Phagocytosis

Two major cell types go on the offensive when a foreign microorganism invades—macrophages and neutrophils. Macrophages are the mature form of circulating monocytes that migrate into tissues and occur primarily around the basement membrane of blood vessels in connective tissue, lung, liver, and the lining of the sinusoids of the spleen and the medullary sinuses of the lymph nodes. They are believed to play a key role in the orchestration of both the innate and adaptive responses, and can recognise invading microorganisms through surface receptors able to distinguishing between self and pathogen. Recognition of the foreign material leads to phagocytosis by the macrophage, followed rapidly by neutrophils recruited from the circulation during the inflammatory process. The activation of the macrophage triggers the inflammatory process through the release of inflammatory mediators. The invading organism is destroyed by fusion with intracellular granules of the phagocyte and exposure to the action of hydrogen peroxide, hydroxyl radicals, and nitrous oxide (Figure 13.1).

FIGURE 13.1 Phagocytosis and the pathways involved in intracellular killing of microorganisms.

The Toll-like Receptor Pathway

A key component of cell-mediated immunity is the Toll-like receptor (TLR) pathway. TLRs are conserved transmembrane receptors which in fruit fly embryos play a critical role in dorsal-ventral development. However, their mammalian homologs function in innate immune responses and microbial recognition (in adult Drosophila, the pathway is responsible for the formation of antimicrobial peptides) and belong to the interleukin-1/TLR superfamily. The superfamily has two subgroups based on the extracellular characteristics of the receptor—i.e., whether they possess an immunoglobulin-like domain or leucine-rich repeats. TLRs typically have extracellular leucine-rich repeats.

There are 10 TLRs in man, each receptor being responsible for recognition of a specific set of pathogen-associated molecular patterns. TLR2 has been well characterised and has an essential role in the detection of invading pathogens, recognizing peptidoglycans and lipoproteins associated with gram-positive bacteria, as well as a host of other microbial and endogenous ligands. TLR2’s primary function is therefore lipoprotein-mediated signaling, and activation of the pathway by recognition of its ligand results in activation of the transcription factor NF-κB, which in turn results in the increased expression of co-stimulatory molecules and inflammatory cytokines (Figure 13.3). These cytokines help mediate migration of dendritic cells from infected tissue to lymph nodes, where they may encounter and activate leukocytes involved in the adaptive immune response. The signaling pathways used by TLRs share many of the same proteins as the interleukin-1 receptor (IL-1R) pathway (Figure 13.2). Activation of TLR leads to recruitment of the MyD88 (this is sometimes known as the MyD88-dependent pathway) which mediates the interaction between IL-1R associated kinases 1 and 4 (IRAK1 and IRAK4).

FIGURE 13.2 The Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) pathways, which share many of the same proteins. Activation of TLR2 and other TLRs, via NFκB activation and gene induction, leads to dendritic cell maturation, upregulation of expression of the major histocompatibility complex and co-stimulatory molecules, and production of immuno-stimulatory cytokines. IKK, I kappa kinase; IkB, NFκB inhibitor; IRAK, IL-1R–associated protein kinases; MKK, MAP kinases; MyD88, adapter molecule; TAB1, TAK1-binding protein 1; TAB2, TAK1-binding protein 2; TAK1, transforming growth factor-β–activated kinase; TRAF6, tumor necrosis factor receptor-associated factor 6.

The activation of the Toll pathway has several important effects in inducing innate immunity. These effects include the production of cytokines and chemokines, including IL-1, IL-6, and TNF-α (tumor necrosis factor-alpha), which have local effects in containing infection and systemic effects with the generation of fever and induction of acute phase responses, including production of C-reactive protein. One important medical condition related to the Toll pathway is septic shock, as activation of the Toll pathway by certain ligands induces systemic release of TNF-α. There are also important health-related consequences that result from TLR2 deficiency or mutation. TLR2 deficient mice are susceptible to infection by Gram-positive bacteria as well as meningitis from Streptococcus pneumoniae.

Extracellular Killing

Virally infected cells can be killed by large granular lymphocytes, known as natural killer (NK) cells. These have carbohydrate-binding receptors on their cell surface that recognize high molecular weight glycoproteins expressed on the surface of the infected cell as a result of the virus taking over the cellular replicative functions. NK cells play an early role in viral infections and are activated by cytokines from macrophages. They recognise virally infected cells through either changes in glycoproteins or in the expression of major histocompatibility complex (MHC) class 1 on virally infected host cells. Attachment to the infected cells results in the release of a number of agents, which in turn results in damage to the membrane of the infected cell, leading to cell death.

