Selective Immunoglobulin Deficiencies in Dogs PDF

Clinical Correlation Selective Immunoglobulin Deficiencies in Dogs At least 80% of Chinese shar pei develop recurrent dermatitis, (staphylococcal), demodectic mange, otitis externa, flea, allergies, cystitis, bronchitis, and atopy. Other dogs such as German shepherds, beagles, Irish wolf...

Clinical Correlation Selective Immunoglobulin Deficiencies in Dogs At least 80% of Chinese shar pei develop recurrent dermatitis, (staphylococcal), demodectic mange, otitis externa, flea, allergies, cystitis, bronchitis, and atopy. Other dogs such as German shepherds, beagles, Irish wolf hounds, and cocker spaniels, also develop similar ailments Clinical laboratory analyses of serum from affected dogs reveal normal levels of IgG and IgM but depressed levels of IgA  What is the common feature of all these diseases? Why would an animal with an IgA deficiency have the symptoms seen in these dogs? How might the IgA deficiency arise?  Adaptive Immunity Humoral Immune Response B lymphocytes, development, activation and differentiation Felix N. Toka DVM, PhD, DSc, DACVM  Objectives Describe the B lymphocytes activation and the role of co- stimulatory molecules Describe the signals necessary for B cell activation and their consequences Describe B lymphocyte differentiation into plasma cells and memory cells Describe B cell response to antigens Explain the dynamics of antibody responses Describe the structure of Immunoglobulins Describe the effector functions of antibodies  Development of B lymphocytes The process that leads to development of B cells is divided into three stages Maturation - generation of mature immunocompetent B lymphocytes. For most vertebrates (particularly humans and mice) B lymphocytes develop in the bone marrow Activation - contact of B lymphocytes with specific antigens Differentiation - activated B cells become plasma cells that produce antibodies or become memory B cells  B lymphocytes (B cells) and antibodies Antigen recognition by surface receptors on B cells initiates proliferation and differentiation of B cells into plasma cells and memory B cells Plasma cells secrete immunoglobulins (antibodies) specific for a given antigen Activation and proliferation of B cells B cell activation is driven by the presence of antigen and leads to proliferation and differentiation into plasma cells and memory B cells  Naive B cells that do not engage an antigen die within a few weeks B cells can be activated by thymus-dependent antigens (TD, T-dependent antigens) or thymus independent antigens (TI, T-independent antigens) Activation of B cells by TD antigens requires the presence of helper T cells (CD4+) www.slideshare.net-  TI antigens directly activate B cells TI antigens are divided into TI-1 and TI-2 TI-1 antigens are polyclonal activators of B cells (mitogens) i.e., they activate B cells non-specifically. They are recognized by the BCR or TLRs (e.g., LPS is recognized by TLR4 or BCR, leading to activation and antibody production) TI-2 antigens activate B cells by extensively cross-linking to multiple membrane Ig receptors (BCRs)  Immune response induced by TI antigens is usually weak and no memory cells differentiate The dominant Ig produced after such stimulation is IgM  Transduction of activating signals All BCRs i.e., IgM and IgD posses a short cytoplasmic tail – that is not enough to transduce a signal The BCR only binds the antigen, but signal transduction is performed by Igα and Igβ chains Igα and Igβ chains, in their cytoplasmic region, have slightly longer tails called ITAMs (Immunoreceptor tyrosine based activation motif)  Humoral immune response Initial contact of an individual with an antigen generates primary immune response Initial contact with antigen leads to antibody production by plasma cells and generation of memory B cells Kinetics of a primary immune response depends on: Nature of the antigen Administration route (vaccine) or infection route Presence or not of an adjuvant (vaccines) Species of infected or immunized individuals  In all instances the primary immune response is characterized by a lag time Events during lag time: Clonal selection - selection of B cells with specificity to foreign antigens Clonal expansion - increase in number of antigen specific B cells Differentiation - development of plasma cells and memory cells Production of antibodies - mostly IgM than IgG is produced  Clonal selection Differentiation Clonal expansion B cell receptor Antigen recognition  B cells primary immune response is characterized by a low antibody level Secondary humoral immune response Subsequent contact with the same antigen generates secondary humoral immune response Secondary immune response is dependent on the existence of memory B and T helper cells  Secondary immune response is generated rapidly compared to primary immune response Secondary immune response is stronger than primary immune response – because of memory B and T cells Secondary immune response lasts longer than primary immune response Antibodies secreted during secondary immune response have higher affinity to antigens Secondary immune response is characterized by high levels of IgG than IgM Comparison of primary and secondary humoral immune responses Property Primary response Secondary response Responding B cells Naive B cells Memory B cells Lag time after antigen Generally 4-7 days Generally 3-4 days administration Peak of response 7-10 days 3-5 days Varies, depending on 100-1000 times more than Magnitude of response antigen type primary response Isotype of antibody IgM dominates IgG dominates produced Type of antigen inducing T-dependent and T- T-dependent antigens the response independent antigens Antibody affinity Low High  Sites of humoral immunity induction Upon entry into the host, antigens become concentrated in various lymphoid organs Antigens from external surfaces are concentrated in lymph nodes, whereas blood borne antigens are concentrated in spleen Sequence of events in humoral immune responses to T cell– dependent protein antigens 1. Immune responses are initiated by the recognition of antigens by B cells and CD4+ T cells. 