INTRODUCTION
1. Blood and immune system
1.1. Blood
Blood is a liquid connectival tissue circulating in the blood vessels of vertebrates; it is characterised by a heterogeneous composition and represents the 8% of the body weight. The blood, when collected in a glass tube, spontaneously forms a clot and a clear and slightly yellow liquid part, the serum. By addition of anticoagulants (i.e.
substances preventing coagulation ) and centrifugation, the blood is separated into a liquid fraction, plasma (55-60%) and a corpuscular part (40-45%).
1.2 Corpuscular fraction
The corpuscular part is constituted by red blood cells or erythrocytes, white cells or leucocytes, thrombocytes.
Erythrocytes
Erythrocytes are the cellular elements in charge for the oxygen transport all over the body through the blood stream. They develop in the bone marrow.
They are disc-shaped and have a diameter of 6.5 – 7.5 µm; their average lyfe is 120 days. During their life cycle nucleus and the endosomal compartments are lost and the mature elements contain only haemoglobin, which binds oxygen through the prostethic group in the lungs. In the peripheral blood stream oxygen is given to the tissues and carbon dioxide is picked up.
Thrombocytes
Thrombocytes play a key role in the clotting events through various functions (transport and storage of clotting factors, phagocytosis etc etc.). Their average life is 10 days.
They look as small rounded or oval bodies (size: 2 – 4 µm), rich of intracytoplasmic organs, which ensure a strong metabolic activity. During the coagulation events, thrombocytes supply a lipidic surface on which the clotting reactions happen; they contribute to these reactions by releasing clotting factors contained in different types of granules. Through the aggregation and activation reactions, thrombocytes ensure the correct clot formation and its removal.
They develop in the bone marrow from the megacaryoblast stem cell, which evolves in a megacaryocyte, in which a intense formation of smooth tubules (which constitute a
reticular compartment) is observed. The fusion of this membranes leads to the delimitation of the future thormboocyte, which is then released in the blood circulation.
The detailed description of the thormbocyte functions is beyond the aim of this introduction.
Leucocytes
The leucocytes, or white cells, are in charge for the immunitary response. They develop in the bone marrow and exert their function in the blood and in the lymph streams. They can be divided in two main types:
1. Granulocytes, which in turn can be divided in (i) neutrophils, (ii) eosinophils and (iii) basophils;
2. Mononuclear cells, which in turn can be divided in (iv) lymphocytes and (v) monocytes.
Neutrophils are rounded cells, having a diameter of 10 µm and a nucleus typically divided in lobes, connected by chromatin bridges; their percentage (calculate don the total amount of leucocytes) is 50 % - 70 %. They are characterized by the presence of different types of granules containing hydrolytic and protease enzymatic activities and other cytoplasmic organs; they can also actively move by pseudopodes formation.
Neutrophils are active during the inflammatory response, removing the responsible agents by phagocytosis and subsequent intralysosomal digestion.
Eosinophils are similar to neutrophils, having a rounded body and a lobe-shaped nucleus. They have specific granules, containing basic proteins and hydrolytic and protease enzymatic activities; their percentage is 2 % - 4%. They present a well developed Golgi apparatus and are specialized in the phagocytosis of antigen-antibody complexes during the inflammatory response caused by parasites or allergic diseases.
Like the neutrophils, they can move by pseudopode formation.
The basophilic granulocytes resemble the neutrophils and eosinophils for shape, nucleus and size. They are rare cells (percentage: 0.5 % – 1 %). They contain acidic granules and polyanionic substances (heparin). They act in the inflammatory response
thromboplastins (triggering the clotting cascade) and inhibiting the intravasal coagulation. They are also called mastcells, very close to the connectival cells mastocytes.
Immunoglobulins E (produced by lymphocytes B under an antigenic induction) are located on the extracellular side of the plasmatic membrane of the mastcells and mastocytes: the formation of the antigen-IgE complex on the plasmatic membrane leads to an activation and a rapid release of the content of the granules.
Lymphocytes are present in the blood, lymph and lymphatic organs. In resting conditions (blood) they are rounded with a big nucleus and a very reduced cytoplasmic portion. In the activated state (lymphatic district) they have a completely different appearance, at both nuclear and cytoplasmic level. They are well represented (20% - 40%) and bigger than erythrocytes and can be distinguished in small, mid and big lymphocytes, with morphological differences and all of them are capable of movement.
Lymphocytes can be divided in B cells and T cells; B cells are involved in the antibody- mediated immune response, while T cells are in charge for the cell-mediated immune response. B and T cells populate different areas of the lymphatic organs.
Monocytes (percentage: 3% – 8%) are precursors of the mobile macrophages of the connectival tissue. They are oval or rounded cells with a diameter of 15 – 20 µm. Like the other types of white cells, they are able to move by pseudopode formation.
Generally monocytes remain in the blood stream from approximately 24 hours and then migrate in the connectival tissues, where they acquire the morphological traits of macrophages. They are involved in the inflammatory response (especially the chronic one) by exerting a strong phagocityc and secretory activity; these two activities lead to a complex series of events, like the release of “defensive” molecules (interpherons), the association and the binding of antibodies-covered particles and complement molecules to the macrophage membrane. The membrane-exposed (“presented”) antigens are recognized by the T cells, which are in turn activated. It is important to underline that B cells are able to recognize soluble antigens, whereas T cells aren’t.
The lymphoid precursors of lymphocytes develop in the liver of the phoetus and then migrate in dedicated organs during the phoetal development. T lymphocytes develop
cells undergo phenotypical modifications and TCR genes rearrangement, while B cells undergo both phenotypical changes and immunoglobulins genes rearrangements. These events lead to the selection of mature clones, generally known as lymphocytes maturation (Rosati, 1989).
1.3. Plasma: general aspects
Plasma is a slightly yellow liquid and is composed of water (90%), in which different types of solutes (10%) are dissolved. The solutes are proteins (7%), inorganic salts as sodium chloride, sodium hydrogencarbonate (0,9%) and organic molecules like aminoacids, lipids, urea, phospholipids, organic acids (2,1 %).
