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You submitted the following rating and review. We'll publish them on our site once we've reviewed them. Continue shopping. Item s unavailable for purchase. When treated with proteases without reduction of disulfide linkages, fragments of antibody molecules with an intact antigen-binding capacity can be generated. Anti-idiotypic antibodies. A Private idiotypic antibody: An anti-idiotypic antibody recognizes the antigenbinding site on the variable regions of an antibody, AB.
B Public idiotypic antibody: An anti-antibody binds to an area on the variable region other than the antigen-binding sites. They have many advantages when used in diagnostic and therapeutic applications because they are smaller than the intact antibody molecules. Antigens, Haptens, and Immunogens A foreign substance which can be recognized by the immune system and can elicit immune responses is called an immunogen.
A substance which can elicit the formation of antibodies with a binding affinity toward its specific recognizable structure is called an antigen. Therefore, an antigen is a substance that not only can elicit the formation of, but also can bind to, the induced antibodies. However, strictly speaking, an immunogen may elicit immune responses from cellular immunity without the formation of antibodies.
Similarly, an antigen may elicit the antibody formation without any immune response. Not all foreign substances are antigens. To be an effective antigen, the substance must have a large molecular weight. The minimum molecular weight for an antigen is dependent on the nature of the substance; generally, for polypeptides the minimum molecular weight as an antigen is 10 kDa. However, there are small molecules such as N-acetyl-Ltyrosine that are antigens to certain species despite their low molecular weights. In addition to the large molecular weight, an antigen molecule must be capable of being catabolized in antigen-presenting cells.
The involvement of antigen degradation in antigen presentation will be discussed later. Antibody fragments. Antibodies can be reduced to produce two identical heavy chains and two identical light chains. These fragments cannot bind the antigen unless they are oxidized and reconstructed into an intact immunoglobulin. However, antibodies can be digested by proteases, e. The structure of a macromolecule is also important for its antigenic properties.
The complexity and steric conformation of an antigen molecule are important factors for the determination of the effectiveness in eliciting antibody formation, because an antibody recognizes not only the primary sequence of an antigenic macromolecule such as a polypeptide or polysaccharide but also its three-dimensional structure. On the other hand, macromolecules with simple structures such as polyethylene glycol, poly amino acids , and starch are poor antigens.
When small molecules such as a drug molecule are conjugated to a macromolecular carrier and the conjugates are used for immunization, antibodies can be elicited to recognize and to bind the structure of the macromolecular carrier as well as the conjugated small molecules Figure 2. Those small molecules are not antigens because they cannot induce the formation of antibodies by themselves, and are called haptens. Anti-hapten antibody formation. A A small molecule is not a covalently linked immunogen and cannot elicit antibody formation. B When the small molecule is coupled to an macromolecule, the conjugate is immunogenic and can elicit the formation of various antibodies, including an antibody that recognizes the structure of the small molecule as well as other epitopes on the macromolecular surface.
Haptens are involved in many drug allergic reactions and are important in the preparation of anti-drug antibodies for the treatment of drug toxicity and for the development of immunoassays in drug analysis. In humans, B cells differentiate from stem cells in bone marrow. The differentiation initiates when the immunoglobulin-encoding genes rearrange into a unique sequence which determines the antigen-specificity of the antibody to be produced by the B cell. Upon being stimulated by the specific antigen, the small number of B cells that bear the specific antibody on their plasma membranes will be induced to proliferate into a larger B cell clone.
Subsequently, the specific clone of B cells will differentiate into plasma cells and produce only the specific antibody which recognizes the specific antigen Figure 2. Such a clone selection process is also involved in the development of antigenspecific T lymphocytes. When an antigen molecule enters into the body, it is recognized first by antigen-presenting cells APC. B-cell differentiation. This process occurs in the bone marrow and on the surface of stromal cells, involving many cell adhesive molecules CAMs and stem-cell factor SCF for binding and a stromal cell-secreted cytokine, interleukin 7 IL-7 , for promoting pre-B cell differentiation.
The antigen specificity of the B cells is already determined at this stage. Subsequently, after the DNA for light chains also rearranged, the monomeric lgM molecules are assembled and expressed on the surface of immature B cells. Before leaving bone marrow and migrating to peripheral lymphatic tissues such as spleen and lymph nodes, immature B cells must be screened for self-antigen recognition. Immature B cells that express surface lgM recognizing self-antigens inside the bone marrow will be eliminated inside the bone marrow.
Once they have migrated to the peripheral tissues, mature B cells express not only lgM but also lgD and lgG on the cell surface. Mature B cells will differentiate into antibody-producing plasma cells and, to a lesser extent, to memory B cells after encountering the specific antigen that is recognized by the surface immunoglobulins. The internalized antigen molecule will be partially degraded into large fragments inside an intracellular compartment, endosomes; those fragments possessing recognizable antigenic structures will be complexed intracellularly with class II MHC proteins and expressed together onto the surface of APC.
These surface-bound antigenrecognizing moieties are immunoglobulins for B-lymphocytes and T cell receptors for T-lymphocytes. A B cell and a T cell may bind to an identical antigen fragment but at distinct sites, hence most antigen molecules possess multiple antigenic recognition sites. The binding of T cells to APC stimulates the secretion of B cell growth factors from T cells which can further promote the proliferation of B cells and the production of antibodies. The antigen-presentation process will be discussed in detail in Chapter 3. Antigen molecules that require the participation of T cells in order to elicit antibody production are called T cell-dependent antigens.