2. The activated lymphocytes migrate toward the B cells and interact with one another, resulting in B cell proliferation and differentiation. Co-stimulation of B cells At least two types of signals are required for the B cells to be activated Signal 1 – antigen engagement (TCR + Ag on MHC II of B cells) Signal 2 – binding of CD40 on the B cell and CD40L on the activated Th cell – induces the B7 that interact with CD28 (co- stimulatory signals)  3. Stimulation of B cells by helper T cells in extrafollicular sites leads to short-lived plasma cell generation and early isotype switchin, while activation of T cells by B cells results in the induction of follicular helper T cells. 4. The late events occur in germinal centers and include the selection of high-affinity cells (affinity maturation), additional isotype switching, memory B cell generation, and the generation of long-lived plasma cells.  B cells in lymphoid organs recognize specific antigens and become activated then begin to proliferate forming what are called germinal centres (GC) What is a germinal center? The germinal center (GC) is a specialized microstructure that forms in secondary lymphoid tissues, producing long-lived antibody secreting plasma cells and memory B cells, which can provide protection against reinfection  Germinal centers in secondary lymphoid organs A. The germinal center is within the follicle and includes a basal dark zone and an adjacent light zone B. The light zone contains follicular dendritic cells, and the dark zone contains proliferating B cells The germinal center reaction in a lymph node  Ig class switching (Isotype switching) In T-dependent responses, B cells undergo heavy chain isotype (class) switching and produce antibodies with heavy chains of different classes, such as γ, α, and ε Ig class switching is a result of somatic hypermutation that leads to changes in Ig class i.e., the cells stop producing IgM and start producing IgG, IgA or IgE - recombination of the VHDHJH unit with C regions of any heavy chain  RECOMBINATION EVENTSIN ISOTYPESWITCHING ACTIVATION INDUCEDCYTADINE DEAMINASE AID EXPRESSIONTRIGGERS DIVERSIFICATION OFTHE  REARRANGED IMMUNOGLOBULIN 19GENES Isotype switching in response to different types of microbes is regulated by cytokines produced by the helper T cells that are activated by these microbes. to activateCD4cells producethecytokines thatworkonBcells thathaveclassswitched  Affinity Maturation Somatic mutation (point mutation, deletion and insertion) of Ig genes and selection of high-affinity B cells its theantigencan likely this dissociatefrom Friable region stopproducinglow affinity soantibodies startproducing highaffinityantibodies affinitymaturation Clonal selection and deletion B cells that bind foreign antigens in germinal centers with high affinity are selected to survive B cells with low affinity to foreign antigens are eliminated through clonal deletion  Follicular dendritic cells play an important role in clonal selection of B cells They present Ag on their cell surface with which B cells interact T CD4+ cells are required during the selection process. They recognize antigen presented by B cells (centrocytes) in MHC class II context After the selection process, B cells begin to differentiate into plasma cells and memory cells Plasma cells lose the BCR and begin to produce antibodies  Generation of Memory B Cells Memory B cells are generated during the germinal center reaction and can make rapid responses to subsequent antigen encounter They can survive for long periods without continuing antigenic stimulation Some memory B cells remain in the lymphoid organ, others recirculate between the blood and lymphoid organs Effective vaccines against microbes and microbial toxins must induce both affinity maturation and memory B cell formation  Antibodies aka immunoglobulins Antibodies are proteins produced by B cells in vertebrates in response to exposure to foreign structures known as antigens  Is it possible for the immune system to produce antibodies needed for protection against all known animal pathogens? Say 100 000 000 !? (10 000 genes Lc X 10 000 genes Hc)  Antibodies are mainly found in serum, but also in milk, tears, saliva, mucus and bile  Antibody structure Antibodies are heterodimers i.e., they are composed of 4 chains L There are 2 light chains designated L, polypeptides with a molecular weight of 22 000 daltons each 2 heavy chains designated H with a molecular weight of 55 000 daltons each H  Antibody structure Each Light chain is associated with a Heavy chain by disulfide bonds and non- covalently by hydrogen and hydrophobic bonds forming a dimer  Antibody structure Each dimer is associated with an region variable identical dimer by disulfide bonds forming a heterodimer The first 