Plasma contains nearly 3000 proteins, of which 300 are characterised and 13 are used in clinical practice. Many plasma proteins are glycoproteins, which are very common among animals, fungi, viruses and bacteria.
The glucidic portion often retains the antigen properties and biological functions of the protein and is generally linked to the peptidic chain by:
- O-glycosilic bond, involving N-acetil glucosammine (GalNac) and serine or threonine residuals, typical of mucines b) mannose and serine or threonine, typical of fungi and yeasts c) oligosaccharides and hydroxilysine or hydroxiproline, typical of collagen.
- N- Acetil glucosammine-asparagine bond involving: 1) simple chains which contain generally a high amount of mannose residuals, typical of ovalbumin 2) complex chains, in which the portion linked to the protein is similar to the simple chain while the external part is often composed by sialic acid-galactose (SAGal), N-Acetil glucosammine-asparagine (GlcNAc), typical of immunoglobulins. The glucidic content of plasma proteins varies substantially between the different proteins (albumin 0%, transferrin 6%, ceruloplasmin 7%, alpha-1 acid glycoprotein 42%).
Plasma proteins have the following are functions: 1) regulation of osmotic pressure, maintenance of volume and viscosity of blood (mainly albumin), 2) transport of other proteins, hormones, vitamins, phospholipids, fatty acids, 3) transport of metallic ions
isoagglutinins) 5) regulation of coagulation and fibrinolysis. Moreover in plasma intracellular enzymes (their presence is a symptom of a disease) and tissue-derived enzymes could be found.
1.4. Plasma proteins
Plasma proteins can be divided in the following groups, normally separated analytically by zonal electrophoresis: Alpha 1 (7%), Alpha 2 (9%), beta (14%), gamma (12%) and albumin (58%).
Figure 1I: Migration pattern of plasma proteins
1.4.1. Albumin
It is the most abundant plasmatic protein, highly soluble in water and alcohol. Structural studies on the aminoacidic sequences of human and bovine albumin indicate that albumin consists of a single peptide chain containing about 580 residues with 17 intrachain disulphide bonds. The distribution of the disulfide bonds and the location of specific residues throughout the polypeptide chain suggest that the albumin molecule folds into 3 structural domains and 9 subdomains. It is free of glucidic chains and poor of triptophan and methionin while cystein and charged aminoacids are well represented.
It has the lowest molecular weight among all plasma proteins (66000 Da) and its concentration is about 50 mg/ml of plasma. A major function of albumin is the osmotic regulation: in fact it is responsible for about 75% - 80% of the oncotic pressure of plasma, because it is representative of slightly more than half of the plasma proteins in weight and has the lowest molecular weight. The osmotic properties are maximal blood pH (7.5). Only about 40% of albumin is located in the blood stream and the remaining is present in the extravascular space of tissues, principally muscles, skin and intestines.
About the 55% of albumin exit from the blood stream and enters the lymph through the
especially those poorly soluble in aqueous environments, as lipids (one of the main effects of analbuminaemia is an impaired lipid transport with high levels of cholesterol, phosphoglycerids and lipoproteins), and free fatty acids from liver to the peripheral tissues. Albumin also binds bilirubin and aids its transport in the liver where it is excreted in the bile. The plasma concentration of different substances such as Ca 2+, steroid hormones and triptophan are regulated to some extent by the binding to albumin.
Albumin concentrates are administered in severe conditions (shock, acute hypotension caused by bleeding, burns, surgical operations, traumas) and it is actually purified from Cohn’s fraction V or by chromatography.
1.4.2. a-globulins
Several proteins are indicated as a1 or a2-globulins, depending on their electrophoretic mobility. Some of them are of unknown function, such as a1-acid glycoprotein, which has an high carbohydrate content (42%) and a structural similarity to the immunoglobulin molecules.
The glycoprotein a-fetoglobulin, is the major protein in the human phoetal plasma and amniotic fluid, but it is normally present in very low amounts in adults. After birth its concentration decreases with concomitant increase of albumin; suggesting that it could play in phoetus the same role of albumin in adults. Indeed, the sequences of these two proteins are highly homologous.
Retinol binding protein (RBP), which is involved in retinol transport, form an equimolar
complex with the protein transthyretin, which also function as a thyroxine-binding protein. It is hypothesized the RBP-transthyretin complex could prevent renal excretion of the small retinol binding protein in the urine.
Ceruplasmin, so named because of its blue colour, contains 3 asparagine-linked
oligosaccharides and has 8 sites for the binding of either Cu+ or Cu2+. It is supposed function as peroxidase, converting Fe2+ to Fe3+ prior to incorporation of Fe3+ in transferrin (the plasma ferric ions transport protein). It also aids the manteinance of Cu2+
homeostasis and Cu2+ transport. In the rare inherited Wilson disease, plasma ceruloplasmin is markedly reduced and Cu2+ levels increase in brain and liver with a resulting neurological changes and liver damage. The complete sequence of human ceruloplasmin reveals 3 internally homologous regions, suggesting that during evolution a structural gene coding for about 350 residues was three-folds replicated to form the
It has a 160 aminoacids sequence homologous to Cu-binding sites of cytochrom oxidase and the cytoplasmic Cu-Zn form of superoxide dismutase.
Haptoglobin constitutes about one-fourth of the a2-globulins and forms specific, stable
1:1 complexes with haemoglobin. Such complexes form in vivo as the result of intravascular haemolysis of erythrocytes. Because of their high molecular weight, the complexes cannot be excreted by the kidney and this prevents the excretion of iron in the urine and at the same time protects the kidney from damage by haemoglobin. The haptoglobin-haemoglobin complexes are degraded by reticuloendothelial cells, and the iron ion is recycled in heme synthesis, after degradation of globins and excretion of degraded heme as bile pigment. Low level of haptoglobin are found in patients with a variety of haemolytic anemias. Human haptoglobin contains two pairs of non identical chains joined by disulphide bonds; the subunit structure is designed as a2 ß2. There are 3 genetic types of haptoglobin Hp 1-1, Hp 2-1, Hp 2-2 differinf only in the structure of their a chain, characterised by high polymorphism.