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There are antigens which do not require the presence of T cells for eliciting antibody formation; these antigens are called T cell-independent antigens Figure 2. T cell dependent antigens are potent antigens and are more effective and sensitive than T cell-independent antigens when they are used for the production of antibodies. In addition to the promotion of B cell proliferation and differentiation, T cells also secrete interleukin 2 IL-2 which can stimulate the expression of IL-2 receptors on T cell themselves and promote their proliferation to develop cell-mediated immunity.
It consists of low technologies such as antiserum production, to the medium range such as highly purified immunoglobulins, and to high technology such as monoclonal and recombinant antibodies. Regardless of the technology used for the production of antibodies, the most critical step in most methods is the immunization of animals with a selective antigen. The response of an animal to the immunization depends on many factors other than properties of the antigen molecule as described in Antigens, Haptens, and Immunogens.
An antigen molecule must be foreign to the host animals and should be pure enough to avoid the immune response to the contaminating substances. Adjuvants can be used to enhance the immune response. The preparation of immunogens and the use of adjuvants in immunization will be described in detail in Chapter 9 Vaccines. The response of an animal to immunization also depends on factors in the host animal such as the age, sex, genetic strain, and physical condition.
In addition, the location and the method of injection can determine the type and extent of the immune response. The amount of a specific antibody in the serum is presented as the titer of the antibody. The titer usually refers to as the extent of dilution of the serum in which the antibody still can be detected by conventional immunological techniques. The titer and the class of antibodies in the serum are both dependent on the schedule of the immunization.
In the primary immunization, i. Furthermore, the class of antibodies from the primary response is predominately IgM. In the secondary immunization, that is when the animal is challenged or boosted with the antigen for the second time, the response is relatively fast; the titer of the antibody increases markedly and the class of the antibody shifts from IgM to IgG Figure 2.
Several booster injections may be required in order to obtain serum with desirable titer and specificity. However, an overboosted animal may produce antiserum with a decrease in both the titer and the specificity. When serum of an immunized animal is used as a source of antibodies, it consists of many antigenbinding immunoglobulins with different specificity and produced from many different clones of B cells Figure 2.
Therefore, this type of antibody is called polyclonal antibody. Another method for the production of antibodies is by using hybridoma technology Figure 2. In this method, a small animal such as a mouse is first immunized with a selected antigen. When the serum obtained from the animal after boost injections indicates a high titer of antibody against the selected antigen, the animal will be sacrificed and cells will be isolated from the spleen.
The splenic cells contain a large number of B-lymphocytes; at least some of the B-lymphocytes can produce the antigen-specific antibodies. The isolated splenic cells, because they are normal cells, have a very short life in cultures. However, these cells can be fused with transformed cells such as myeloma cells in the presence of fusogens such as polyethylene glycol to produce half-normal-half-transformed cells, which are called hybridomas.
If a hybridoma is formed by the fusion between an antibody-producing B-lymphocyte and a myeloma cell, this hybridoma will possess both the antibody production capability of a B cell and the unlimited proliferation ability of a cancer cell. After careful selection of the fusion products, a single clone of hybridoma can be picked and propagated.
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This clone of hybridoma will continuously produce a single type of antibody as regards the specificity and class of immunoglobulin. This type of antibody is called monoclonal antibody because it is produced by a single clone of B-lymphocyte. A comparison of the properties of polyclonal and monoclonal antibodies is shown in Table 2.
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The choice of either a monoclonal or a polyclonal antibody depends largely on the purpose of the use. If the purity and specificity of the antibody is important such as the use of antibody as a drug, monoclonal antibodies are usually preferred. The processing of T cell-dependent A and T cell-independent B antigens. T cell-dependent antigens usually consist of more than one antigenic determinants. After processing by antigen-presenting cells APC , the surface-expressed, MHC-complexed antigen determinants will be recognized by the antigen-specific B and T cells.
T cells will release interleukins, e. T cell-independent antigens, such as a polymeric antigen with repeating determinants, can bind directly to the immunoglobulins on the surface of a B cell. This type of cross-binding on the cell membrane, often in the presence of other mitogens, will induce the proliferation and differentiation of the B cell. T cell independent antigens usually elicit the production of only IgM antibodies with low titers. Table 2. Comparison of polyclonal and monoclonal antibodies. Primary and secondary antibody responses. When a host is exposed to an antigen for the first time, i.
This primary response usually produces IgM first followed by a low titer of IgG, and only lasts for a short period of time. When the host is exposed to the same antigen again, i. The secondary response of antibody production not only is with higher titers but also lasts much longer than the primary response. In addition to the production from serum and hybridomas, antibodies can also be made by using the recombinant technology. Two of the most important features for the development of recombinant antibodies are 1 the preparation of chimeric or humanized antibodies that consists of human constant regions to minimize the hypersensitization to animal immunoglobulins in patients Figure 2.