110 aa on the N-terminal end of the L and H chain are highly variable for each antibody This region is called the VARIABLE region and is designated V (VL for the light chain and VH for the heavy chain)  Antibody structure Amino acid sequences of the region below the variable is constant, and so it is called the CONSTANT region in both L and H chains. The regions are designated C Digestion of IgG with a proteolytic enzyme papain produces 3 fragments, 2 identical and 1 different The 2 identical fragments have the capability to bind antigens and are called “fragment, antigen binding” Fab The third fragment does not bind antigens, but because it crystallizes at low temperature it is called “fragment, crystalizable” Fc  There are two types of L chains, kappa and lambda Antibodies can only posses one type of L chains i.e., kappa or lambda, never two different chains at once There are 5 types of H chains, alfa, gamma, delta, epsilon and mu (α, γ, δ, ε and μ, respectively) The type of H chain determines the class of the immunoglobulin e.g., IgA, IgG, IgD, IgE or IgM Immunoglobulins with different heavy chains are called isotypes  Classes and subclasses of Ig Small differences that can be found in amino acid sequences of heavy chains determine the subclass of immunoglobulins within each isotype  Immunoglobulin classes and their biological activity Each Ig class is characterised by a unique amino acid sequence for the H and L chain in the C region  IgG About 80% of immunoglobulin in serum is IgG IgG posses 2 γ H chains and 2 L chains κ or λ gamma lambda The number of IgG subclasses differ in different animal species (see table)  In humans IgG1, 3 and 4 can pass through placenta to the foetus and play an important role in protection of the growing foetus IgG3 activates the complement IgG3 binds to Fc receptors on macrophages which enhances opsonization and phagocytosis  IgA About 10-15% of total immunoglobulin in serum is IgA It is the dominating immunoglobulin in body secretions such as milk, tears and mucous in mucosal of the respiratory, digestive and reproductive tracts It is mostly found as monomer, but may be found as a dimer, trimer or tetramer As a dimer, two units of IgA are associated through the Fc and the joining chain  The IgA found in external secretions is a dimer – called secretory IgA (sIgA) The two units of this IgA dimer are connected by Fc, joining chain and a secretory component Plasma cells that produce secretory IgA are found in the submucosal of the mucous membranes Secretory IgA is formed during translocation through epithelial cells of the mucosal The IgA dimer binds to the poly-Ig receptor on the epithelial cell surface The poly-Ig receptor is found on the basolateral surface of many mucosal epithelia such as the digestive tract and mammary, salivary and lacrimal glands IgA is then transported through endocytosis together with poly-Ig receptor across the cell to the opposite side of the cell surface – luminal side e.g., intestines The poly-Ig receptor is cleaved enzymatically and becomes part of the secretory IgA dimer  The secretory component protects the hinge regions of IgA from digestion by proteolytic enzymes That is why high IgA levels are maintained at surfaces rich in proteolytic enzymes IgA protects from pathogens (bacteria, viruses) that may enter the body through mucosal surfaces Antigen-antibody complexes formed at such surfaces are removed by local mechanisms in place e.g., ciliated epithelial cells of the respiratory tract or peristalsis in the digestive tract  IgM About 5-10% total immunoglobulin in serum is IgM Free IgM is always a pentamer, however monomeric IgM is found as a receptor on the surface of the B cell (as part of the B- cell receptor complex, BCR) Molecular weight 900 000 daltons Five subunits are arranged in a star-like fashion and each subunit is connected through the Fc region and an associated joining chain It has a total of 10 antigen binding regions i.e., 10 Fab fragments  IgM is the first immunoglobulin synthesized by newborns and the first immunoglobulin produced during primary humoral immune response Despite the 10 Fab fragments, IgM can only stably bind 5 large antigens (only small antigens such as haptens can occupy all 10 antigen binding sites) IgM is effective in binding antigens that have repetitive epitopes such as viruses and red blood cells IgM is more effective in activating the complement than IgG. Two Fc fragments in close proximity are required to activate the complement  IgE Is found in small amounts in serum Plays an important role in allergic reactions Binds to Fc receptors on like cells these basophils and mast cells When antigen binds two IgE molecules, mast cell granules translocate to the cell surface and degranulate Contents of granules are usually mediators of inflammation  IgD About 0.2% total immunoglobulin in serum Together with IgM, IgD is the immunoglobulin found on the cell surface of naïve B lymphocytes as B cell receptors No biological effector function has been identified for IgD  Antigenic determinants on immunoglobulins Ig are immunogens as well i.e., antigens They can induce an immune response when administered to other animal species - producing anti-Ig antibodies Such antibodies are useful in studying development of B lymphocytes and for diagnostic purposes  Antigenic determinants on Ig Antigenic determinants fall into three categories: Isotypic – determine the isotypes of the immunoglobulin