UEU-co logo

Ovid: Oxford Handbook of Medical Sciences

Editors: Wilkins, Robert; Cross, Simon; Megson, Ian; Meredith, David Title: Oxford Handbook of Medical Sciences, 1st Edition Copyright ©2006 Oxford University Press, 2006, except ‘Clinical aspects’ section of Chapter 2 (Copyright by Keith Frayn) > Table of Contents > Chapter 12 – Infection and immunity Chapter 12 Infection and immunity Pathogens Bacterial structure General features Bacteria are prokaryotes, ~1–3µm in size, and lack a nuclear membrane and membrane-containing organelles. The cytosol contains ribosomes, inclusions such as glycogen or endospores, organic and inorganic molecules, plus a nucleoid (a nucleus that lacks a nuclear membrane). Cell wall Bacterial cell wall contains peptidoglycan, a sugar heteropolymer of cross-linked N-acetylglucosamine and N-acetylmuramic acid molecules. Inside this lies a lipid bilayer cell membrane containing proteins and carbohydrates (Fig. 12.1). Gram-positive bacteria have a thick cell wall containing lipoteichoic acid and an inner cell membrane. Gram-negative bacteria have a phospholipid outer cell envelope containing a high concentration of lipopolysaccharide and protein, a thin cell wall, and an inner cell membrane. Additional features Some bacteria have a polysaccharide capsule on the outside. Surface structures may include flagella aiding motility and pili or fimbriae contributing to adherence. Additional specific proteins and enzymes contribute to virulence or represent vaccine targets. Some bacteria contain spores and their position aids speciation. Genomic organization Bacterial genomes contain 1–6 million base pairs (Mb). DNA is linear and closed. In addition to genomic DNA, bacteria contain mobile genetic elements transferred between bacteria. Mobile DNA contains genes encoding resistance to antimicrobials. Examples of mobile DNA elements are:

  • Plasmids: small circular DNA molecules containing up to 0.1Mb
  • Transposons: contain insertion-sequence elements—enzymes that facilitate transposition of DNA between sites on chromosomes or plasmids
  • Bacteriophages: viruses that infect bacteria and transfer bacterial DNA.

Protein synthesis DNA-dependent RNA polymerase allows DNA to be copied into RNA. mRNA binds to an initiation site on the 30S ribosomal subunit. The 50S ribosomal subunit is bound to form a 70S complex to allow protein synthesis. tRNA is also present and used in protein synthesis.

Fig. 12.1 Bacterial structure.

P.781
P.782
Bacterial growth Minimal requirements are water, carbon, and inorganic salts. Many bacterial pathogens lack enzymes required for synthesis of key amino acids, nucleosides, or vitamins and these must be added. Synthesis of organic macromolecules allows growth and cell division. Assuming balanced replication, the daughter cell will contain the same amount of macromolecules as the parental bacterium. Growth phases

  • Lag phase: bacterium adapts to new environmental conditions and replication does not occur
  • Exponential or logarithmic phase: bacteria replicate
  • Stationary phase: limited availability of nutrients prevents further replication
  • Death phase.

Regulation of gene transcription during growth Changing environmental conditions require methods to adjust gene transcription:

  • Nutrient sensing: gene transcription regulated by detection of specific nutrients
  • Two-component signal transduction: sensor detects an environmental factor and phosphorylates a regulator that activates gene transcription
  • Quorum sensing: changes in cell density alter levels of diffusible autoinducers that bind to transcriptional activators inducing gene transcription—in effect, quorum sensing is a mechanism of communication between bacteria.

P.783
Bacterial classification During gram staining, the initial reagents, crystal violet and Gram’s iodine, interact with bacterial ribonucleotides to give a purple colour. In Gram-positive bacteria, this remains but in Gram-negative bacteria, this colour is washed away by alcohol and acetone and the bacteria stain red after counter-staining with safranin. This stain also allows separation into cocci (round) and bacilli (rod-shaped). Anaerobes require an environment lacking oxygen. Metabolic tests, motility patterns, and spore formation allow further features for classification. Molecular techniques (sequencing genes and analysis of DNA) aid classification. Ricketsiae and chlamydiae are obligate intracellular parasites that are Gram-negative cocco-bacilli. Most ricketsiae are unable to survive outside the cells they infect. Mycoplasma sp. are unusual bacteria in that they lack a cell wall. P.784
Bacterial pathogenesis Bacterial pathogenesis Reflects the capacity of bacteria to cause disease. Influenced by host factors and microbial factors. Some bacteria replicate in the host without causing disease (commensals). Others penetrate tissues or elaborate toxins causing clinical disease (pathogens). Bacterial adaptations contribute to pathogenesis (virulence factors) but may only be expressed in a particular environment e.g. inside macrophages. Disease results from both microbial factors and the host response to bacteria. A dysregulated inflammatory response leads to tissue damage. Colonization First stage in human diseases is often the ability to adhere to cutaneous or mucosal surfaces (colonization). Not unique to pathogens. Pili, fimbriae, and polysaccharide capsules aid adherence. Colonization may necessitate killing commensal flora by release of antibacterials (e.g. bacteriocin) or scavenging iron, hence limiting its availability for other bacteria. Invasion Tissue invasion facilitated by enzymes that degrade matrix (e.g. hyaluronidase, elastase). Other bacteria may invade by allowing internalization by host cells and subsequently escaping these intracellular locations. Avoidance of host defence Host defence against bacteria includes opsonization of bacteria and phagocytosis. For intracellular bacteria, T-cells prime macrophage killing. Humoral immunity is also important. Capsule inhibits complement-mediated opsonization and phagocytosis. Fc-binding proteins or IgA proteases inhibit immunoglobulin. Some intracellular bacteria are efficiently phagocytosed but escape the phagolysosome or are able to resist antimicrobial molecules (e.g. dentrification inactivates phagocyte nitric oxide). Toxins Exotoxins are secreted virulence factors. Toxin-releasing bacteria need not invade tissue (e.g. intra-luminal toxin release by colonizing bacteria or ingestion of preformed toxin in food). Two main groups of toxins are:

  • Cytolytic: directly damage cell membranes and kill host cells
  • Bipartite (A-B toxins): the B subunit binds to the host cell receptor and the A unit acts on the intracellular substrate. For example, cholera toxin B unit binds to GM1, a sialoganglioside; the A unit induces ADP ribosylation and hence cAMP-mediated fluid secretion by enterocytes. Other toxins enter target cells by direct injection, as occurs with the type III secretion system of many gram-negative bacteria. Some toxins act on extracellular targets (e.g. streptococcal pyrogenic toxins bind to the T-cell receptor Vβ chain to induce cytokine release and toxic shock).

P.785
Endotoxin Bacterial constituents, not secreted. Antimicrobials may enhance endotoxin release. The lipid A portion of lipopolysaccharide is responsible for many of the pathogenic features of gram-negative bacteria including fever, shock, and activation of clotting. In this case, it is the host response to the bacterial component that results in dysregulated cytokine production (IL-1β, TNF-α, IL-6). Lipoteichoic acid in the gram-positive cell wall induces similar effects. Diagnosis Commonly used techniques in the diagnosis of bacteria are summarized in Chapter 14. The most commonly employed diagnostic techniques are:

  • Detection of bacteria in specimens by gram staining (or other specialized stain) and visualization by microscopy
  • Bacterial culture and detection of specific bacteria using diagnostic algorithms.

In specific cases, other methodologies involving detection of bacterial antigens, serology, molecular techniques, and analysis of biopsy specimens are employed. P.786
Antibiotics These are antimicrobials produced by living organisms. Many antimicrobials are synthetic and the term antimicrobials is preferable. Pharmacodynamics Antimicrobials may be bactericidal (bacteria are killed) or bacterostatic (growth is inhibited but bacteria are not killed). Bactericidal antimicrobials include β-Lactams, aminoglycosides, and quinolones. Bacterostatic antimicrobials include macrolides, tetracyclines, sulfonamides, and chloramphenicol. The lowest concentration of an antimicrobial agent that inhibits growth of bacteria after 18–24 hours of culture is called the minimal inhibitory concentration (MIC). For some antimicrobials, the time the antibiotic concentration is above the MIC determines efficacy (time-dependent killing) (e.g. penicillin and macrolides). For others, the peak dose achieved, Cmax, is most important and the ratio of Cmax/MIC predicts outcome (dose-dependent killing) (e.g. aminoglycosides and quinolones). Antimicrobials that demonstrate dose-dependent killing may also exhibit a post-antibiotic effect (ability to inhibit antibiotic growth after the concentration has fallen below the MIC). Some antimicrobial combinations demonstrate synergy (combined effects greater than predicted from the sum of individual effects). Drug levels in specific sites (e.g. cerebrospinal fluid) may be low—knowledge of antimicrobial penetration of selected locations predicts efficacy. Antimicrobials may fail to reach adequate concentrations in locations such as abscesses and a combination of surgery and antimicrobial therapy is required. In the case of prosthetic materials (prosthetic joints or cannulae), resolution occurs in most cases by foreign body removal in addition to antimicrobials. Pharmacokinetics Some antimicrobials are well absorbed after oral administration, while others require intravenous administration. Antibiotics may need dose adjustment in individuals with liver or renal impairment, depending on the route of elimination. In addition, use in pregnancy or paediatrics may require dose adjustment because of altered metabolism or drug toxicity. Allergy and drug toxicity Antimicrobials are a frequent cause of allergy. This can range from mild skin reactions to life-threatening desquamation or anaphylactic shock. Adverse reactions vary but include fever, abnormal liver function tests, renal impairment, and decreased production of polymorphonuclear cells, lymphocytes, or other blood cells. P.787
Mechanisms of action Ideal antimicrobial targets are specific for prokaryotes. Table 12.1 summarizes common targets. A brief overview of the spectrum of action of these antimicrobials and of some of the bacteria inhibited is provided, but for more detail, the reader is referred to more specialized texts. Molecular targets for inhibition include:

  • Cell wall synthesis: makes the bacteria susceptible to osmotic rupture, as bacteria are hyperosmolar compared to the host environment
  • Bacterial protein synthesis: exploits selective differences between prokaryotic and eukaryotic ribosomes
  • Folic acid synthesis: selectively inhibited in bacteria
  • DNA synthesis: DNA gyrase is required for negative supercoiling of bacterial DNA, a prerequisite for DNA replication. Prokaryotic RNA polymerases may also be selectively inhibited.