Genetic Basis of Antibody Diversity It has been estimated that in humans there are at least types of antigenic structures that can be recognized by the immune system. This large diversity of antibody specificity has been a puzzle for immunologists for many years, and many theories have developed for its explanation. It is only during the last ten years that, due to the advent of modern molecular biology, immunologists have finally been able to understand the genetic basis that control the diversity of antibody formation in B cells.
However, many mechanisms involved in the antibody formation such as the class switch and the fine tuning of specificity are still not fully understood at the present time. The most important event in antibody biosynthesis is the rearrangement of genes to form DNA sequences which upon expression, transcription and translation, will produce specific antibodies. Polyclonal antibodies are produced by many different clones of B cells which can recognize different epitopes on the surface of an antigen molecule.
As illustrated in this figure, different antibodies recognize various shapes of epitope on a spherical antigen. Therefore, polyclonal antibodies such as antisera isolated from immunized animals are essentially mixtures of many types of antibodies with regard to the classes of immunoglobulin and the specificity and affinity toward antigens. V and C regions correspond to the variable and constant regions in the polypeptide chain, and J joining region encode a small peptide segment which joins the V and C peptides to form the complete light chain.
For heavy chains, there are four regions of genes encode the polypeptide. In addition to the V, J, and C regions, there is a D diversity region of small segments of genes which can be inserted between V and J regions to further increase the diversity of the heavy chain amino acid sequence. In human B cells, there are approximately V genes, 12 D genes and four J genes. In addition, there are nine C genes that encode the nine isotypic variations of the heavy chain, i.
Because the heavy chain and light chain are encoded in different genes and in different chromosomes, their pairing to form an immunoglobulin molecule is random. Therefore, when a light chain and a heavy chain combine together to form an antigenic binding site Fab , there are approximately 7. Monoclonal antibody production, 1. Mouse is immunized with the specific antigen. After anti-antigen antibody can be detected in the serum, mouse will be sacrificed and spleen cells will be isolated. Spleen cells consist of a large number of B cells.
A myeloma cell line is selected which is deficient in the enzyme hypoxanthine-guanine phosphoribose transferase HPRT. This cell line cannot survive in a medium containing aminopterin, a thymidine synthetase inhibitor, because the cell cannot use purines in the salvaged pathway for DNA synthesis. The spleen cells and the HPRT-negative myeloma cells are fused in the presence of fusogens such as polyethylene glycol. After fusion, cells will be maintained in a medium containing hypoxanthine, aminopterin and thymidine HAT.
Only cells that are fused between one spleen cell with enzyme HPRT and one myeloma cell immortal , i. Hybridomas will be diluted and seeded in small well plates e. After many days in culture, each well will have enough cells for the screening of the production of antibodies. Wells that produce desirable antibodies will be identified. Cells in those wells will be transferred and proliferated into a large number of hybridoma cells. The culture medium will contain a high concentration of the monoclonal antibody against the specific antigen. If necessary, the antibody can be purified to homogenity by using various immunoglobulin isolation techniques such as affinity chromatography.
For large scale production, the hybridoma cells can be injected intraperitoneally into mice, and the ascite fluids can be collected and, if necessary, purified to obtain the monoclonal antibody preparation. Examples of antibodies produced by genetic engineering technology. By substituting the Fc region in a mouse immunoglobulin, e. B Fv fragments cannot be obtained by degradation of immunoglobulins because there is no disulfide linkage to hold the two polypeptides together.
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However, recombinant DNA technology can produce single chain Fv polypeptide sFv which consist of VL and VH peptide sequences connected by a linker peptide which provide a linkage of the two polypeptides without interference of the antigen-recognition activity. In addition to the gene rearrangement and light chainheavy chain combination, the diversity of antibody can also be generated from point mutation of gene.
This mutation usually occurs in the hypervariable regions by altering one amino acid residue that leads to a marked increase of the affinity toward the antigen. The blue domain indicates the constant region and the white domains indicate the variable region. The specificity of the immunoglobulin is determined as early as in the DNA deletion process; for example, the linking of V3 and J4 segments will determine the antibody specificity as shown in this diagram. Complement Antibodies are factors in the humoral immunity for the recognition of pathogens via antibody-antigen interaction.
In very few cases, antibody-binding on the surface of target cells alone can cause the killing of the antigen-bearing cells. However, in most cases, subsequent events are required in order to generate cytotoxicity and to destroy the target pathogens. One of the mechanisms involved in antibody-mediated cytotoxicity is by complement activation which causes the lysis of the target cells. Complement factors are a group of serum proteins which are designated by symbols as C1 to C9. Once the activation of complement factors is initiated, the cascade will proceed to form a complex, i.
As described in Chapter 1, the innate immune response can initiate the complement cascade, a process which is called the alternative complement pathway. However, in acquired immunity, the complement cascade is initiated by the interaction of complement factors with antibody-antigen complexes, and this process is called the classical complement pathway.
A comparison of these two complement pathways has been described in Figure 1. Classical complement pathways and its biological activities. The classical complement pathway is initiated by the binding of C1 factor to the antigen-antibody complexes of IgG or IgM. The enzyme cascade C1 to C5 , followed by a sequential aggregation C6 to C9 , will form a membrane-attack complex MAC which is a cylindrical transmembrane pore and is capable of lysing the antigen-bearing target cell.