Allotypic – determine the difference between isotypes of the immunoglobulins within a given species Idiotypic – determine the difference between specificities of antibodies  Isotypic – these are constant regions found on heavy-chain class and subclass and each light chain type and subtype within a species They are encoded by separate genes for non-variable regions Each species has different isotypes, so antibodies of one species are foreign antigens for another species and will induce an immune reaction  Allotypic – Each species has the same genes for isotypic antigens, but alleles can exist for certain genes Alleles encode small differences in amino acid sequences, which become allotypic determinants, i.e., the differences in sequences encoding isotypes within a species Allotypic antibodies can be produced by injecting immunoglobulin from one member of a species into another member of the same species or blood transfusion  Idiotypic - these are antigenic determinants found in the variable regions of both light and heavy chains of a given antibody  Effector functions mediated by antibodies Apart from binding antigen, antibodies have other biological roles Antibodies do not kill pathogens and do not remove pathogens from the body They induce effector activity of other components of the immune system which in turn kill and remove pathogens The variable part of an antibody binds the antigen, but the constant part (Fc) interacts with other cells to induce the effector mechanisms Two main functions of antibodies Binding of foreign antigens recognized within the host Mediation of effector functions that lead to elimination of foreign antigens i.e., infectious agents  Since antibodies do not kill pathogens, what do they do? canneutralize http://bio1151b.nicerweb.net/Locked/media/ch43/  Opsonization An antigen is said to be opsonized when it is covered by antibodies Opsonization facilitates phagocytosis by macrophages and neutrophils – a role in antimicrobial protection Macropages and neutrophils have Fc receptors on their cell surfaces Phagocytosis is initiated when Fc receptors on the macrophages become engaged by Fc fragments on antibodies Antibody-dependent cellular cytotoxicity - ADCC Target recognition through CD16 will occur only when antibodies are present  Fc receptors Fc receptors are glycoproteins found on the surface of many cells They are responsible for many biological functions of antibodies such as: Transcytosis Mediation of phagocytosis Antibody dependent cell cytotoxicity Antibodies bound to Fc receptors on cell surfaces can induce regulatory signals that affect activation or inhibition of cells bearing the Fc receptors  Types of Fc receptors Poly-Ig receptors – responsible for transport of IgA and IgM through epithelial cells FcRn – neonatal Fc receptor – transports IgG through placenta to blood circulation of the foetus and participates in regulating the level of IgG in serum FcαR binds IgA, FcεR binds IgE FcγR – is found in several forms – RcγR (RI, II-A, RII-B1, RII- B2, RIII) they have capability to bind IgG and its subclasses  Types of Fc receptors Poly-IgR FcγR1 FcγR2 FcγR3 FcαR FcεR1 FcRN CD32 CD64 CD16 CD89  Monoclonal antibodies Most antigens posses many antigenic epitopes and induce proliferation and differentiation of many different clones of B cells that produce different antibodies Serum generated in such circumstances is heterogenous. It contains a mixture of antibodies with specificities for different antigens Such antibodies are called polyclonal antibodies Polyclonal antibodies are suited for various effector functions that antibodies carry out in the host e.g., mediation of phagocytosis and activation of the complement  Polyclonal antibodies are not suitable for diagnostic or therapeutic procedures Currently there is no feasible way to purify polyclonal antibodies in order to prepare monoclonal antibodies Monoclonal antibodies – are antibodies produced by a single clone and are specific for a single antigenic epitope Nobel Prize Award in 1984 for discovery of the principle for production of monoclonal antibodies" Niels K. Jerne Georges J.F. Köhler César Milstein In 1975 Jerne, Köhler and Milstein derived a method to produce monoclonal antibodies by fusion of normal plasma cells and myeloma cells The fusion process produced hybrid cells that are immortal and have capability to continuously produce antibodies of single specificity Zdjęcia: http://www.nobelprize.org/nobel_prizes/medicine/laureates/1984/ Monoclonal antibody production procedure Application of monoclonal antibodies (mAb) In diagnostics of infectious diseases and cancer In human therapy: 80 FDA-approved monoclonal antibody therapies for the human health canine mAb for pruritus – CYTOPOINT neutralizes IL-31, a key mediator of allergic itch The suffix „-umab” in the name of the drug denotes mAbs that are of human origin In scientific research. Common Antibody INN Representative Origin Substem Examples Abciximab, Rituximab, Chimeric -xi- Infliximab, Cetuximab Palivizumab, Trastuzumab, Humanized -zu- Bevacizumab, Natalizumab Adalimumab, Panitumumab Human -u- Golimumab, Ipilimumab  What is the common feature of all these diseases? Associated with body surfaces Why would an animal with an... deficiency have the symptoms seen in these dogs? = a deficiency of IgA antibodies in the skin, and mucosal surfaces. How might the... deficiency arise? inherited genetic defect in the gene segment encoding IgA H chains