Seven protease inhibitors have been found in human plasma and, among these, Antithrombin III and a2-antiplasmin aid the regulation of homeostasis by inhibiting blood coagulation proteases. The others prevent unwanted proteolysis, presumably by tissues proteases that could enter the blood stream but their physiological role is yet not fully understood.
a2-macroglobulin is capable of inhibiting proteases of all classes, whereas the others
primarily inhibit serine-hystidine proteases such as, trypsin, chymotrypsin and proteases of blood coagulation. a 2-macroglobulin contains two pairs of disulphide-linked identical subunits in which one or two peptide bonds are hydrolyzed by the protease to be inhibited. After cleavage the conformation of a2-macroglobulin changes, resulting in a tighter binding between a2-macroglobulin itself and the protease. Once formed, the protease- a2-macroglobulin complex is bound to macrophages and is then internalised by endocytosis via coated pits.
a1-protease inhibitor is often called a1-antitrypsin, and is reactive against a broad
spectrum of serine proteases.
Other a-globulins are: a1-T-glycoprotein, transcortin, a1-antichymotrypsin, a1-B- glycoprotein, vitamin D-biding protein, a1-lipoproteins, Factor X, transcobalamin, C9, thyroxine-binding globulin.
1.4.3. ß-globulins
ß-globulinic fraction consists of a variety of proteins including several lipoproteins.
Transferrin is a major component constituting about 3% of the total plasma proteins.
Although it may combine with Cu2+ and Zn2+ the major function of transferrin is to bind and transport Fe3+ to tissues, particularly those of the reticuloendothelial system where Fe3+ is released without altering the protein. Transferrin also helps to regulate the concentration of the free iron in plasma, thereby preventing iron accumulation in tissues and urinary loss of iron. Transferrin binds 2 atoms of Fe3+ per molecule but only in the presence of CO2; in physiological conditions only one-third of the total amount is saturated with iron. Characterised by a molecular weight of 76000 Da, a pI of 5.5-5.9, it is formed by 630 aminoacids and circulates in plasma in a concentration of 2.8 g/L. 21 variable genetic forms are known.
Hemopexin binds heme and prevents its urinary excretion, retaining heme iron for
further use. The hemopexin isolated from normal individuals does not show a full biing capacity for heme, whereas the haemopexin isolated from patients with haemolytic anemia is almost fully saturated, containing one heme per haemopexin molecule. The haemopexin-heme complex is removed from blood by the liver, where the iron recovered from the heme can be reused.
C-reactive protein is normally present at a concentration less than 1mg/dl in adults but
increases markedly after acute infections. Its name derives from the ability to form precipitates with a group C polysaccharide of Pneumococcus in the presence of Ca2+. It has been suggested that it promotes phagocytosis.
ß2-microglobulin is present in very small amounts in plasma; because of its molecular
weight, it is excreted in the urine, where it is found normally in a concentration of about 0.1 mg/L. Its sequence of 100 residues shows a remarkable degree of homology to single domains of immunoglobulins. ß2-microglobulin is the smaller subunit of HLA histocompatibility antigen complex, which regulates the self/non-self recognition, as the rejection of transplanted tissues.
The protein binds to membrane of several types of lymphoid cells as well as to cells of other tissues.
Other ß-globulins are: steroid –binding ß-globulins, factor V, factor VII, factor IX, plasminogen, many proteins of complement (C1r, C2, C3, C4, C5, C6,C3 activator),
1.4.4. ?-globulins
This fraction of plasmatic proteins contains the immunoglobulins, which are considered in the following paragraph. Included in this group are the so called cryoglobulins, immunoglobulins that precipitate at low temperatures. Cryoglobulins are often found during inflammatory illnesses. The so called “cold insoluble globulin” behaves as a cryoglobulin, and it is closely related to fibronectin.
1.4.4.1. Immunoglobulins
All immunoglobulins (gammaglobulins or antibodies, Ig) share a common skeleton, made by two identical light chains (molecular weight: 25 KDa) and two identical heavy chains (molecular weight: 55 – 70 KDa). Each heavy chain is linked to a light chain and the two heavy chains are linked together too. Both the light chains and the heavy chains contain a series of homologous repeated units, 110 aminoacids long, that fold up in a globular structure called the immunoglobulin domain, that contains in its turn two layers of planar ß-sheets with three or four anti-parallel peptidic sequences. In spite of their structural similarity, Ig can be easily divided in different classes and subclasses, on the basis of chemical-physical properties (as size, electric charge and solubility). In the human species the classes of antibodies are named IgA, IgD, IgE, IgG and IgM.
Members of the same class have the same isotype. The isotypes of IgA and IgG can be further divided into strictly correlated subclasses, named respectively IgG1, IgG2, IgG3, IgG4 and IgA1 and IgA2. The isotype is attributed on the basis of the heavy chain classes, that are identified by letters of the greek alphabet: for example the IgA1 contains the a1-heavy chain, the IgA2 contains the a2 heavy chain and so on.
Figure 2I: Structure of Immunoglobulins
The heavy chains of all Ig belonging to the same isotpe/subclass show extended regions of identity but differ significantly from the heavy chains of other isotypes/subclasses.
A single human being has more than 1*107 types of antibody molecules structurally different in terms of the unique aminoacidic sequence in the antigen binding site. This extraordinary diversification of the structure explains why differences in the aminoacidic sequence can affect the antigen binding site. The variable regions are relegated in three small zones of the N-terminal domain of both heavy and light chains.
Structural studies of monoclonal Ig demonstrate that the aminoacidic sequences of the amino terminal domains are organised in variable regions (V) and more conservative constant regions (C), located in the remaining part of the chain (Barnikol et.al. 1982).
The sequences characterised by high variability are called hyper-variable regions, and are kept together by the framework regions (FR). In the native Ig molecule, the three hyper-variable regions of each heavy chain are located in order to built a structure able to bind the antigen, called Fab site (binding antigen fragment). Since these sequences constitute a complementary surface for the tri-dimensional structure of the antigen, the hyper-variable regions are called “Complementary Determining Regions (CDR, fig.)”.
The Fc fragment is responsible of different physiological functions of Ig.
