IMMUNOLOGY - Roit A. - Mir 2000

Chapter 15. Evolution of the Immune System

IMMUNITY IN VERTEBRATES

Compared to the vast diversity of invertebrate forms, the Organization of vertebrates follows a fairly uniform general plan, with all of them belonging to a single phylum, Chordata. Although the vertebrate evolutionary tree encompasses numerous levels and branches, including jawless fish, cartilaginous fish, bony fish, amphibians, reptiles, birds, and mammals, the core CELLULAR AND MOLECULAR components of innate Immunity are remarkably conserved across all modern jawed vertebrates (Gnathostomes). However, the Increasing complexity of body Structure is mirrored by a greater specialization of lymphoid tissue and lymphocyte Functions, as well as an expansion in The Diversity of immunoglobulin classes. Mammals possess the most structurally and functionally complex immune system.

T Cells and the Evolution of the MHC

Cytotoxic and helper T lymphocytes (T_c and T_h cells, respectively) in mammals, bearing αβ T-Cell receptors (TCRs), recognize most foreign Antigens only when these antigens are presented in the appropriate context by self-polymorphic MHC molecules. Therefore, the phylogenesis of certain T-cell populations [e.g., T_c cells and lymphocytes participating in the mixed leukocyte reaction (MLR)] and the evolution of the MHC should be considered together (Fig. 15.14).

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Fig. 15.14. This Phylogenetic Tree of vertebrates illustrates various aspects of the evolution of the MHC and T cells. Two functional Criteria for the presence of the MHC are shown (the existence of cytotoxic T lymphocytes (T_c) and the mixed leukocyte reaction (MLR)), alongside biochemical and molecular-biological data demonstrating the expression of class I and class II MHC Proteins and genes. An empty square indicates a lack of available data.

Functional criteria and/or Molecular and genetic evidence establish the presence of the MHC in all jawed vertebrates, from cartilaginous fish upwards. [The functional criteria are that the MLR and rapid allograft rejection are regulated by a single polymorphic Gene region, and that phenomena such as T- and B-cell cooperation, the generation of antigen-specific cytotoxic responses (e.g., against allogeneic cells), and T-cell "education" in the Thymus are under MHC control.]

The MHC has been studied in detail in the poikilothermic vertebrate Xenopus. Recent studies have identified MHC genes in cartilaginous fish, whose ancestors diverged from the main vertebrate Lineage over 400 million years ago. However, MHC genes and proteins have been most thoroughly investigated among poikilotherms in the African clawed frog, Xenopus laevis. Figure 15.15 illustrates the organization of its MHC (XLA) in comparison with the avian MHC (B locus) and the murine MHC (H-2 locus).

Fig. 15.15. The MHC can be found in all jawed vertebrates. The putative ORGANIZATION OF THE MHC loci in the clawed frog (Xenopus) and chicken (Gallus) is shown. The structure of the murine H-2 complex (Mus) has been fully mapped. Intergenic distances in Xenopus and Gallus are shown arbitrarily. (Diagram kindly provided by Dr. L. Du Pasquier.)

Classical MHC antigens (class Ia) in Xenopus. Class Ia proteins in Xenopus are polymorphic and encoded by approximately 20 alleles. They are expressed On the surface of all mature cells, but most abundantly on hematopoietic cells. The α-chains of these proteins have a Molecular Weight of 40–44 kDa, fold into 3 domains, and are non-covalently associated with β₂-microglobulin. Class I MHC proteins in Xenopus are unusual in that they are encoded by a single gene locus (as opposed to three in humans and two in mice).

Non-classical MHC antigens (class Ib) in Xenopus. Among the class I genes in Xenopus, the first to be identified was a large family of monomorphic non-classical (class I) MHC-like molecules. The genes encoding these molecules and the classical MHC genes (class Ia) are located on different Chromosomes. The class Ib gene apparently encodes a molecule homologous to the peptide-binding domains of heat Shock proteins 70 (HSP70). A recently proposed hypothesis suggests that the peptide-binding region of class I MHC molecules evolved from pre-existing HSPs. (HSPs are evolutionarily conserved molecules found in all organisms that act as "chaperones," assisting in Protein folding and Intracellular Transport.) Epithelium-associated non-classical MHC-like proteins have been found in all vertebrates studied; it is hypothesized that they may serve various functions, such as recognizing HSPs from pathogens or infected/stressed self-cells and subsequently presenting these conserved Peptides to T cells with restricted TCRs.