Binding membrane-fixed C3b fragments can induce the phagocytosis of the target cells by C3b-receptor bearing phagocytes, a process referred to as opsonization. In addition, the release of C3a and C5a fragments can attract phagocytes to the inflammatory site via chemotactic effects, and cause anaphylaxis via binding to endothelium and mast cells. A bar above the components indicates an active complex.
There are nine factors involved in the classical complement pathway Figure 2. Functionally, these nine factors can be divided into two categories of proteins: proteins with proenzymatic activities such as C1, C2, C3, and C4, and proteins with aggregating activities such as C5, C6, C7, C8, and C9. During the complement cascade, factors C1 to C5 require enzymatic transformation into the active forms while factors C6 to C9 do not need activation. In the classical complement pathway, the Fc domain of an antigen-bound antibody can be recognized by complement protein C1.
The six binding units in C1q can form a crosslinkage between two IgG molecules. Activated C1r subsequently converts C1s to an active enzymatic form and initiates the cascade in the classical complement pathway. C4 is the first complement component in the blood which is activated by C1s. C4 is split into two polypeptides, the shorter one is C4a and the longer one is C4b. C4b, as an activated enzyme, can convert complement C2 component into a larger polypeptide with enzymatic activity, C2b, and a shorter polypeptide, C2a. C2b and C4b form a complex with C1s on the antigen-bearing surface, known as C3 convertase, which can convert C3 into C3a and C3b.
As can be seen in Figure 1. C3b binds to the membrane with antibody-antigen complex and can enhance the phagocytosis of the target cell by phagocytes via the C3b receptors present on the surface of the phagocytes. The process of enhancing phagocytosis by the attachment Table 2.
Effects of opsonization on phagocytosis. The marker molecules recognized by phagocytes are opsonins. Besides C3b, antibody-antigen complexes when bound to the surface of target cells can also behave like opsonins by enhancing phagocytosis via Fc receptors on the surface of phagocytes Table 2.
The membrane-bound complex of C4b, C2b, C3b is called the C5 convertase which produces a membrane-bound C5b fragment by cleaving off a small C5a fragment from the C5 component. During the activation process from C1 to C5, three small polypeptide fragments are generated, i. Although these three small peptides are not directly involved in the complex formation on the surface of target cells that eventually produce the cell death, they are important factors for the induction of inflammatory responses.
C4a, C3a, and C5a are anaphylatoxins because they bind to mast cells and basophils and stimulate the release of granules from these cells. The degranulation of mast cells or basophils releases several important inflammatory mediators including histamine. These mediators cause the anaphylatic response: the contraction of smooth muscles and leakage of the endothelium. These anaphylatoxic peptides are inactivated by an anaphylatoxin inhibitor, carboxypeptidase B, in the serum which removes the carboxyl terminal arginine residue.
In addition to the anaphylaxis, C5a also acts as a factor of chemotaxis and an activator of neutrophils see Chapter 1. However, the chemotactic activity of C5a is not reduced by the action of carboxypeptidase B. Thus, intact C6, C7, and C8 molecules aggregate sequentially around C5b to form a membrane-associated complex which, in turn, polymerizes C9 molecules to form a transmembrane channel.
This transmembranous channel, known as the membrane attack complex, consists of an average of 15 molecules of C9 and acts as a pore to cause the leakage of electrolytes and other cytoplasmic components from the antigen-bearing cells. Eventually, the target cells are killed by this cytolytic action.
Mechanism of V D J recombination. Gene rearrangement and B-cell development. Antibody-antigen complexes. Immunoglobulin class switching: Molecular and cellular analysis. Immunoglobulin Genes, 2nd ed. Complement-immunoglobulin interactions. Physiology and pathophysiology of complement: progress and trend. The genetic engineering of monoclonal antibodies.
Single-chain Fvs. The Company is interested in developing an anti-BX antibody for the potential applications in breast cancer diagnosis and treatment. BX has been characterized by the Chemistry Group in the Company as a glycopeptide with a molecular weight of 2, daltons. When BX-human serum albumin conjugate was used as an immunogen, anti-BX antiserum was obtained from immunized rabbits.
The Company decided to immunize mice with BX-conjugate for the production of monoclonal anti-BX antibodies for further development of breast cancer diagnostic kits and, potentially, breast cancer immunotherapeutic drugs. Why was human albumin, instead of rabbit albumin, used for the preparation of the conjugate? Question 2: What are the possible immunoglobulin isotypes present in the rabbit antiserum as anti-BX antibodies? Question 3: Why did the Company decide to produce monoclonal anti-BX antibodies instead of using the current anti-BX antiserum for new product development.
Answer 1: The effectiveness of an antigen to elicit antibody formation is dependent on many factors. BX, with a molecular weight of 2. In order to produce an antibody that will recognize the structure of BX, this small glycopeptide must be linked to a large molecular carrier as a hapten immunogen. When rabbits are used as hosts for immunization, rabbit proteins such as rabbit albumin should not be used as immunogens. Answer 2: The anti-BX antiserum produced from the immunization of rabbit is a polyclonal antibody which consists of all possible classes of rabbit immunoglobulins including IgG, IgM and IgA.