Complement

The complement system is a complex of 20 or so plasma proteins that cooperate to attack extracellular pathogens. Although the critical role of the system is to opsonize pathogens, it also recruits inflammatory cells and kills pathogens directly through membrane attack complexes. The complement system can be activated through three pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway (see Figure 13.3).

FIGURE 13.3 The classic and alternative pathways of complement activation. The main functions of complement are recruitment of inflammatory cells, opsonization of pathogens, and killing of pathogens.

MB, mannose-binding

Complement nomenclature, like much else in immunology, can be confusing. Each component is designated by the letter C, followed by a number. But they were numbered in order of their discovery rather than the sequence of reactions. The reaction sequence is C1, C4, C3, C5, C6, C7, C8, and C9. The product of each cleavage reaction is designated by letters, the larger fragment being ‘b’ (b = big), and the smaller fragment ‘a’. In the lectin pathway, MBL in the blood binds another protein, a serine protease called MASP (MBL-associated serine protease). When MBL binds to its target (for example, mannose on the surface of a bacterium), the MASP protein functions like a convertase to clip C3 into C3a and C3b. C3 is abundant in the blood, so this happens very efficiently. The other two complement pathways also converge toward C3 convertase, which cleaves C3. C3a mediates inflammation while C3b binds to the pathogen surface, coating it and acting as an opsonin. The effector roles of the major complement proteins can be summarized according to function as follows (Figure 13.4):

FIGURE 13.4 Overview of the main components and effector actions of complement. Note that the MBL pathway involves the MBL protein, MASP-1, MASP-2, C4, and C2. MASP acts as a C3 convertase, creating a C3b fragment from C3. C3b attaches to the pathogen surface and binds to receptors on phagocytes, leading to opsonization. C3b can also combine with other proteins on the pathogen surface and form a membrane attack complex.

There are clinical consequences relating to mutations in the genes of these pathways. The frequency of mutations of the MBL2 gene in the general population may be 5% to 10%. Although most individuals with MBL deficiency from mutations and promoter polymorphisms in MBL2 are healthy, there is an increased risk, severity, and frequency of infections and autoimmunity. The deficiency has been reported to be particularly common in infants with recurrent respiratory tract infection, otitis media, and chronic diarrhea.

Immunoglobulins

The immunoglobulins, or antibodies, are one of the major classes of serum protein. Their function, both in the recognition of antigenic variability and in effector activities, was initially revealed by protein studies of their structure, and later by DNA studies.

Immunoglobulin structure

Papaine, a proteolytic enzyme, splits the immunoglobulin molecule into three fragments. Two of the fragments are similar, each containing an antibody site capable of combining with a specific antigen and therefore referred to as the antigen-binding fragment or Fab. The third fragment can be crystalized and was therefore called Fc. The Fc fragment determines the secondary biological functions of antibody molecules, binding complement and Fc receptors on a number of different cell types involved in the immune response.

The immunoglobulin molecule is made up of four polypeptide chains—two ‘light’ (L) and two ‘heavy’ (H)—of approximately 220 and 440 amino acids in length, respectively. They are held together in a Y-shape by disulfide bonds and non-covalent interactions. Each Fab fragment is composed of L chains linked to the amino-terminal portion of the H chains, whereas each Fc fragment is composed only of the carboxy-terminal portion of the H chains (Figure 13.5).

FIGURE 13.5 Model of antibody molecule structure.

Generation of Antibody Diversity

It could seem paradoxical for a single protein molecule to exhibit sufficient structural heterogeneity to have specificity for a large number of different antigens. Different combinations of H and L chains could, to some extent, account for this diversity. It would, however, require thousands of structural genes for each chain type to provide sufficient variability for the large number of antibodies produced in response to the equally large number of antigens to which individuals can be exposed. Our initial understanding of how this could occur came from persons with a malignancy of antibody-producing cells—multiple myeloma.

DNA studies of antibody diversity

In 1965 Dreyer and Bennett proposed that an antibody could be encoded by separate ‘genes’ in germline cells that undergo rearrangement or, as they termed it, ‘scrambling’, in lymphocyte development. Comparison of the restriction maps of the DNA segments coding for the C and V regions of the immunoglobulin λ light chains in embryonic and antibody-producing cells revealed that they were far apart in the former but close together in the latter. Detailed analysis revealed that the DNA segments coding for the V and C regions of the light chain are separated by some 1500 base-pairs (bp) in antibody-producing cells. The intervening DNA segment was found to code for a joining, or J, region immediately adjacent to the V region of the light chain. The κ L-chain was shown to have the same structure. Cloning and DNA sequencing of H-chain genes in germline cells revealed that they have a fourth region, called diversity, or D, between the V and J regions.