1.4.5. Structure of light chains
The light chains of antibodies are distinguished in two classes, ? and ?, approximately present in equal ratios. Each light chain, either ? or ?, is folded in separated V and C domains, corresponding respectively to N-terminal and C-terminal portion of the molecule; each domain is composed by 110 aminoacids. Each CDR is 10 aminoacids long and there are three CDR, respectively named CDR1, CDR2, CDR3; the last one is the most variable. Also the C-terminal end of C region in the light chain folds in a immunoglobulin domain. Even though C? and C? differ in the aminoacidic sequence, both are structurally correlated (or homologous) to each other, and to V? and V? to a smaller extent.
1.4.6. Structure of the heavy chains
All heavy chains (a, ?, d, e, µ), independently from the antibody isotype, contain a series of homologous sequences, each composed by roughly110 aminoacids, and fold in a 12KDa immunoglobulin domain.
As for the light chains, the Vh N terminal domain shows the highest variability in the sequence, the most highly variable residuals being concentrated in the CDR1, CDR2, CDR3 regions. The remaining part of the heavy chain forms the constant region, which shows variability among the different isotypes; nevertheless this region does not change in the antibodies of a certain isotype.
In the heavy chains ?, a, d a non globular portion (a1, a2, ?1, ?2, ?4) composed by 10 ÷ 60 aminoacids or more (?4 and d) is located between the first and the second domain of the constant region (CH1 and CH2 respectively) and it is called “zip region". The antibodies of those subclasses which contain flexible zip regions, can engage more sites to bind a particular antigen and such involvement increases the binding strength. The last CH domain of heavy chains, depending on the aminoacidic sequence, can determine:
- the secretory form, present in plasma ending with a sequence containing polar hydrophilic amnoacids;
- the membrane form, present only on the cellular membrane of B lymphocytes, containing a C-terminal sequence characterised by 26 non polar hydrophobic aminoacids, followed by a variable number of polar aminoacids (usually basic), which form the intracellular portion of the molecule.
The secretory form of ?, a, d chains contains an additional non globular sequence at the C-terminal end of the last CH domain; this region is called “tail sequence” and supports interactions between different units in multimeric Ig molecules such IgA and IgM.
Particularly, the IgM forms pentamers, constituted by 10 heavy chains and 10 light chains, whereas the IgA can form dimers (containing 4 heavy chains and 4 light chains) or trimers (containing 6 heavy chains and 6 light chains).
The multimeric IgM and IgA contain another 15 KDa polypeptide, called joining chain (J), linked to the tail sequence by disulphide bonds, which stabilises the multimeric complex. Unlike the secretory form and independently from the isotype, all the membrane Ig are monomeric and contain only 2 light chains and 2 heavy chains. The heavy chains are glycosylated in the N position: the polypeptide chain contains oligosaccharide residuals linked by a N-glicosylic bond with N-linked asparagines residuals. The position of the oligosaccharides can change in the different isotypes of IgG.
1.4.7. Association between heavy chain and light chain
In the fundamental model of association between the chains, a light chain binds each heavy chain and the 2 heavy chains are paired. The association between light chains and heavy chains involves either covalent or non covalent bonds. The covalent bonds are disulphide bridges between the C-terminal of the light chain and the C-terminal region of VH or CH1 domain of the heavy chain. The exact portion on the heavy chain that participates to the formation of the disulphide bridge varies according to the isotype.
Principally, the non covalent bonds are hydrophobic interactions between VL and VH domains, and CL1 and CH1 as well. This association allows the juxtaposed domains to contribute to the formation of the antigen binding site.
1.4.8. Distribution of antibodies
The antibodies can be found in many anatomic districts:
- The endosomal cytoplasmic compartment (endoplasmic reticulum, Golgi apparatus);
- The extracellular side of B lymphocytes plasmatic membrane;
- Plasma and in less amounts in the interstitial fluids;
- On the extracellular side of the cell membrane of some effector cells of immune system, as mononuclear phagocytes, Natural Killer (NK) cells and mastocytes;
- In the secreted fluids as mucus and milk, where certain types of antibody molecules are specifically accumulated.
2. The immunological system
The function of the immunological system is to protect the host from the infection of pathogen micro-organisms and substances which can cause a state of disease. When the functionality is optimal, the immunological system is able to recognise specifically antigens belonging to the body (self antigens), and external to the body (non self antigens) The protection from the infections and from the diseases is provided by two main components: the innate immunological system and the acquired immunological system.
2.1. The innate immunological system
The innate immunological system is the first defence of the body against the antigens
components (macrophages and neutrophils). The cellular types involved in the immune response have been described in paragraph 1.2., page 2.
2.2. The acquired immunological system
If the innate immunological response is not able to solve the infection, the acquired immunological system will be activated. The acquired or specific immunity can be classified as:
1) Cell-mediated immunity;
2) Humoral immunity.
Both systems have a lot of different properties, which contribute to the successful elimination of pathogens. The specific immunological responses can be divided in different phases:
- Recognition phase, consisting of the capacity to answer to different antigens, in a very specific way;
- Activation, in which discrimination of the non-self antigens from the self ones occurs;
- Effector phase, during which a vigorous response of memory against already met antigens is activated.
2.2.1. Cell-mediated immunity
The generation of a specific immune response requires the participation of cells presenting the antigen (APC), like macrophages, dendritic cells, Langherans cells.
These cell types have a crucial role in the immune response because they (i) engulf exogenous antigens and digest them in the lysosomal compartment (ii) transfer the antigen fragments to the plasma membrane (iii) and show the antigen fragments in association with Major Histocompatibility Complex molecules, type II (MHC-II). The MHC-II-antigen complex is immunogenic.
Receptors on the T cells are able to recognize the immunogenic complexes and to bind the APC cell membrane and the result of this interaction is the so-called “lymphocyte activation”.
Following the activation of Helper T-lymphocytes (CD4+), effector molecules, called cytokines (Katzung et al, 2000) are secreted. The effect of such molecules is the
activation of immunogenic complexes phagocytosis by APC cells and the activation of IgG secretion by B-lymphocytes. The IgG binds then the antigen.
Generally the “lymphocyte activation” starts with the generation of secondary messenger molecules, which stimulate the transcription of genes involved in the clonal proliferation of T-cells and the development of effector functions.