Answer 3: Monoclonal antibody technique can continuously produce a large quantity of pure and well-characterized immunoglobulins. These criteria are important especially if the antibodies are to be used in therapeutic applications. However, monoclonal antibodies usually do not possess the high affinity and high cytotoxicity of polyclonal antibodies, and sometimes they are less stable than antiserum. Furthermore, mouse monoclonal antibodies when used as therapeutic agents may elicit the formation of anti-mouse immunoglobulin antibodies HAMA in humans.
Genetic engineering technology can make chimeric antibodies, humanize antibodies, and single chain Fv fragments sFv that are potentially superior to mouse monoclonal antibodies as therapeutic agents. The pluripotent stem cell is primarily found in the bone marrow of long bones and the pelvis. Although these PSC make up only 0.
One daughter cell will mature and differentiate to form circulating cells, whereas the other daughter cell will retain its quiescent state, and rejoin the pool of stem cells, thus maintaining the same number of parental cells Figure 3. The differentiation and maturation process into mature circulating cells is under the control and regulation of hematopoietic cytokines or growth factors.
These cytokines may also determine the type of circulating cell formed. Activated stem cells can evolve into either myeloid or lymphoid cells. The process as to how the progenitor cell knows what type of cell to form is still unclear. Progenitor cells are pluripotent and can differentiate into a lymphoidal progenitor cell. The maturation of lymphoid progenitor cells is influenced by lymphokines which determine whether the progenitor will form either T- or B-lymphocytes. Cells in the Circulation Each cell plays an important role in maintaining homeostasis.
The various cells found in the circulation are listed in Table 3. There are various types of lymphocytes and they are divided into either bursa- B or thymus- T lymphocytes. B-lymphocytes or plasma cells produce and secrete antibodies, which are used to neutralize foreign invaders and serve as a chemoattractant for cellular elements of the immune defense. All circulating cells are derived from a common parental cell called the hematopoietic stem cell or pluripotent stem cell.
In this diagram, the evolution of the various circulating cells is influenced by the various cytokines that regulate their maturation and differentiation process. T-lymphocytes can be further divided into either regulatory or effector cells. However, one of these daughter cells will retain its quiescent state and rejoin the pool of stem cells, thus maintaining the same number of parental cells. Table 3. Peripheral blood cells. Figure 3.
Antibody dependent cellular cytotoxicity ADCC is where antibody binding will initiate cellular cytotoxicity. The effector cells such as cytotoxic T-lymphocytes CTLs and natural killer cells NK are responsible for eradication of virally-infected or tumor cells. Although the origin of NK cells is still uncertain, the role of NK cells is to eliminate antibody coated pathogen by antibody dependent cell-mediated cytotoxicity ADCC.
NK cells are not phagocytic in nature, rather they carry out their cytotoxic function through formation of perform complexes that allow the leakage of intracellular contents. Presently, the exact mechanism by which NK cells exert its cytotoxic activity is still unclear. CTLs eliminate cells that express foreign antigens on their surfaces, such as virus-infected host cells and tumor cells.
In order for a CTL to acquire cytotoxic activity, it must receive two signals. The first signal is the induction of high affinity IL-2 receptors, which is followed by IL-2 binding onto these receptors, thus initiating cellular cytotoxic activity. After cellular binding, the CTLs will release specific proteins that assemble on the cellular membrane.
The mechanism of cytotoxic T-lymphocyte CTL is shown in this figure. CTLs will bind onto the pathogen via T-cell receptors that are specific for antigen. The presence of MHC I will indicate to the CTLs that the organism is indeed foreign, and thus will release a series of cellular toxins that will eliminate the intruder. Unlike cellular cytotoxicity, apoptosis is where the chromosomes are fragmented by the activation of cellular endonucleases. Myeloid cells have a wider variety of biological activities. Erythrocytes or red blood cells RBCs , due to the presence of hemoglobulin which give rise to its red pigmentation, transport oxygen from the lungs to the tissues, and remove the oxidative byproduct, carbon dioxide.
Platelets are derived from megakaryocytes, and are important in the repair of vascular damage, through initiation of the coagulation cascade during vascular integrity breakdown. The remaining myeloid cells are referred to as either granulocytes or monocytes due to their morphology. Granulocytes are so called because of pigmented granules found in the cytoplasm of these cells. Granulocytes and monocytes are responsible for cellular response to the presence of antigen.
Eosinophils have granules loaded with histamine which is released during allergic reactions. Release of histamine will result in vasodilation and pulmonary constriction. In addition, eosinophils provide host defense against parasitic infections. Similar to eosinophils, basophils provide inflammatory response to allergic reactions but the exact role of these cells is still unclear.
The other important granulocyte is the neutrophil, which plays a seminal role in the defense against bacterial infections. Most notable of these infections is gramnegative bacteremia, which is responsible for the majority of deaths associated wth severe neutropenia. Macrophages are antigen presenting cells APCs , which break down the antigen to an identifiable form for immune recognition. Host Defense Mechanism The body is constantly bombarded with infectious agents, however in the immunocompetent host, only a small percentage of these pathogens actually enter into the circulation and cause clinical symptoms.
This is due to the various defense mechanisms, which when functioning normally, will enable the host to maintain a sterile environment. Barrier defense apparatus such as the skin and mucosal membrane represent the largest mechanism preventing the entrance of pathogens into the circulation. Once the pathogens have evaded the barrier defense, the body will mount an attack consisting of both humoral and cellular immunity.