Antibiotic resistance A global problem that limits the efficacy of antimicrobial chemotherapy and adds to the cost. The major factor in the development of resistance is inappropriate use of antimicrobials in humans and in animals. Resistance may be intrinsic (e.g. Gram-negative bacteria are not susceptible to glycopeptides). Acquired resistance involves mutation of existing genes or acquisition of new genes. New genes are spread by mobile genetic elements (plasmids, transposons, bacteriophages) exchanged between bacteria. Antimicrobial use exerts a selective pressure that allows resistant strains to proliferate at the expense of susceptible strains. Some transposons contain both virulence factors and antimicrobial resistance genes in the same transposonaiding propagation. Examples of resistance are summarized in Table 12.1. Mechanisms include:

  • Production of enzymes that inactivate the antimicrobial agent
  • Mutation in the molecular target for the antimicrobial agent
  • Decreased penetration of the agent into the bacterium
  • Active efflux of the drug out of the bacterium.

Increasingly, bacteria are resistant to multiple antimicrobials e.g. methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase (ESBL) producing Gram-negative bacteria and multi-drug resistant strains of Mycobacterium tuberculosis (MDRTB).

Table 12.1 Antibacterial agents
Cellular target of inhibition Specific targets Class of antimicrobial Examples Spectrum Mechanisms of resistance
Cell wall Transpeptidases (Penicillinbinding proteins) that mediate cross-linking of peptidoglycans β-Lactams Penicillin cephalosporins, carbapenams Broad, some more specific for GPC, others for GNB. Specific β-lactam antibiotics for Staphylococcus aureus and Pseudomonas aerugniosa. Enzymatic; β-lactamases. Altered pencillin-binding proteins.
Decreased permeability of GNB Efflux.
Addition of sub-units to peptidoglycans Glycopeptides Vancomycin GPC Alterations of D-alanine-Dalanine component of stempeptide.
Protein synthesis Binds to ribosomal 50S subunit Macrolides Erythromycin Mainly GPC; Also Mycoplasma sp., Chlamydia sp., Legionella sp., Helicobacter sp. Ribosomal methylation to inhibit binding. Efflux from bacteria.
Lincosamides Clindamycin GPC and anaerobes Ribosomal methylation.
Chloramphenicol Chloramphenicol GPC and GNB but widespread resistance now limits use Enzymatic; chloramphicol acetyl transferase.
Oxazolidinones Linezolid GPC, Mycobacteria sp. Alteration of ribosomal target.
Streptogramins Quinupristin/Dalfopristin GPC Alteration of ribosomal target.
Efflux.
Drug inactivation
Binds to ribosomal 30S subunit Tetracyclines Doxycycline GPC and GNB including Mycoplasma sp., Chlamydia sp., Ricketsia sp., Legionella sp., Brucella sp., and spirochetes Efflux.
Alteration of ribosomal target.
Aminoglycosides Gentamicin GPC and GNB. Particularly used for GNB in combination with β-lactam Enzymatic; antimicrobial modification by amino-glycosidemodifying enzymes.
Decreased uptake into bacteria.
Efflux.
Cell metabolism Inhibition of folic acid synthesis Sulphonamides Co-trimoxazole (trimethoprim-sulfamethoxazole) Mainly GNB. Some GPC. Also some parasites and Pneumocystis; irovecii (carinii). Modification of targets to bypass metabolic block.
DNA synthesis Inhibit A subunit of DNA gyrase and topoisomerase IV Quinolones Ciprofloxacin GNB, some activity against GPC. Altered DNA gyrase. Also altered permeability to drug or enhanced efflux.
  Generation of DNA damage due to generation of reactive-intermendiates. Imidazoles Metronidazole Anaerobes and some parasites Can occur but precise mechanisms undefined.
  DNA-dependent RNA polymerase Rifamycin Rifampicin Mycobacteria, Staphylococcus aureus, Legionella sp., Brucella sp., and Rhodococcus equi.
Always in combinations
Mutations in RNA Polymerase.
Cell membrane Cationic polypeptides inserted into bacterial membranes and permeabilize bacteria Polymyxins Polymyxin B GNB Unknown
Key: GPC; Gram-positive cocci, GNB; Gram-negative bacilli.

P.788
P.789
P.790
Viral structure and classification Viruses contain nucleic acid in the form of linear or circular RNA in a single strand (most RNA viruses) and DNA in a double strand (most DNA viruses) and in a (+) or (-) sense orientation. Viral genomes encode from three to > 100 proteins. The nucleic acid is surrounded by a protein coat (capsid), made of subunits that form a helical, icosohedral, or complex structure (Fig. 12.2). Nucleic acid and capsid form the nucleo-capsid. Nucleic acid may complex with nucleoprotein. Many viruses contain a lipid envelope. Unenveloped viruses are resistant to acid and desiccation and typically cause infection via the oro-faecal route. Respiratory or parenterally acquired viruses typically contain an envelope. The envelope contains proteins that are glycosylated in the external portion. Classification is by presence of RNA or DNA, size of the genome, capsid configuration, or presence of envelope (Table 12.2). Grouped into families with individual members (e.g. the herpesviridae include herpes simplex virus 1 and 2, Varicella—Zoster virus, Epstein—Barr virus, and cytomegalovirus).