2.2.2. Humoral immunity
B-lymphocytes undergo selection in the bone marrow: the reactive lymphocytes against self antigens are eliminated by the clonal deletion, whereas the B clones specific against non self antigens are amplified. Unlike the specificity of T cells for the non self antigens (that is determined genetically), the specificity of B cells is due to the fact that they produce and expose antibodies on the extracellular side of the plasma membrane. For B and T cells this committment occurs before they meet the antigen. The antigen binds the Ig (membrane IgG, IgM or IgD) on B cells, and the complex is brought into the cell cytoplasm by endocytosis, digested and presented to the CD4+ T-Helper cells. These T- Helper cells are then stimulated to produce IL-4 and IL-5 interleukins. IL-4 and IL-5 activate the proliferation of B cells and their differentiation into B memory cells or into plasmacytes secreting antibodies. The normal antibody response consists most of all in the production of IgM. The next antigenic stimulation leads to a vigorous increase of the response, followed by the change of the Ig class (isotypic switch) and the consequent production of IgG, IgA, IgE, characterised by different and specific effector functions.
The antibodies simultaneously increase the binding specificity for the antigen. The antibodies regulate the immunitary functions either increasing the phagocytic and the cellular cytotoxicity or activating the complement cascade, mediating the inflammatory response and bacterial lysis.
2.3. Effector functions of the antibodies
Generally the antigen molecules (substances specifically recognized by an antibody) are much bigger than the antibody combining site; the antibody binds afterwards a little specific portion of the macromolecule, called determinant or epitope. Macromolecules usually contain a large amount of determinants and each one can be bound by its specific antibody. On the basis of the antigen-antibody interaction, the antibody can express different effector functions:
- Antibody expression: the antibody can be expressed on the surface of B lymphocytes after the contact with the antigen;
- Steric mechanism: the secreted antibodies, through the binding of antigen determinants, can contrast sterically the interaction of the antigen with the receptors on the surface of target cells;
- Specificity: different types of Ig can have the same binding specificity of the antigen due to the isotypic switch of the heavy chains on B lymphocytes;
- Opsonization: the Ig can make the phagocytosis more efficient by coating antigen particles. The bound IgG are then recognized by Fc?R (cell surface receptor for the Fc fragment of Ig) located on the surface of leucocytes, which engulf more efficiently the antigen;
- Antibody dependent cell-mediated cytotoxicity (ADCC): Ig can coat the target cells and then trigger cytotoxic T-lymphocytes, which in turn induce the cellular lysis.
- Protective role: the maternal IgG can protect foetus against diseases by circulation through the placenta. IgA can be carried selectively in the lumen of organs, where they neutralize pathogenic agents, and can be secreted in the maternal milk, for the protection of the bowel in newborns;
- Anti-inflammatory role: after antigen binding the IgE can aggregate and cause the release of inflammatory mediators and vasoactive substances from the mastocytes and basophilic granulocytes. Moreover, they induce the ex novo synthesis and secretion of mediators produced by lipidic metabolism and cytochines. The consequence of the release of these mediators is an inflammatory vascular response called immediate hypersensitivity (Abbas et al., 1994).
In table 1I the principal functions of the antibodies, the daily half life and the plasmatic level in adult population are summarized.
Table 1I: Plasmatic levels, daily half life, principal functions and effects of Ig
Class Plasmatic level in
adults (g/L) Daily half life Principal functions and effects
IgM 1.0 5
Complement fixation.
Prevalent in the primary immunitary response
IgG 12.0 25
Complement activation.
Activation of cellular receptor for the constant
portion inducing phagocytosis
IgA 1.8 6
Protection of nasal mucosa. Activation of cellular receptor for the
constant portion inducing phagocytosis
IgD 0.03 2.8 Unknown function
IgE 0.0003 2
Stimulation of mast cells. Elimination of
parasites
2.4. Immunodeficiency syndromes
Immunoglobulin deficiency syndromes are a group of disorders which involve defects of any component of the immune system or a defect of another system that affects the immune system, leading to an increased incidence or severity of infection. In these disorders, specific disease fighting antibodies (immunoglobulins such as IgG, IgA, and IgM) are either missing or present at reduced levels. Children who have immunodeficiency syndromes may be subject to infections, diseases, disorders, or allergic reactions to a greater extent than individuals with fully functioning immune systems.
About 70 % of immunoglobulin deficiencies involves B lymphocytes and 20–30 % involves T-lymphocytes. Another 10 % may involve both B and T-lymphocytes.
Many of the infections occurring in children with immunoglobulin deficiency syndromes are caused by bacterial organisms or microbes. Children with immunoglobulin deficiencies are also prone to viral infections, including echovirus, enterovirus, and hepatitis B.
They may also develop infection after receiving live (attenuated) polio vaccine. This is one of the reasons that live polio vaccine is no longer used routinely in the United States.
There are two types of immunodeficiency diseases: primary and secondary.
Immunoglobulin deficiency syndromes are primary immunodeficiency diseases. They account for 50 % of all primary immunodeficiencies and are the largest group of immunodeficiency disorders. Some are well defined and some are not fully understood.
Secondary disorders occur in normally healthy people who are suffering from an underlying disease that weakens the immune system. Successful treatment of the disease usually reverses the immunodeficiency.
Examples of well defined immunoglobulin deficiency disorders include the following:
• X-linked agammaglobulinemia is an inherited disease stemming from a defect on the X chromosome, consequently affecting more males than females. The defect results in absence or reduced number of B-cells, functionally impaired.
All classes of immunoglobulins are decreased in agammaglobulinemia.
• Immunoglobulin heavy chain deletion, a form of agammaglobulinemia, is a genetic disorder in which part of the antibody molecule is absent. This condition results in the loss of several antibody classes and subclasses, including most IgG antibodies and all IgA and IgE antibodies. The disease occurs because part of the gene for the heavy chain has been lost.
• X-linked hypogammaglobulinemia can occur in combination with growth hormone (GH) deficiency, producing short height and delayed puberty, primarily in boys but also occurring in girls.