Although the immune system is divided into cellular and humoral compartments, it works in concert to orchestrate an attack that will preserve sterility. The humoral immunity includes neutralizing antibodies and the complement cascade. Additionally, humoral mediators include stimulatory cytokines, such as interleukin-1 IL-1 and tumor necrosis factor TNF will activate immune cellular response. These immunological defenses are summarized in Table 3. Immune response to antigen presentation.
Antigens that have evaded the barrier defense are neutralized by humoral factors such as antibodies and complement factors. Antigen binding will activate cellular migration to the affected site. The processed antigen is then presented to a naive T-lymphocyte, and is activated by a secondary signal such as cytokine activation.
The first cell that arrives at the affected site is usually an APC, such as a macrophage or primed Blymphocyte. Once the antigen is processed, this will activate macrophage expression of cytokines. The processed antigen along with the MHC II molecule is then presented to a naive T-lymphocyte, which will activate the cell to upregulate expression of interleukin-2 IL Along with antigen presentation, IL-1 and TNF are produced by macrophages, which are required to co-stimulate naive T-lymphocytes.
Paracrinic factors are stimulatory cytokines that are produced by neighboring cells. There are various monocyte-derived cytokines. IL-1 is a primary inflammatory mediator that can stimulate the active recruitment of cells to the affected site. There are two ways this can be accomplished. One mechanism is through the demarginalization of immune cells that adhere onto the walls of the endothelium of the microvasculature.
The other method is to stimulate maturation and differentiation of stem cells to differentiate and expand to form circulating cells. In addition, IL-1 is also able to induce expression of CSFs and lymphokines, which regulate the differentiation, proliferation, and maturation of myeloid and lymphoid cells, respectively. In order for IL-2 to exert its activity, it must first bind onto a receptor which activates a series of intracellular signals that will result in cellular activation.
TH1 regulate cellular immunity, whereas TH2 regulate humoral immunity. In addition, IL-2 and IL will increase their cytotoxic effects. IL-4 is able to induce the production of TH2 cells, such as eosinophils, and mast cells. In addition, IL-4 will promote IgE expression, suggesting that it plays an important role in allergic reactions.
Either IL-4 or IL are able to suppress the induction and function of TH1 cells, which may be the mechanism of downregulating cellular immunity. These cytokines not only stimulate the release of IL-2, but also increase recruitment of immunological cells and stimulate the release of local chemotactic agents.
Additionally, TNF and IL-1 are both able to stimulate accessory cells such as fibroblasts, endothelial, and activated T cells to produce other cytokines such as colony stimulating factors CSFs. IL-1 is able to mobilize progenitor cells into the peripheral blood compartment, which can mature into circulating lymphoidal or myeloidal cells.
TH1, regulate cellular immunity responses such as mobilization and expansion of CTLs, whereas TH2 regulate humoral immune responses such as increased levels of antibodies and complements factors. Biological activity of tumor necrosis factor and IL Biological activity of interleukin The expansion, proliferation, and differentiation of myeloid cells are under the strict control of colony stimulating factors CSFs. CSFs are classified by their capacity to sustain the proliferation and differentiation of various myeloid progenies in colony forming unit CFU assays Figure 3.
Primarily produced and secreted by either activated T-lymphocytes or macrophages, however endothelial cells and fibroblasts have also been described as producing CSFs. These glycoproteins have variable molecular weights which are dependent on the degree of glycosylation. With the exception of macrophageCSF, CSFs are single chain molecules with intrachain disulfide bond s ; the disulfide bond s must be intact for full biological activity.
Other means to classify CSFs have been to divide them according to their potential biological activity Table 3. They are classified as either pluripotent or unipotent CSFs. An important caveat is that all CSFs have been shown in vitro to possess the capacity to stimulate the proliferation and expansion of other lineages; this is usually concentration dependent.
The active GM-CSF molecule consists of a single chain amino acid polypeptide, that was derived from a amino acid precursor protein. The secondary structure of the molecule is maintained by two internal disulfide linkages. GM-CSF has unique regulatory activity, enabling it to influence a wide variety of progenies. Although in vitro studies suggested that GM-CSF has the capacity to maintain megakaryocytic, eosinophilic, and erythroid lineages, these results have not been confirmed in clinical studies. This downregulation of other CSF receptors may be the mechanism by which progenitor cells are selected into a specific differentiation pathway.
Colony forming assays are performed by adding purified bone marrow cells into a semi-solid medium containing either agarose or methylcellulose. Within 7—14 days, distinctive hematopoietic colonies are formed. IL-3 is primarily produced and secreted by activated T-lymphocytes and macrophages. It has a molecular weight of 14—28 kDa. Secondary structure is influenced by the presence of a single disulfide bond.
Carbohydrate complexes are anchored onto the two arginine residues found in its amino acid polypeptide backbone. MCSF is a heavily N-glycosylated dimer that exists as either a soluble or membrane-bound glycoprotein. Its biological activity is lost following reduction of the homodimer, yielding two identical monomeric polypeptides with a MW of 15 kDa each.