Class II MHC antigens in Xenopus. Class II MHC molecules in Xenopus are polymorphic (encoded by approximately 30 alleles) and constitutively expressed only on certain mature cells, including thymocytes, B AND T lymphocytes, and various APCs, such as Langerhans-like Cells of the epidermal Skin (Fig. 15.16). Class II proteins consist of MHC-encoded α- and β-chains; both chains are transmembrane Glycoproteins with a molecular weight of 30–35 kDa. The genes for the β-chains of class II MHC molecules in Xenopus encode Polypeptides that share nearly 50% Homology with the class II β-chains of mammals. During synthesis, class II MHC proteins temporarily associate with the invariant chain. Like humans, Xenopus possesses three class II MHC β-gene loci.

Fig. 15.16. Immunofluorescence of Xenopus dendritic cells bearing surface class II MHC molecules. APCs resembling Langerhans cells (L) are frequently found in the basal epidermal layer of the skin. Class II-positive cells of the skin gland ducts traversing the epidermis (D) and subepidermal Skin glands (G) are also visible, ×100.

MHC expression in Xenopus varies at each stage of The life cycle. An interesting feature of ontogenetic MHC expression in Xenopus is that classical class I MHC molecules are not expressed on The surface of any cells prior to metamorphosis. In contrast, class II molecules appear early in tadpole development on B cells and certain epithelial cells that are in direct contact with the external environment. This indicates that the expression of classical class I molecules is not essential for early developmental stages or for immune system function during the tadpole stage. However, non-classical class I proteins may play an important role in tadpole immunity. At this stage of ontogenesis, cell-mediated immunity restricted by class II MHC molecules may be of primary importance. The broader representation of class II MHC molecules in tadpoles compared to adult frogs suggests that in earlier evolutionary stages, within a more primitive immune system, these very molecules may have served the antigen-presentation function.

The MHC in other vertebrates. Class I and class II MHC proteins, as well as polymorphic class II genes, have recently been discovered in cartilaginous fish. Among teleost fish, rainbow trout have been shown to possess class I MHC genes and β₂-microglobulin, while carp possess class II MHC genes.

Axolotls, which are characterized by relatively weak T-cell responses to alloantigens, possess class II MHC α- and β-chains with limited polymorphism. These amphibians also express MHC-encoded erythrocyte antigens similar to class I α-chains (44 kDa) and to polymorphic class IV molecules present on nucleated chicken erythrocytes. These may also be present in Xenopus. Class I α-chains and heterodimeric class II molecules have likewise been found in various reptiles.

T cells are phenotypically and functionally identified in various vertebrates. Avian species have been found to possess αβ and γδ TCRs complexed with the coreceptor molecules CD3, CD4, and CD8. Evidence is now emerging for the presence of some of these receptors or their constituent chains in fish and amphibians. For instance, genes from thymocytes and splenocytes of the Mexican axolotl show significant homology with the TCR β-chain genes of birds and mammals. A 55 kDa protein, similar in Amino Acid Sequence to the TCR δ-chain, is present on the surface of thymocytes and thymic tumor lymphoid cells in Xenopus. Currently available anti-Xenopus Monoclonal Antibodies detect markers potentially corresponding to CD5 (71–88 kDa, expressed on all T cells) and CD8 (35 kDa, expressed on cytotoxic T cells). In rainbow trout, gene segments encoding TCR β-chains have recently been identified, although monoclonal antibodies specific for fish T cells have not yet been produced. Four distinct types of T-cell receptor genes have been found in cartilaginous fish. The cellular and Molecular Basis of the MLR observed in hagfish remains to be elucidated.

Temperature is of paramount importance for immune responses in poikilothermic animals. In catfish, low temperatures inhibit the proliferation of T cells (but not B cells). These effects are attributed to the low content of certain Unsaturated Fatty acids (such as oleic acid) in fish T cells and the consequent membrane fluidity. Therefore, a diet rich in these fatty acids may help fish adapt better to low temperatures. Oleic acid also reverses the low-temperature suppression of mammalian T-cell responses in vitro (the exact Nature of the thermosensitive processes has not yet been established).

Evolution of B cells and IMMUNOGLOBULINS

Heavy and light chains of immunoglobulins are present in various vertebrates. Proteins discovered in hagfish that were previously considered antibodies are now identified as Complement proteins C3–C5. So far, molecules belonging to the immunoglobulin superfamily have not been detected in cyclostomes (hagfish and lampreys).

All jawed vertebrates produce antibodies against a wide range of antigens. However, antibodies produced by poikilothermic vertebrates are characterized by low affinity and weak immunological memory compared to those of homeothermic vertebrates. The Structure of antibodies is evolutionarily conserved; across all animals, these proteins consist of multidomain heavy and light polypeptide immunoglobulin chains (see Ch. 6), which can either be expressed on the surface of B cells to function as receptors or be secreted into the Blood by activated B cells.

All jawed vertebrates possess polymeric IgM (Fig. 15.17), and in fish, antibodies predominantly belong to this class. Each heavy μ-chain consists of four constant domains and one variable domain; heavy and light chains are linked by disulfide bridges. The μ-chain family exhibits considerable phylogenetic diversity; for instance, The amino acid Sequence homology between catfish and mouse μ-chains is only 24%.

Fig. 15.17. Distribution and putative phylogenetic relationships of vertebrate immunoglobulins. Polymeric IgM is found in all jawed vertebrates, but, as illustrated, with a varying number of basic units (consisting of 2 heavy + 2 light chains). Monomeric IgM is also present in the blood of cartilaginous (elasmobranchs) and teleost fish. Heavy chains not belonging to the μ-isotype have been discovered in various groups, though The Role of these immunoglobulin isotypes frequently remains elusive. (Table kindly provided by Dr. G. Warr.)

Certain cartilaginous fish, such as rays and sharks, possess low-molecular-weight antibodies lacking μ-chains (see Fig. 15.17), yet the evolutionary relationship of IgR to other H-chain isotypes remains unclear. Amphibians, reptiles, and birds possess a heavy-chain isotype comprising four constant domains, designated as IgY. It is presumed to be the precursor of mammalian IgG and IgE, sharing Structural and functional similarities with them. In the axolotl, IgY may also function as a secretory immunoglobulin, as it associates with secretory-like molecules in the gut. Interestingly, despite the apparent absence of IgE in fish, teleost fish exhibit type I hypersensitivity reactions; they may possess tissue-bound homocytotropic antibodies. In Xenopus, the IgX isotype—whose production, unlike that of IgY, is thymus-independent—may be the functional equivalent of mammalian secretory IgA, given that this isotype is predominantly localized to the gut. The IgA isotype likely makes its first phylogenetic appearance in birds.

Light chain diversity is also characteristic of many poikilotherms. Two antigenically distinct types of light chains, one of which resembles the k-chain, have been identified in Xenopus, alongside two types in catfish, turtles, and alligators. Sharks possess both k- and λ-chains, indicating that the divergence of ancestral light chains occurred prior to The Emergence of cartilaginous fish.

The nurse shark has recently been shown to possess a previously unknown molecule of the immunoglobulin superfamily that may be an evolutionary precursor to immunoglobulins and TCRs. This molecule, termed the new antigen receptor (NAR), consists of one variable and five constant domains and circulates in the serum as a dimer. NAR is encoded by a gene locus that undergoes rearrangement and somatic mutation. The discovery of this novel class of chimeric antibodies in cartilaginous fish challenges the long-held assumption that IgM represents the primordial Ig isotype.

Four types of immunoglobulin Introduction/29.html">Gene Organization are found in lower vertebrates. Active investigations of the immunoglobulin gene locus in poikilothermic vertebrates using Recombinant DNA technology have recently revealed four distinct types of genomic organization.

Amphibians and teleost fish. In these animals, the IgH locus is organized in a manner similar to that of mammals (the "translocon" model). Xenopus, for example, possesses 80–100 V_H segments, 15 D_H segments, and 9 J_H segments (Fig. 15.18). Both structural and complementarity-determining regions have been identified. The constant regions for each chain (IgM, IgX, IgY) in Xenopus are encoded by four C_H exons. Light chains are encoded on two different chromosomes, each containing V_L, J_L, and C_L segments. In teleost fish, immunoglobulin light chain genes exhibit a "multicluster" organization, which is typically exemplified by their arrangement in sharks (see below).

Fig. 15.18. The STRUCTURE OF THE IgH locus in Xenopus resembles that found in teleost fish and, to some extent, mammals. Recombination signal sequences flank all gene segments. Initially, D segments join to J_H, after which V_H genes join to the D-J rearrangement. Finally, the rearranged V-D-J genes join with the constant region gene to form functional IgM, IgX, or IgY genes. (Based on data from Dr. L. DuPasquier.)

During B-cell maturation in Xenopus, as in mammals, multiple rearrangements of immunoglobulin genes take place, and allelic exclusion operates to generate monospecific B lymphocytes. Although recombinase-activated genes (involved in immunoglobulin gene rearrangement) have been identified in Xenopus, antibody diversity (within V regions) remains quite limited; adults possess only about 5 · 10⁵ distinct antibody molecules. The restricted affinity maturation observed following B-cell activation in Xenopus (and other poikilotherms) is apparently not due to a lack of somatic hypermutation in immunoglobulin genes. Rather, it is more likely attributable to inefficient mutant Selection resulting from the absence of structured germinal centers in the Lymphoid Organs of cold-blooded animals. Lymph Nodes containing germinal centers are found exclusively in birds and mammals. Although Xenopus tadpoles possess the same three Ig isotypes present in adults, the antibody repertoire differs between the two life stages. The adult Ig repertoire is shaped by gene rearrangements occurring during a secondary wave of B-cell maturation following metamorphosis. In adults, the third hypervariable region acquires additional diversity through the random addition of N-terminal residues, a process that does not occur in tadpoles.

Generation of antibody diversity in birds. In this lineage, antibody diversification is linked to a distinct type of immunoglobulin gene organization and takes place within a specialized microenvironment unique to birds—the bursa of Fabricius, located near the cloaca (Fig. 15.19). The chicken light-chain locus contains a single V gene that subsequently rearranges and joins to a single J-C complex (Fig. 15.20). The IgH locus also contains a region of multiple D genes. Gene rearrangement occurs only during a restricted window of early development when stem cells colonize the bursa of Fabricius; in contrast, immunoglobulin gene rearrangement in mouse and chicken pre-B cells continues throughout life. Subsequently, segments (10–120 bp in length) of the rearranged immunoglobulin genes in chickens are replaced by nucleotide sequences derived from pseudogenes flanking the unique V gene. Gene Conversion events occur at a high frequency (similar to rabbits) throughout the period of B-cell proliferation within the bursa.

Fig. 15.19. The two primary Organs of the avian immune system are the thymus (2) and the bursa of Fabricius (1). Lymphocytes developing in the thymus are designated as T cells, whereas those developing in the bursa are designated as B cells. Lymphoid follicles (L) and the duct lumen (P) are indicated in the bursa, while the cortical (C) and medullary (M) zones are shown in the thymus. Hematoxylin and eosin staining, × 20.

Fig. 15.20. The germline immunoglobulin light-chain locus in chickens spans less than 30 kb of DNA. A single functional V gene (V_L) is located 2 kb upstream of a unique J-C element, flanked by a cluster of 25 pseudogenes (P) spanning 19 kb. Rearrangement occurs during a brief developmental window early in B-cell ontogeny. Antibody diversity is generated via gene conversion between the pseudogenes (P) and the rearranged sequence. The illustrated rearrangement (P1, P2, and P24) serves merely to exemplify the process; within the modified V gene segment, pseudogenes are not necessarily arranged in this exact order.

A third type of Ig gene locus organization is found in cartilaginous fish. In these animals, heavy (μ) and light (λ- and k-like) immunoglobulin chains are encoded by numerous small, separate clusters (cassettes) (Fig. 15.21) that incorporate all V (D), J, and C genes. Each immunoglobulin gene cluster differs in its DNA sequence from the others. These sequences exist in a germline configuration. Shark antibodies appear to possess an extraordinarily diverse (multimillion) repertoire of binding specificities; however, because this diversity is genetically encoded in the germline DNA rather than generated through somatic mechanisms, individual sharks show virtually no interindividual variation in their immunoglobulins. Thus, generating antibody diversity via somatic gene rearrangement (as seen in teleost fish, amphibians, birds, and mammals) is not a universal feature of all vertebrates. Cartilaginous fish possess a vast array of innate antibodies directed against diverse antigens; these antibodies are analogous to the polyspecific (and frequently autoreactive) mammalian IgM antibodies secreted by CD5⁺ B cells during ontogeny. It remains unknown whether the clustered organization of immunoglobulin genes in sharks can support clonal restriction of B cells. Nevertheless, specific antibody titers can rise in these animals without a corresponding overall increase in serum Ig levels, pointing to the existence of clonal selection.

Fig. 15.21. Sharks possess approximately 200 heavy-chain gene clusters, each containing a single set of V, D, J, and C gene segments. The fourth and ninth clusters are shown in an expanded view. The V_H, D_H, and J_H segments are tightly linked, spanning a region of approximately 1.3 kb. Together with the C_H segment, they occupy a region of only about 10–15 kb. The arrangement of genes within the cluster appears to be germline-encoded and independent of somatic rearrangement mechanisms, which may account for the absence of interindividual differences in immune responses within this species. (Based on data from Drs. J. Marchalonis and G. Litman.)

Preliminary data suggest that coelacanths—evolutionary "relatives" discovered living in the Indian Ocean—may possess a novel IgH locus (comprising V–D gene clusters dispersed along the chromosome length).

Cells of the Innate Immune System

Natural killer (NK) cells are present in most vertebrates. In mammals, NK cells constitute a population of large granular lymphocytes distinct from T AND B cells. Unlike cytotoxic T cells, they can spontaneously lyse transformed cells that fail to express MHC antigens. NK-like lymphoid cells have also been identified in several lower vertebrates, including birds, reptiles, amphibians, and teleost fish. Furthermore, nonspecific cytotoxic cells have recently been discovered even in protochordates, where they have been shown to destroy mammalian tumor cells. Macrophages from both cartilaginous and bony fish exhibit spontaneous cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) reactions have been documented in sharks.

Monoclonal antibodies directed against catfish NK-like cells modulate the cytotoxic activity of both fish and human NK cells against human tumor cell lines, highlighting the evolutionary conservation of the corresponding antigen receptors. Birds possess cells whose cytotoxic activity is not MHC-restricted, which may represent functional equivalents of NK cells. These cells resemble mammalian NK cells in that they contain cytoplasmic CD3, lack the TCR–CD3 complex on their surface, and frequently express CD8. Such characteristics point to a close evolutionary relationship between NK and T cells. However, both in mammals and birds, NK cells are of extrathymic origin.

Phagocytic activity in fish. The problem of Disease resistance in fish is of paramount importance for aquaculture. Consequently, factors capable of enhancing phagocytic activity in fish are being thoroughly investigated. To facilitate this research, developing Methods for the long-term in vitro culture of fish leukocytes, such as those from catfish and carp, is critical. Enhancement of fish phagocyte activity against bacterial antigens (likely mediated through the release of macrophage-activating factors) can be readily achieved by administering killed pathogenic microbial cells and their derivatives. To boost phagocyte-mediated immunity in fish, β-glucans (Polysaccharides from The Cell walls of Yeast and other Fungi) are also employed; they have proven to be effective vaccine adjuvants, although their precise MECHANISM OF ACTION remains to be fully elucidated. Cytokines, such as fish T cell-derived "gamma-interferon" and human TNF-α, synergistically enhance the respiratory burst activity of rainbow trout macrophages, leading to The production of bactericidal oxygen metabolites (superoxide anion and hydrogen peroxide). Macrophage activation in fish can be inhibited by mammalian TGF-β. Chemokine-like factors capable of influencing macrophage motility have also been identified in fish. Because stress-induced immunosuppression is a frequent challenge in aquaculture, the recently described ability of the immunoactive peptide FK-565 to counteract such suppression is of considerable interest.

Leukotrienes and other lipid mediators (collectively termed Eicosanoids) typically participate in various inflammatory processes in mammals. It has now been established that eicosanoids are also synthesized by fish (as well as amphibians) and play a vital role in their inflammatory responses. For example, leukotriene B₄ enhances leukocyte migration in rainbow trout, and eicosanoids likewise influence T-cell proliferation in these fish. Dietary lipid content can modulate eicosanoid synthesis in fish, making its optimization crucial when designing vaccination protocols—an issue currently under active investigation.

Antigen-nonspecific molecules

Vertebrates possess well-developed classical and Alternative pathways of complement activation. Jawless vertebrates feature antibody-independent, complement-like proteins. In hagfish, these are homologous to mammalian C3, C4, and C5 complement components and act as opsonins; a specific 105 kDa receptor for them has been identified on phagocytic leukocytes. Representatives of all other vertebrate classes possess both classical and alternative complement activation mechanisms (though the existence of The alternative pathway has not been proven in cartilaginous fish).

Complement components C1 through C9, as well as factors B and D, have been detected in carp. Significant homology has been established between Xenopus and mammalian C3 genes. In anuran amphibians, the complement components C1q, C4, C5, the membrane attack complex, and factor B have also been characterized. Fish and amphibian complement components share the same fundamental properties (thermolability and the requirement for Ca²⁺ and Mg²⁺ ions for activation) as the corresponding mammalian proteins. Naturally, the temperature range for complement activity in poikilothermic animals is considerably broader, with activity persisting even at 4 °C. At the same time, thermal inactivation can occur at lower temperatures. In Xenopus, for instance, complement activity is completely abolished after 40 minutes of incubation at 45 °C. Guinea pig complement can be used for the in vitro hemolysis assay of antibodies from adult amphibians. However, for most fish and tadpoles, complement from the same or closely related species is required.

Lower vertebrates possess cytokines functionally similar to mammalian cytokines. The Study of cytokines and, in particular, their receptors in lower vertebrates lags significantly behind the successful molecular investigations in the evolution of immunoglobulins, TCRs, and MHC. Nevertheless, biological methods have established that specific cytokine groups are present in many vertebrate classes. These include interleukins, interferons, tumor necrosis factor, colony-stimulating factors, and chemokines.

For example, T-cell growth factors (TCGFs) that stimulate T-lymphoblast proliferation in vitro have been isolated from culture supernatants of stimulated T lymphocytes from teleost fish, tailed and tailless amphibians, snakes, and chickens (Fig. 15.22). Purified Xenopus TCGF is a 16 kDa protein with biochemical and functional similarities to mammalian IL-2. The gene encoding this “IL-2” and its receptors remain unknown.

Fig. 15.22. Culture supernatant from PHA-stimulated Xenopus splenocytes possessing activity (conditioned medium, CM) was compared with culture medium from control cultures (control medium, CC) (both preparations were subjected to preliminary partial purification). The CM evidently contains cytokines, as it clearly stimulates T-lymphoblast proliferation (while exerting a lesser stimulatory effect on resting splenocytes) and promotes the growth of alloreactive T-cell lines; the CC exhibits no such effects. Culture medium from mixed leukocyte cultures exhibits similar, albeit lower, activity. Xenopus T-cell growth factor activity is mediated by a 16 kDa protein which, judging by its biochemical and functional properties, may well represent amphibian IL-2. (Figure kindly provided by Dr. N. Cohen.)

“IL-2-like” activity has been detected in macrophages of teleost fish, amphibians, and birds. In poikilothermic vertebrates (such as farmed fish populations that can be decimated by Viruses), “interferon-like” factors that activate macrophages and exhibit antiviral activity have also been identified. The interferon of the flounder has recently been sequenced, although it bears little resemblance to any other interferons. This lack of homology may be why oligonucleotide probes (conservative sequence domains of genes encoding mammalian cytokines) do not always successfully identify cytokine genes in poikilothermic animals. However, this has been achieved for amphibian fibroblast growth factor, TGFβ (Xenopus TGFβ5, which can inhibit T-cell proliferation, and chicken TGFβ4), and flounder IL-2. Available data showing that the activation of rainbow trout macrophages by human TNFβ is blocked by preincubation with antibodies against the human TNF receptor (p55) indicate the evolutionary conservation of TNF receptors.

Antimicrobial peptides. Antimicrobial peptides, which are structurally similar to the invertebrate antimicrobial peptides described above, perform an important function in the vertebrate immune system. For instance, cecropins have been found in the intestine of pigs, while defensins that affect Microbial growth are present in phagocytes and certain intestinal cells of mammals. The granular skin and intestinal glands of Xenopus secrete peptides belonging to another family known as magainins. They possess a broad spectrum of biocidal activity against Gram-negative and Gram-positive Bacteria, fungi, and Protozoa. In addition, they exert a cytotoxic effect on cells of various human malignant tumors. Artificially synthesized magainins have already been obtained, and their potential use as therapeutic agents is currently being explored. Another candidate for medical application is squalamine, a steroid derived from sharks that exhibits broad-spectrum antibiotic activity.