• Transient hypogammaglobulinemia of infancy is a temporary disease of unknown cause. It is believed to be caused by a defect in the development of T helper cells. As the child ages, the number and condition of T helper cells get better, up to a complete recovery. Hypogammaglobulinemia is characterized by low levels of antibodies in the blood. During the disease period, children may have decreased levels of IgG and IgA antibodies. In some infants laboratory tests are able to show that the present antibodies do not react properly with infectious bacteria.
• IgG subclass deficiency is a disorder associated with a poor ability to respond and make antibody against polysaccharide antigens, primarily the Pneumococcus antigen.
• Selective IgA deficiency is an inherited disease characterized by a failure of B cells to switch from IgM production to IgA production. The amount of produced IgA is limited in either serum or the mucosae of organs. This condition may result in more infections of mucosal surfaces, such as the nose, throat, lungs, and intestines. This syndrome is often asymptomatic.
• IgM deficiency is characterized by the absence or low level of total IgM antibodies. This condition results in slow response to infective organisms and slow response to pharmacological treatments.
• IgG deficiency with hyper-IgM content is a disorder due to the failure of switching from IgM to IgG. This condition produces an increase in the amount of IgM antibodies and a decrease in the amount of IgG and IgA antibodies and is due to a genetic mutation.
• Severe combined immunodeficiency (SVID) is not precisely an immunoglobulin deficiency, but a combined deficiency resulting from a T-cell disorder. The T- cell dysfunction can either be X-linked, affecting more males than females and characterized by the absence of T-lymphocytes, or it can occur through autosomal inheritance, resulting in an absence of both T and B lymphocytes and a deficient thymus gland.
Common variable immunodeficiency (CVID) is a primary immunodeficiency with symptoms typically occurring in the second or third decade of life. It is never diagnosed before two years of age and is diagnosed only after drug toxicity and other primary immune deficiencies have been ruled out. IgG and IgA and/or IgM will be measured at about two standard deviations below normal. Typically the individual will not produce antibodies against proteins, polysaccharide antigens or incompatible blood group antigens (hemoagglutinins).
Primary immunoglobulin deficiency syndromes occur rarely. The X-linked ones occur more in males than females; other immunoglobulin deficiencies occur equally in both sexes. Detection of the syndromes usually occurs in childhood. Numbers of new cases of specific syndromes are difficult to estimate because many deficiencies are not
The incidence rates are known for IgA deficiency (one on 500–700), agammaglobulinemia (one on 50,000–100,000), severe combined immunodeficiency or SCID (one on 100,000–500,000), and common variable immunodeficiency or CVID (one on 50,000–200,000).
Primary immunoglobulin deficiencies are primarily the result of congenital defects that affect the development and function of B lymphocytes (B cells). Defects can occur at two main points in the development of B-cells. First, B cells can fail to develop into antibody-producing cells: X-linked agammaglobulinemia is an example of this disease.
Secondly, B cells can fail to produce a particular type of antibody or fail to switch classes during maturation. Frequent symptoms are persistent infections, particularly of the respiratory system. Frequent digestive disturbances and diarrhoea may lead to incorrect absorption of essential nutrients and failure to thrive. Children with primary immunoglobulin deficiency syndromes will exhibit some of the following characteristics:
• signs of infection in the first days or weeks of life;
• a slow response to treatment;
• infection suppressed by appropriate treatment but not cured;
• common bacterial or viral organisms causing increasingly acute recurring infections;
• uncommon bacterial or viral organisms causing infection;
• multiple simultaneous infections in different sites;
• delays in growth and development;
• development of unexpected complications such as anemias and chronic diseases.
An immunodeficiency disease is suspected when children become ill frequently, especially repeat illness caused by the same organisms. Diagnosis will begin with a detailed history of the child's illnesses (dates, duration, and infection site) and review of all prior medications and immunizations and results of diagnostic tests performed.
Determining which immunoglobulins are present and which are absent or present in reduced amounts is critical for diagnosis. Diagnostic testing may include routine blood tests such as a complete blood count (CBC) and differential (peripheral blood smear) to evaluate overall health and determine the type and number of red cells, white cells, and
of bacteria or virus causing recurring infections. B lymphocytes and T lymphocytes may be quantified. When an immunodeficiency is suspected, levels of the classes of immunoglobulins are measured in blood serum by using a clinical laboratory procedure called electrophoresis. This procedure both quantifies the amount of each antibody present and identifies the various classes and subclasses of antibodies. Deficiencies may be noted in one class or subclass or in combinations of antibodies. Genetic testing may be done to help identify the type of immunodeficiency disease.
Immunoglobulins deficiency diseases cannot be cured, but treatment that replaces or boosts specific immunoglobulins can support the immune function in affected children.
Immune serum, obtained from donated blood, containing adequate levels of IgG antibodies, may sometimes be transfused as a source of antibodies to boost the immune response, even though it may not contain all antibodies needed and may lack antibodies specific for some of the recurring infections. The preferred treatment is to give specific immunoglobulins intravenously (intravenous immunoglobulin therapy or IVIG) or subcutaneously. No replacement therapy is available for the treatment of IgA deficiencies.
Treatment will also focus on controlling infections in immunodeficient children.
Immunization against frequent infection can be achieved in some children by administering polysaccaride-protein conjugate vaccines shown to improve immune response in certain types of infection. Antibiotics are used routinely at the first sign of an infection to eliminate infectious organisms. Antifungal drug therapy may be administered to treat fungi infections. Few drugs are effective against viral diseases, and each viral illness will be evaluated and treated differently, depending on the virus and on the overall health of the child. Bone marrow transplantation may correct immunodeficiencies in some cases.
Several nutritional supplements are reported to help build the immune system. Regular medical observation, treatment of symptoms, and appropriate immune system boosting usually produces a good result in children with immunodeficiencies. Prognosis is related to the immune system's ability to produce the specific antibodies that are missing or present in reduced amounts. Individuals with immunodeficiency syndromes may have a normal life span although a variety of complications could occur, including
autoimmune, gastrointestinal, granulomatous, and malignant conditions as a result of progressive immune deficiency disorders and/or repeat infections.
Immunodeficiency cannot be prevented; however, challenges to the immune system can be reduced and infections avoided in immunodeficient individuals.