The average circulation life span of neutrophils is estimated to be less than about 10 hours. Therefore to maintain basal levels of neutrophils, at least 6 billion new neutrophils must be formed daily. The primary amino acid sequence of G-CSF does not include any arginine linked carbohydrate sites; thus only O-glycosylation appears in this molecule. It is also, to a lesser extent, able to support monocytic, megakaryocytic, and erythroid progenies at higher concentrations.
Similar to other blood components, erythrocyte production is under the strict regulation of CSFs. There are various cytokines that regulate the differentiation of early erythroid progenitor cells such as IL-3 and interleukin-II. However terminal maturation into red blood cells is under the control of erythropoietin EPO.
Human erythropoietin is a 30 kDa glycoprotein with amino acids and four carbohydrate side chains. EPO originates from peritubular cells, possibly fibroblasts that are found in the renal cortex. Trace amounts of EPO can be produced from parenchymal cells in the liver. Endogenous EPO production is regulated Table 3.
Activity of colony stimulating factors. Megakaryocytopoiesis is regulated by a number of cytokines with overlapping biological activity. However, the elusive cytokine that regulates terminal differentiation of platelets could not be isolated for 35 years. Although a factor regulating platelet formation was suspected, not until recently was this cytokine isolated and its gene cloned.
It was initially called c-Mpl-ligand. This receptor is expressed on megakaryocytes and platelets. The TPO gene was located on the long arm of human chromosome 3. Genetic analysis suggests that TPO had a polypeptide backbone consisting of amino acids with striking homology to erythropoietin throughout the N-terminal half. Recombinant TPO has profound effects on megakaryocyte growth and development. These effects appear to include the expansion of megakaryocyte progenitors and induction of megakaryocyte maturation to functional platelet.
It is evident that the maturation, proliferation and survival of circulating blood cells are controlled by the combination of various CSFs. Cytokine networks with infection: mycobacterial infections, leishmaniasis, human immunodeficiency virus infection, and sepsis. The Immune Response. T-lymphocytes and Natural Killer Cells. Clinical effects of biologic response modifiers. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Haemopoietic growth factors. The molecular control of granulocytes and macrophages.
Proinflammatory cytokines, nutritional support, and the cachexia syndrome: interactions and therapeutic options. Inflammatory cytokines in animal health and disease. He tells you that he has been experiencing these symptoms the last 3 days. However, he states that he was not around anyone with colds or flu-like symptoms for at least 7 days. Can you explain the lag period when JQ was initially exposed in comparison with onset of symptoms? Answer 1: Although JQ was probably infected 7 days ago when he came into contact with infected individuals, symptoms normally do not develop immediately.
This may be attributed to the time required for the pathogenic organism to by-pass the barrier defense e. Once the foreign intrusion has occurred, immunological response must ensue. Fevers, chills, and rigors occur because macrophages and activated lymphocytes produce interleukin-1 and tumor necrosis factors, which can induce pyrogenic effects. In order for the clinical signs to occur, enough immune stimulus is required. Two factors can occur here, 1 the pathogen burden on the body, and 2 immune response to the intrusion.
The lag time may occur because time is required for the pathogens to replicate and induce an immune response. However, the therapeutic applications of antibodies became realistic only after the introduction of monoclonal antibodies and the advances of biotechnology in recent years. Several antibodies have already been produced and marketed by pharmaceutical companies as therapeutic drugs for the treatment of human diseases.
It can be anticipated that more antibodies, particularly genetically engineered antibodies or antibody derivatives, will be introduced as new drugs in the next few years. Generally, antibodies can be considered as therapeutic agents in two ways: as drugs and as drug carriers. From the description of humoral and cellular immunity in previous chapters, we should recognize that there are many obstacles when using antibodies for the treatment of various diseases.
There are general limitations of using antibodies as therapeutic agents Table 4. However, there are additional limitations that are associated only with the use of antibodies either as drugs or as drug carriers Table 4. Table 4. General limitations in antibody therapy. Limitations Problems Impermeability of endothelium to antibody molecules Poor tissue penetration of antibodies Antigen heterogeneity and modulation Lack of specific antigen on target cells Lack of human monoclonal antibodies Inaccessible to target tissues Inaccessible to entire tissue Ineffective to antigen-negative or antigen-modulated cells Side effects to normal cells Hypersensitivity towards animal immunoglobulins Table 4.
Comparison of antibodies as drugs and as drug carriers. As Drugs Antibody isotype Antibody modification Host immunity Target Internalization Isotype dependent Intact antibody Dependent on host immunity Antigen-bearing cells As Drug Carriers Isotype independent Antibody conjugation Independent on host immunity Can be designed to kill bystanding nonantigen cells.
These complexes are cleared rapidly from the blood via either the reticuloendothelial system or, if small Fab fragments are used, the kidneys. Such a process can eliminate selectively the pathogens or toxic substances from the blood or tissues. When using intact antibodies as neutralizing agents in the treatment of human diseases, type III hypersensitivity serum sickness is one of the major adverse reactions. This type of hypersensitivity will be discussed in Chapter 5. Traditionally, antibodies have been used in the form of antiserum to save lives from severe infections or poisoning.
For example, rabbit antisera against snake venoms are effective antidotes for poisonous snake bites and anti-diphtheria toxin antisera are used for the treatment of diphtheria infection. It must be emphasized that antigen-antibody complex formation may not result in a total elimination of pathological events.