There are estimated to be some 60 different DNA segments coding for the V region of the H-chain, 40 for the V region of the κ L-chain, and 30 for the λ L-chain V region. Six functional DNA segments code for the J region of the H-chain, five for the J region of the κ L-chain, and four for the J region of the λ L-chain. A single DNA segment codes for the C region of the κ L-chain, seven for the C region of the λ L-chain and 11 functional DNA segments code for the C region of the different classes of H-chain. There are also 27 functional DNA segments coding for the D region of the H-chain (Figure 13.6).

FIGURE 13.6 Estimated number of the various DNA segments coding for the κ, λ, and various heavy chains.

The genomic regions in question also contain a large number of unexpressed DNA sequences or pseudogenes (p. 17). Although the coding DNA segments for the various regions of the antibody molecule can be referred to as ‘genes’, use of this term in regard to antibodies has deliberately been avoided because they could be considered an exception to the general rule of ‘one gene–one enzyme (or protein)’ (p. 167).

Antibody gene rearrangement

The genes for the κ and λ L-chains and the H-chains are located on chromosomes 2, 22, and 14, respectively. Only one of each of the relevant types of DNA segment is expressed in any single antibody molecule. The DNA coding segments for the various portions of the antibody chains on these chromosomes are separated by DNA that is non-coding. Somatic recombinational events involved in antibody production involve short conserved recombination signal sequences that flank each germline DNA segment (Figure 13.7). Further diversity occurs by variable mRNA splicing at the V–J junction in RNA processing and by somatic mutation of the antibody genes. These mechanisms readily account for the antibody diversity seen in nature, even though it is still not entirely clear how particular DNA segments are selected to produce an antibody to a specific antigen.

FIGURE 13.7 Immunoglobulin heavy-chain gene rearrangement and class switching.

Class Switching of Antibodies

There is a normal switch of antibody class produced by B cells on continued, or further, exposure to antigen—from IgM, the initial class of antibody produced in response to exposure to an antigen, to IgA or IgG. This class switching involves retention of the specificity of the antibody to the same antigen. Analysis of class switching in a population of cells derived from a single B cell has shown that both classes of antibody have the same antigen-binding sites, having the same V region but differing only in their C region. Class switching occurs by a somatic recombination event that involves DNA segments designated S (for switching) that lead to looping out and deletion of the intervening DNA. The result is to eliminate the DNA segment coding for the C region of the H-chain of the IgM molecule, and to bring the gene segment encoding the C region of the new class of H-chain adjacent to the segment encoding the V region (see Figure 13.7).

The Immunoglobulin Gene Superfamily

Several other molecules involved in the immune response have been shown to have structural and DNA sequence homology to the immunoglobulins. This involves a 110–amino acid sequence characterized by a centrally placed disulfide bridge that stabilizes a series of antiparallel β strands into an ‘antibody fold’. This group of structurally similar molecules has been called the immunoglobulin superfamily (p. 16). It consists of eight multigene families that, in addition to the κ and λ L-chains and different classes of H-chain, include the chains of the T-cell receptor (p. 16), the class I and II MHC, or human leukocyte antigens (HLA) (p. 200). Other molecules in this group include the T-cell CD4 and CD8 cell surface receptors, which cooperate with T-cell receptors in antigen recognition, and the intercellular adhesion molecules-1, -2, and -3, which are involved in leukocyte-endothelial adhesion and extravasation, T-cell activation, and T-cell homing.

Antibody Engineering

At the beginning of the 20th century, Paul Ehrlich proposed the idea of the ‘magic bullet’—the hope that one day there might be a compound that would selectively target a disease-causing organism. Today we have monoclonal antibodies (mAb) and, for almost any substance, it is possible to create a specific antibody that binds that substance. Monoclonal antibodies are the same because they are made by one type of immune cell which are all clones of a unique parent cell.