On the basis of the above considerations, replacement therapy plays a very important role in assuring a good quality of life in immunodeficient patients. In the following paragraph the production of immunoglobulin concentrate and all the related problems are reported.
3. Plasma as IgG source material
Due to the complex molecular structure, IgG cannot produced by synthetic routes: a natural source of Immunoglobulins is represented by blood, and particularly by plasma.
Therefore the production of IgG concentrates suitable for administration in humans (intravenous, intramuscular or subcutaneous) must take into account the characteristics of the source material.
In fact plasma must be collected from volunteer donors, separated by blood (if it is the case), frozen, stored and finally processed for IgG extraction. During all these steps safety and manufacturing issues must be solved to achieve a “final IgG concentrate”
suitable for administration in patients. In the following paragraphs the achievement of this target is described.
3.1. Plasma collection 3.1.1. General aspects
The production of immunoglobulin concentrates start from the collection of the blood, the source material, from volunteer donors. The liquid part of the blood, plasma, is separated from the corpuscular part either by centrifugation or by specific techniques named plasmapheresis. According to the method of separation (centrifugation vs plasmapheresis) and to the time lag between collection and freezing, different cathegories of plasma can be distinguished (see table 2I). It is important to underline that in the plasmapheresis the corpuscular part is simultaneously given back to the donor.
Table 2I: Classification of collected human plasma for fractionation
Type of plasma Method of separation
Time before
freezing Characteristics
A Plasmapheresis 6 hours
B Centrifugation 7 hours
C Centrifugation 72 hours
D Plasmapheresis 6 hours Anti-hepatitis B
Apart from the final classification, the plasma is a solution composed by proteins, lipids, sugars, vitamins and hormones; the composition is extremely heterogeneous and it is affected by many factors such as genetic variability, environmental conditions, food habits. Infectious agents like viruses can be found in the plasma. The possible presence of infection agents makes the compilation of guidelines mandatory. The aim of these guidelines is to guarantee the safety of plasma derivatives by monitoring the collection, separation, transfer to the industrial production and treatment/analysis. These procedures include control of source materials and production process, including viral removal and inactivation steps. Because the quality of the final product depends on the different phases of manufacturing and collection of blood, all the operations should be done in compliance with a proper quality system and Good Manufacturing Practices (GMP) in which the quality system of production is described.
In addition, it is necessary:
- to avoid the transmission of infectious disease (ICH Q5A Step 4 on 5 March 1997, CPMP/BWP/269/95 rev.03 25 January 2001, CPMP/BWP/268/95 14 February 1996);
- to apply the requirements and the specification stated in the monographs of European/US Pharmacopoeia, regarding human plasma for fractionation (Ph.Eur. 01/2008:0853) and guideline on the investigation of manufacturing processes for plasma-derived medicinal products. (CPMP/BWP/269/95 rev.03 25 January 2001).
3.1.2. Donations
3.1.2.1. Collection centres
The facilities for plasma or blood collection must have adequate dimensions and must
maintenance and cleaning. The collection of source material, the manufacturing and the analytical controls, as well as the anamnesis of the donors must be performed in proper and segregated zones in order to protect the privacy of the donors, to avoid cross contamination between collected source materials. Collection, manufacturing and analysis must follow proper requirements and must not be dangerous for the operators.
The maintenance and the calibrations must be done according standard procedures and must be properly documented.
3.1.2.2. Collection procedures
The collection procedures start with the identification of each donor after the acceptance in the transfusion centres and immediately before the withdrawal. The labels in which the code numbers of each donation are reported must be controlled separately, in order to guarantee the match between the bags, the vial tubes, and the data sheet registrations.
The bags and the devices used for the apheresis must be controlled to verify their integrity before the withdrawal.
3.1.2.3. Equipment for plasma collection
The equipment used for plasma separation from the whole blood is classified on the basis of their functionality. Two different systems can be used:
1) centrifugation;
2) filtration;
Cellular separators that work by centrifugation can operate in a continuous or a discontinuous flow. The continuous flow separators are equipped with a rotant bowl made by a ring tube containing 80 ml of blood and the haematic flow rate is about 30-40 ml/min. The separators working in discontinuous flow are equipped with a rotant recipient made by a chamber containing various volumes of blood and the haematic flow rate is 40-60 ml/min. Both continuous and discontinuous flow separator systems are connected to an extracorporal circuit where the withdrawn blood is collected.
The gravity force generated by rotation of the chamber/tube (about 4800 rounds per minute) separates the blood components in a density dependent pattern. In this way the liquid component, plasma, is recovered in the centre of the centrifugation chamber/tube, while the corpuscular elements are recovered in the periphery. The separation by
centres. In addition, resuspension of the corpuscular elements occurs in the extracorporal circuit with no significant variations of intravasal plasmatic osmolarity (which could cause haemodinamic instability). During the separation by centrifugation potential dispersion of platelets in the plasma could cause a platelets activation due to the presence of damaged erythrocytes.
In plasmapheresis blood is engaged through synthetic membranes (polypropylene, polysulphone, polyvinilalcohol, polymethylmetacrilate) arranged on plate or capillary patterns. The porosity generally ranges between 0.2 and 0.8 microns. Corpuscular elements are retained by the filter, while proteins, salts and water pass through. In the cellular separators that work by filtration, the progressive stratification of macromolecules (cryoglobulins, IgM) on filter membranes could limit the performance by clogging of the pores. Activation/dispersion of platelets is limited and the survival of the erythrocytes is not significantly affected. The plasma obtained by plasmapheresis and dedicated to fractionation must be frozen at temperatures less or equal than -30°C within 72 hours from the collection, while the plasma obtained from whole blood is separated from cellular components and frozen at temperatures less or equal to -20°C within 72 hours from the collection (Ph. Eur. 01/2008:0853).
3.1.2.4. Screening of donations/plasma pools and traceability of donations
Quality systems are designed in order to trace the path of each donation from its collection in the transfusion centre to the final product(s). In such way non-suitable donations and donors are promptly found due to a planned, efficient and quick communication between the transfusion centre and the manufacturer.