In fact, recent attempts by several biotechnological companies to produce monoclonal antibodies against endotoxins from gram-negative bacteria for the treatment of sepsis and bacteremia in humans have not been very successful. One of the applications of antibodies as neutralizing agents is for the treatment of drug toxicity. In this case, antibodies are raised against drug molecules as haptens. The preparation of anti-hapten antibody has been discussed in Chapter 2. Both polyclonal and monoclonal antibodies are suitable for this use.
Fab fragments are more practical than the intact antibody because they are smaller in size and therefore, their toxic drug complexes can be excreted from the kidneys. Consequently, the half-life of Fab fragments in the blood circulation is much shorter than that of the intact antibody. In humans, the plasma half-life of Fab fragments from a foreign source such as an animal antibody or a mouse monoclonal antibody is approximately 9 hours, which is significantly shorter than that of the intact IgG, i.
Besides the pharmacokinetic advantages, the dose of antibody required for neutralizing a certain amount of a toxic substance is another important factor to be considered. Due to the relatively large molecular weight of antibody kDa for IgG and 50 kDa for Fab as compared to toxic substances less than 1 kDa for most drugs , a large quantity of antibody or Fab fragments must be used in order to bind stoichiometrically the toxic agent in the blood or tissues.
Theoretically, Fab fragments can be used at a lower dose than that of the intact antibody. Another advantage of using Fab as a drug is that this fragment is less immunogenic than the intact antibody, and, therefore, is less likely to develop hypersensitive reactions if the treatment has to be repeated in the future. The most successful example of using antibody to neutralize toxic substances is the treatment of digoxin poisoning which could be either accidental or intentional overdose. Several pharmaceutical companies have marketed digoxinspecific Fab fragment from animal immunoglobulin or monoclonal antibody as an antidote of digitalis poisoning.
The Fab fragment is given by i. Free digoxin in the serum of the patient completely disappears during the infusion. However, protein-bound digoxin concentration actually increases rapidly which is due to the removal of tissue-associated digoxin by anti-digoxin Fab.
The half-life of the protein-bound digoxin in the serum is approximately 20 hours. However, there are four possible mechanisms that antibodybinding can lead to cell death or growth inhibition Figure 4. This effect can be achieved when an antibody binds to either a growth factor or its cell surface receptor. This approach is particularly attractive in cancer therapy because it is generally believed that many tumor cells produce growth factors as autocrines that stimulate their own proliferation.
Antibodies against these autocrine growth factors or receptors for tumor cell proliferation conceivably could inhibit selectively the growth of the tumor Figure 4—1Aa. Alternatively, antibody binding on surface markers may promote the differentiation of tumor cells to normal cells and therefore is potentially useful in differentiation therapy.
This approach has been proposed for the treatment of B-cell lymphomas. An anti-idiotypic antibody, which recognizes and binds the variable region of immunoglobulin molecules on a B-lymphoma cell, may mimic the action of an antigen molecule to promote the differentiation of the lymphoma cell into an antibody-producing cell.
Such a differentiation process may terminate the proliferation of B-lymphoma cells Figure 4. Antibodies facilitate the phagocytic process by opsonizing the target cells for either Fc or C3b receptors on the surface of phagocytes, a process that has been described in Chapter 2 Figure 4. This complement activation can cause the cytotoxicity to the target cells by the formation of membrane attack complex Figure 4.
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The mechanisms involved in this killing process most likely involve cell-cell contact and the releasing of cytotoxic substances from the effector cell to the target cell Figure 4—1D. ADCC is considered to be the major function of macrophages or K cells in killing of tumor cells because the size of these target cells is too large to be phagocytosed by RES cells. Mechanisms of antibody-mediated cytotoxicity. Regulatory growth control: Antibodies against growth factors or receptors, such as autocrine growth factors in tumor cells, can inhibit cell proliferation.
Alternatively, antiidiotypic antibodies against B-cell surface immunoglobulins may induce the differentiation of the B-cell and subsequently, induce their differentiation into plasma cells. Reticuloendothelial clearance: Antibody-binding on the surface of the target cells can enhance the phagocytosis by macrophages, a process of opsonization which is mediated via Fc receptors. Complement-mediated cytotoxicity: Complement fixation can cause the lysis of antibody-bound target cells.
Antibody-dependent cell-mediated cytotoxicity ADCC : The binding of antibody-bound target cells to effector cells such as K cells via Fc-receptor can cause the death of the target cells due to releasing cytotoxic factors from the effector cells. Human immunoglobulins obtained from the general population can be used for the treatment of patients with immunodeficiency syndrome and for the prevention of infection in patients with chronic lymphocytic leukemia.
Immunoglobulins obtained from healthy donors, e. Human immunoglobulin solutions containing a high titer of antibody to hepatitis B surface antigen can be prepared and purified from the plasma of antibody-carrying donors. These immunoglobulin solutions, e. The use of human immunoglobulin solutions, for the prevention of either general or specific infections, is called passive immunization. The route of administration of these solutions as indicated on the labels, i. It reduces the circulated T-lymphocytes and produces a suppressive effect in both cell- and humoral-mediated immunities.
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