In the 1970s it was understood that the B-cell cancer multiple myeloma produced a single type of antibody—a paraprotein. The structure of antibodies was studied from this but it was not possible to produce identical antibodies specific to a given antigen. Myeloma cells cannot grow because they lack hypoxanthine-guanine-phosphoribosyl transferase, which is necessary for DNA replication. Typically, mAb are made by fusing myeloma cells with spleen cells from a mouse (or rabbit) that has been immunized with the desired antigen. They are then grown in medium which is selective for these hybrids—the spleen cell partner supplies hypoxanthine-guanine-phosphoribosyl transferase and the myeloma has immortal properties because it is a cancer cell. The cell mixture is diluted and clones grown from single parent cells. The antibodies secreted by different clones are assayed for their ability to bind to the antigen in question, with the healthiest clone selected for future use. The hybrids can also be injected into the peritoneal cavity of mice to produce tumors containing antibody-rich ascitic fluid, and the mAb then has to be extracted and purified.

To overcome the problem of purification, recombinant DNA technologies have been used since the 1980s. DNA that encodes the binding portion of mouse mAb is merged with human antibody-producing DNA. Mammalian cell culture is then used to express this DNA, producing chimeric antibodies. The goal, of course, is to creat of ‘fully human’ mAb, which has met with success in ‘phage display-generated’ antibodies and mice that have been genetically modified to produce more human-like antibodies.

Specific mAb have now been developed and approved for the treatment of cancer, cardiovascular disease, inflammatory diseases, macular degeneration, and transplant rejection, among others. A mAb that inhibits TNF-α has applications in rheumatoid arthritis, Crohn disease, and ulcerative colitis; one that inhibits IL-2 on activated T cells is used in preventing rejection of transplanted kidneys; and one that inhibits vascular endothelial growth factor (VEGF) has a role in antiangiogenic cancer therapy.

T-Cell Surface Antigen Receptor

T cells express on their surface an antigen receptor, which distinguishes them from other lymphocyte types, such as B cells and NK cells. The antigen consists of two different polypeptide chains, linked by a disulfide bridge, that both contain two immunoglobulin-like domains, one that is relatively invariant in structure, the other highly variable like the Fab portion of an immunoglobulin. The diversity in T-cell receptors required for recognition of the range of antigenic variation that can occur is generated by a process similar to that seen with immunoglobulins. Rearrangement of variable (V), diversity (D), junctional (J), and constant (C) DNA segments during T-cell maturation, through a similar recombination mechanism as occurs in B cells, results in a contiguous VDJ sequence. Binding of antigen to the T-cell receptor, in conjunction with an associated complex of transmembrane peptides, results in signaling the cell to differentiate and divide.

The Major Histocompatibility Complex

The MHC plays a central role in the immune system. Its role is to bind antigen peptides processed intracellularly and present this material on the cell surface, with co-stimulatory molecules, where it can be recognised by T cells. MHC molecules occur in three classes: class I occur on virtually all cells and are responsible for presenting cytotoxic T cells; class II occur on B cells and macrophages and are involved in signaling T-helper cells to present further B cells and macrophages; and the non-classic class III molecules include a number of other proteins with a variety of other immunological functions. The latter include inflammatory mediators such as the TNF, heat-shock proteins, and the various components of complement.

Structural analysis of class I and II MHC molecules reveal them to be heterodimers with homology to immunoglobulin. The genes coding for the class I (A, B, C, E, F, and G), class II (DR, DQ, and DP) and class III MHC molecules, or what is also known as the human leukocyte antigen (HLA) system (p. 200), are located on chromosome 6.

Transplantation Genetics

Organ transplantation has become routine in clinical medicine and, with the exception of corneal and bone grafts, success depends on the degree of antigenic similarity between donor and recipient. The closer the similarity, the greater the likelihood that the transplanted organ or tissue (the homograft), will be accepted rather than rejected. Homograft rejection does not occur between identical twins or between non-identical twins where there has been mixing of the placental circulations before birth (p. 50). In all other instances, the antigenic similarity of donor and recipient has to be assessed by testing them with suitable antisera or monoclonal antibodies for antigens on donor and recipient tissues. These were originally known as transplantation antigens but are now known to be a result of the MHC. As a general rule, a recipient will reject a graft from any person who has antigens that the recipient lacks. HLA typing of an individual is carried out using PCR-based techniques (p. 56).