Each individual donations is tested for Hepatitis B surface antigen (HBsAg), human immunodeficiency type 1 and type 2 antibodies (anti HIV1/2), Hepatitis C antibodies (anti-HCV) and for HCV RNA, using either a minipool strategy or testing single donations by PCR techniques properly validated for the sample size, in accordance to the monograph on Human plasma for fractionation (0853) of the European Pharmacopoeia (current version). Furthermore the manufacturer could request individual donations to be NAT tested for HIV 1RNA, HBV DNA, HAV RNA and Parvovirus B19 DNA. The same criteria as for HCV NAT must be followed.
The in compliance donations are transferred to the manufacturing site. The transport is normally done at controlled temperature, and the maintenance of the cold chain must be
demonstrated and validated through the monitoring of the temperatures and the documentation provided for each delivery to the production site.
At industrial level, the tests carried out are in accordance with the applicable European Pharmacopoeia monographs depending on the finished products to be manufactured:
monographs Human plasma for fractionation (0853), Human anti-D immunoglobulin (0557), Human plasma (pooled and treated for virus inactivation, 1646). Plasma pools
could also be tested for HBV DNA, HIV-1 RNA, HCV RNA, HAV RNA and B19 virus DNA by different laboratories depending on the site of manufacturing.
Analytically positive pools must be discarded; nowadays, through robotic machines which simulate the production pool formation it is possible to find out the positive donation. Regarding donations traceability a system must be in place in order to enable the path taken by each donation to be traced for the donation centre to finished product and vice versa. At the collection site, each unit of plasma is assigned a unique identifying number which is applied to the unit, any sample and all documentation which accompanies the plasma unit is placed in the donor’s file.
To reduce at the lowest level the risk of microbiological contamination and the introduction of unwanted material in the manufacturing route, thawing and pooling of plasma donations must be performed in a pharmaceutical grade classified area with validated/qualified equipment.
When a potential risk is identified through risk assessment procedures, it is mandatory to inform the health authorities and to take proper measures to eliminate the risk (recall of the lot from the market and eventual destruction).
4. IgG manufacturing methods
IgG are generally extracted from human plasma by a method known as Cohn and Oncley fractionation, which will be extensively described in the next sections. The method, based on the protein separation by precipitation, was developed by Cohn and coworkers in the ‘40s and, although some modifications have occurred, the basic process skeleton remains substantially unchanged.
In the last 10-15 years manufacturers explored alternative IgG extraction techniques, based on chromatography, which were applied directly on plasma or on fractionation solid intermediates. Despite of this different technical approach the starting phases of plasma processing are common to nearby almost all manufacturing procedures.
4.1. General aspects
Plasma donations are generally maintained in a frozen state (-35°C) to the production site. On the one hand, only plasma which is compliant with the Ph. Eur monograph Human plasma for fractionation (n° 0853, current edition.) can undergo the production cycle; on the other end manufacturing facilities must be built and operate according the current Good manufacturing Practices Guidelines.
The production cycle of plasma consists in subsequent extractions of proteins for therapeutical application according to methods that must separate the proteins themselves (purification) but preserve their 3D conformation: in other words the antigenic structure and the biological integrity must be preserved. The purpose has to be accomplished by techniques potentially applicable at the industrial scale: the most employed are: (i) precipitation (ii) chromatography. In the next paragraphs these two techniques will be explored.
Plasma thawing is performed in three steps:
1) Removal of envelopes from each donation;
2) Collection of solid frozen plasma in a proper vessel at controlled temperature;
3) Heating of plasma at 0-3°C and subsequent centrifugation.
1) Removal of envelopes from each donation: donations are generally frozen in a plastic envelope or bottles. The removal is achieved by cutting away the envelope and extracting the solid frozen plasma.
2) Collection of solid frozen plasma in a proper vessel at controlled temperature: it is very important to collect a number of donations coming from an high number of donors in order to assure the homogeneity of the chemical-physical properties of the starting material.
3) Heating of plasma at 0-3°C and subsequent centrifugation: during the heating, plasma turns from solid state to a semisolid state: centrifugation through continuous flow devices allows the separation of a solid precipitate (cryoprecipitate or cryopaste), which settles down at the rotor wall from a liquid supernatant, called cryo supernatant or cryo-poor plasma, which is collected in a cooled vessel.
Cryopaste is enriched in cold insoluble globulins and represents the starting material for the manufacturing process of Factor VIII, Fibrinogen, Von Willebrand factor; it is stored in a frozen state. Cryo supernatant undergoes Cohn’s fractionation.
4.2. Precipitation
Precipitation can be used to separate different plasma proteins. In fact the most famous and used method, the so called Cohn’s Fractionation, is based upon selective precipitation of different solid fractions in different conditions of ethanol concentration, pH, ionic strength and temperature. Cohn’s fractionation process was firstly published in 1946 (Cohn et al. 1946) and introduced a new technique of plasma proteins selective precipitation. In fact, the alcoholic precipitation was proposed as an alternative to salting out of proteins, based on the increasing of saline concentration.
Ethanol water-mixture enhances different types of interaction (ion-ion, ion-protein, protein–protein) due to the lowered (by ethanol) dielectric constant of the medium. For this reason, a “target protein” can be maintained in a solution, in a high range of chemical and physical parameters, by changing simultaneously the solubility of other proteins in the same solution. However pH and ethanol concentration are the most important factors, although plasma fractionation is regulated by five parameters: pH, ionic strength, temperature, alcohol and protein concentration (Kistler and Friedli, 1980).
4.2.1. Cohn and Oncley method
Cohn and coworkers developed six different methods aiming at the production of albumin. This method has two main advantages: (i) operations are carried on at low temperatures (ranging from 1-2°C to-8°C/-10°C) (ii) high amounts of ethanol are added.
These combined features prevent bacterial growth. Moreover, ethanol is well soluble in water, does not form gaseous explosive mixtures in working conditions, has a low molecular weight (46 Da), it is volatile, quite inert and poorly toxic, cheap and easily available. On the contrary the use of huge amount of ethanol and purified water and the high progressive dilution of plasma could represent negative characters, in terms of material consumption.
Basically Cohn’s fractionation can be divided in the following steps:
