – horses, pigs, camel; does not allow the passage of the antibodies; chorionic epithelium is in direct contact with the intact uterine mucosa. TRANSFER THROUGH COLOSTRUM:The immune effectors are transmitted to the newborns due feeding with colostrum.The accumulation of immunoglobulin in the mammary gland begins 1-2 weeks before parturition and is accompanied by an adequate decrease in the blood serum. Young animals that suckle soon after birth take colostrum into their digestive tract.Unsuckled animals have very low levels of Ig in their serum. The successful absorption of colostral Ig supplies the new born with serum IgG at a level approaching that is found in adults. Immunoglobulins received through the colostrum are kept in young animals circulation for a variable period of time, between 3 and 4 weeks. After this period their concentration drops and starts the production of self immunoglobulins. Colostral transfer of immunity is essential for the survival of young animals. Failure of maternal antibodies transfer through colostrum – 3 major reasons: - Production failure: the mother produce insufficient or poor-quality colostrum; - Ingestion failure: colostrum is sufficient, but intake by the new-born is insufficient; - Absorption failure: the colostrum and its intake are sufficient, but the absorption does not take place because of: lesions of intestinal mucosa; protease high activity (degrades immunoglobulins); the colostrum is administered too late. Passive immunity (in the chicken) Immunoglobulins (IgY) are actively transported from the hen’s serum to the yolk while the egg is still in the ovary. In yolk, IgY can be found at levels at least equal with those in hens’ serum.IgM and IgA are added with the albumen (white) when the egg passes the oviduct. 5. Phagocytosis and pinocytosis PHAGOCYTOSIS AND PINOCYTOSIS The body allows free access of nutrients and oxygen, stopping in the same time the access of some dangerous microorganisms. In order to accomplish this, a few defense mechanisms are evolved in the body. In the body, there are mechanisms that recognize and destroy any substance or material that manages to pass the primary defense at the skin and mucosa level.These systems imply cells that can connect,ingest and destroy foreign materials through phagocytosis. Phagocytosis and pinocytosis are components of the endocytosis process. Phagocytosis represents the ingestion of particulated material, containing pathogenic microorganisms (bacteria, viruses, parasites). Migration of phagocytic cell, attracted by chemotactic factors, to inflamation locus. Bacteria adhering to the phagocytic cell, process favored by opsonins. Phagocytic cells emit pseudopods and the bacteria are internalized . Bacteria are disintegrated by lysosomal enzymes. 1. Chemotaxis - an orientated movement of the phagocytic cell, induced and controlled by different substances or particles with positive chemotactic effect Chemotactic factors: bacteriological polysaccharides; component C5a of the complement;the kinin-kallikrein system; fibrin; antigen-antibody complexes; some enzymes (e.g. proteases) 2. Adherence phase -this process (i.e. adherence of bacteria to the phagocytic cells) is favored by natural opsonins or other substances. Opsonization is a process following which opsonins from serum reacts with the microorganism, preparing it for phagocytosis. 3. Ingestion (inclusion) phase - After adherence, there follows the process of inclusion (ingestion). Polymorphonuclear leukocytes engulf microbes, and there is formed a vesicle called phagosome in the cell. 4. Intracellular digestion phase - Lysosomes in the cytoplasm fuse with the phagosome, releasing digestive enzymes into the phagosome. The structure resulting from this fusions is called phagolysosome In the process of intracellular digestion two mechanisms are involved: -oxygen-independent system; -oxygen-dependent system. 6. Cellular effectors of the immune response – general properties, common to all cells of the immune system - The cells of the immune system are: 1. Lymphocytes (T and B cells) 2. Monocytes and macrophages 3. Polymorphonuclear cells: a. neutrophils b. eosinophils c. basophils 4. Antigen presenting cells (APC) 5. Natural killer cells (NK) - General features, common to all cells of the immune system: 1. are concentrated in organs and tissues, fact that allows the cooperation between them. 2. the majority of the cells are motile – they are moving between blood / lymph and lymphoid organs & tissues. 3. Between the cells of the immune system, there are established bidirectional relationships. Therefore, two different types of cells cooperate, mutually transmitting certain activating information 4. Generally, these cells are found in two situations: - resting (in hold); - in the activation phase. Transition from resting phase to activation is triggered by a stimulus, which can be: - a bacterial antigen; - a viral antigen; - a parasite antige; - a tumor cell; - + a supplementary stimulus . 5. Immune cells participate differently, sometimes highly specialized in: innate immunity mechanisms or/and adaptive immune mechanisms; There are cells that participate in both types of immunity. 6. These cells are essential elements of the immune response, participating both in the humoral and cellular immune response . 7. Monocyte-macrophage system CELLULAR EFFECTORS OF THE NONSPECIFIC IMMUNITY / Monocyte-Macrophage system The monocyte-macrophage system is constituted from the totality of mononuclear phagocytic cells derived from the pluripotent stem cell, on the myeloid line. - STAGES: Development of monocytes takes place in bone marrow; - STEM CELLS - monoblasts - MONOBLASTS – leave the bone marrow and go in blood ; - PROMONOCYTE (immature cell) - MONOBLAST; - PROMONOCYTES - MONOCYTES Monocyte – blood; - A part of the monocytes leave the blood and locate in different tissues, becoming resident cells, called MACROPHAGE - Macrophage – bone marrow; Names of the resident macrophages - Kupffer cell (blue) – in liver - alveolar macrophage – in lungs - mesangial cell – in kidney - microglia – in nervous system - osteoclast – in bone - histiocyte – in connective tissue MONOCYTES - morphology: - large cells - 15-20 μm; - nucleus – bean or horseshoe shape; - cytoplasm rich in: lysosomes, phagosomes, Golgi apparatus, mitochondria and endoplasmic reticulum, actin and myosin filaments (for motility and phagocytosis) MACROPHAGES- morphology: - large cells - 15-20 μm; - small pleated nucleus; - cytoplasm rich in cells organelles: Golgi apparatus, mitochondria, lysosomes, phagosomes, microtubules. On the surface of monocytes and macrophages there are found many molecules with the property of reacting and forming non covalent bonds with diverse molecules recognized as foreign. These molecules, recognized as non-self, can be free molecules or molecules in the structure of bacteria, viruses, tumor cells, etc. On the surface of monocytes and macrophages there are: 1. Receptors molecules: for certain carbohydrate molecules (mannose and fructose); for non specific immunoglobulins and antibodies; receptors for IL-2(CD25) and transferrin (CD71). 2. MHC class II molecules: protein and glycoprotein molecules located on the membrane; transports and express on the surface of the cells the antigens resulted from phagocytosis; These molecules have an aspect of „plates” or „treys”,with small size cavities where the antigen is presented. 3. Cellular markers: molecules characteristic for each cellular type; - have different functions in immune mechanisms and are noted as CD (cluster determinant); - there are known more than 350 CD molecules in the present. PHAGOCYTOSIS IN MACROPHAGES (PHASES) Functions of the monocytes and macrophages: 1. Recognition and phagocytosis of some foreign complex antigens; 2. To eliminate dead or aged cells - elimination of some own cells that have become nonfunctional; Example – removal of senescent (aged) red blood cells by the macrophages in the spleen. 3. Destruction particles and phagocitated cells ; 4. Presentation of antigens on the surface of the cell ; 5. Secretion of cytokines, complement, lysozyme, prostaglandins, various enzymes, halogens, oxygen radicals; 6. Secretion of fibroblasts and epithelia growth factors; 7. Accomplishment of some Antibody-dependent cell-mediated cytotoxicity mechanisms ; 8. Regulation of the immune response through cytokines. Example of cytokines produced by macrophages. 8. Polymorphonuclear leucocytes CELLULAR EFFECTORS OF THE NONSPECIFIC IMMUNITY -Called also granulocytes,come from the myeloid stem cell which is differentiating in promyelocyte,metamyelocyte, non-segmented granulocyte and polymorphonuclear leucocyte. Are formed in the bone marrow, and leave it as mature polymorphonuclear leucocytes,to do their functions. Are relatively large cells (10-15 µm) and are grouping, depending on the color of the cytoplasmic granules, in 3 large categories: - basophils - eosinophils - neutrophils -NEUTROPHILS most important cells of the myeloid system, formed in the bone marrow, pass into circulation and after 12 hours arrive in tissues; 60–75% of total white blood cells in all mammals, except ruminants, with 20 – 30%; almost spherical cells (1215 µm), fine granular cytoplasm; nucleus has lobed appearance; In the cytoplasm two types of granules, rich in enzymes: primary granules and secondary granules ; Neutrophils granulocytes have on surface a series of molecules, among them receptors for immunoglobulins and for the activated components of the complement; The neutrophils, have an important role in defense against bacteria and this fact is demonstrated on animals which have deficits in formation of neutrophils granule. Unlike monocytes and macrophages, neutrophils break all the bacteriological chemical structures. The quantitative assessment of these cells is very important for the characterization of the immune status: neutrophilia (the increase of the absolute number of neutrophils) indicates the evolution of a bacteriosis or an inflammatory reaction; neutropenia (abnormally low count of neutrophils) is observed in some viral infections and also in intoxications and some secondary immunodeficiency. -EOSINOPHILS the second polymorphonuclear granulocyte as importance, after neutrophils; represent 2-5% of total leukocites, in healthy animals;relative large cells (10-15 μm); bilobed nucleus;cytoplasm- rich in large granules, classified in 2 types: primary;- secondary; both types of granules contain enzymes and a specific basic protein.FUNCTIONS: Eosinophil can engulf immune complexes as well as a series of microorganisms (bacteria, fungi, protozoa);phagocytic capacity of these cells is inferior to the one of neutrophils and macrophages; Eosinophils contribute to the anti-parasite defense through several mechanisms, among which the release of cationic proteins from granules is very important; This property can be important also in the limitation of tumoral growth but it can have negative effects also over the own cells; Eosinophils have also an important role in allergies (type I hypersensitivity) -BASOPHILS The least numerous cells of the myeloid system (0.5% of total leucocytes); normally they are not found in the extra vascular tissues, but, under the influence of lymphocytes, they can infiltrate tissues; In tissues, basophils trigger inflammation, because their granules contain amines like histamine and serotonin; The phagocytic activity and the bactericide capacity of these cells are of reduced intensity or absent; Basophils have a major role in type I hypersensitivity and in the basophilic type IV hypersensitivity. -PLATELETS minor role in immune defence;they can attach and ingest some bacteria, but this is a pseudo phagocytosis, because the bacteria can enter in the cytoplasmic canaliculi of the thrombocytes;Bacteria attached to the thrombocytes can be destroyed by the system of the mononuclear phagocytes. -MAST CELLS Cells located in the connective tissues and in mucosa; cytoplasm rich in large metachromatic granules which contain serotonin, proteolytic enzymes, cationic proteins rich in arginin, chemotactic factors for eosinophils; on the surface, mast cells have receptors for IgG and IgE, and some components of the complement system. 9. Antigen presenting cells CELLULAR EFFECTORS OF THE NONSPECIFIC IMMUNITY TYPE I HYPERSENSITIVITY ANTIGEN PRESENTING CELLS (APC) -The antigen presenting process is represented by the property of all these cells to expose on their surface antigens with the purpose of their recognition by the most important cells participating to the specific immune response, respectively by T cells. - The APC group contain: Langerhans and dendritic cells - have only APC function of presenting the antigen; B cells, monocytes and macrophage which have other functions. ANTIGEN PRESENTATION 1Antigen transport. After phagocytosis and processing (cleavage) of foreign antigens, MHC molecules are produced within the cell, and then they move to the cell surface carrying antigenic determinants (epitopes). 2Antigen presentation. The main role of MHC molecules is to present antigen to specific cells of the immune system.T cell can only be activated by antigenic peptides that are presented together with MHC molecules, on the surface of antigen-presenting cells or target cells.MHC molecules present peptide of small size. 3Genetic restriction.Cells cooperation in generation of both cellular and the humoral immune response is governed by MHC complex.This control is called genetic restriction because it allows a restrictive interaction between cells expressing MHC molecules and cells expressing complementary TCR, MHC molecules expression being genetically determined. 4Graft rejection.Graft rejection is an immune response which always possesses attributes of Specificity, Memory, Recognition (of self or non-self).The main mechanism involved in graft rejection is a cellular immune response that is initiated mainly by MHC molecules expressed on the surface of grafted cells Central role in graft rejection is held by T cells, being involved both helper and cytotoxic T limphocytes. Transplantation is defined as the transfer of cells, tissues or organs from one individual to another.It is already known that organ transplantation between two genetically different individuals is followed by rejection of that organ (the function of the organ is lost) 10. T lymphocytes: origin and classification General info regard Lymphocytes are CELLULAR EFFECTORS OF THE SPECIFIC IMMUNITY - small cells – 5-10 µm diameter; - nucleus - large, round, intensely stained, even with hematoxylin; - cytoplasm – thin pellicle with mitochondria, ribosome and a reduced Golgi apparatus. -Electron microscopy - some lymphocytes present smooth surface, while others have on their surface small protrusions. -Lymphocytes are found in a variable rate in lymph nodes, spleen, bone marrow, thymus, thoracic duct, blood and MALT (mucosa associated lymphoid tissue). l T cells have a relatively long life, living from six months to ten years l B cells have a much shorter life (e.g. 5-7 weeks in mice). T Lymphocytes are CELLULAR EFFECTORS OF THE SPECIFIC IMMUNITY -Origin: pluripotent stem cell (bone marrow) -Development - complex process; with the participation of thymus “T lymphocyte / cell” name or “thymus-dependent cell”. Initially called „pretymocites”, these young cells leave bone marrow and arrive in thymus where they transform in „thymocytes”. In thymus takes place the complicated process of maturation of these cells that are proliferating and differentiate in „mature T cells”.The maturation process is characterized by the appearance on the surface (membrane) of the characteristic molecules. -T cells are in majority circulatory lymphocytes, their number reaching up to 80% from the total of lymphocytes in the peripheral blood. -A part of these cells circulate permanently between lymph nodes, through the lymphatic system. -T cells, although they have an identical morphologically, they are very different according to their functions, being described the following subpopulations: 1. Helper T cells (LTh or LTH) : - are the main regulatory cells of the immune response - the “helper” name refers to the stimulation of the most diverse functions of other cells of the immune system (e.g. monocytes and macrophages) - activate specifically the B cells (LB) which is differentiating in plasma cells (produce antibodies) and help LT in performing their immunologic actions 2. Suppressor T cells - LTS: - also participate in the immune response, but they act negatively, decelerating the development of the response mechanisms (down-regulation); -Suppressor T cells can influence T or B cells, limiting their clonal expansion, after antigenic stimulation 3. Cytotoxic T cell - LTC: - cytotoxic or cytolytic function against the cells infected with viruses or tumor cells; - LTC cells act specifically and dependent on the presence of MHC molecules. - LTC attack on tumor cell. 4. Amplifier T cells - LTA: - up-regulation of the immune response; more intense and more efficient than LTH; - activates selectively the differentiation and multiplication of B cells that express certain membrane receptors for antigens the immune response against certain simple antigens will be dominated by antibodies with certain idiotypic characters. 5. Contra-suppressor T cells - LTCS: - countervail the activity of TS cells, suppressing their activity and unblocking the inhibitor effect of these over the immune response. 6. Delayed hypersensitivity T cells - LTD: - participate in delayed hypersensitivity (type IV); 11. T lymphocytes: membrane (surface) molecules, functions SURFACE MOLECULES OF THE T CELLS: Initiation of the immune response (cellular or humoral) is the consequence of the interaction between the antigen and antigenreactive cells, this interaction is facilitated by surfaces molecules.Each T or B cell has receptors for a single type of antigen.That is why only the cells that possess specific receptors for the respective antigen will be stimulated by its presence, will proliferate and will differentiate in effector cells. Membrane molecules of T cells are: 1.Receptor for antigen (TCR) – molecule able to recognize antigens.TCR is in the membrane of the lymphocytes, capable to bind a given antigen and to initiate an immune response against this antigen. It is also involved in the recognition of the complex constituted from an MHC molecule and an antigenic peptide, located on the surface of the antigen presenting cells and of the target cells (infected or tumor cells). 2.MHC class II - are found exceptionally on LT and only on the surface of activated LT. Presence of MHC molecules says that T cells can be also antigen presenting cells. 3.Accessory molecules -Cellular markers named CD (cluster of differentiation, classification determinants), molecules through which certain cellular populations are differentiate.There have been identified more than 350 CD molecules, present not only on the surface of T cells but also on other cells. - Main markers of T cells are:CD2; CD3; CD4; CD8; CD5,CD28,CD44. [CD3 on all T cells – located (in pair) near TCR, participates on the TCR function; CD2 on all T cells - important because connects LT with other cells. It has the property of fixing erythrocytes of sheep or mouse on the surface of LT forming rosettes, property used in the diagnostic for counting lymphocytes; CD4 on helper T cells - implicated in the recognition of the complex MHC class II molecule – antigen by its connection to the MHC molecule, with functions in adhesion and transduction of the signals; CD8 on cytotoxic and suppressor T cells – recognition of the complex MHC class I– antigen ; CD5, CD28, CD44 on different subtypes of LT - with functions in adhesion and transduction of the signals.] 4.Adhesion molecules - molecules through which T cells bind correspondent molecules located on the APC surface; ensure a tight contact, sufficient for the transmission of the antigenic information. The most important adhesion molecules are:LFA,VLA. [LFA (Lymphocyte function-associated antigens) – on the surface of LT; bind molecules on the surface of APC, named ICAM (Intercellular adhesion molecule); VLA (very late antigen) – bind VCAM molecules (vascular cell adhesion molecule) on the vascular endothelium (endothelial cell can be also APC)] 5.Receptor molecules for cytokines and for various stimulatory substances 6.Receptor molecules specialized for certain viruses. 7. Receptor for the human immunodeficiency virus (CD4) FUNCTIONS OF T CELLS 1. Both cellular and humoral immune response are not possible without T cells. LT is the most important cells in specific immune response; their functions are fulfilled primarily through the receptor for antigen. 2 . Following the recognition of the antigen, T cells synthesize and secrete a series of substances that are part of cytokines family (lymphokines). The main cytokines produced by T cells are the interferons and interleukins 12. B lymphocytes: ontogenesis (origin), clasification General info regard Lymphocytes are CELLULAR EFFECTORS OF THE SPECIFIC IMMUNITY From the functional point of view immunity can be divided in two components: cellular immunity, humoral immunity CELLULAR IMMUNITY is accomplished by: T cells; NK cells; other types of cells capable of recognizing and directly neutralizes certain antigens (for e.g. tumoral cells, cells infected with viruses, bacteria, parasites, etc.) HUMORAL IMMUNITY - is accomplished by B cells through the immunoglobulins (Ig) present: on the cellular surface as antigen receptors (BCR); under soluble form, in the serum. B cell is only cell of the body capable to produce antibodies (Ig) -B cells ontogenesis: LB comes from the hematopoietic stem cell – lymphoid line - maturation of these cells is accomplished in the:bone marrow in mammals,bursa of Fabricius in birds. -MATURATION several stages, in the end of which mature B cells are exported from the organs through blood and populate the secondary lymphoid organs, especially areas called B dependent areas. -MORFOLOGY B cells resemble T cell. After the activation, a mature B cell become lymphoblast. -Lymphoblasts: large cells - 12-15 µm; are considered a transition stage because can evolve in 2 directions: either towards plasma cell; or towards memory B cell. -Plasma cells: the final stage of B cells evolution; large cells- about 20 µm; eccentric nucleus, like a “bike wheel”; produce about 5.000 - 10.000 molecules of Ig/sec. ;life span – few days -3 subpopulations of B cells – functionally and phenotipically differentiated: - Classical B cells or B2, responsible for the primary immune response find in spleen and lymph nodes; - Memory B cells; - CD5+ B cells or B1 – these cells, after activation, become plasma cells, but not memory B cells. 13. B lymphocytes: membrane (surface) molecules, functions Surface molecules of B cells 1. The main surface molecules of B cells are Ig (IgM and IgD) which are antigen receptors (BCR - B cell receptor) – recognize and bind foreign antigens (bacterial, viral etc. components). Recognition of foreign antigens is followed by a chemical reaction between the antigenic structures and BCR. Due to this reaction takes place the defense process – respectively:B cells activation;cell cycle; B cells proliferation; transformation in cells which accomplish the main function of B cells ; antibodies synthesis. 2. Receptor molecules for complement, Fc fragment of Ig, ILs (IL-2, IL-4, IL-5, IL-6) ; 3. MHC class II molecules; 4. Receptor molecules for viruses. FUNCTIONS OF B CELLS 1. Synthesis and secretion of Ig and antibodies: the main function of B cells; is permanent and amplifies following the contact with a foreign antigen. 2. Recognition of foreign antigens. Is accomplished through the receptors for antigen (BCR) On the surface of B cell (or T cells) there are receptor for a single antigen Using BCR, B cells bind foreign antigens, and the complec “antigen-receptor” is formed 3. Presentation of foreign antigens,B calls have also the capacity to engulf foreign antigens,which will be processed in the cells and exposed again on their surface. These antigens will be then recognized by other B cell (and by T helper cells) 4. Synthesis and secretion of cytokines ; B cells can produce some cytokines (like IL), but this functions has a lesser extent that in T cells. 14. Large granular lymphocytes (lymphocytes with big granulations) Types: - NK – natural killer cells; - LAK – lymphokine-activated killer cells; - NK-like – natural killer like cells; - K – killer. Main characteristics of there cells are: 1 - They differentiate form the hematopoietic stem cell, being considered primitive cytotoxic T lymphocytes which are not undergo maturation in thymus 2 - Can be found in blood and lymphoid tissues 3 - Their cytoplasm contains numerous granules 4 - These cell do not have specific receptors for antigens (like TCR and BCR) their receptors for antigens lack specificity, so they do not have a complex biochemical structure 5 – They have receptors for Fc fragment of IgG and for complement 6 - Their activation can be achieved through cytokines (IFN γ, IL-2, IL-7, TNF), the previous contact with the antigens is unnecessary 7 – They can destroy tumor cells and cells infected with viruses through non specific mechanisms, without the partici-pation of MHC molecules NK cells – are effectors in ADCC - antibody dependent cell mediated cytotoxicity, with the participation of CD16 – receptor that recognize the targets bind by antibodies. 15. Complement system Is a group of over 30 serum and membrane proteins, whose concentration is not increasing after immunization. It plays an essential role in the body defense and in the inflammatory processes It becomes active in the cases of some bacteriological infections or under the action of various pathogenic factors;It is synthesized by various cells (hepatocytes, monocytes, and macrophages) and tissues. Complement components – can be found in an inactive state in serum, and the transition to active state is accomplished through three pathways: - classical pathway; - alternative pathway; - lectinic pathway. Structurally complement is a multi-molecular system, consisting in 15 componentst that are the following: - C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, - initiation factor; - properdin; - B factor; - D factor. CLASSICAL PATHWAY: - discovered long before the alternative pathway; - the activation of complements is mainly the result of antigen-antibody reaction ALTERNATIVE PATHWAY: - the activation of complement is a result of the presence of antigens on the microbes surface (in the absence of antibodies) - is less efficient, but of great importance, because intervenes very fast, before the formation of specific antibodies 16. Classical pathway of complement activation It is a mediator of the specific humoral immune response (antibodies) Begins with the interaction of the first component of the complement - C1q - with Fc fragment of the antibodies (IgG or IgM) that have connected previously an antigen During this classical activation pathway, there are participating 11 complement component, grouped in three functional units: - recognition unit (C1q, C1r, C1s); - activation unit (C4, C2, C3); - attack unit (C5, C6, C7, C8, C9). Recognitions: - is triggered due to the interaction of antigen with IgG or IgM - antigen attaches at the level of the Fab fragment of immunoglobulin; - then first component of the complement – C1 – binds the Fc fragment of Ig. C1 is present in circulation as a macromolecular complex, consisting in three subunits: C1q, C1r and C1s. Furthermore the activation unit (C4C2C3) enters in action: - first, C1s of recognition unit acts over C4 component, splitting it in C4a and C4b; - the resulted C4b is absorbed on the cellular membranes and couples with C2a also coming from the splitting of the fraction C2 (cleaved by C1s). This way there is formed a complex molecule - C4b2a – which is an enzyme (C3convertase) Its enzymatic activity is exerted over the component C3 , cleaving it in C3b and C3a, resulting an more complex molecule C4b2a3b or C5-convertase. Then, C5-convertase cleaves C5 component in C5a and C5b, activating the attack unit C5b6789 (MAC membrane attack complex) RESULT – cytolysis l MAC - constitutes the main factor through which there are accomplished the functions of the complement. l There has been proved that each molecule C9 contains two α-helixes that form the walls of a transmembrane pore (channel). There is thus formed a channel of approximately 103 Å diameter and 100 Å length that appears electron microscopically surrounded by a ring of 5 nm diameter 17. Alternative pathway of complement activation ALTERNATIVE PATHWAY OF COMPLEMENT ACTIVATION - an old system from phylogenetic point of view - the complement activates in the absence of the antigen-antibody reaction; The alternative way is initiated by the: - endotoxins of the Gram negative bacteria - teichoic acids from the pneumococi wall - bacterial polysaccharides (LPS) - fungal polysaccharides (zymosan) - vegetal polysaccharides (insulin) - agarose - snake venom (cobra) - certain cells of mammals - immunoglobulin aggregates. -The alternative pathway of complement activation starts with the C3 component -Properdin activates C3 through B factor, like a C3 - proactivator-convertase, cleaving C3 in C3a and C3b. -In the same time, both C3b and D factor acts over B factor, cleaving it in Ba şi Bb fragments. -C3b and Bb form C3bBb (C3-convertase) which react with other C3 components, cleaving them in C3a and C3b. -C3bBb and C3b form C3bBb3b (C5-convertase) which cleaves C5 in C5a and C5b, in the end resulting C5b6789 or MAC. 18. Biological function of the complement system 1. Opsonization function – establishes the link between phagocytic cells and bacteria through C3b. 2. Function of neutralization and/or destruction of some cells infected with viruses, parasites, and bacteria. 3. Function of neutralization of some circulatory toxins (ex. toxins produced by Clostridium spp.) 4. Cytolysis of some cells (tumor cells, aged cells) through membrane attack complex, with or without participation of antibodies. 5. Clearance (elimination of immune complexes ) -Immune complexes are harmful only if they fall within a limited range of sizes: if the complexes are very large, they are efficiently removed by phagocytic cells; if they are very small, are normally removed by erythrocytes. 6. Pro-inflamatory and anaphylactogen function : Inflammation is a complex process that includes a series of events: active local hyperemia; vascular increases of vessels permeability ; formation of an exudate. Complement participates to the inflammatory phenomenon through the C3a, C4a and C5a components , which are chemoattractants and anaphylatoxins. - C3a and C4a components: induce contraction of the smooth muscles; increase of the vascular permeability and degranulation of basophiles, with the release of histamine and the mucous secretion. -C5a component: has similar effects with C3a, but is 200 times more potent in inducing the release of histamine and of the spasmogenic response; it is also chemoattractant for phagocytes; increases adherence and determines the aggregation of neutrophils; stimulates the oxidative metabolism and release of lysosomal enzymes to a variety of phagocytic cells. 7. Regulation of the immune response: -Component C3 has a key role in the immune response regulation, by the depositing on the antigens and immune complexes, the C3 component intervenes in the processing and presentation of antigen. - complement participates in the recognition of antigens by B cells (LB); 8. Complement contribute in coagulation process The complement system is activated during process of blood coagulation, being important in some species (rabbit). 9. Immunostimulatory function – Is achieved as a result of the partial alteration of the immunocompetent cells membrane , that enhance metabolic exchanges. 19. Immunogens and antigens: definitions, chemical structure ANTIGENS - chemical structures that can interact with the ANTIBODIES which have been released during the immune response and with SPECIFIC RECEPTORS FOR ANTIGENS - TCR and BCR, located on the surface of T and B cells. The property of antigens to specifically bind T and/or B-cell surface receptors is called antigenicity. IMMUNOGENS - structures which, after their recognition by the immune system can induce the initiation of a detectable specific immune response; this property is called immunogenicity - immunogenicity – define the ability to induce and immune response. - antigenicity - property to react with the efectors of this response, respectively with the antibodies and the cells possessing specific receptors for antigen. CHEMICAL STRUCTURE OF ANTIGENS AND IMMUNOGENS -In biochemical terms, antigens primarily consist of protein, and secondly of carbohydrates. -A large number of antigens (highly active) are glycoproteins and consist of polypeptides on which are attached the carbohydrate residues. -To a much lesser extent, for the immune response are important the nucleoproteins and lipids, they usually having stimulatory function. -The majority of the antigens are proteins, and their structure is organized on 4 levels: primary structure - represented by linear sequences (chains) of amino acids. secondary and tertiary structure - resulting from the folding of these chains (spatial conformation). quaternary structure - results after the association of several proteic molecules. -Antigens consist in segments called antigenic determinants or epitopes EPITOPE - represents the smallest structure or "minimal structure" of an antigen capable to induce an immune response.Consists of at least five-six amino acids located either along the polypeptide chain. The action of each epitope is performed after being recognized by a receptor molecule which is located on the surface or on the membrane of a T or B lymphocyte. The immunogenic properties of a protein molecule are dependent on two types of epitopes: sequential epitopes - represented by a continuous segment of amino acids, and are dependent on the primary structure of the protein; conformational epitopes – consisting in amino acids located at a distance in the primary structure, amino acids that become close after folding of the polypeptide chain, in the secondary and tertiary structure of the protein. - The number of epitopes of a molecule is variable, being correlated with the size of the molecule. - An antigen must have at least two epitopes HAPTENS - small molecules or chemical groups that can act as epitopes when are bound to other molecules. The antigenic molecule to which it binds is called carrier.Many drug allergies occur due to their binding to normal proteins, drugs thus functioning as haptens. 20. Classification of immunogens and antigens 1. According to origin- antigens are categorized in: - endogenous antigens - structures with antigenic function, existing in the body; - exogenous antigens – originate outside of the body - bacteria, viruses, parasites, etc. 2. According to their formations – antigens can be: - natural antigens – they exist in nature without any human intervention - bacteria, viruses, parasites, cells, various proteins, etc. - artificial antigens – obtained in experimental conditions (lab) - synthetic antigens – obtained in experimental conditions; they are not exist in the nature . 3. According to the complexity of the antigen molecule: - complete antigens - incomplete antigens (haptens) 4. According to the relationship with the recipient - heterologous or xenogeneic antigens - derived from different species than recipient organism; - homologous or allogeneic antigens - derived from individuals of the same species as the recipient, of which it is differing in allotypic terms; - autologous antigens – normal constituents of the body; in normal circumstances are not immunogenic; - isologous antigens – derived from an identical twin or from an individual genetically identical ; - congenic antigens - derived from animals which differ by a single locus in MHC. 5. According to chemical structure: - protein antigens - the largest group of substances. 6. According to the intensity of the immune response there are: -proteins with low immunogenicity (e.g. hemoglobin) - proteins with high immunogenicity (e.g. albumin, serum glycoproteins, etc.). - polysaccharide antigens - an important group of substances found in nature; - lipid antigens - include various lipids, alone or in combination with other substances. Lipids alone don’t have immunogenic properties, but may have hapten qualities, especially if are coupled with proteins. - nucleic antigens – nucleic acids, DNA and RNA, with low immunogenic properties 21. Factors influencing the immunogenicity of a substance Generally, for a substance to be a good immunogen, certain conditions have to meet, among which the most important are the following: 1. Molecular complexity - a substance is a better immunogen if it contains a large number of different biochemical structures. 2. Size of the molecules – expressed as molecular weight (MW). Usually there is a direct relationship between the size of the antigen molecule and its ability to induce immune responses, considering that the bigger a molecule is, the more intense response it causes. 3. Rigidity of the antigen molecule - antigen molecules are more active if they are stable (rigid), allowing interaction with cell surface receptors 4. Optical isomerism - antigen processing plays an important role in the case of protein antigens If the antigen id easily cleaved into its component parts, their immunogenicity is greater, and the stimulatory informations can be transmited faster and more efficiently by lymphocytes Macromolecules that cannot be degraded into these cells are poorly immunogenic. 5. Degradability – not all non self molecules are able to induce an immune response. 6. Foreignness (non self) - in order to be immunogenic, determinants (epitopes) of the antigen should not be present on molecules which are normally in the body The term "non self" describes the quality of a substance to be foreign to the body it which penetrates (enters) and which has to discriminated between the antigens and own components designated as self (proper) 7. Genetic factors - Some substances are immunogenic in one species but not in another 8. Administration route – immunogenicity of a substance depends also on the administration route Efficient administration routes: intravenous, subcutaneous and oral intraperitoneal, intradermal, 9. Dose - generally the begining of the immune response to an immunogen requires a minimum dose that varies from one substance to another. Then the response is proportional to the amount (dose) of immunogen administered, up to the critical dose, from which increasing the dose is not followed by the increasing of the response, and in some cases even followed by the installation of the tolerance state to that particular antigen. For example, intravenous administration of high doses of immunogens can induce the installation of tolerance to that immunogen. A single dose of immunogen will not induce a strong response, but repeated administration of the immunogen produces absolutely superior immune responses. 10. Functional status of the immune system Usually the very young and the very old animals have a diminished ability to mount and immune response in the presence of an immunogen 11. Adjuvants - are substances or mixtures of substances which, administered together with an immunogen, enhances the immune response to it. Adjuvants can enhance both the humoral and cellular immune response, depending on their own properties and the antigens properties Their mode of action is not fully understood, but several mechanisms have been described: -prolonged retention of the immunogen at the site of inoculation; -increase the size of antigenic molecule (faster phagocytosis); -increase local influx of inflammatory cells, macrophages and dendritic cells; -non-specific stimulation of the lymphocytes; -can be a costimulatory signal necessary for T cell activation. Practical aspects of the adjuvants use is obvious in vaccination. In order to increase the persistence in the body of certain antigens (especially bacterial vaccines), these are associated with substances able to form deposits (e.g. aluminum hydroxide, mineral oils, etc.), thereby allowing a prolonged contact with the elements of the immune system, thus being increased the immunogenicity 22. Antibodies: definiţie, distribution in the body, general chemical structure ANTIBODIES – glycoproteins; their synthesis is induced by an immunogen or by a hapten combined with a carrier, and which react specifically with the immunogen or the hapten that induced its formation ANTIBODIES – produced by mature B cells differentiated (after the antigenic stimulus) in plasma cells. Antibodies have different names, depending on their effects on the antigens: - agglutinins - agglutinate the corpuscular antigens; - precipitins - precipitate soluble antigens; - lysins (hemolysins) - induce lysis; - opsonins - facilitate phagocytosis; -reagins – induce the type I hypersensitivity reaction; -sensibilizine - sensitize the surface of some cells and make possible the complementdependent lysis The distribution of antibodies and immunoglobulins - in cytoplasm of the plasma cells – polypeptide chains and complete immunoglobulins; - on the B cells membrane – receptor for antigen (BCR – B cell receptor); - in secondary lymphoid organs and in mucosa associated lymphoid tissue; in mammals, antibodies can be found also in bone marrow, where differentiation of B lymphocytes reaches the plasma cell stage; - on the surface of the cells which have receptors for the Fc fragment of Ig. GENERAL CHEMICAL STRUCTURE OF IMMUNOGLOBULINS AND ANTIBODIES Immunoglobulins –consists of four polypeptide chains which also include in their structure carbohydrate residues bound together by non-covalent bonds (S-S, disulfide bonds) -The four characteristic immunoglobulin chains are: -two heavy chains, identical, which are marked with the letter H and -two light chains (L) also identical -the antibody molecules consists of two light chains and two heavy chains bound to each other by disulfide bonds -two disulfide bonds between the heavy chains -one bond between a light chain and a heavy one. HEAVY CHAIN consist of:450 amino acids,50 KDa (molecular weight) LIGHT CHAIN– about 220 amino acids, molecular weight between 22 and 24 KDa. 23. Immunoglobulin classes. Generation of antibody diversity IMMUNOGLOBULIN CLASSES -Ig are classified into five classes, noted as follows (according to their concentration): IgG;IgA; IgM; IgD; IgE. IMMUNOGLOBULIN G (IgG) 70 - 80% of the total serum Ig, both in mammals and birds; concentration of about 20 mg/ml serum , is also found in: lymph, ascites fluid, milk, and is the only Ig capable to cross placenta, is produced, during immunization, after IgM; production of IgG increases in secondary immune response (achieved after the second vaccination ); involved in neutralization of toxins, viruses, and bacteria, phagocytosis, antibody-dependent cytotoxicity (ADCC) and complement activation; IMUNOGLOBULIN M (IgM): is the most prevalent immunoglobulin among species, being present in all vertebrates, including cartilaginous fish; IgM concentration in serum varies among the species – 1.5 mg/ml in human, 2.5-2.8 mg/ml in cattle and 1-1.3 mg/ml in pigs; functions is the first class of Ig produced by mature B cells and the main Ig class produced during primary immune response; important role in defense – is the first Ig produced in an infection or after first vaccination; providing protection of the mucosa. IMUNOGLOBULIN A (IgA):represents 10-15% of serum Ig and is the main Ig class; found in secretions (milk, saliva, tears, digestive and respiratory secretions); IgA can be fount in two different forms: serum IgA; secretory IgA. Serum IgA: can be found in serum at a concentration of 1-2 mg/ml; weak activity as antibody for the antigens introduced through systemic immunization; does not have the ability to fix the complement;Secretory IgA : present in large amount in: saliva, tracheobronchial secretions, digestive secretion, tears, colostrum, milk, and genitourinary secretions; involved in mucosal immune defense, preventing attachment of microbes by fixing them; prevents penetration of bacteria in epithelial cells; Facilitates microbial lysis by lysosomal enzymes; In the antiviral defense ; IgA is secreted in colostrum and maternal milk, ensuring adequate protection of the newborn IMUNOGLOBULIN D (IgD) It is hardly detectable in the sera due to the low concentration and high sensitivity to proteolytic enzymes; represents about 0.2% of total Igs and has a low concentration - under 0.03 mg/ml;IgD soluble molecules circulating in plasma together with IgM. Itdoes not fix and activate the complement,does not cross the placenta, does not cause mast cell degranulation,does not bind the macrophages and lymphocytes receptors; It is intervening in cell differentiation and establishment of immunological memory. IMUNOGLOBULIN E (IgE):extremely low serum concentration under 0.0005 mg/ml. It does not cross the placenta, does not activate the complement, role in the immediate hypersensitivity reaction (asthma, anaphylactic shock) and helminth infestations, When an allergen enters the body, is specifically recognized by IgE antibodies, fixed on the surface of mast cells and basophils.Formation of the immune complexes (IgE + allergen) represent a signal for the mast cells and basophils, and induce the degranulation of these cells, which release vasoactive amines like histamine and serotonin.These substances will cause smooth muscle contraction, will increase vascular permeability, resulting the typical symptoms of type I hypersensitivity (immediate) GENERATION OF ANTIBODY DIVERSITY The immune system is able to synthesize 109-1010 antibody molecules with different specificities, which in turn can recognize an unlimited number of antigens from the surrounding antigenic universe. As a result, the immune system consists of a number of sub-population (clones) of the antigen reactive cells, each engaged in the synthesis of a particular type of antibody, with a unique specificity. Depending on the antigens’ nature, that are multivalent, and because of the large number of antigenic determinants (epitopes), the immune response is polyclonal,it is carried out by several clones of B cells which produce antibodies specific to different antigenic determinants of the same antigen. Antibody diversity is determined by the diversity of amino acid sequence of the variable region of the heavy and light chains.There are several mechanisms that explain how and when antibody diversity is generated, mechanisms that can be applied also for T and B cells receptors for antigen. A single lymphocyte produces a single type of antibodies with unique specificity and is able to transmit this unique features to all daughter cells resulting after its activation and proliferation. The law "one clone - one antibody" refers only to the variable regions (V) of the antibody molecule. 24. Regions and fragments of immunoglobulins. Functions of immunoglobulins and antibodies REGIONS AND FRAGMENTS OF IMMUNOGLOBULINS Each heavy and light chain consists in two different types of regions: - constant region (C); - variable region (V). V and C regions are encoded by genes that are expressed in different combinations. VARIABLE REGION - Within the variable region there are segments called hypervariable, comprised of 2030 amino acids that interact with the antigen itself. - These amino acids, characterized by extremely pronounced variability are organized into three regions called hypervariable regions or COMPLEMENTARITY-DETERMINING REGIONS - CDRs. - V and CDR regions form a portion of the antibody, called the antigen binding site / fragment or "Fab" CONSTANT REGION - placed at the COOH ends of the chains (H & L); - has the same structure in all chains from the same class, subclass, and type of Ig. -Fab fragment (antigen binding) fragment that binds the antigen can be found on both chains (H & L). -Fab fragments of H and L chain forms the antigen binding site. -Fc fragment (fragment crystallizable) placed in the constant region of H chains, at COOH end. FUNCTIONS OF IMMUNOGLOBULINS AND ANTIBODIES Antibodies are essential factors in the acquired immune response,of which the most important are: 1.function of immunoglobulins is to bind the antigen in the antigen-antibody reaction (soluble form) or on the surface of B cells (Ig = receptors) IgM on IgD on the surface of B cells (specific receptors for antigens) bind the antigen, that leads to the activation B cells, initiating the cell cycle and the transformation in plasma cells. 2. Complement activation some structures of IgG and IgM (located on the C region of H chains – Fc fragment) are responsible for complement activation through classical pathway. 3. Opsonin role or opsonisation function - Ig are linker molecules between APC (e.g. monocytes, macrophages) and etiologic agents (e.g. bacteria). Binding of the antibodies on the surface antigens (on viruses or bacteria) is followed by agglutination, precipitation, neutralization and disposal of the respective microbes 4. Direct neutralization of the toxins- immunity in bacterial diseases caused by toxigenic bacteria is mostly accomplished by the antibodies, which bind the toxins and neutralize their negative effects 5. Participate in the mechanisms of antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity 6. In diagnosis: - antibodies are the main elements in serological assays, assays used in most of the infectious diseases diagnosis; - normal levels of non specific Igs are indicators of the normal activities of B cells - total Igs is an important parameter for the assessment of immune status 25.Monoclonal antibodies(mAB) A contact with a simple antigen has the effect of mobilization of several B cell clones which produce antibodies with different specificity, affinity and avidity (because each immunogen has multiple epitopes) having a low efficiency specially because the absence of a particularly high specificity, the researchers developed the monoclonal antibody technology. Production of the immune sera by immunization of laboratory animals with different antigens is a current practice in immunology laboratories, but products obtained present a great heterogeneity regarding the antibody content. The antigens used for immunization in conventional methods have a different antigenic determinants (epitopes) and, therefore, stimulate multiple clones of lymphocytes, which produce various types of antibodies with different characteristics and reaction capabilities Polyclonal antibodies "family" includes immunoglobulins with specificity to different antigenic determinants on the surface of an antigen, as well as for some related antibodies, differing in binding mode and avidity. It is impossible to isolate a particular mAb from a polyclonal serum because Ig molecules have properties close related. In monoclonal antibody technology it is selected only the desired clone, which is further multiplied in vitro and will produce antibodies with the specificity for a unique epitope, because the resulted cells come from a single clone, namely from a lymphocyte population generated by a single clone. 26. Major histocompatibility complex (MHC) – generalities It is represented by a distinct genetic region that is part of the genome of all cells of the organism. MHC genes encode a group of proteins called histocompatibility molecules or antigens. These molecules are found in a differentiated manner on the surface of all cells. In transplantation, MHC molecules act as a potent immunogen, inducing graft rejection in a short time. It was found that graft acceptance depends on the presence on the surface of all cells of MHC molecules with the same structure both in donor and the recipient. MHC molecules are called : -Of histocompatibility because they determine the compatibility in organ and tissue transplantation -Major because their function exceed the importance in transplantation -The name "histocompatibility complex" is still preserved today, although this is not its primary role -MHC has evolved in relation to the defense function against various “intruders”, presenting non-self antigens to T cells and priming specific immunity in this way. -MHC genes are induced in a number of regions: MHC class I and II region, highly polymorphic and involved in antigen presentation; MHC class III region -with other immune functions (genes for complement, tumor necrosis factors, etc.); -genes that encode proteins, involved in the processing of antigens, in transmembrane transport of processed peptides and in their fixation to the MHC presenting molecule; -genes with non-immune or unknown function (encoding ribonucleoproteins etc.) and non-functional genes. 27. MHC class I, MHC class II, MHC class III MHC CLASS I - integral membrane proteins, expressed in number of about 104-105 molecules/cell - present on the surface of all nucleated cells; - absent on red blood cells and - limited distribution on spermatozoa, hepatocytes, neurons, endocrine glands, skeletal and smooth muscle General structure of MHC class I - is similar in all mammalian species, despite the enormous variability of the sequence of amino acids. MHC cl. I consists of 2 chains bonded monovalent : heavy chain with 3 domains, called α1, α2 and α3; light chain, called β2 microglobulin. -Heavy chain – consists of 3 segments: - extra membrane segment - 3 domains (α1, α2 and α3), with 90 AA each; - transmembrane segment - 40 AA; - cytoplasmic segment - 30 AA. Between α1 and α2 there is an area for presenting the antigen, called antigen binding cleft, this locus has exact size that allows binding of an antigenic determinant (epitope), with a size required for the transmission of antigenic information to T cells. Antigens linked to the major histocompatibility complex molecules, are very small and – about 5-10 amino acids. MHC CLASS II - heterodimer of the membrane, of glycoprotein nature, consisting of two polypeptide chains, α and β, joined by non-covalent bonds - Each chain has 3 regions: - extramembrane - 2 domains (α1, α2 and β1, β2); transmembrane 20-25 AA; cytoplasmic 8-10 AA. Between α1 and β1 segments there are created a place - antigen binding site. The difference between the antigen binding site of MHC class II and MHC class I - the binding site is open at both ends in MHC class II molecules, which allows the accommodation of antigenic peptides with different lengths which end above antigen binding site (up to 20 AA) The number of MHC molecules on the cell surface greatly increases also as a result of the action of cytokine, and, in particular of interferon. Out of antigen-presenting cells, the following do have MHC class II molecules: Monocytes, Macrophages, B cells, Langerhans cells, Dendritic cells. MHC CLASS III MOLECULES MHC class III molecules are not really a part of the MHC, being encoded by genes interspersed between the genes of this complex Are proteins that are not linked to the cell membrane and do not have a role in antigen presentation to T cells. However, a number of these molecules have a role in immune processes The most important MHC class III molecules are components of complement (C2, C4 and B factor); cytokines (tumor necrosis factor α and β), two heat shock proteins, one hydrolase. 28. MHC – functions MHC FUNCTIONS- ANTIGEN PRESENTATION 1Antigen transport. After phagocytosis and processing (cleavage) of foreign antigens, MHC molecules are produced within the cell, and then they move to the cell surface carrying antigenic determinants (epitopes). 2Antigen presentation. The main role of MHC molecules is to present antigen to specific cells of the immune system.T cell can only be activated by antigenic peptides that are presented together with MHC molecules, on the surface of antigen-presenting cells or target cells.MHC molecules present peptide of small size - MHC class I molecules: primarily present endogenous peptides.They have major role in the presentation of tumor antigens and antigenic peptides of some microorganisms with intracellular habitat (viruses, rickettsiae, mycoplasmas etc.) - MHC class II molecules: primarily present exogenous peptides (peptides resulted after the phagocytosis and processing of foreign antigens in antigen presenting cells). 3Genetic restriction.Cells cooperation in generation of both cellular and the humoral immune response is governed by MHC complex.This control is called genetic restriction because it allows a restrictive interaction between cells expressing MHC molecules and cells expressing complementary TCR, MHC molecules expression being genetically determined. 4Graft rejection.Graft rejection is an immune response which always possesses attributes of Specificity, Memory, Recognition (of self or non-self).The main mechanism involved in graft rejection is a cellular immune response that is initiated mainly by MHC molecules expressed on the surface of grafted cells Central role in graft rejection is held by T cells, being involved both helper and cytotoxic T limphocytes. Transplantation is defined as the transfer of cells, tissues or organs from one individual to another.It is already known that organ transplantation between two genetically different individuals is followed by rejection of that organ (the function of the organ is lost). 29. Cytokines (definition, classification); Interleukins (IL); Interferons (IFN) Cytokines - soluble mediators of the immune response, involved in differentiation and proliferation of cell lines, that influence the motility and intervene in the inflammatory response by specific action on specific target cells. In chemical terms - protein or glycoprotein molecules synthesized and secreted by a variety of lymphoid and nonlymphoid cells. Classifications: lymphokines (produced by lymphocytes); monokines (produced by monocytes and macrophages). Cytokines are produced by lymphocytes. Cytokines act through cellular receptors, activating or inhibiting the cell functions. Usually their concentrations in the serum is very low, due to synthesis maintained at a low level, when the cells are unstimulated and, rapid inactivation and elimination from the blood. INTERLEUKINS (ILs) biologically active molecular mediators that in chemically terms are soluble proteins or glycoproteins. Initiate, maintain, enhance or block the activation, growth, proliferation and effector functions of T or B cells. Interleukins are costimulatory molecules. In the absence of interleukin costimulation, the antigen alone does not activate lymphocytes or other cells. Interleukins are capable to activate, regardless the presence / absence of the antigens, the majority of the cellular effectors of the immune system. INTERFERONS (IFNs) belong to the network of immune-modulating cytokines, contribute to the anti-viral defense, respective "interfere" with viral infection, have cellular antiproliferative and antitumor activity. IFN are glycoproteins, are destroyed at 60°C in about one hour.Their synthesis are induced especially by viruses.Currently there are known 5 types of interferons with different physicochemical properties. Classifications of IFNs: - type1 (IFN-α, IFN-β, IFN tau, IFN omega);- type 2 (IFN-γ) IFN-α synthesized by leucocytes (white blood cells). IFN-β It is produced mainly by fibroblasts, which is why it is called fibroblast interferon. IFN-γ known as immune interferon synthesized lymphocytes, and by the NK cells. IFN-tau isolated from sheep,cow,goat,humans.It roles is to protect the corpus luteum during pregnancy. Biological actions of interferons They have: antiviral effects, have antiproliferative effect, stimulate the proliferation of activated B cells and immunoglobulin synthesis, increase MHC class I molecule expression and inhibit the expression of MHC class II molecules, stimulates natural-killer cell activation, activator factor of monocytes-macrophages, destruction of intracellular parasites, inhibits the proliferation of various types of tumors; 30. Tumor Necrosis Factors (TNF); Colony Stimulating factors (CSF). TUMOR NECROSIS FACTORS (TNFs) -TNFs are members of the cytokines family with an important role in the inflammatory response to pathogens (bacteria, viruses, parasites) and anti-tumor activity. -This molecule exerting an antitumor activity. -TNF group includes 3 molecular populations:TNF-α(cachectin);TNF-β(lymfotoxin α);lymfotoxin β. -Main sources of TNF-α: monocytes and macrophages; activated T cells, B cells, NK cells. -Minor sources of TNF-α: neutrophils; mast cells; keratinocytes, endothelial cells smooth muscle cells. ; -Sources of TNF-β – ONLY lymphocytes - Biologic activity of TNF-α acts both on the immune system cells and on the other cells that are not part of this system has the following effects: 1. Antitumor effects: TNF produce tumor necrosis, their action in most cases is not due a direct cytotoxic effect on tumor cells, but due to bleeding and hypoxia in the tumor. 2. Immunological effects:TNF-α is a key mediator of the immune response;TNF-α activates macrophage differentiation, chemotaxis, expression of adhesion molecule, activation and the production of interleukins; TNF-α increases the cytocide capacity of macrophages, and the nonspecific immunity, by activating neutrophils, increases the expression of MHC class I molecules,stimulates cytotoxicity against virus infected cells and tumor cells, activation of T cells, stimulates the proliferation and differentiation of activated B cells. COLONY-STIMULATING FACTORS (CSFs) Called also hematopoiesis stimulating factors.Molecules synthesized by bone marrow stromal cells and other cells activated by IL, TNF and other cytokines 1. Granulocyte colony-stimulating factor (G-CSF)is produced by activated macrophages, which are the main cellular source,is an important regulator of differentiation and proliferation of myeloid precursor cells to granulocyte line and stimulates granulocytes maturation,it is enabled the production in other cell types also: fibroblasts, endothelial cells and bone marrow stromal cells. 2. Granulocyte-macrophage colony-stimulating factor (GM-CSF)is one of the first of hematopoiesis regulatory cytokines.Its secretion is induced in the presence of stimulating factors (cytokines, mitogens-inflammatory agents) which activate T and B cells, monocytes, macrophages, fibroblasts and endothelial cells. It is the main stimulator of granulocyte colony proliferation, of monocyte/macrophages, and eosinophils in the blood marrow, stimulates effector functions of granulocytes and mature macrophages which include:phagocytosis, chemoattraction and antibody dependent cellular cytotoxicity. 3. Macrophage colony-stimulating factor (M-CSF)is a member of the hematopoietic cytokines family that stimulate the formation of colonies of mature myeloid cells, monocytes and macrophages, from myeloid precursors. Stimulate the proliferation, viability, differentiation and functions of mature macrophages. It is synthesized by a variety of cell types: macrophages, endothelial cells, fibroblasts, bone marrow stromal cells, osteoblasts, thymic epithelial cells and keratinocytes. 31. General properties of cytokines Cytokines - soluble mediators of the immune response, involved in differentiation and proliferation of cell lines, that influence the motility and intervene in the inflammatory response by specific action on specific target cells. In chemical terms - protein or glycoprotein molecules synthesized and secreted by a variety of lymphoid and nonlymphoid cells. Classifications: lymphokines (produced by lymphocytes); monokines (produced by monocytes and macrophages). Cytokines are produced by lymphocytes. Cytokines act through cellular receptors, activating or inhibiting the cell functions. Usually their concentrations in the serum is very low, due to synthesis maintained at a low level, when the cells are unstimulated and, rapid inactivation and elimination from the blood. Cytokines are produced by lymphocytes -Cytokines act through cellular receptors, activating or inhibiting the cell functions -Usually their concentrations in the serum is very low, due to synthesis maintained at a low level, when the cells are unstimulated and,rapid inactivation and elimination from the blood. Cytokines classification: - interleukins (IL-1 to IL-39) - mediators which provide communication between different populations of leukocytes; - interferons (IFNs); - tumor necrosis factors (TNFs); - colony stimulating factors (CSFs); - chemokines. 32. Immune response: definition, phases, effectors The immune response is how the body recognizes and defends itself against microorganisms and substances that appear foreign and harmful.The foreign microorganisms / substances are neutralized, destroyed and removed from the body through effectors and specific mechanisms which constitute the immune response Phases:the immune response consist in 3 phases: 1.The initial phase includes the following events :antigen enters in the body the body and binds on specific receptors on the lymphocyte membrane, in this stage takes place the recognition of foreign antigen, the internalization, processing and presentation of the antigen in a manner accessible to other cells. 2. The second stage is the most complex and comprises:receiving information about the nature of the antigen and initiation of biochemical reactions cascades, completed by cell proliferation and activation, with protein synthesis (immunoglobulins). 3. In the third phase occurs the neutralization of antigen and the activation of control mechanisms that will bring all the immune system in the initial stage of "calm“, but with an increased number of antibody molecules or memory T and B cells that are ready to react after the contact with the same antigen. Effectors of the immune response -Cells that receive the antigenic information are T lymphocytes in most cases, whether the final answer will be cellular or humoral type. The difference is that in the case of the cellular immune response remain engaged only T cells, while in the case of the humoral immune response, T cells transmits the antigenic information to B lymphocytes -Initiation of cellular or humoral immune response is the result of interaction of antigen with antigen-reactive cells. This interaction occurs on the cell membrane and is determined by specific structures called antigen receptors, able to recognize and bind the antigen -Antigen reactive cells belong both to T and B cell type. Each T or B cell is unipotent, i.e. has receptors for only one type of antigenic determinant -For this reason, at the entry of an antigen into the body, only cells which have specific receptors for the given antigen will be stimulated, will proliferate and differentiate into effector cells -The first contact with an antigen results not only in appearance of effector T and B cells, but also of memory T and B cells. -Memory T and B cells are able to maintain "immunological memory" and, after the second contact with the same antigen, they are quickly mobilized, proliferate and respond by increased synthesis of immune effectors. SUBJECTS 33-43 33-Cell-mediated immune response Definition: Cell-mediated immunity is the response to various etiological agents, which involves in a dominant way the cells of the immune system and the molecules produced by these cells Principles: installation of immunologic memory after primary stimulation and intense and rapid reaction after the second contact with the antigen responsible for primary stimulation CELLS participating in the cellular immune response are: 1. Antigen-presenting cells – APC: Monocytes, Macrophages, Langerhans cells, Dendritic cells and B cells Role: Recognize and retain foreign antigens, and some of them (macrophages and B cells), even to process antigens. After recognition and processing; all cells in this group, present antigens on the surface associated with MHC molecules 2. T lymphocytes: all populations of T cells, and in particular cytotoxic T cells. Role: They receive antigenic information, and after that become active and determine the inhitiation and development of cellular response. Cytotoxic T cells, are able to destroy tumor or virally infected cells and even allogeneic non self cells. 3. NK lymphocytes (natural killer) Role: During a typical viral infection, NK cells are activated by IFNα and β. Consequently, are developed a type NK cytotoxic response in the first three days of infection, which selectively destroys the viral infected cells and prevent propagation of the infection. MOLECULES participating in the cells immune response are: 1. Antigens: The main stimulating factor with a determinant role in initiating the immune response 2. Cytokines: They are intermediary messengers which ensure cooperation between cells. The most important are interleukins, in particular IL-1 and IL-2 ,the interferon group, IFNγ, which has an important immunoregulatory role and TNF. PHASES OF CELLULAR MEDIATED IMMUNITY 1. Recognition of the antigen by the antigen presenting cells and processing of the Antigen (presentation through MHC class II molecules to T helper cells) 2. Activation of T cells: Antigenic information is taken up, first by the T helper cells, but also by the other T cells populations types. It leads to proliferation and secretion of specific proteins (cytokines). TWO Types of T lymphocytes are formed: àEffector T cells àMemory T cells that will intervene in defense after a subsequent contact with the same antigen. 3. Effector phase : Destruction of the antigen. The elimination of the target antigen can occur through àDirect cytotoxicity: direct attack of cytotoxic cells (The cells involved are: Natural killer and cytotoxic àAntibody-dependent cell-mediated cytotoxicity: Involves nonspecific cells that attack target cells using immunoglobulins already attached on the surface of the target. (The cells involved are: NK cells, monocytes and macrophages, neutrophils and eosinophils) àDelayed hypersensitivity:Inflammatory reaction. The cells involved are subpopulation of T cells. 34.ANTIBODY-MEDIATED IMMUNE RESPONSE/ Humoral IR Definition: The activity of B cells and their progeny, which produce circulating antibodies in response to the presence of a foreign substance and recognize the substance upon renewed exposure. Synthesis of antibodies to most antigens requires intervention of helper T cells. CELLS involved in humoral immune response are: àCell cooperation is of particular importance: Antigen-presenting cells and T cells, together with B cells MOLECULES involved in humoral immune response are: 1. Antigen molecules: which in this immune response, are of two types: l T-independent antigens: Received directly by B cells, without intervention of T cells l T-dependent antigens: Activate B cells. l Receptor molecules: BCR, represented by the membrane immunoglobulins (IgM and IgD classes). l Molecules of the major histocompatibility complex class II l CD molecules: CDs with receiver role for complement and immunoglobulins l Cytokines: Interleukins and interferons l Adhesion molecules PHASES OF THE HUMORAL IMMUNE RESPONSE Study of humoral immune response focuses mainly on B cells ontogenesis, which comprises two successive stages: 1. Antigen independent stage: Represented by the differentiation in the bone marrow from haematopoietic stem cell, followed by the development of mature resting B cells, equipped with all markers and mechanisms which allow them to respond to antigenic stimulation. 2. Antigen dependent stage: Activation, proliferation and differentiation of mature B cells in the presence of specific antigen (in peripheral lymphoid organs and tissues). àB cells act in the activation phase as an antigen presenting cell. Then, helper T lymphocytes interact through TCR with the complex antigen - MHC class II molecules of B cells, and become activated cells, expressing new surface markers and producing cytokines àActivation of antigen-specific helper T cells generates an activator signals for B cells,which differentiate in l Plasma cells l Memory B cells 3. Effector phase consist in: l Neutralization of toxins and other substances l Microorganisms destruction and destruction of certain cells 35. Primary and secondary immune response l The immune response, primary and secondary, involves both cell mediated antibody mechanisms. l They are initiated sequentially, by the first contact between the immune system and the antigen (primary response) and the further contacts (secondary response) of the immune system with the same antigen PRIMARY IMMUNE RESPONSE It starts with the first contact of the immune system with the antigen, and evolves in four successive stages, with the production of two types of immunoglobulins, namely IgM and IgG. 1. Lag Phase l Can be named “period of incubation” l Activated B cells are differentiating into plasma cells BUT l The level of antibodies is undetectable l 2-3 days, but often is longer – up to 14 days l At the end of this phase begins the antibody synthesis, first IgM in a larger quantity, then IgG in a smaller amount 2. Log Phase l Rapid increase in the concentration of antibodies to a possible maximum level depending on the antigen l Maximum titer is reached in: à4-5 days for red blood cells à9-10 days for soluble proteins à 3 months for toxoids 3. Plateau Phase l Antibody concentration is maintained without significant changes at a constant level (maximum level) l 4-6 weeks 4. Decline Phase l Gradual decrease in the concentration of antibody, initially slower, then fast and steadily SECONDARY IMMUNE RESPONSE Appears at the second contact of the immune system with the same antigen and is initiated by the specific memory B cells generated at the first contact Differences between secondary and primary immune response àSecondary immune response: l Requires a lower dose of antigen for production of antibodies l It is initiated/triggered very quickly - within a few days the concentration of antibodies is higher than in the primary response l Antigens that initiate the secondary immune response are strictly T cellsdependent l Synthesis of the antibodies is prolonged and antibodies persist longer, so, in many situations, secondary immune response offer a lifetime protection l Antibodies produced belong to IgG l It can be repeated several times and each time is occurs more quicker and more intense. Qualitative and quantitative differences between the two types of immune response are due to the phenomenon of "immunological memory". l Memory cells (one precursor cell can also produce 1000 daughter cels) "remember" that they have been in contact with the respective antigen, and will respond by increasing production of antibodies. 36.Regulatory mechanisms of the immune response – regulation through antigen and antibodies Immune reactions are controlled by a complex set of mechanisms, of at least equal complexity to those underlying their initiation and expression. Through these mechanisms, the immune response is maintained between normal limits. 1. Regulation through Antigen (Ag) Antigen molecule is first regulator of immune reactions and is the basis for the onset of these reactions, but it can also induce a state of tolerance or suppression. l The regulatory potential of the antigenic molecule depends on: l Physical form l Route of administration l Dose l Accessibility of the receptors on the cell membrane, etc. PROCESS involved: l Initially, the antigen is in excess, favoring the synthesis of IgM, and later the switching to IgG. When IgG reaches the excess, will intervene in the negative control of the signals received by T lymphocytes from the Fc receptor l Repeating the contact with the same antigen, in the case of a high concentration of antibody, it is not followed by the stimulation of the immune response. l Elimination of the antigen is an important mechanism for immuno regulation. l When the antibodies concentration reach an optimum level, they block the antigenic determinants, antigen-antibody complex formed neutralizing the stimulatory function of the antigen. Then complexes are quickly eliminated by phagocytosis 2. Regulation through Antibodies (Ab) Antibodies are, in particular, inhibitors of the immune response, as their presence in the circulation can result in the neutralization of antigens, so that antigens do not cause an immune response PROCESSES involved: Circulating immune complexes are acting in two ways, as stimulants and inhibitors of the immune response: l Amplification is achieved by binding of immune complexes to an antigen presenting cells, followed by the presentation of the antigen, activation of T cells and, later, of B cells l Inhibition occurs after the the binding of immune complexes to Fc and BCR receptors. 37. Regulatory mechanisms of the immune response – regulation through cytokines and membrane receptors 3. Regulation through cytokines Cytokines are the main regulatory factors of the immune reactions, involved in determining both in activation and suppression. PROCESSES involved: l Cytokines, are biochemical messengers of the IR. They form a true network of biochemical messages that control proliferation, differentiation and cell functions. l Self regulation and self control: When large quantities of interleukin are produced (IL-1, IL-2, IL-4, IL-6, IL-8, etc.) higher than normal, they are able to inhibit their own synthesis, both through their own receptor expression and by activation of immunosuppresive hormones, such as progesterone or glucocorticoids. 4. Regulation through membrane receptors This regulation is done through Antigen receptor and/or Fc receptor A. Regulation through antigen receptors l The intensity of the humoral immune response is conditioned on a dynamic balance between l Amount of antigen l Antigen-antibody complexes l Antibody levels in circulation. l In the early phase of stimulation, when the antigen is in excess compared to the antibody molecules, stimulation of the cell will be achieved primarily through the membrane immunoglobulin receptor, followed by cellular activation. The highaffinity receptors become saturated, and antigenic determinants remain available also for low affinity receptor cells. l Being in large quantities, antibodies block the antigen epitopes, making them inaccessible to cellular receptors. The higher their activity, the stronger their blocking effect is, inhibiting the immune response. B. Regulation through Fc receptors l This receptor plays an important role in differentiation of B lymphocytes in cells with effector or memory function. l B cell need at least 2 signals to become plasma cell: l The antigen – through antigen receptor (BCR) l Soluble factor (called TRF) – secreted by helper T cells – which are able to bind Fc receptor l When the antigen is in excess, is primarily bound by the BCR on the B-cell surface, while the Fc receptor will bind the soluble factor TRF, achieving a true blockage B cell are oriented to effector activity, producing immunoglobulins (antibodies). l When the antibodies reach the excess level, a large number of antigen-antibody complexes are produced, and these complexes will bind the Fc receptor l Fc receptor cannot bind TR, and B cell is directed to memory function, and antibody synthesis stops 38. Cellular cooperation in the immune response Induction and expression of immune response involves not the recognition of antigen and the interaction of cell populations, especially lymphocytes and macrophages. àThe intensity of the immune response, expressed by synthesis of antibodies or activity of effector cells must be controlled so that it does not exceed the normal limits. 1.Lymphocyte T – lymphocyte B cooperation (T-B) àB cells - immunoglobulin-producing cells – become specific and effective only after their activation in the lymph nodes or other secondary lymphoid organs àTH lymphocytes (helper) play a crucial role in this process, and the molecular features of how these two types of cells interact are subject of a lot of recent research PROCESSES involved: l Foreign antigen enters the body àIt is engulfed and processed by APC , presented in association with MHC class 2à activation of helper T cellsàactivation of B cells à cells proliferate and differentiate into plasma cells and memory B cells l B cells can be also APC !!! Models proposed for T-B cells interaction A.Direct interaction between T and B cells l T cells are involved only passively in the transmission of antigenic information l In the case of antigens T cells-dependent l If helper T cells responsible for that antigen are absent (congenital disease or removed), B cells cannot become activated, and the immune response to that antigen is absent B. Direct interaction between T cells – Macrophages and B cells (T-M-B) l Macrophage is a mediator between T and B cell l Cooperation can be done simultaneously, the same macrophage binding both a T and B cells and presenting the antigen to both type of cells C. Indirect interaction between T cells, Macrophages and B cells (T-M-B) l Requires the intervention of soluble molecular mediators 2.Lymphocyte T – lymphocyte T cooperation (T-T) This type of cooperation was demonstrated in lymphocytes belonging to structurally and functionally distinct subpopulations PROCESSES involved: l T – T cooperation was detected in most of the immune processes in which T cells are involved such as, for example: l Cell-mediated cytotoxicity – between helper T cells and cytotoxic T cells l Between helper T cells and suppressor T lymphocytes l Cooperation between helper T cells and cytotoxic T cells 3.Macrophage - lymphocyte T - lymphocyte B (M-T-B) cooperation Macrophages do not have specific receptors for antigen, but have the capacity to express the antigen on the surface (in association with MHC molecules) and to present it to T and B cells, in the cooperation process. PROCESSES involved: l B and T lymphocytes, can not be stimulated by T-dependent antigens in the absence of macrophages l The activation of T cells depends on the ratio of T lymphocyte/macrophage. When the ratio is greater than five, there is T cell activation, and when that ratio is around one, no activation occurs. 4.Cooperation between antigen presenting cells (APC) In cellular cooperation process, there are also involved APC populations, which interact with each other for efficient antigen processing and presentation. Such cooperation may be sequential, being interested in the same time two or several cellular populations. PROCESSES involved: l These interactions were described for: l Macrophage - dendritic cells l Macrophage - lymphocyte B – lymphocyte T l Dendritic cells - lymphocytes B 39.Immunological tolerance – definition, classification Definition:Immunological tolerance is an active process of suppression or inactivation of effectors cells, which is manifested by the inability of the body to produce an immune response towards a given antigen. It consist in a lack of a specific immune response or poor response to a given antigen. Characteristics: l It is antigen-specific, because the body can react normally to other antigens. Classification: Depending on the origin of the antigen, immunological tolerance is classified into two major categories: 1. Natural tolerance (to self antigens, for example RBC antigens) 2. Acquired tolerance (to non-self antigens). According to the site where is taking place: 1. central 2. peripheral The following particular situations can be distinguished: A.Immunological tolerance to self antigens, which are in contact which the immune system; essential phenomenon that occurs during the ontogenesis of each individual. B.Tolerance to “hidden” organ or tissues, which has no contact with the immune system effectors in normal conditions (some eye structure, sperm, central nervous system, etc) C.State of natural tolerance to other antigens than self antigens due to the expression of genetic control on the immune response. D.Absence of immune response, thus "acceptance" of foreign antigens without initiating appropriate defense reactions in primary and secondary immunodeficiencies. E.Tolerance induced after inoculation of immunosuppressive products or applying other methods (irradiation, removal of lymphoid organs, blocking cell receptors etc.) F.Tolerance after administration of an antigen which cause the "paralysis" of the immune system to that antigen; the effects of antigen administration dependent on the quality of antigen, dose (high doses are the most effective in inducing tolerance), the number of inoculations, the age of the animal (fetuses and young animals are more prone to develop tolerance) etc. G.Tolerance found in some pathological conditions (infectious diseases, cancer, nutritional deficiencies, etc.). 40.Natural and acquired immunological tolerance Natural I.T. Definition: Lack of expression of the immune response to endogenous antigens (self/own) is known as natural immunological tolerance or self tolerance. It is recognized that every individual has a natural tolerance to all his components, which become antigenic if are administered or transplanted to another individual (same species or another species). The mechanisms that allow maintaining of tolerance to self antigens are: 1. Self antigen isolation from the immune system 2. Elimination of self reactive T cell clones during development (negative selection) 3. Absence of processing and presentation of self molecules 4. Activity of suppressor T cells Acquired I.T. Definition: The absence of immune response following administration of exogenous (non-self) immunogen is known as acquired immunological tolerance. (To cure autoimmune disease, or to facilitate organ transplantation) Installation of acquired tolerance depends on many factors, the most important being: 1. Age of the animal 2. Qualities and dose of the immunogen 3. Acquired immunological tolerance is installed usually in young animals. 4. Also is easier to induce tolerance to antigens that have a structure similar to host’s components. 5. The more complex the antigen is, the more difficult is to induce the tolerance for it. Classification depending on the dose of antigen: 1. High-dose tolerance: May be induced by inoculation of a single dose of concentrated antigen solution and dispersed in the presence of certain detergents. Once established, this tolerance can be maintained throughout adult life by repeated administration of the same antigen in low doses. 2. Low-dose tolerance: It was reported only in certain species (rat, human) who were treated with low doses of antigen (doses unable to induce an immune response) in neonatal life. After the installation of tolerance, the administration of the same antigen, in high doses, is not followed by installation of an immune response. INDIVIDUALS. l Embryos: In correlation with the development of the thymus and bursa of Fabricius (in birds l After birth / hatching l The interval when a foreign substance is not recognized as non self is called immunological immaturity period. (During this period the body does not have the ability to differentiate between own and foreign structures) !!!Important aspects of the acquired immunological tolerance are: l Animals tolerant to microorganisms are carriers of those microorganisms. l Tolerance = absence of immune response, thus serological test cannot detect carriers. l Carriers kept in collectivity = a permanent source for outbreaks. l Example of tolerance to microorganism – in brucellosis, avian leukosis, lymphocytic choriomeningitis in mice, etc. 41.Autoimmunity – general aspects The possibility that an individual’s immune system may react against autologous antigens and cause tissue injury was appreciated by immunologists since the moment when specificity of the immune response for foreign antigens was recognized. We now know that the key events in the development of autoimmunity are: l The recognition of self antigens by auto reactive lymphocytes l The activation of these cells to proliferate and differentiate into effector cells l The tissue injury caused by the effector cells and their products. General concepts: 1. Autoimmunity results from a failure or breakdown of the mechanisms normally responsible for maintaining self-tolerance in B cells, T cells or both. TOLERANCE to self antigens is normally maintained by: l Selection processes that prevent the maturation of some self antigen-specific lymphocytes and l Mechanisms that inactivate or delete self-reactive lymphocytes that do mature. l It is believed that failure of T cell tolerance is an important mechanism of autoimmune diseases. FAILURE of self-tolerance in T cells may result in autoimmune diseases in which the lesions are caused by: l Cell-mediated immune reactions l Autoantibody production because helper T cells are necessary for the production of high-affinity antibodies against protein antigens 2. The major factors that contribute to the development of autoimmunity are genetic susceptibility and environmental triggers, such as infections. l Susceptibility genes and infections contribute to the breakdown of selftolerance, and infections in tissues promote the influx of autoreactive lymphocytes and activation of these cells, resulting in tissue injury. l Infections and tissue injury may also alter the way in which self antigens are displayed to the immune system, leading to failure of self-tolerance. 3. Autoimmune diseases may be either systemic or organ specific l For example, the formation of circulating immune complexes consisting in self antigens and specific antibodies typically produces systemic diseases. l Autoantibodies or T cell responses against self antigens with restricted tissue distribution lead to organ-specific diseases 4. Various effector mechanisms are responsible for tissue injury in different autoimmune diseases l Epitope spreading: Autoimmune reactions initiated against a self antigen that injure tissues may result in the release and alterations of other tissue antigens, activation of lymphocytes specific for these other antigens, and exacerbation of the disease. It may explain why once an autoimmune disease has developed, it tends to be chronic and often progressive. 42.Systemic autoimmune diseases Definition: Systemic autoimmune diseases are characterized by formation of autoantibodies directed to a wide variety of self antigens including: Nucleic acids, Surface molecules, Intracellular proteins The most important systemic autoimmune diseases found in animals (especially in dogs and cats) are: Systemic lupus erythematosus, Rheumatoid artritis , Sjögren's syndrome. 1.SLE Definition: is characterized by production of antibodies directed toward various autoantigens and the presence of a high amount of circulating immune complexes. ETIOLOGY: The primary cause of SLE is not fully understood. Several factors are considered favorable: genetic factors; some environmental factors, especially UV light; viral infections; drug administration (e.g., contraceptives, anticonvulsants. penicillamine); vaccination; immunodeficiency disorders. PATHOGENESIS: There are several types of autoantibodies produced in SLE, as follows: anti-nuclear antibodies (ANA) directed towards cells nuclei; anti-double stranded DNA (dsDNA) antibodies (dsDNA); anti-Smith ribonucleoprotein antibodies, (Smith ribonucleoprotein is located in cell nucleus); anti-histones antibodies; anti-ribosomal proteins autoantibodies, etc. The autoantibodies are directed toward red blood cells, leukocites and platelets. Age/Breeds/Species SPECIFICITY: Breed (Dogs): German shepherd, Afghan Hounds, Beagle, Shetland Sheepdog, Old English Sheepdogs Age: Any age, but higher frequency in middle-aged dogs (mean age 6 years). Breed (Cats): SLE is very rare in cats, Himalayan, Persian and Siamese. CLINICAL SIGNS Most of the pets develop autoimmune hemolytic anemia, thrombocytopenia, symmetrical polyarthritis, and glomerulonephritis. The clinical signs associated are: Lethargy, Anorexia, Fever, Lymphadenopathy, skin lesions (alopecia, crusting, erythema, furunculosis, seborrhoea on: face, ears, limbs, abdomen, etc), Oral ulcers, Footpads ulcers, Recurrent shifting lameness,Polydipsia and polyuria,Pneumonia and pleuritis, etc. Diagnosis :Complete bood count (CBS) should be conducted to hightlight the anemia, neutrophilia and low platelet count. Urinalysis is made, to check for proteins and cells in the urine. Detection of anti-nuclear antibodies by indirect immunofluorescence .Treatment Treatment of SLE includes: Corticosteroids - prednisone, prednisolone, methylprednisolone. Other immunomodulating drugs: eg. azathioprine, cyclophosphamide chlorambucil sometimes in combination with corticosteroids. Levamisole. Aspirin has been beneficial in some canine cases .Vincristine has been used if thrombocytopenia is severe. Splenectomy. Management of secondary disease(renal failure). 2.Rheumatoid arthritis (RA)Definition:Rheumatoid arthritis is an autoimmune disease that affects the joints. The immune system fails to distinguish between self oriìoteins and non self proteins and produces antibodies that attack the cartilage surface, causing inflammation and pain. RAis a type of "erosive arthritis", which means that in addition to inflammation, cartilage and the bone in the joint surfaces are also destroyed. ETIOLOGY: The primary cause is not fully understood. Secondary causes: infection (possible with Canine distemper virus, Mycoplasma, Erysipelothrix and Borrelia), digestive system disease, cancer , genetic disorder. Age/Breeds/Specie SPECIFICITY: Canine RA is relatively rare and usually occurs in toy and small breed dogs, such as Miniature Poodles and Shetland Sheepdogs- 4 y.o.. However, it can affect dogs of all ages and breeds. PATHOGENESIS Development of autoantibodies towards IgG called RHEUMATOID FACTORS, is the main pathological process. CLINICAL SIGNS: Synovitis: lymphocytes in the synovial membrane; neutrophils in the joint fluid; The synovial membrane proliferates and eventually extends into the joint cavity; This synovial membrane consists in fibrous and vascular tissue and release enzymes (proteases) that erode the joint cartilage and ultimate the neighboring bone tissue. SYMPTOMS : Dogs with RA may present:chronic depression,anorexia, pyrexia, lameness – tends to be more severe after rest (e.g., immediately after waking in the morning), The RA affects usually peripheral joints, which show symmetrical swelling and stiffness.In severe cases – joints may fuse as a result of bony ankyloses. RADIOLOGICAL FINDINGS: Subchondral rarefaction, Cartilage erosion, Narrowing of joint space. DIAGNOSIS Lab exam: Agglutination reaction using sheep red blood cells coated with canine anti-sheep serum mixed with serum of suspected patient (for rheumatoid factor); Synovial fluid analysis - increased protein concentration and the presence of nucleated cells, and cytokines. TREATMENT RA is a chronic lifelong disease for which there is no known cure. There are a variety of measures which can lead to a significant improvement of the patient's condition. They are Non specific treatment, which aims to reduce the active, destructive inflammatory processes. Rest and physiotherapy are important aspects of treatment. Drugs used include: salicylates, corticosteroids ,nonsteroidal anti-inflammatory drugs,immunosuppressive drugs. 3.Sjögren's syndrome Definition: In this syndrome, antoantibodies attack salivary and lacrimal glands and lead to conjunctival dryness (keratoconjunctivitis sicca), mouth dryness. SIGNS: Gingivitis , Dental caries, Excessive thirst, It is often associated with RA, SLE, polymiositis and autoimmune thyroiditis. Dogs and Horses: Autoantibodies directed towards epithelial cells of the 3rd lid, lacrimal & salivary glands and pancreas, and these organs may be infiltrated with lymphocytes and other mononuclear cells. TREATMENT: Medicaments: Optimmune - an eye ointment that contains cyclosporine; corticosteroids; nonsteroidal anti-inflammatory drugs; immunosuppressive drugs. 43. Immune Response In Bacteriosis Antigenic structures of the bacteria Antigenic structures are different among bacteria species. Ag located in the following cell structure are: Cell wall: l Peptidoglycans (in Gram-positive bacteria) l Antigen O (polysaccharides, lipids and proteins in Gram-positive bacteria) l Glycolipids, mycolic acids, arabinogalactan and lipoarabinomanan in mycobacterium. Bacterial capsule: It consists in polysaccharides-(antigen K). Pili: include F antigens (from fimbriae). Flagella: Antigenic proteins (antigens H). Resistance and protective mechanisms Three main types of protection: 1. species resistance (genetically determined); 2. non-specific immune response; 3. specific immune response (adaptive). 2.Nonspecific defense mechanisms A.Physical and biochemical barriers: - skin (with participation of saturated and unsaturated fatty acids on the surface); - mucosa; - unfavorable pH (stomach); - antagonistic action of normal bacterial flora on the skin and mucosa. B.Complement, polymorphonuclear leukocytes, mast cells etc C. Enzymes (including lysozyme),basic proteins and peptides etc. The main processes for the protection of non-specific defense against bacterial pathogens are: - phagocytosis; - complement activation - mast cell degranulation and eliminating factors involved in inflammation; - blood clothing. 3.Specific (adaptive) defense It is achieved by participations of the effectors and mechanisms that compose the immune system. (Capture and presentation of the antigenic information, activation of selected cell clones,production of chemical mediators that provide cellular cooperation,antibody synthesis,neutralization and destruction of invading bacteria). Cells and molecules involved in defense against bacteria l The cells of the monocyte-macrophage system are activated by a number of bacterial products, including endotoxins, murein (from peptidoglycan) and polymerized carbohydrates. These cells ensure phagocytosis, cleavage of the complex antigens and presentation in simple form (epitopes) to effector cells of the adaptive response. l T lymphocytes have an important role in both cellular and humoral immune response. (TH and T cytotoxic cells) l B cells are essential effectors of the humoral immune response. Following their differentiation, they become plasma cells able to produce antibodies. l Antibodies. The intervention of antibodies is recognized in various stages of bacterial attack Protection strategies in bacteria Like other pathogens, bacteria possess properties and structures that ensure their protection against the body’s defense. The most important mechanisms by which bacteria are operating in order to avoid the immune system are: l Presence of capsule, cell wall of Mycobacterium l Production of soluble factors with different actions: suppression of chemotaxis, inhibition of phagocytosis, destruction of macrophages and lymphocytes l Resistance to complement-mediated lysis l Release of proteolytic enzymes that cleave some immunoglobulins; l Antigenic variability. S 44. IMMUNE RESPONSE TO VIRAL INFECTIONS Etiological agents of viral infections Definition: Viruses are microorganisms able to multiply only in host cells. Their life cycle include: attachment to cell receptors, entering in the cytoplasm, replication of viral components and the assembly of the new viruses within the host cell, release from host cells (with or without cell lysis). Virions:(entire virus particles) consist of: viral genome, capsid, and viral envelope. Genome and capsid form the nucleocapsid of the virus. Capsid.:The capsid is composed of proteins that cover and protect the viral genome.. The viral genome:consists of a linear or circular DNA or RNA molecule Viral antigens:The envelope (present in some viruses) include a lipid bilayer, glycoproteins and protein Prions: They are class of subviral infectious agents, consisting of proteins, lacking of nucleic acids. The immune response is totally different and largely unknown in infections caused by prions. They are called slow viruses, because they cause disease of progressive evolution, usually fatal, in which the lesions of the central nervous system are predominant. They do not induce alterations in the immune status and detectable cellular or humoral response. Diseases caused by prions are among others (example bovine spongiform encephalopathy,Parkinson-Guam, Alzheimer). Protective mechanisms in viral infections Antiviral defense of the body is ensured both by specific and nonspecific factors. Body resistance and effectiveness of the immune response are dependent on the morphological and functional integrity of the immune system, which is significantly influenced by: nutrition, microclimate, stress, certain drugs, the evolution of other diseases, etc. Very virulent viruses are pathogenic for animals, regardless their immune status. Immune response to viral infection Participation of a large number of cellular and molecular effectors. The main mechanisms involved in the defense are: Early phase of the infection, where the viral replication occurs in the epithelia and lymph nodes, Effectors are interferon, secretory IgA and NK cells Viremic phase - antibodies; Virus multiplication in target organs - activated complement, activated cells, Ab, IFs Destruction of infected cells phase: activated cytotoxic cells Effectors involved Antiviral defense mechanisms are T-dependent, even if the final expression of the immune response is antibodies synthesis; many subpopulations of T cells (T-helper and cytotoxic T cells) Cytotoxic cells include lymphocytes, macrophages and neutrophils that possess Fc receptors. NK cells (natural killer) are also involved, and play an important role in some viral infections Antibodies neutralize viruses and block cell infection, stimulate phagocytosis by macrophages. Interferons are molecular mediators whose synthesis is induced in many viral infections. They work as inhibition of virus replication and activation of NK cells, macrophages and B lymphocytes etc. Tumor necrosis factors (TNF) have also antiviral effects, similar to those of IFN. Complement plays an important role in preventing and limiting viral infections. It is involved in lysis of viruses and infected cells and mediates several immune mechanisms. Defense strategies described in viruses Avoiding the immune response by the virus is realized in several ways, including: - antigenic variability; - viral adaptation so that antibodies do not neutralize the virions; - persistence of viruses in host cells (latent viral forms, which are not subject of immune response). S 45. IMMUNE RESPONSE TO PARASITES Etiologic agents of parasitic diseases represent foreign elements that immune cells recognize as non-self. Further, they trigger the development of immune response mechanisms that assures the body’s nonspecific or specific defense. Immunological effectors and mechanisms involved in the immune response to parasites Activation of: T cells, cytotoxic cells action,complement activation; Increasing the number of eosinophils, T and B cells, activated macrophages/monocytes and all antigen presenting cells; Specific immunoglobulin synthesis, in particular IgG and IgE; Antibodies and complement functions are: direct destruction of helminths or protozoa and phagocytosis mediated by antibodies Specifically for the Ab: They participate both in cellular and humoral mechanisms. The main action of the antibody is applied directly to parasites, by binding the surface antigens and damaging them. Also, antibodies mediate complement lysis and cellular cytotoxicity. Eosinophils and Mast cells: They are activated by soluble mediators released by parasites, and by lymphokines (cytokines) secreted by activated T cells. Both cell populations are involved in triggering the inflammatory reactions. Immune reactions in the intestinal mucosa Mechanism - “acquisition” of the antigens and its presentation to competent cells by antigen presenting cells; - pronounced hyperplasia of immune cell populations and their specific activation; - T and B cell activation - secretion of cytokines, respectively antibodies - stimulation of mast cells(release of inflammation mediators) and eosinophils Defense strategies described in parasites Factors: Parasites size. Great size variability Biologic cycle and location: Complex Bio-cycle, makes the immune system to react against an enormous number of antigens, with sequential mobilization of defense mechanisms – cellular and humoral. Enhanced motility of most parasites makes immunological recognition more difficult, especially when parasites change their locations, populating various organs and tissues during their biological cycle. Also, the immune response is poorly expressed against parasites located in digestive tract. The capacity of multiplication: It is very variable, depending on species of parasites. Rapid multiplication of some of them (especially protozoa) is an important mean of defense against the immune system. Avoiding the immunity effectors: Most protozoa and helminths have the ability to diminish and even cancel, their recognition by the immune system. Also, most of the parasites (the large one especially) have very complex antigenic structure, and repetitive antigenic determinants situated on the surface, which induce activation of a large number of cell clones and synthesis of antibodies in high concentrations, but with low affinity. S 46. IMMUNE RESPONSE TO TUMORS Neoplastic diseases are characterized by uncontrolled development of tumor cells that induce lesions and dysfunctions of the invaded organs. Tumor cells do not respond to the regulatory signals, have an independent and invasive development, are capable form metastases, and in generally express different antigens than the normal cells. Immune response can be specific and non specific, its efficiency is affected by several factors as: - General characteristics of the tumors, their etiology and the expression of the new antigens - Immune status (the capacity of recognition and response of the immune system); - endogenous and exogenous factors Tumor antigens: The immune response to tumors is triggered when the effector cells recognize the tumor antigens. The absence of "foreign" antigens will determine the characteristic evolution to malignancy, to which the immune system does not develop an appropriate response. A.Classification of Tumor Ag (Ags): 1. Ags strictly limited to a single tumor (developed in one individual) 2. Ags common to tumor cells in many individuals 3. Ags present on many types of normal or malignant cells. B.Another classification includes (depending on their association): 1. Tumor specific Ags (unique, individual) found only on tumor cells 2.Tumor-associated Ags (common, non-specific), present both on the tumor cells and normal cells C.Another class. (Depending on the causes of their expression and molecular structures involved -Viral Ag. Cells infected with oncogenic viruses express on the surface two types of neoantigens: (V o T Ags) -Embryonic Ags (oncofetal Ags) - molecules synthesized during embryogenesis but absent in normal adult humans or animals. -Specific transplantation antigens (TATA – Tumor-Associated Transplantation Antigens) detected mainly in tumors induced by the action of chemical agents. -Idiotypic determinants (unique) - expression of antigenic specificity of each cell; even if a substance is capable to induce two different primary tumors in the same animal, they will not have the same antigens. Specific immune response (adaptive immune response) In neoplastic diseases there are four essential factors of which depends the existence (expression) of an appropriate/effective response of the immune effectors: (1) presence of the tumor antigens (Ags); (2) Ags recognition by specialized cell receptors; (3) activation of cells critical for adaptive immune response; (4) lack of the tolerance phenomenon to tumor antigens. Cells involved in the adaptive immune response are: T cells, B cells, antigen-presenting cells (APC), macrophages, cytotoxic cells (cytotoxic T cells, K – killer cells and NK – natural killer cells). Participating molecules are: Ab, cytokines (IL, TNF and IFN), complement, molecules involved in cytotoxic phenomenon; amolecular processes of T and B cell activation and molecular mechanisms of phagocytic cells.. Adaptive mechanisms: Humoral immunity result effector cells and molecules: -Cytotoxic T lymphocytes that recognize and destroy directly the target cells; K cells (killer), participating in the mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC) -NK cells (natural killer) whose function is Ag-nonspecific, but these cells are stimulated during the adaptive immune response -Macrophages and polymorphonuclear leukocytes. Non-specific immune response Cells that have the ability to kill tumor cells spontaneously. These cells are: - Macrophages (MF) recognize the tumor antigens by receptors for neoantigens or by Fc receptors which bind the antibodies attached on the surface antigens of the tumor cells. MF destroy target cells following the release of cytolytic/cytostatic (cytokines, enzymes, reactive oxygen species) factors. - Neutrophils act in the framework of inflammatory mechanisms with the help of lytic molecules released. - NK cells recognize the antigenic determinants present on target cells, including tumor cells. Defense strategies: In developing an insufficient/ineffective immune response, the factors involved respectively of tumor cells are: - selection and development of cells resistant to immune effectors; e - existence of different antigenic structures depending on the cell cycle or mutations that occur during successive cell proliferations; - reduced expression of tumor antigens; - tumor antigens - synthesis of blocking factors - development of tumors in lymphoid organs and/or cells involved in the immune response. S 47. HYPERSENSITIVITY – DEFINITION, CLASSIFICATION, MECHANISMS Definition: Represents a deformed reaction of the immunological system, which causes disturbances that may extend from minor inconvenience to a fatal disease. It include immune processes characterized by significant changes in the way the body responds to an antigen. These processes may be harmful during repeated recognition of an antigen that is in excess or which produces intense responses. Allergic phenomenon conditions: 1.Prior sensitization of the organism with the antigen; 2.Existence of a latency period between the time of sensitization and the manifestation of sensitivity; 3.Installation of allergic condition, with development of humoral or cellular effectors, like in the active immunization; the body produces immune effectors that are strictly specific for the antigen (allergen) Differences between Normal I.R. and Hypersensitivity: Terminology: At the beginning of this century, Immunitythe body's reaction to stimuli called "antigens" Anaphylaxis and allergy reactions to "allergens". After that it was introduced the term "atopy", which defines an abnormal intensity reaction to a normal antigenic stimulus. However these terms - hypersensitivity, allergy, atopy - are synonymous. Gell and Coomb Classification: It takes into account, the rate of occurrence of clinical events triggered by contact with the allergen and the immune effectors responsible for installing these events. type I immediate hypersensitivity - anaphylactic type; type II immediate hypersensitivity - cytotoxic-cytolytic type; type III immediate hypersensitivity - produced by immune complexes; type IV delayed hypersensitivity - cell-mediated type. Added to these, there is also the stimulatory hypersensitivity (type V). Another Classification: Depending on the main participant effectors and the time necessary for clinicalanatomical expression Immediate, with molecular effectors (types I, II, III and V); Delayed, with cellular effectors (type IV). Not all allergic diseases can be strictly classified into one of the five types, some being the result of the combined action of two or even three types of allergy. S 48. TYPE I HYPERSENSITIVITY Definition:Type I hypersensitivity includes the most intense clinical manifestations of all hypersensitivity mechanisms. This type hypersensitivity was described in 1902 by Richet and Portier Also called “Immediate hypersensitivity”, allergy or anaphylaxis (ana = no/absence; philaxia = protection), which is its systemic form. The mechanism of type I hypersensitivity development: 1.The first contact of the body with a heterologous antigen (allergen) followed by the synthesis of IgE antibodies; 2.Attaching IgE to the receptors FcεRI and FcεRII of mast cells and/or basophils; 3.A new contact with the allergen and its binding to two adjacent IgE molecules on the surface of mast cells and/or basophils; 4.Degranulation of mast cells and/or basophils; the granules content is released in surrounding tissue and in general circulation (blood); the chemical mediators (vasoactive amines - leukotrienes, thromboxanes, etc.) are released mainly by mast cells located in mucosa; Predisposing factors: Vasoactive amines such as histamine, serotonin, heparin, bradykinin (anaphylatoxins) cause capillary vasodilatation, increased permeability of their walls, smooth muscle contraction, increased secretion of mucus and other disorders that characterize the anaphylactic shock. 5.Development of the symptoms and lesions characteristic to different forms of type I hypersensitivity. The main biological active mediators released from granules are: Proteases: trypase; Vasoactive factor; Chemoattractants; Anticoagulants; Also, after activation of mast cells: kallikreins; prostaglandins; thromboxane; platelet activating factor. Etiology: Antigens, such as: Animal proteins (serum proteins, albumin, etc.) or bacterial antigens, various enzymes, vaccines of very diverse origins.Anaphylactic phenomena develop after intravenous administration of antigen, causing the general phenomena of anaphylactic shock (general anaphylaxis) or by local administration, which leads to localized symptoms (local anaphylaxis). CLINICAL MANIFESTATIONS: Systemic anaphylaxis: The most striking phenomenon that characterizes type I hypersensitivity is the shock, also known as anaphylactic shock. Most of the times is induced (caused) and is rarely spontaneous. The most frequent and violent, the shock is triggered after intravenous inoculation of allergen, followed by massive degranulation of mast cells and basophils. The clinical manifestations vary from one species to another as follows: - In cattle, it is manifested by systemic hypotension and pulmonary hypertension.Dyspnea, Tachycardia, diarrhea are present; animals remain in the lateral / sterno-abdominal decubitus or die by collapse. - In sheep, horses, and pigs the target organ are the lungs, so prevalent clinical signs are pulmonary (cough, dyspnea) and digestive (diarrhea). - In dogs, the most affected organ, unlike other species, is the liver, especially liver vessels. Animals exhibit restlessness, excitation phenomena, are following digestive disorders (vomiting, diarrhea) then hypersalivation, swollen nose, cyanosis of mucous membranes, pruritus. In the final stage, comatose state, and the animals die within an hour, sometimes death is preceded by convulsions. - In cats, the target organs are the lungs. Because facial pruritus, animals are scratching and scratching in the head zone; then hypersalivation, dyspnea, impaired balance, and collapse. Specific allergic states Food allergy is a syndrome diagnosed in humans, horses, pigs, dogs and rarely in cattle and cats. Clinical expression of the dominant form of food allergy is urticaria. Laminitis (or pododermatitis aseptica diffusa) is the aseptic inflammation of the laminae of the foot - the soft tissue structures that attach the coffin or pedal bone (3rd phalanx) of the foot to the hoof wall. The disease can be found in all ungulates, including wildlife, and was noticed especially in cattle and horses. Milk allergy is the reaction to alpha-casein. Is commonly found in cows, but was described also in females of other species, like mares or bitches. Allergic rhinitis is more common in humans, but there are also reports of cases of dogs, pigs, cattle and horses. Allergens responsible for this condition are: in dog - mites, in pigs - dust released from the protein-rich fodder, and in cattle - pollen and dust. Allergic dermatitis is the most common canine allergy. Very rarely has been reported in cattle and horses. Hypersensitivity is induced by allergens of ectoparasites (fleas, mites) and other dusts such as human dandruff, house dust, spores of fungi, helminthes, etc. The main symptoms are: licking and biting the paws, rubbing the head, alopecia, sometimes blisters. DIAGNOSIS METHODS -Direct skin testPrausnitz-Küstner test (PK). The test consists of intradermal inoculation of serum taken from an animal to be tested in a normal animal. After 24-48 hours, in the same place, the allergen is inoculated. The positive reaction develops rapidly (1-2 minutes) and consists in intense erythema with edema. -Schultz-Dale test is an experimental in vitro test for hypersensitivity in which a strip of smooth muscle is removed from a sensitized animal and exposed to specific antigen. The positive reaction consists in the muscular contraction. -To highlight the concentration of IgE (total and/or specific) is used immunoenzymatic assay - ELISA (enzyme-linked immunosorbent assay). Prevention and Treatment - Food allergies caused by hyperproteic diet requires a hypoallergenic diet. - Deparasitation is a preventive and curative measure mandatory in allergy induced by fleas or other ectoparasites. - The treatment of anaphylaxis (to reduce edema, increase blood pressure, reduce muscle contractions) - administration of simpatico-mimetic drugs, antihistaminic drugs, anti-inflammatory drugs, etc. S 49. TYPE II HYPERSENSITIVITY Active mediators: IgG and IgM class antibodies, which specifically recognize different antigenic determinants present on the cells’ surface. Nucleated and anucleated cells that contain these antigens are the target of the antibodies, which may be exercised in two ways: 1. With predominant involvement of the complement. The antibodies (IgG and IgM) bind the surface antigens of the target cells and play a role of opsonins for phagocytes. Simultaneously, the antigen-antibody complexes trigger the activation of complement through the classical pathway, leading ultimately to the formation of the membrane attack complex (MAC - C5b6789) which determines the target cell lysis. 2. Through antibody mediated cytotoxicity Associated with the action of the complement. This mechanism is based on the action of antibodies on the cell membrane, which attract many types of cells that have Fc receptors on their surface. These are macrophages, neutrophils, eosinophils, natural killer cells, and platelets. They will exert an attack on the target, using cytotoxic compounds at their disposal. The mechanisms involved in this type of hypersensitivity are: - phagocytosis mechanisms; - activated complement activity; - cytotoxic mechanism. ETIOLOGY: The main forms of type II hypersensitivity are accidents caused by incompatible blood transfusions and hemolytic diseases of the newborn - Haemolytic disease of the newborn, known in humans, was described in Rhnegative mothers immunized against Rh-positive fetal erythrocytes. Phatogenetic mechanism of the disease is the same as in transfusion shock, cause by incompatible transfusion – the synthesis antibodies against blood group antigens of the newborn. - Ab ingested through colostrum (their presence there is the result of maternal previous sensitization, by incompatible transfusion or by the fetus red blood cells which cross the placenta during gestation and enter in the maternal bloodstream, causing an antibody immune response against fetal red blood cells). After colostrum consumption, antibodies arrive in digestive tract of the newborn, cross the intestinal barrier and reach the circulation, where they cause agglutination and lysis of red blood cells, as in transfusion shock. Hemolytic disease of the newborn can be found in almost all species of domestic animals. Clinical manifestations Usually occur in a range from 12 hours to 5 days after the first suckle and consist of: weakness, pale mucous membranes, and later jaundice (icterus), tachypnea, tachycardia, soft pulse, haemoglobinemia, haemoglobinuria, hypothermia, and in final phase, convulsions caused by cerebral hypoxia. In acute severe forms, death can occur within 24 hours. - In lambs, haemolytic disease of the newborn is rarely encountered. - In dogs, haemolytic disease of the newborn is a rarity. It is triggered only after an incompatible transfusion and mating with an A positive male (the most powerful antigen of blood group system). Diagnosis For the diagnosis of Type II hypersensitivity may be used: - Serological tests for blood groups, hemolysis test in the presence of complement, agglutination reactions, antiglobulin test; - Detection of anti-erythrocyte antibodies using direct or indirect Coombs test. Prevention and Treatment Treatment of hemolytic diseases of the newborn: oReplacement of maternal colostrum with colostrum from another cow, mare, bitch etc.; oPerforming blood transfusions from compatible donors; oImmunosuppressive drugs; oControl of blood groups to prevent hemolytic anemia and transfusion accidents. S 50. TYPE III HYPERSENSITIVITY Definition: It is also known as "immune complex hypersensitivity" because responsible for the lesions are not molecules of IgE class antibodies, as in type I hypersensitivity, or IgG in type II, but the immune complexes formed after the combination of antibodies with soluble antigens. Antigen - antibody complexes exist normally in the body but in very small quantities, These complexes are, however, quickly removed by phagocytosis, or by excretion at kidney level. However, if the body has large amounts of antigens and specific antibodies, a large amount of immune complexes will be formed. The mechanisms of removal become inefficient, and the excess of complexes is deposited on the blood vessels walls, especially where the blood flow is low - kidneys, joints and the skin. There are known three types of immune complexes: - with microbial antigens in the case of persistent infections - with self antigens in the autoimmune mechanisms - with antigens from the environment CLINICAL MANIFESTATIONS A. Arthur Reactions-Local 1. Like all immune responses, this reaction is specific to an antigen; 2. Like all hypersensitivity reactions, it evolves in three phases: -Sensibilization phase - caused by first inoculation of the antigen; -Latency phase - during which immunological mechanisms are initiated; -Lesion phase - triggered by a new contact with the same antigen. 3. Delay in the lesion within the category of semi-late response; 4. The local hypersensitivity reaction at the site of injection of antigen (in this case the skin) is characterized by the following clinical symptoms: swelling, hemorrhage and necrosis. 5. Humoral mediated reaction because sensibilization can be transmitted to another animal with the serum; 6. Reaction requires the presence of neutrophils and the serum complement; reaction disappears if the animal is deprived artificially from one or other of these elements. Examples -Extrinsic allergic alveolitis is a pneumonia caused by an Arthus-type reactions. The responsible allergens are spores of Micropolyspora faeni, spores of fungi from genera Aspergillus and Fusarium, as well as vegetal powders -Anterior uveitis (blue eye disease) Clinically it is manifested by the inflammation of the anterior chamber eye and corneal edema. -Staphylococcus allergy is a pustular dermatitis described in dogs, being a consequence of immune complexes deposition in skin. B. Generalized reactions Can be reproduced in experimental conditions by the intravenous inoculation of antigen in animals with high concentrations of circulating specific antibody. -Serum sickness. The antigen- antibody interaction is carried out in the blood. -Immune complex glomerulonephritis. The development of the disease is influenced by many factors, like the presence of allergens (viruses, bacteria, parasites) ecc. DIAGNOSIS METHODS IN TYPE III HYPERSENSITIVITY The main techniques that allow highlighting immune complexes are: - in organs, immune complexes are detected by immunofluorescence test GENERAL PRINCIPLES OF PREVENTION AND TREATMENT The only possible prevention is to avoid contact with responsible antigens. It is recommended desensitization when the exposure cannot be avoided. Treatment microbial diseases or other diseases in which may occur hypersensitivity (antibiotics, chemotherapy) S 51. TYPE IV HYPERSENSITIVITY Definition: Antibodies are not involved, but there is the action of the cell effectors.This type of reactions are based on the interaction between the antigen, the dendritic cells and receptors on the surface of a T lymphocyte sub-family specifically the so called " TD "(delayed). Mechanisms: TD lymphocytes sensitized by an antigen, after the second contact with it, will release interleukins which will recruit other types of cells, in particular macrophages, their intervention causing damages that will become apparent after 24-48 hours. Local changes are alteration of the vascular permeability, dilation followed by mononuclear cell infiltration of the area. Differences: - type IV hypersensitivity mechanisms are cell-mediated; - it develops in a time exceeding 12 hours; - is not transmitted from one animal to another by humoral effectors but by TD lymphocites; - lesions are localized exclusively on the site of antigen inoculation. Classification: It is based on the maximum time of occurrence after a new contact with the same antigen. -Jones-Mote hypersensitivity. -hypersensitivity. -Tuberculin hypersensitivity. -Granulomatous hypersensitivity. It is the most important clinical form of this group of reactions being involved in a number of pathological conditions. It is based on the presence of a causative agent (microorganism) within macrophages, as a result of their inability to destroy it. The result is the formation of epithelioid cell granulomas. Besides epithelioid cells, in this reaction giant cells are also present, which appear as a result of chronic stimulation produced by living infectious agents or foreign substances. In some cases, in the center area of granulomatous lesions appears an area of necrosis. The main antigens responsible for this type of hypersensitivity are species of Mycobacterium, which cause tuberculosis in humans and animals, leprosy bacillus (M. leprae), Brucella species, some parasites (Schistrosoma) and some non-antigenic inert substances (e.g. talcum powder). DIAGNOSTIC METHODS IN TYPE IV HYPERSENSITIVITY To highlight the delayed hypersensitivity the following tests may be used: - intradermal test (eg. allergic reaction to tuberculin); - determination of blast transformation of T in the presence of antigen; - non-specific lymphocyte blast test. S 52. IMMUNODEFICIENCIES: DEFINITION, CLASSIFICATION Definition: Group of disorders in which part of the immune system is missing or defective. As a result, an animal with an immunodeficiency disorder has frequent infections that are generally more severe and last longer than in a healthy animal. Regardless of their etiology, immunodeficiencies involve morphological and functional changes affecting one or more effectors of the immune response, disturbing mechanisms by which is ensured the balance of the immune system. Classification: Immunodeficiencies can be classified according to several criteria: 1. According to the etiology and mode of transmission: - primary immunodeficiencies – caused by an inherited flaw in the immune system that increases the susceptibility to infections (congenital immunodeficiencies) - secondary immunodeficiencies - the primary etiologic factor is obvious and may be of great variety: virus, bacteria, parasite, stress, nutritional factors, etc. 2. According to the nature of the effectors involved: - immunodeficiencies of the natural immunity effectors, with nonspecific action (complement, enzymes, phagocytes, K cells) - immunodeficiencies of the organs and cells of the immune system (bone marrow, thymus, bursa of Fabricius, T cells, B cells, etc.) 3. By the nature of immune deficiency involved in the genesis of primary immunodef.: - partial - affecting the cellular or humoral immune response; - mixed - with concomitant damage to both cellular and humoral immune response. S 53. PRIMARY IMMUNODEFICIENCIES – DEFICIENCIES OF HUMORAL AND CELLULAR MEDIATED IMMUNITY 1-DEFICIENCIES OF THE HUMORAL MEDIATED IMMUNITY Definition:Humoral immunodeficiencies are a group of diseases caused by disorders of B cells. They have a relatively high incidence and significant impact on the health of humans and some species of domestic animals. The main form of expression involves the disruption of immunoglobulins or antibodies synthesis. Depending on the concentration of immunoglobulins, are reported pathological conditions such as agammaglobulinemia or hypogammaglobulinemia. The main mechanisms involved in their production are: - absence of precursor cells; - inability of B cells to maturate; - reduced synthesis of immunoglobulins; - enzymatic disorders; - action of toxic metabolites; - mutations in genes that encode the synthesis of immunoglobulins. Examples of diseases: -X-linked agammaglobulinemia – Burton syndrome. It was described in a boy with respiratory infections. It was found later that the syndrome is genetically determined and generally occurs in boys around the age of 5-6 months. Death occurs from 2 to 18 months of age. -Transient hypogammaglobulinemia. It was described in children (two years old), horses (2-5 months old foals) and dogs. In foals, transient hypogammaglobulinemia results from a delay in endogenous IgG production, which brings a risk of bacterial infection and poor development -Selective immunodeficiencies. They are immunodeficiencies characterized by decreased synthesis of either a single immunoglobulin class or isotype .Disorder appears as a clonal disorder, by the dissociation of helper and suppressor T cells cooperation against B lymphocyte clones that follow to secret a certain class of immunoglobulins. Selective IgA immunodeficiency - in humans, dogs, and birds. Immunodeficiency in IgA may be associated with various diseases,such as Ataxia telangiectasia , respiratory infections, viral hepatitis, chromosomal deficiencies etc.In dogs, IgA deficiency was described shepherd dogs and Beagles. In birds, the disease was diagnosed in a line of obese chickens. Selective IgM immunodeficiency is extremely rare and has a well-defined clinical manifestation. In animals, the deficit has been described in horses and dogs. IgM is absent, while the in other immunoglobulins level is normal. Clinical signs are more severe in the young, and include recurrent fevers, pneumonia, arthritis, enteritis and generalized lymphadenopathy Selective IgG Immunodeficiency is very rare, and is not sufficiently characterized from clinical point of view. Hereditary transmission is not fully demonstrated. It has been reported in humans, horses, cattle (Danish Red breed) and birds. 2.DEFICIENCIES OF THE CELL-MEDIATED IMMUNITY Definition:Selective deficiencies involving certain cellular effectors of immunity have a great importance due to the severity of the associated pathological conditions and possible complications. They affect cell-mediated adaptive immune mechanisms. Examples of diseases: Thymic hypoplasia (DiGeorge syndrome) is a primary immunodeficiency, often but not always, characterized by cellular (T-cell) deficiency because of thymus hypoplasia or aplasia, characteristic facies, congenital heart disease, parathyroid gland abnormalities, and hypocalcemia. DGS is caused by abnormal formation of certain tissues during fetal development. Thymic dysplasia (Nezelof syndrome) is an abnormal condition characterized by absent T cell function, deficient B cell function, fairly normal immunoglobulin levels, and little or no specific antibody production. It affects both male and female siblings, indicating that it may be transmitted as autosomal-recessive genetic disorder. The cause is unknown. Ataxia telangiectasia syndrome is disease reported in children is transmitted through an autosomal-recessive mechanism. It is characterized by the presence of: thymic hypoplasia, lack of response to mitogens, but also disruption of IgA and IgE synthesis. Clinically it manifests as progressive ataxia (lack of coordination) due to a defect in the cerebellum. S 54. PRIMARY IMMUNODEFICIENCIES – COMBINED IMMUNODEFICIENCIES AND COMPLEMENT DEFICIENCIES 1.COMBINED IMMUNE DEFICIENCIES Definition:Are immune deficiencies with severe evolution, involving associated disorders of the cell and humoral mediated immune response. A-Reticular dysgenesis It is a rare congenital disease with an extremely severe evolution and fatal ending in the first days after birth. Deficiency occurs in all cellular effectors and immune mechanisms. Severe disorder of cells differentiation in myeloid and lymphoid strains result in serious damage of the local defense specific and nonspecific capacity, B-Severe combined immunodeficiency In dogs, affected animals lack both T and B cells, which makes it impossible for the body to fight foreign invaders. Cases have been seen in Bassett Hounds, Toy Poodles, Rottweilers, and.Affected dogs are healthy during the first several months of life but become progressively more susceptible to bacterial infections as the antibodies they received during nursing disappear. No treatment is available and the longterm outlook is poor. 2.COMPLEMENT DEFICIENCIES The complement system is crucial for defense against pathogens, removal of unwanted materials such as dying cells or immune complexes as well as for development of adaptive immune responses. Genetically determined deficiencies of components of the complement system are usually relatively rare, but they result in many severe diseases such as an increased susceptibility to recurrent, severe bacterial infections, autoimmune disorders (systemic lupus erythematosus), etc.. All complement deficiencies are autosomal recessive traits, except C1 deficiency, which is autosomal dominant disease. The main pathological consequences are: - failure of the normal immune processes: opsonization, hemolytic activity, leukocyte mobilization, phagocytosis, killing of the bacteria, biochemical processing and presentation of antigens; - absence or reduced intensity of cytotoxic mechanisms essential for destruction of certain cells infected by viruses; - decrease resistance to infections, especially those with bacterial etiology; - high incidence of immune complex diseases. In animals, cases of complement fractions deficits have been reported in dogs and lambs. In dogs, it described an almost total deficit of component C3 of the complement. S 55. PRIMARY IMMUNODEFICIENCIES – FUNCTIONAL DEFICIENCIES OF PHAGOCYTES FUNCTIONAL DEFICIENCIES OF PHAGOCYTES eficient expression of the phagocytic function, which inevitably leads to decrease the body's defense capacity, is linked to some immunological mechanisms such as action of various suppressive factors or deficient participation of immunoglobulins. Functional deficits of phagocytes could manifest as: - decrease of total number; - alteration of mobility function; - alteration of bactericidal function. Examples of diseases: Chronic granulomatous disease Leads to recurrent or persistent intracellular bacterial and fungal infections and to granuloma formation. In approximately two thirds of patients, the first symptoms appear during the first year of life in the form of infections, dermatitis, gastrointestinal complications, diarrhea due to colitis. The clinical picture can be quite variable, with some infants having several of these complications and others appearing to be far less ill. In dog was a similar syndrome diagnosed characterized by suppurative dermatitis, lymphadenitis, osteomyelitis, gingivitis and pododermatitis. Haematological examination revealed a marked leukocytosis. Canine cyclic neutropenia Cyclic hematopoiesis in grey collie dogs is an autosomal recessive disorder in which regular 11-13-days cycles of the neutrophil count lead to severe infections and early death. The disease is caused by a defect in regulation of marrow stem cell proliferation. The cyclic fluctuation of the neutrophil count is reproducible and is reliably associated with fluctuations in eosinophils, monocytes, lymphocytes, platelets, and reticulocytes.The disease is manifested also by depigmentation of the skin, eye damage, respiratory infections. Chédiak–Higashi syndrome Chédiak–Higashi syndrome (CHS) is an extremely rare form of partial albinism that is accompanied by problems in the immune and nervous systems, described is humans, cat, mink, cattle, mice, and the killer whale. In cats was detected especially in individuals with yellow eye color and "blue smoke" hair color. Most of the cats had bilateral nuclear cataracts as early in life as 3 months of age. Abnormally large eosinophilic granules were observed in the neutrophils of the granulocytic series of blood and bone marrow by electron and light microscopy. Granules of eosinophils and basophils were also enlarged. Light microscopic studies of hair and skin revealed enlarged melanin granules. Leukocyte adhesion deficiency Leukocyte adhesion deficiency (LAD) is caused by deficiency of adhesive glycoproteins on the surfaces of WBCs; these glycoproteins facilitate cellular interactions, cell attachment to blood vessel walls, cell movement, and interaction with complement fragments. Deficiencies impair the ability of granulocytes (and lymphocytes) to migrate out of the intravascular compartment, to engage in cytotoxic reactions, and to phagocytose bacteria. Severity of disease correlates with degree of deficiency. S 56. SECONDARY IMMUNODEFICIENCIES (virus) In adult animals, immunodeficiencies often occur as a consequence of virus infections, malnutrition, stress, or toxins. These are called secondary immunodeficiencies.Virusinduced secondary immunodeficiencies are the most important of these. VIRUS-INDUCED IMMUNODEFICIENCIES One way in which viruses survive in infected animals is by immunosuppression. For example, canine distemper virus infects and kills lymphocytes, causing a profound combined immunodeficiency in affected puppies.This infection is associated with a progressive decline in immunoglobulin levels and increased susceptibility to organisms normally controlled by cellular immunity ex: -Parvoviral infection- in both dogs and cats also causes a profound depression in the resistance to fungal infections such as aspergillosis, mucormycosis, or candidiasis in the immediate postrecovery period. Example of disease: Feline Leukemia Virus (FeLV) FeLV is associated with an increased susceptibility to secondary and opportunistic infections. Acquired immunodeficiency in FeLV infection is multifactorial. Infected cats can have deficiencies of neutrophils, decreased synthesis of antibodies (especially to bacterial antigens), decreased cellular immunity, and reduced complement levels. Feline Immunodeficiency Virus (FIV) FIV has been identified in domestic and wild felids. The infection is endemic in cats throughout the world. Virus is shed in the saliva, and biting is the principal mode of transmission. As a result, free-roaming, male, and aged cats are at greatest risk of infection. After infection, there is a transient fever, lymphadenopathy, and neutropenia. Most cats then recover and appear to be clinically normal for many months or years before progressive immunodeficiency develops. Cats with acquired immunodeficiency induced by FIV then develop chronic secondary and opportunistic infections of the respiratory, GI (including mouth), and urinary tracts, as well as the skin. Simian Type D Retrovirus This viral infection of macaques has a similar pathogenesis to that of FeLV infection of cats but can induce even more severe immunodeficiency. Simian Immunodeficiency Virus (SIV) This lentivirus is closely related to human immunodeficiency virus. Many strains of SIV exist in nature. The common hosts are African primates such as African green monkeys, sooty mangabeys, mandrills, baboons, and other guenons. IMMUNOPROPHYLACTIC METHODS AND PRODUCTS Immunoprophylaxis represents the prevention of a disease by administration of vaccines or sera (induction of active or passive immunity). Immunoprophylactic methods have an important place in veterinary medicine. The methods and biological products used are: - conventional methods and products: serums and vaccines applied in veterinary medicine and human medicine; - modern, unconventional methods and products: immunoprophylaxis using vaccines produced by genetic recombination, chemical synthesis, anti-idiotypic antibodies (idiotype-based vaccines). S 57. Vaccination: definition, routes of administration of the vaccines, general contraindications, adverse reactions Definition:The vaccination means the administration of vaccines in strict compliance with requirements of vaccination leaflet prepared by the producing company. Factors very important in order for the vaccination to properly stimulate the general immunological reactivity: - Nutrition which thru the quality and the balance of the main nutritive substances (proteins, carbohydrates, lipids, vitamins, and minerals) The alteration of the immune response which occurs when food deficiency occurs involves both components of the immune response ( cellular and humoral). In these conditions,the organism will yield to the attack of microorganisms. - Negatively influenced by microclimate deficiencies or zoo-hygienic factors. Excessive temperatures, cold air currents, gas or toxins accumulation, overcrowding or not respecting growth technologies are other factors. Only healthy animals can be vaccinated. Vaccination can be preventive vaccination– in this case biological products are administered in the absence of the illness ( eg: antiswine fever, anthrax, Newcastle disease, etc) or necessity vaccination, when the biological products are administered to healthy animals which are healthy in disease outbreak areas (eg: anti- paratuberculosis, listeriosis, avian cholera, etc) Routes of administration: - A-Parenterally (subcutaneous, intraperitoneal, intramuscular) - B-Natural routes (oral, nasal, conjunctival route) and by deposit on the scarified dermis. A-Parenteral vaccination ensures both the certainty of effectuation of the operation and the inoculation of the prescribed dose. Usually it is hard to apply in big farms, especially in the case of birds where the big number of animals make individual interventions almost impossible. B-Vaccination using natural routes offer the advantage of collective administration of the vaccines advantage which makes them the preferred method in the case of big collectivities of animals. This method is efficient especially in the case of attenuated live vaccines, beeing able to induce local immunity which is useful.( ex: vaccination for swine infectious pleuropneumonia using aerosolization). Vaccination by derm scarification consists in sprinkling derm scarifications with pads soaked in the vaccine. After vaccination, immunity installs after 10 – 14 days and sufficient for a variable period of time, most often for a period of 6 up to 12 months. Immunity is maintained by the administration of rappel vaccines dependent of the vaccine category. In the case of inactivated vaccines follow up vaccines are usually a most in order to achieve a secondary immune response while in the case of live vaccines follow up vaccines will be administered after an exact period of times in order for the immune response to not be affected by the presence of antibodies. Postvaccinal immunity reduces quantitative progressively if the organism hasn’t had any other contact with the antigen. General contraindications of vaccines. Contraindications for the use of vaccines were generated by the appearance of complications after the administration of biological products. Contraindications can be : - Temporary contraindications: fever (secondary to benign infections), infectious disease (in evolution or Conv convalescence), Animals which can be related to any of these categories can not be vaccinated. - Definitive contraindications are serious organic disease, evolutive neurological affections, major allergies to the proteins included in the vaccine and organic immunodeficiencies. Animals which can be related to any of these categories can’t be vaccinated and in the case, they are vaccinated the protective effect is zero. Post-vaccinal adverse reactions (events caused by the vaccine). Post-vaccinal adverse reactions are different depending on the type of vaccine, manifestation and the individual . They can occur after the administration of the inactivated vaccines and after the administration of live vaccines. Post vaccinal reactions can be: - Local reactions are the most numerous,The most common local reactions are redness, edema, abscesses, nodes, necrosis, and lymphangitis. - General reactions are fever, allergic reactions, convulsions, arthralgia, myalgia, and paralysis. In any of this cases, immediate treatment will be applied. The presence of general reactions in a large number of vaccinated animals will be reported to the producer. - Focal reactions include the reactivity of old infectious processes most frequently in the pulmonary area. DESCRIPTION OF THE MAIN TYPES OF VACCINES: The description of vaccines will be carried out considering the biological state of the etiological agent included in the vaccine. S 58. LIVE VIRULENT VACCINES Def.:This kind of vaccines were used on a large scale in the past but in the present, they present two major disadvantages: They can cause vaccination accidents which will clinically be carried out exactly like a normal outbreak of the disease; They artificially create carriers and as a consequence outbreaks of infection with the potential broadcast. n the present, the usage of this kind of vaccines finds applicability in necessity interventions, in the case of diseases in which there are no alternatives. Protocol: 1. Simultaneous vaccination and serum therapy (sero-vaccination) It consists out of the simultaneous administration of separate points of the unmodified vaccinal agent ( live and virulent) and a certain quantity of immune serum, the last one ensures protection both against a wild strain and vaccinal agent. The serum attenuates the potential pathogenicity of the vaccinal agent. In time, the effect of the antibodies administered with the specific serum decreases and the organism starts to synthesize it’s own antibodies under the inductor action of the vaccine. The disadvantages this method presents include the difficulty in obtaining a correct dose, especially in the case of bacterial infections, and the optimal report between germs and serum. A report which is not balance is a real risk for the virulence of the germs to out-do the passive immunity and as a consequence, the disease can occur or the virulence can be neutralized and the obtained immunity will not be satisfying in terms of intensity and durability. On the other side, vaccine organisms using the pathogen agent used as a live vaccine can become carriers and germ eliminators (especially in the case of antiviral vaccines) for long periods of time leading to the artificial creation of stationary disease outbreaks. 2. Vaccination using pathways non specific for the pathogenic agent’s tropism It has been applied in the case of infectious diseases produced by pathogenic agents with limited tropism for certain tissues and which, when inoculated on other routes than those on which they naturally produce the disease they will produce o benign, localized infection with the result of a solid immunity. in the case of animals inoculated this way a local reaction appears and a solid immunisation is induced. The disadvantages of this procedure consist of the possibility of accidents occurring with the development of the disease and carriers. 3. Immunization using guided infections are used in the case of some infectious diseases for which no efficient vaccines have been elaborated. In the case of swine transmissible gastroenteritis, content from the infected piglet's gut will be administered to the pregnant sows. The sows get immunized and transmit immune effectors to the piglets which have proved to be effective. A similar procedure has been used in the case of aviar contagious coryza in which, the healthy birds have been given mucus from the sick birds. The disadvantages of this procedure consist of the possibility to spread other pathogenic agents with the gut content or with the mucus. S 59. LIVE ATTENUATED VACCINES This type of vaccines is the most commonly used for the prophylaxis of infectious disease. Live vaccines are represented by attenuated or neutralized strains of microorganisms which maintained their multiplication capabilities and immunological proprieties. Methods: The attenuation of microorganisms has been made spontaneously, through the influence of hazard and their simple isolation from natural hosts or it has been induced as a result of a meticulous laboratory work, using repeated passages on different culture substrates (on avian and rabbit cell cultures or by cultivation in suboptimal conditions) or in the presence of some selective agents (antibio-independent bacteria). In the case of attenuated viruses, there are more methods that can be used: using succesive passages on cellular cultures; adaptation to certain tissues and the change of the characterists; adaptation to species which in natural conditions are not receptive. Examples of viral diseases in which attenuate strains are used for the making of vaccines are: swine fever – strain C (Chinese) which is totally apathogenic, strains Hudson and Koprowsky which have an accentuated residual pathogenicity and which are used only as necessity vaccines. Immune response: -Live vaccines induce a prompt immune response with a fast installation of postvaccinal immunity (4-7 days) which makes their use possible in the case of necessity vaccinations. -The immunity which results is solid, stable and durable, between 6 – 12 months or even more, and is assured by the concomitant stimulation of humoral and cellular immune responses, and implicitly through protection mechanisms of the mucosa with the production of secretory IgA. Characteristics: -Attenuated vaccines are easy to administer and can be administered in the drinking water or through aerosols. -Conservation methods: this kind of vaccines can be lyophilized, which assures a longer life. -Live vaccines are contraindicated in the case of animals treated with immunosuppressive drugs or with antibiotics which can determine the neutralization of the postvaccinal effect. DISADVANTAGES: Lower genetical stability of some strains (reverse to the initial form is possible) Strains with residual pathogenicity can cause postvaccinal reactions or vaccination accidents Sometimes vaccination with attenuated strains has unfavourable effects on diagnosis of occult infections The biggest disadvantage is represented by the risk of a reverse to the initial virulence. Categories: A special category of vaccines is live heterologous vaccines. Examples are: bovine Rotavirus strains and the avian influenza viruses which assure protection against the infection with Rotavirus and influenza virus in humans, Shope fibroma virus and turkey herpesvirus which are used for the immunisation of rabbits against myxomatosis and chicken against Marek disease as well as the BCG vaccine (Mycobacterium bovis) used in the antituberculosis immunisation in humans. The advantages of live heterologous vaccines are: - reduced risks of infection; - the possibility of beeing administered to young animals with maternal antibodies; - immunity with high intensity and long lasting; - reduced local effects or no effects. S 60. Inactivated vaccines Def:Inactivated vaccines consist of germs which, after being treated with different physical or chemical procedures, lost their ability to multiplicate or replicate in the body, but their antigenic structure has been preserved. Inactivation characteristics: Inactivation constitutes a safe procedure after which germs from inactivated vaccines become harmless regarding their innocuity. The main purpose of inactivation is, on one hand, to totally annihilate the pathogenic capacity of the vaccine agent, and on the other hand to maintain the antigenic structures which are responsible for the induction of the immune response.The absence of the multiplication or replication capability of the germs inside the organisms makes the immune response to correlate with the quality and quantity of the incorporated antibody in the inactivated vaccine. The attenuation of the immunogenicity of the vaccine strains after the inactivation is compensated by the utilization of germs with superior immunogens proprieties and by an adequate concentration. Inactivation using physical procedures: Among the physical factors used for the inactivation of germs, the following can be mentioned: heat, ultrasound, ultraviolet light, and radiation. -Heat as an inactivation factor has been and is largely used in the case of most vaccines inactivated by using physical methods. High temperatures over 37°C, which are active through their own actions, are inefficient in the preparation of vaccines, because of the destruction of microorganisms’ antigenic structures. -Ultrasound, ultraviolet light, or ionizing radiation, have an action that can be properly dosed, is possible when current methods of inactivation don’t preserve the antigenic structure of microorganisms. The products obtained using these methods have a good immunogenicity, can be stored for long period of time, and are well tolerated. -Chemical products is the most commonly used method and it consists out of the treatment of microorganisms with different substances: antiseptic products (formalin, phenol, sodium methionate), dyes (methylen blue, toluidin blue, crystal violet) and organic compounds (beta propiolactone and ethylenediamine derivatives). The advantage of inactivated vaccines are: they can be obtained from microorganisms in which attenuation is not possible; after inactivation, there is no risk of reversion to the initial, pathogenic properties; they can be used in almost all categories of animals. The disadvantages are: compared to attenuated live vaccines, a higher dose of antigens is required to obtain a similar response; the absence of multiplication or replication of the microorganisms in the body correlates the immune response with the quantity and quality of the antigens incorporated in the vaccine; these vaccines have a lesser effect on cellular immunity thus the resulted immunity is less stable and shorter (up to 3-6 months); the onset of immunity is delayed: 10 – 14 days after vaccination and for consolidation, follow up vaccines are required after 1-3 weeks. the stimulation local immunity processes is frequently absent, thus the level of mucosa IgA is insufficient, creating a risk for colonization of the mucosa and replication of viruses; relatively high content of unessential substances, immunologically inactive, which can generate secondary phenomena like pain, local reaction, hypersensitivity or even the inhibition of the immune response; S 61. VACCINES PREPARED BY MODERN TECHNOLOGY There are many restrictions regarding the production of conventional biological products. Among these restrictions the most quoted are: the difficulty or impossibility to replicate some viruses in vitro, complications regarding the obtaining of pure virus strains, without foreign proteins or other viruses, high rate of mutations and the continuous change of antigenic determinants in some viruses, the reverse to initial pathogenic form or apparition of virulent mutants strains in case of live vaccines obtained using the classical methods. Along all these, it is important to obtain a sufficient quantity of active principles (bacterial, viral or parasitic antigens), in acceptable economic conditions, following the principles of environmental protection against eventual spreading of infectious agents and to protect the people who handle them (e.g., rabies virus).It is very important and necessary that by chemical synthesis, biosynthesis and other modern biotechnological methods, to obtain proteins or polypeptides which imitate useful antigenic determinants, obtaining specific, active and harmless components. Ribosomal vaccines are prepared using bacteria in active growth phase, while the number of ribosomes is at its maximum. The destruction phase of the microorganisms, in a homogenizer with glass pearls, is followed by centrifugation and filtration using sterile membranes. Their immunogenicity is due to the surface cellular polypeptides recently translated to the level of ribosomes, which remain temporarily attached to the mRNA. The advantages of using ribosomal vaccines: they have a well-defined chemical composition compared to vaccines obtained from intact bacteria; they confer, in some cases, cross protection against the serotypes of a microorganism, eliminating the necessity to use multivalent vaccines; they are less toxic than intact cells which have toxic components on their surface; they are more immunogens than whole bacteria, probably due to the adjuvant effect of mRNA. Examples.Salmonella enteritidis, Pseudomonas aeruginosin, Pasteurella multocida, Brucella abortus, Candida albicans, Cryptococcus neoformations. Anti-adhesin vaccines. This kind of vaccines is capable to stop bacteria adhesion to the mucous surfaces and is also capable of stimulating phagocytosis before colonization takes place. Such vaccine has been proven to be effective against colibacillosis caused by Escherichia coli K88 in piglets. Synthetic vaccines. The isolation, characterization and the possibility of synthesizing in laboratory conditions of some of the peptides which contain a limited number of amino acids has opened a fundamentally new direction in the research area regarding the prophylaxis of both human and animal infectious diseases. The first observations regarding this topic started from the finding that the proteins or lipoprotein macromolecules found in viruses, bacteria, and parasites carry a large number of antigenic determinants. Production of synthetic vaccines implies the usage of complex technologies which include: identification of the immunogenic determinants, their analysis and their purification, synthesis of analogous peptides, checking the immunogenicity of the synthesized products.Important breakthroughs have been accomplished with the hepatitis B virus, foot and mouth disease virus, influenza and polio in humans, as well as some bacteria such as Vibrio cholera. With all successes recorded until now regarding the production of immunogenic peptides, their immunizing potential is generally below that of the whole antigens. Genetically engineered vaccines Genetic engineering involves the use of techniques in which the genome of a cell, bacteria or a virus is modified as a result of the insertion of foreign genes. The main techniques used in this context are genetic recombination, DNA cloning, cell fusion, induction or repression of gene expression - Subunitary vaccines obtained using recombinant DNA. For the preparation of recombinant viral vaccines, most convincing results have been obtained using vaccinia virus as vector. The vaccinia virus is non-pathogenic (harmlessness) and has a stable structure. For the preparation of recombinant bacterial vaccines, special attention from researchers in this field enjoys the bacteria Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosin, and yeast Saccharomyces cerevisiae. - Vaccines containing bacterial or viral subunits. It was demonstrated that the proteins isolated from the surface structures of viruses and bacteria have the ability to induce the synthesis of antibodies and protection against the original etiologic agent, thus confirming the findings of experimental whereby active antigenic determinants are represented by a small number of amino acids, situated on the surface area of protein structures. S 62. PASSIVE IMMUNIZATION (SERUM THERAPY) Def: Passive immunization is an important mean in diseases prevention and treatment and also in inducing immunity that is immediate but short-lived by administering antibodies derived from an immunized animal. Biological products used for passive immunization of animals by the parenteral administration are known as immune and hyperimmune sera which contain antibodies to a fairly high concentration. Immune sera are represented by the sera or plasma (from animals immunized for this purpose or animals that were infected and recovered after the disease) which, by inoculation to healthy animals, induce passive immunity and in the case of inoculation to sick animals have a therapeutic role. Hyperimmune sera, which are prepared in biofactories on animals are most widely used, and they are standardized products which are characterized by a high content of antibodies. Preparation of hyperimmune sera is based, in principle, on the administration of large and repeated doses of antigen to donor animals. Hyperimmunization of the donors translates into an intense immune response expressed by the elevated serum antibodies, exceeding the values existing in spontaneously immunized animals, which have been ill or vaccinated. The main stages for obtaining a hyperimmune serum are: 1. Choosing the donor animals. Donor animals can be horses, cattle, sheep and swine. Homologous sera (obtain from the same species as the recipient) have a higher value. Heterologous serum, present the disadvantage of heterogeneity, the immunoglobulins of other species inoculated to other animal species having antigenic functions.Most of the sera used in veterinary medicine are obtained from horses or cattle, and those which are necessary only in small doses from rabbits, rats or chickens.Donor animals have to be clinically healthy and free of microbial infections. 2. Preparation of antigens. The antigens can be corpuscular or soluble depending on the nature of antibodies whose production is intended. For subcutaneous or intramuscular inoculations, usually antigens are associated with adjuvants (aluminum hydroxide gel, Freund's complete or incomplete adjuvant, oil adjuvants, etc.).Antigens for the first inoculations must contain germs or toxins in a form that does not jeoparize the animal's life. 3. Inoculation of the antigen. Antigen doses used vary depending on the donor antigen and, if the protocol provides several inoculations, are increasingly larger from one to the next inoculation. The route of inoculation may be the same for all inoculations or may be different. Some protocols require a first inoculation or multiple subcutaneous or intramuscular inoculations of the antigen usually associated with an adjuvant, followed by intravenous inoculation, significantly increasing the concentration that stimulates antibody production.The number of inoculation is very different depending on the serum being prepared. In some cases, a single administration of antigen is sufficient, the interval between the inoculations is of several days. Usually between the first two inoculations range is higher (e.g., 14-21 days) and smaller between subsequent inoculations (e.g., 3-10 days). 4. Obtaining the serum samples. To obtain serum, immunized animals are subject to partial or total bleeding. Each donor can be exploited to obtain up to 5-7 batches of serum. Partial bleeding involves collecting the maximum amount of blood compatible with life. The interval between the last inoculation of antigen and bleeding is about 1430 days. After each partial bleeding, donors receive the best possible care and diets. 5. Serum control. Each batch of serum is controlled in terms of sterility, harmlessness, and protective value. Sterility is controlled by seeding culture media for the normal and aerobic and anaerobic bacteria and fungi. Innocuousness control is performed by inoculating the serum to experimental animals (mouse, guinea pigs, and rabbits) and local reaction, the general state of the animals, and possibly other parameters are checked.Protective value control (antibody titer) is made by inoculating naturally susceptible animals with decreasing doses of serum, followed by an infection with the minimal lethal dose of bacteria for which the respective serum is prepared for. Depending on the number of dead and surviving animals DP-50 (the dose protecting 50% of animals) is then calculated. The final product is treated with a preservative which has the purpose of protecting for subsequent contamination without altering the antibodies titer. S 63. APPLICATIONS OF THE IMMUNE SERA In order to be used, immune sera must meet the following conditions: - To be active, i.e. to possess neutralizing capacity against germs or toxins that led to the immunization of the recipient animal; - To be free of microorganisms that could lead to the onset of other diseases; for this reason, they have to be controlled in terms of sterility and obtained f rom animals free of diseases; - their administration should not induce side effects, especially anaphylactic reactions. Hyperimmune sera have three major applications: 1. Serum therapy. It is the use of immune sera in the treatment of various infectious diseases, preferably at an early stage. A good therapeutic efficacy was found in antibacterial sera for smallpox, tetanus, gas-gangrene (produced by Clostridium spp.), hemorrhagic enteritis and other infections produced by Clostridium spp., pasteurellosis, infections with E. coli, etc., especially the youth sheep and other species. Serum therapy is usually associated most with antibiotics. 2.Prophylaxis. Consists of administration of immune sera in prophylactic purposes, in healthy animals or in disease outbreaks with the main goal of protecting animals against diseases. Immunization against tetanus using sera is a general measure applied to all receptive species – humans, horses, pigs in the case of particular lesions, before surgical interventions or before neutering animals.The prophylactic doses are generally ten times lesser than the therapeutic doses. After the administration of an immune serum in prophylactic purposes, a passive immunity state onsets in a few hours up to 24 hours and lasts for about 7 – 20 days. 3.Serovaccination. It is a mixed active-passive immunization method which is applied in the control of swine fever in outbreak areas. The method consists in administrating swine fever virus – an attenuated strain (Hudson or Koprovski Rovac strains with residual pathogenicity) or even wild strains under the protection of the serum against the virus. Even though it is a pretty brutal method it presents a great efficacy as an emergency measure in order to stop morbidity in the situation of dangerous outbreaks. • Diagnostic. It is a laboratory practice in which immune sera are needed for titrations, control reactions and identification of bacterial and viral antigens, genera, species, subspecies, serotype being established based on positive reaction with specific known antibodies. S 64. REVELATORY PRODUCTS USED IN DIAGNOSIS Def:Revelatory products are biological products used for the diagnostic of disease in which state of allergy (type IV delayed hypersensitivity) installs. For the initiation, development and manifestation of the specific state of hypersensitivity, two contacts with the same antigen are necessary. The specific immunological character of the phenomenon is demonstrated by the necessity of a prior antigen sensitization (infection with M. tuberculosis, M. paratuberculosis, Pseudomonas mallei, Brucella spp., parasites) and by the onset of the reactions only after a new contact with the same antigen or extracts from the same microorganism. In the case of the above mentioned bacteria, the first inoculation is induced by the infection itself and the onset of the first lesions, which in the beginning can’t be clinically noticed. Installation of state of awareness is made within two to three weeks since the conflict between microorganisms and the responsive host. Relevant inoculation (usually artificially induced) allows detection after intradermal inoculation of revealing products with interpretation within 24-72 hours. The revelatory products used in veterinary medicine are: 1-Tuberculin. It is the product used in the allergic diagnosis of tuberculosis in humans and animals. Yellowish color in the case of bovine tuberculin and reddish color in the case of avian tuberculin. 2-Paratuberculin. It is a revelatory substance used for the allergic diagnosis of cattle’s and sheep’s paratuberculosis. The product is administered intradermally. 3- Mallein. It is used in the allergic diagnosis of glanders in horses using the intradermic and subcutaneous administration. 4-Brucelohydrolyzate. It is a purified protein product, specific and antigenic, obtained from selected strains of Brucella sp. It can be found under two forms: - Brucelohydrolyzate for cattle obtained from B. abortus strains; - Brucelohydrolyzate for swine obtained from B. suis strains. Both products can be found as transparent liquids, colorless or yellow colored. It is used in the allergic diagnosis of brucellosis in - cattle, which will be inoculated intradermally on the side of the neck, and pigs, by intradermal inoculation on the external ear.