The HLA system is highly polymorphic (Table 13.2). A virtually infinite number of phenotypes resulting from different combinations of the various alleles at these loci is theoretically possible. Two unrelated individuals are therefore very unlikely to have identical HLA phenotypes. The close linkage of the HLA loci means that they tend to be inherited en bloc, the term haplotype being used to indicate the particular HLA alleles that an individual carries on each of the two copies of chromosome 6. Thus, any individual will have a 25% chance of having identical HLA antigens with a sibling, as there are only four possible combinations of the two paternal haplotypes (say P and Q) and the two maternal haplotypes (say R and S), i.e., PR, PS, QR and QS. The siblings of a particular recipient are more likely to be antigenically similar than either of his or her parents, and the latter more than a non-relative. Therefore, a sibling is frequently selected as a potential donor.

Table 13.2 Alleles at the HLA Loci

HLA, human leukocyte antigen.

Although recombination occurs within the HLA region, certain alleles tend to occur together more frequently than would be expected by chance, i.e. they tend to exhibit linkage disequilibrium (p. 138). An example is the association of the HLA antigens A1 and B8 in populations of western European origin.

HLA Polymorphisms and Disease Associations

The association of certain diseases with certain HLA types (Table 13.3) should shed light on the pathogenesis of the disease, but in reality this not well understood. The best documented is between ankylosing spondylitis and HLA-B27. Narcolepsy, a condition of unknown etiology characterized by a periodic uncontrollable tendency to fall asleep, is almost invariably associated with HLA-DR2. The possession of a particular HLA antigen does not mean that an individual will necessarily develop the associated disease, only that the relative risk of being affected is greater than the general population (p. 384). In a family, the risks to first-degree relatives of those affected are low, usually no more than 5%.

Table 13.3 Some HLA-Associated Diseases

HLA, human leukocyte antigen.

Explanations for the various HLA-associated disease susceptibilities include close linkage to a susceptibility gene near the HLA complex, cross-reactivity of antibodies to environmental antigens or pathogens with specific HLA antigens, and abnormal recognition of ‘self’ antigens through defects in T-cell receptors or antigen processing. These conditions are known as autoimmune diseases. An example of close linkage is congenital adrenal hyperplasia from a 21-hydroxylase deficiency (p. 174) from mutated CYP21, which lies within the HLA major histocompatibility locus. This form of congenital adrenal hyperplasia is strongly associated HLA-A3/Bw47/DR7 in northern European populations. Non-classical 21-hydroxylase deficiency is associated with HLA-B14/DR1, and HLA-A1/B8/DR3 is negatively associated with 21-hydroxylase deficiency.

Disorders of Innate Immunity

Primary disorders of innate immunity are considered under humoral and cell-mediated immunity categories.

Defects in NFκB signaling

Inappropriate activation of nuclear factor kappa-B (NFκB) has been linked to inflammation associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. Conversely, persistent inhibition of NFκB has been linked directly to apoptosis, abnormal immune cell development, and delayed cell growth.

Since 2000, mutations have occasionally been found in the X-linked IKK-gamma gene, part of the TLR pathway (p. 193), in children demonstrating failure to thrive, recurrent digestive tract infections, often with intractable diarrhea, and recurrent ulcerations, respiratory tract infections with bronchiectasis, and recurrent skin infections, presenting in infancy, suggesting susceptibility to various gram-positive and gram-negative bacteria. Sparse scalp hair is sometimes a feature and in older children oligodontia and conical-shaped maxillary lateral incisors have been noted. Survival ranged from 9 months to 17 years in one study. IgG is low and IgM usually high. Interestingly, IKKg is the same as NEMO, the gene that causes X-linked dominant incontinentia pigmenti (p. 117–118). However, in this condition of the immune system mutations occur in exon 10 of the gene.

IRAK4 is another component of the TLR pathway and deficiency leads to recurrent infections, mainly from gram-positive microorganisms, though also fungi. There is a reduced inflammatory response. Infections begin early in life but become less frequent with age, some patients requiring no treatment by late childhood. It follows autosomal recessive inheritance.

DiGeorge/Sedláčková Syndrome

Children with the DiGeorge syndrome (also well described by Sedláčková, 10 years earlier than DiGeorge) present with recurrent viral illnesses and are found to have abnormal cellular immunity as characterized by reduced numbers of T lymphocytes, as well as abnormal antibody production. This has been found to be associated with partial absence of the thymus gland, leading to defects in cell-mediated immunity and T cell–dependent antibody production. Usually these defects are relatively mild and improve with age, as the immune system matures, but occasionally the immune deficiency is very severe because no T cells are produced and bone marrow transplantation is indicated. It is important for all patients diagnosed to be investigated by taking a full blood count with differential CD3, CD4, and CD8 counts, and immunoglobulins. The levels of diphtheria and tetanus antibodies can indicate the ability of the immune system to respond. These patients usually also have a number of characteristic congenital abnormalities, which can include heart disease and absent parathyroid glands. The latter finding can result in affected individuals presenting in the newborn period with tetany due to low serum calcium levels secondary to low parathyroid hormone levels. This syndrome has been recognized to be part of the spectrum of phenotypes caused by abnormalities of the third and fourth pharyngeal pouches (p. 95) as a consequence of a microdeletion of chromosome band 22q11.2 (p. 282).