Fig. 12.2 Viral structure.
Table 12.2 Classification of viruses
Type of nucleic acid Genome size (kilobases) Virus Family Envelope Capsid
RNA 7–8 Poliovirus Picornaviridae Absent Icosohedral
7–8 Norwalk virus Calciviridae Absent Icosohedral
7–11 HIV-1 Retroviridae Present Icosohedral
10–12 Rubella virus Togaviridae Present Icosohedral
10–13 Yellow fever virus Flaviviridae Present Unclear
10–14 Influenza virus Orthomyxoviridae Present Helical
13–16 Rabies virus Rhabdoviridae Present Helical
16–20 Measles virus Paramyxoviridae Present Helical
19 Ebola virus Filoviridae Present Helical
16–27 Rota virus Reoviridae Absent Icosohedral
20–30 Corona virus Coronaviridae Present Helical
DNA 3 Hepatitis B virus Hepadnaviridae Present Icosohedral
5 Parvovirus B-19 Parvoviridae Absent Icosohedral
5–8 Human papillomavirus Papillomaviridae Absent Icosohedral
36–38 Adenovirus Adenoviridae Absent Icosohedral
120–240 Herpes simplex virus Herpesviridae Present Icosohedral
130–380 Vaccinia virus Poxviridae Present Complex
Key: HIV-1 (Human immunodeficiency virus-1).

P.791
P.792
Viral life cycle Viruses require a host cell in which to replicate by fission. Stages in the cycle include: Attachment to host cell Capsid proteins (rotavirus) or envelope glycoproteins (influenza virus) bind to host cell surface targets (receptors). The range of cells that express these viral receptors is a factor determining the range of cells capable of infection (tropism). Attachment is often a multi-step process involving several receptors (HIV-1 binds to host cells via the interaction of glycoprotein gp120 with CD4 and chemokine receptors). Viral entry Mechanisms of internalization include:

  • Conformational changes: allows fusion with host membrane
  • Receptor-mediated endocytosis involving clathirin-coated pits
  • Direct penetration.

Internalized virus is uncoated and envelope and capsid are degraded by cellular proteases and/or acidification in cellular organelles to allow release of nucleic acid for viral replication. Viral replication

  • (+) sense RNA viruses: RNA functions as mRNA for translation by host ribosomes into protein or is copied by viral-encoded RNA polymerase into intermediate (-) sense RNA that acts as a template for (+) sense RNA generation during replication
  • (-) sense RNA viruses: RNA is copied into (+) sense RNA by preformed RNA polymerase to allow transcription or function as an intermediate in transcription of (-) sense RNA for incorporation into progeny virions
  • Double-stranded RNA viruses use the (-) sense RNA to produce (+) sense RNA
  • Retroviruses contain RNA-dependent DNA polymerase; RNA is copied into DNA that integrates into the host genome to allow transcription (e.g. HIV-1). This strategy permits persistence of infection
  • DNA-containing viruses transcribe DNA in the nucleus using host transcription machinery. Transcription follows a temporal pattern: early transcripts encode regulatory proteins and proteins involved in DNA replication while late transcripts encode structural proteins. Some DNA viruses also persist in the host cell but are present as small extra-chromosomal DNA-termed episomes rather than integrated into the host chromosomes (e.g. herpesviruses).

Viral assembly Viral proteins are usually transcribed as a precursor that requires cleavage by specific proteases into individual proteins. Viral nucleic acid allows initiation of capsid nucleation. Capsid subunits combine to form a complete capsid surrounding nucleic acid. Enveloped viruses acquire envelope as the virus buds through nuclear, cytoplasmic, or endoplasmic reticulum P.793
membranes. Surface proteins are incorporated and modified by processes such as glycosylation in the golgi apparatus. Matrix or tegument proteins are added. Mature virions are released from the host cell. Often, the maturation process is inefficient and the majority of viruses produced are incomplete and unable to infect further cells. P.794
Viral pathogenesis Cellular effects Viral replication modifies host cell gene transcription with loss of normal cellular homeostasis. Expression of multiple host cell kinases is altered. Genes induced include host response genes including cytokines. Cell cycle may be modulated. Cell proliferation occurs with certain viruses. The immune response to viral proliferation contributes to cytopathology inducing cell death of directly infected and uninfected bystander cells. Cytopathic effects include induction of apoptosis. Viruses can modulate HLA antigen, chemokine, or cytokine expression and induction of apoptosis enabling immune evasion. Possible outcomes of acute lytic infection include:

  • Chronic infection: persistent replication continues and viruses are shed from infected cells
  • Latency: virus persists in the host genome but is not transcribed until reactivated by specific stimuli
  • Reactivation: specific stimuli induce transcription of latent virus. Immunosuppression may be an important trigger
  • Transformation: ability to promote immortalization of cells leading to cancer formation. Chronic pro-inflammatory effects may contribute to malignant transformation by some viruses.

Systemic effects Local replication may allow systemic spread. Detectable virus in the blood (viraemia) produces further replication in the reticulo-endothelial system resulting in increased levels of viraemia before seeding of target organs. Some viruses spread via other routes (e.g. peripheral nerves). Clinical symptoms result from the cytokines produced by viraemia or tissue damage locally or in target organs. Host immunologic response Includes humoral and cell-mediated immunity (cytotoxic T-lymphocytes; CTL). Modified by age, nutritional status, immunosuppression, and vaccination status. Results in the production of cytokines that induce symptoms and facilitate viral clearance. Viruses have developed adaptations to allow immune evasion (e.g. alteration of surface proteins recognized by antibodies or CTL). P.795
Viral infection Subclinical or results in clinical disease. The ability of a virus to cause human infection or disease is determined by viral and host factors. Transmission Various routes including oro-faecal, respiratory, touching an infected lesion, insect or animal bites, transfusion of blood, transplantation of organs, sexual exposure, or vertical transmission from mother to child. Some routes of infection (e.g. transfusion) are associated with a larger infecting dose and more severe disease. Virulence Determined experimentally by the quantity of virus required to cause cytopathic effects in cell culture or death to experimentally infected animals. Reflects host susceptibility and viral factors. Viral mutations alter virulence. Host susceptibility Individuals may be immune due to prior infection or immunization. This may prevent or modify infection so disease does not occur. Genetic make-up may modify susceptibility. P.796
Viral therapy Antiviral agents are described in Table 12.3. Viruses may develop resistance to antiviral agents by developing mutations in genes encoding the targets.