Ataxia Telangiectasia

Ataxia telangiectasia is an autosomal recessive disorder in which children present in early childhood with signs of cerebellar ataxia, dilated blood vessels on the sclerae of the eyes, ears, and face (oculocutaneous telangiectasia), and a susceptibility to sinus and pulmonary infections. Low serum IgA levels occur and a hypoplastic thymus as a result of a defect in the cellular response to DNA damage. The diagnosis is made by the demonstration of low or absent serum IgA and IgG as well as characteristic chromosome abnormalities on culture of peripheral blood lymphocytes—a form of chromosome instability (p. 288). Patients have an increased risk of developing leukemia or lymphoid malignancies.

Wiskott-Aldrich Syndrome

Wiskott-Aldrich syndrome is an X-linked recessive disorder in which affected boys have eczema, diarrhea, recurrent infections, thrombocytopenia, and, usually, low serum IgM levels and impaired T-cell function and numbers. Mutations in the gene responsible have been shown to result in loss of cytotoxic T-cell responses and T-cell help for B-cell response, leading to an impaired response to bacterial infections. Until the advent of bone marrow transplantation, the majority of affected boys died by mid-adolescence from hemorrhage or B-cell malignancy.

Rhesus Hemolytic Disease of the Newborn

A proportion of women who are Rh-negative have an increased chance of having a child who will either die in utero or be born severely anemic because of hemolysis, unless transfused in utero. This occurs because if Rh-positive blood is given to persons who are Rh-negative, the majority will develop anti-Rh antibodies. Such sensitization occurs with exposure to very small quantities of blood and, once a person is sensitized, further exposure results in the production of very high antibody titers.

In the case of an Rh-negative mother carrying an Rh-positive fetus, fetal red cells that cross to the mother’s circulation can induce the formation of maternal Rh antibodies. In a subsequent pregnancy, these antibodies can cross the placenta from the mother to the fetus, leading to hemolysis and severe anemia. In its most severe form, this is known as erythroblastosis fetalis, or hemolytic disease of the newborn. After a woman has been sensitized. there is a significantly greater risk that a child in a subsequent pregnancy, if Rh-positive, will be more severely affected.

To avoid sensitizing an Rh-negative woman, Rh-compatible blood must always be used in any blood transfusion. Furthermore, the development of sensitization, and therefore Rh incompatibility after delivery, can be prevented by giving the mother an injection of Rh antibodies—anti-D—so that fetal cells in the maternal circulation are destroyed before the mother can become sensitized.

It is routine to screen all Rh-negative women during pregnancy for the development of Rh antibodies. Despite these measures, a small proportion of women do become sensitized. If Rh antibodies appear, tests are carried out to see whether the fetus is affected. If so, there is a delicate balance between the choice of early delivery, with the risks of prematurity and exchange transfusion, and treating the fetus in utero with blood transfusions.

Molecular Basis of the Rh Blood Group

There are two types of Rh red cell membrane polypeptide. One corresponds to the D antigen and the other to the C and E series of antigens. We now know that two genes code for the Rh system: one for D and d, and a second for both C and c and E and e. The D locus is present in most persons and codes for the major D antigen present in those who are Rh-positive. Rh-negative individuals are homozygous for a deletion of the D gene. Therefore, an antibody has never been raised to d!

Analysis of complementary DNA from reticulocytes in Rh-negative persons who were homozygous for dCe, dcE, and dce allowed identification of the genomic DNA sequences responsible for the different antigenic variants at the second locus, revealing that they are produced by alternative splicing of the mRNA transcript. The Ee polypeptide is a full-length product of the CcEe gene, very similar in sequence to the D polypeptide. The E and e antigens differ by a point mutation in exon 5. The Cc polypeptides are, in contrast, products of a shorter transcript of the same gene through splicing. The difference between C and c is four amino-acid substitutions in exons 1 and 2.