Table 12.3 Antiviral agents
Description Agent Spectrum Target
Inhibitors of entry and disassembly T-20
(enfuvirtide)
HIV-1 Blocks viral fusion with host cell membrane
Rimantidine Influenza A virus Blocks viral uncoating after entry
Pleconaril Enteroviruses, rhinoviruses Binds to capsid, blocks attachment and uncoating of the genome
Inhibitors of viral replication Acyclovir, valacyclovir HSV, VZ Phosphorylated drug inhibits DNA polymerase
Ganciclovir CMV, HHV-6 As for acyclovir
Foscarnet HSV, VZ, CMV, HHV-6 Directly inhibits DNA polymerase
Cidofovir CMV, HSV, VZ, HHV-6, HHV-8 Phosphorylated drug inhibits DNA polymerase
Zidovudine HIV-1 Inhibits reverse transcriptase
Lamivudine HIV-1, HBV Nucleoside reverse transcriptase inhibitor of HIV-1 and DNA polymerase inhibitor of HBV
Tenofovir HIV-1, HBV As for Lamivudine but nucleotide inhibitor
Ribavirin RSV, HCV, Lassa fever virus Not fully elucidated; involves depletion of guanosine triphosphate and inhibition of nucleic acid synthesis
Formivirsen CMV Antisense nucleotide; intravitreal injection inhibits ocular replication of CMV
Inhibitors of viral assembly and release Zanamivir oseltamivir Influenza A virus
Influenza B virus
Inhibits viral neuraminidase and hence release of virus
Protease inhibitors (e.g. Lopinavir) HIV-1 HIV protease inhibition blocks cleavage of polyproteins and inhibits production of mature virions
Immunomodulators Interferon-α HBV, HCV, HPV, HHV-8 Stimulates antiviral host responses that inhibit viral protein synthesis
Imiquimod HPV Binds to toll-like receptor 7; stimulates interferon-α expression
Key: HIV-1; Human immunodeficiency virus, HSV; herpes simplex virus, VZ; Varicella—Zoster virus, CMV; Cytomegalovirus, HHV-6/-8; human herpesvirus-6/-8, HBV; hepatitis B virus, HCV; hepatitis C virus, RSV; Respiratory syncytial virus, HPV; human papillomavirus

P.797
P.798
Prions Proteinaceous infectious particles that lack nucleic acids. Resistant to procedures that hydrolyse nucleic acids. Cause chronic, progessive neurologic diseases characterized by reactive astrocytosis and often spongiform cellular changes. Result in accumulation of an abnormal protein—prion protein (PrP). Examples of prion diseases include Kuru, Ceutzfeldt—Jacob disease (CJD), and new variant CJD. Diagnosis may involve identification of PrP in biopsy material. Treatments are investigational. Fungi Classification

  • Yeasts (e.g. Candida or Cryptococcus sp.)
  • Moulds—filamentous fungi (e.g. Aspergillus sp. and Trichophyton sp.)
  • Dimorphic fungi—yeasts in tissue but grow in vitro as moulds and include the North American dimorphic fungi (e.g. Histoplasmosis).

Dermatophyte infections are superficial fungal infections of the skin and related structures caused by moulds. Yeasts are oval structures that replicate by budding. Moulds are tubular structures—hyphae that grow by longitudinal extension and branching. Fungal replication involves the production of spores that may be either asexual or sexual. Structure and pathogenesis Fungal cell walls contain chitin and polysaccharides and fungal endotoxin stimulates pro-inflammatory cytokines. Cryptococci contain a polysaccharide capsule, but most fungi do not. Inside the cell wall is the sterol-containing cytoplasmic membrane. Enzymes involved in sterol (in particular, ergosterol) synthesis are targets of antifungal drugs. Fungi produce exoenzymes; proteases digest tissue components such as keratin to facilitate invasion, and phospholipases also contribute to pathogenicity. Immunologic defects predisposing to fungal infection include decreased polymorphonuclear phagocyte function, defects in physical barriers (often associated with prosthetic devices such as cannulae), and impaired cell-mediated immunity. Diagnosis and therapy Diagnosis includes microscopy after special staining, culture, serology, antigen detection, molecular techniques, and analysis of pathologic specimens. The principal antifungal agents and their targets are summarized in Table 12.4. New antifungal targets are being identified but resistance is also emerging.

TABLE 12.4 Antifungal agents
Class Agent Spectrum Target
Echinocandins Caspofungin Yeasts and moulds Inhibition of B1, 3 glucans in the cell wall
Polyenes Nystatin, Amphotericin B Yeasts and moulds Bind to membrane sterols such as ergosterol
Azoles Fluconazole Yeasts Blocks C-14α methylase and inhibits ergosterol synthesis
Itraconazole, voriconazole Yeasts and moulds Same as fluconazole but spectrum extended to moulds
Allylamines Terbinafine Moulds (dermatophytes) Inhibits squalene epoxidase required for ergosterol synthesis; concentrated in nails and hair
Other Griseofulvin Dermatophyte infections Concentrated in nails and hair; inhibits microtubule polymerization
Cytosine analogue Flucytosine Yeasts Inhibits thymidylate synthetase and so DNA synthesis
Immunomodulators GM-CSF Moulds and yeasts Stimulates macrophage microbicidal killing of fungi

P.799
P.800
Parasites Spectrum Unicellular organisms, protozoans (e.g. Plasmodium falciparum), and multicellular organisms (e.g. helminths—worms and flukes) and ectoparasites (including insects whose life cycle only involves interaction with the external surface of the host). Many parasitic infections are geographically restricted to tropical counties and occur in temperate climates due to travel or immigration. Parasites cause major global illness (e.g. malaria). Life cycles Unique—often with stages of development in hosts other than humans. Knowledge of intermediate hosts (helminths, insects, and other mammals) is important in prevention. P. falciparum causes malaria and is spread by infected Anopheles sp. mosquitoes that release sporozoites when biting. These pass to the liver hepatocytes and mature into schizonts and are released from the liver as merozoites that infect erythrocytes. Within erythrocytes, maturation to ring trophozoites and schizonts occurs. Asexual erythrocytic schizonts mature and are released as merozoites that infect other erythrocytes. Alternatively, sexual development in the erythrocyte allows formation of male and female gametocytes. Ingestion of these by a mosquito results in gamete fusion, formation of a diploid zygote, oocyst formation, and release of sporozoites to the salivary gland for infection of further humans. Pathogenesis Genomics and proteomics provide molecular insights. For example, in malaria, red cells express P. falciparum-infected erythrocyte membrane protein-1 (PfEMP1) inducing cytoadherence to endothelial cells, obstruction of small capillaries, and leading to complications such as cerebral malaria. Host responses to parasites involve cell-mediated immunity. Many protozoan infections are particular problems in individuals with HIV infection. Intracellular protozoans such as Leishmania sp. require Th1 responses with TNF-α, IFN-γ, and IL-12 production to prime macrophage killing of intracellular parasites. For other parasites, a Th2 cytokine bias with release of IL-4, IL-5, and IL-10 predominates. Increased levels of eosinophils are observed with many helminthic infections. Humoral immunity is important for some enteric protozoan infections such as Giardia lambdia. Intact splenic function is required in protection against some parasites, including erythrocytic parasites. Diagnosis and treatment Diagnosis involves microscopy, antigen detection, serology, molecular techniques, and analysis of pathology specimens. A selection of antimicrobial agents and the targets they act upon are summarized in Table 12.5.

TABLE 12.5 Antimicrobials active against parasites
Agent Parasite Target
Quinine, chloroquine, mefloquine Plasmodium sp. Haeme polymerase inhibitor, blocks conversion of free haemoglobin into malarial pigment. Full mechanism of quinine unclear
Atovaquone Plasmodium sp. Inhibits coenzyme Q and hence cellular respiration and pyrimidine synthesis
Sodium stibogluconate Leishmania sp. Dysregulation of parasitic metabolism, but exact mechanism unclear
Suramin Trypanosoma brucei Inhibits glycerol-3-phosphate oxidase and dehydrogenase and hence energy metabolism
Nifurtimox Trypanosoma cruzei Generation of reactive oxygen species that damage the trypanosome
Sulfadiazine plus pyrimethamine Toxoplasma gondii Inhibits dihydropteroate synthetase and dihydrofolate reductase to block parasitic folic acid synthesis
Metronidazole Giardia lamblia, Entamoeba histolytica DNA damage
Praziquantel Schistosoma sp. and tapeworms Stimulates calcium influx, leads to paralysis
Albendazole Multiple worms Numerous targets including inhibition of microtubule assembly and impaired uptake of glucose
Ivermectin Some nematodes e.g. Strongyloides stercoralis and Onchocerca volvulus Opens glutamate-gated chloride channels, paralysing pharyngeal pumping activity
Diethylcarbamazine Tissue nematodes Multiple activities, hyperpolarization leading to paralysis and membrane damage
Niclosamide Tapeworms Uncouples oxidative phosphorylation leading to break-up of the scolex
Permethrin, malathion Ectoparasites including Sarcoptes scabei Kill adults and ovicidal

P.801
P.802
Defence Against Infection Innate immunity Evolutionarily conserved; present in invertebrates and vertebrates. Allows differentiation of microbial non-self from self. Rapid response. Germ-line encoded receptors recognize a limited number of highly conserved microbial products. Physical barriers The skin and mucosa block invading micro-organisms. Breached by prosthetic appliances (e.g. catheters) or by tissue damage (e.g. burns). Epithelial cells can internalize micro-organisms and secrete cytokines. Commensal flora block colonization by pathogenic micro-organisms. Secretions contain antimicrobial factors such as lysozyme, defensins, collectins, iron-binding proteins, natural antibodies (antibodies against colonizing micro-organisms that may cross-react with a pathogenic strain) and have a low pH. Complement pathways >30 proteins that contribute to innate host defence, link with adaptive immunity and aid clearance of immune complexes. Cascade of sequentially activated serine proteases (Fig. 12.3).

  • Classical pathway: C1 complex binds to antibody on the bacterial surface
  • Lectin pathway: mannose-binding lectin (MBL) binds to bacterial mannose
  • Alternative pathway: C3b binds to surface hydroxyl groups on bacteria.

These pathways activate cleavage of other components, in particular C3 and C5. Complement enhances phagocytosis of opsonized bacteria (C3b fragments), chemotaxis (migration of leukocytes to sites of infection; C5a), polymorphonuclear leukocyte (PMN) activation (C5a and C3a), and bacterial lysis (C5b-9 membrane-attack complex). Complement also enhances antibody response and immunologic memory, hence linking innate and adaptive responses. Collectins Contain a collagenous domain linked to a calcium-dependent lectin; include C1q, MBL, and surfactant proteins —A and —D. MBL binds to microbial carbohydrates and this activates two associated serine proteases that cleave C3. Pathogen-associated molecular patterns (PAMPs) PAMPs are conserved patterns of molecules on microbial structures recognized by the immune system via pattern-recognition receptors (PRRs). PAMPs include: lipopolysacchaide (LPS), lipoteichoic acid (LTA), peptidoglycan, flagellin, mannan, glucan, bacterial DNA, and double-stranded RNA. PRRs are secreted, endocytic, or signalling and widely expressed in a non-clonal pattern.

Fig. 12.3 Complement pathways.

P.803
P.804
Toll-like receptors (TLRs) Activate transcription factors of the nuclear factor-κB (NF-κB) family and induce pro-inflammatory and antimicrobial gene transcription in response to PAMPs. Signal via multi-component molecular complexes that may contain other molecules functioning as the PRR (e.g. LPS binds to CD14 and this complex binds to MD-2 and the TLR4 molecule to induce signalling). For other TLRs, such as TLR2, heterodimers of different TLR are required for signalling. TLRs contain a leucine-rich extracellular domain and a cytoplasmic domain similar to the interleukin-1 (IL-1) receptor. TLRs and their ligands include: TLR2 (LTA and lipopeptides), TLR3 (double-stranded RNA), TLR4 (LPS), TLR5 (flagellin), and TLR9 (CpG motifs in bacterial DNA). Signal transduction involves IL-1 receptor associated kinases and mitogen-activated protein kinase kinases. Nucleotide-binding oligomerization domain (Nod) proteins Nods are intracellular receptors that respond to bacterial components such as components of peptidoglycans. Nod signalling results in NF-κB induction. Phagocytes In addition to humoral factors, the principal effectors of innate immunity are phagocytes.

  • Mononuclear phagocytes are monocytes in the blood and differentiate into macrophages in tissue. The resident phagocyte is the long-lived tissue macrophage
  • PMNs are short-lived phagocytes and, along with monocytes, are recruited to sites of infection
  • Eosinophils contribute to host defence against parasites (p.826).

Phagocyte functions include: Internalization of micro-organisms Macrophages internalize non-opsonized micro-organisms using PRR (mannose receptor, macrophage scavenger receptors, etc.). Viruses may be internalized by receptor-independent endocytosis involving direct membrane invagination. Phagocytes internalize opsonized microorganisms using Fc receptors that bind immunoglobulin (FcγRs also bind Creactive protein or serum amyloid protein) and complement receptors (CR) that bind C3 complement cleavage products (CR1 also binds C1q and MBL). Integrins also contribute to phagocytosis. Internalized microorganisms are contained in phagolysosomes, but some micro-organisms have developed adaptations to escape the phagolysosome and avoid intracellular killing. Other organisms are resistant to phagocytosis due to factors such as a polysaccharide capsule. Opsonization may reverse a micro-organism’s resistance to phagocytosis. Cytokine production Phagocytes produce cytokines to activate (or downregulate when appropriate) innate and adaptive immune responses. Primes phagocyte killing of micro-organisms and regulates the immune response. P.805
Killing of micro-organisms Micro-organisms are killed intracellularly and extracellularly. Antimicrobial mechanisms include:

  • Reactive oxygen species (ROS). Superoxide generated by the NADPH oxidase system, undergoes further reactions to generate ROS. Myeloperoxidase (MPO) catalyses the reaction between hydrogen peroxide and halide ions to produce potent antimicrobial molecules. PMN are major producers of ROS. Some of ROS effects may be via activation of proteases
  • Reactive nitrogen species (RNS). Nitric oxide, produced by inducible nitric oxide synthase (iNOS), reacts with ROS to produce RNS. Important for killing of intracellular pathogens in macrophages, although the importance of NO in human defence mechanisms is questionable
  • Proteases. Acid or neutral. Include cathepsins such as cathepsin G
  • Cationic proteins. Proteins of different size, positively charged to interact with the negatively charged surface of micro-organisms (e.g. bactericidal/permeability-increasing protein (BPI; a component of PMN granules, active against gram-negative bacteria), defensins (small arginine-rich peptides), and cathelicidins)
  • Lactoferrin. Bacterostatic; deprives bacteria of iron required for growth
  • Low pH. Contributes to microbial killing but many bacteria can withstand low pH.

Antigen presentation Although not as effective at antigen presentation as dendritic cells (DC), macrophages also function as antigen-presenting cells (APC). TLRs play a role in upregulating the expression of co-stimulatory molecules (CD80, CD86 on APC) and help link innate to adaptive immune responses. TLRs also aid DC maturation. Natural killer cells Effectors of cellular innate response, especially antiviral responses. Large granular lymphocytes that do not contain T-cell receptors (TCR) and do not require prior stimulation. Possess surface receptors—killer-cell inhibitory receptors (KIR)—that bind to major histocompatibility complex (MHC) class I antigens expressed on almost all normal nucleated cells. This prevents target killing. Viral-infected (and tumour) cells may down-regulate MHC class I antigen and, in the absence of KIR engagement, NK cells kill targets by lysis or apoptosis. NK cells can also kill viral-infected cells coated with antibody. P.806
Adaptive (acquired) immunity Present only in vertebrates. Development is unique to every individual and not passed on to offspring. Specific recognition of small details of molecules (termed antigens) results in immunologic memory. Allows response to foreign antigens but also response to self-antigens (controlled by deleting auto-reactive somatic cells). Requires stimulation and resultant clonal expansion before maximal immune response occurs— hence delayed. Diversity generated by somatic rearrangement of genes encoding antibody and the T-cell receptor (TCR). P.807
Antigens Description An antigen is a specific compound binding specific antibody or TCR. If it induces an immune response, it is termed an immunogen. Characteristic features of immunogens include:

  • Non-self, recognized as foreign antigens
  • Chemical complexity
  • High molecular weight (small molecular weight compounds that are immunogenic—haptens—need to be complexed to a carrier protein)
  • Susceptibility to antigen processing by degradation of parent molecule (proteins undergo proteolysis).

Epitopes A portion of the antigen (the epitope) binds non-covalently to a unique antigen binding site on antibody or TCR. The epitope is usually 5–15 amino acids long (or equivalent size for non-peptides). Peptides are strongly immunogenic. Carbohydrates are immunogenic, particularly when present in structures such as glycoproteins. Lipids are only immunogenic when combined with a lipid carrier. Nucleic acids may function as immunogens in auto-immunity but rarely in infection. B-cells bind soluble, non-processed antigens present in microbial proteins, carbohydrates, or lipids, while T-cells recognize processed antigen derived from peptides presented by APC after proteolytic degradation. Superantigens activate many T-cells with different antigen specificities but common characteristic TCR Vβ segments, resulting in high level cytokine production. Streptococcus pyogenes and Staphylococcus aureus can express superantigen toxins and induce toxic shock. P.808
Major histocompatibility complex (MHC) MHC genes and gene products T-cells respond to antigen bound to a MHC molecule on the APC (MHC restriction of T-cell responses). MHC molecules bind to only selected antigens, so contributing to the selection of antigen for presentation. MHC class I molecules Human leukocyte antigens (HLA) A, B, and C. Consist of an α chain bound to β2 microglobulin. Expressed on all nucleated cells. MHC class II molecules HLA DR, DQ, DP. Consist of an α and a β chain. Expressed constitutively on B-cells, DC, and thymic epithelium and inducible on T-cells, macrophages, and endothelial cells. Expression Each individual expresses both parental genes for each HLA antigen (codominant expression). There are seven HLA DR genes, so an individual expresses six class I and up to 20 class II molecules on each cell expressing class I and II molecules. HLA genes differ between individuals due to genetic polymorphism, and these variants are termed alleles. This has consequences in transplantation, where it is highly unlikely two individuals will have identical HLA haplotypes. HLA typing describes each allele by locus, allele type (which corresponds largely to serologic group), and subtype (determined by PCR) (e.g. DRB1*0702). Certain HLA haplotypes are associated with increased risk of certain diseases (e.g. tuberculosis). MHC presentation of peptide MHC molecules bind peptide fragments in the polymorphic region (peptide groove). This region also binds the TCR. MHC class I molecules bind peptides derived from endogenous antigen (e.g. from intracellular viruses or parasites) and present antigen to CD8 T-cells (MHC class I binds CD8). MHC class II molecules bind peptides derived from degradation of exogenous peptides (e.g. phagocytosed bacteria or viruses) for presentation to CD4 T-cells (MHC class II binds CD4). Most large antigens, as occur in micro-organisms, have multiple different epitopes, at least one of which will bind successfully to a HLA allele ensuring an immune response. P.809
Antigen presenting cells DC, macrophages, and B cells perform antigen presentation. DC are the only antigen presenting cell (APC) capable of inducing primary immune responses and, hence, immunologic memory. Immature DC capture antigen in peripheral tissue. Subsets of immature DC contribute to innate immunity by producing interferon (IFN) α. After antigen capture, DC migrate to regional lymphoid tissue, where DC maturation is associated with upregulation of MHC molecules, and antigen presentation occurs. DC antigen presentation to naive T-and B-cells results in activation and the subset of DC involved in antigen presentation can determine whether a type 1 (Th1) or type 2 (Th2) immune response is generated. DC are separated by surface markers in humans; myeloid (CD11c+) and plasmacytoid (CD11c-) DC exist. DC include interstitial DC of the dermis and Langerhans cells of the epidermis. Macrophages presentation is less efficient but occurs during secondary immune responses. B-cell antigen presentation occurs when B- and T-cells have both already been primed by antigen. P.810
T-cells Thymic-derived lymphocytes that induce cell-mediated immunity. Split into subsets (e.g. C4+ and CD8+). Directly interact with antigen via TCR. TCR complex structure Transmembrane protein with one α and one β chain, each containing constant and variable regions. The variable regions interact to form three hypervariable regions (complementarity determining regions—CDRs 1, 2, and 3). The TCR complex is formed by two seperate heterodimers of the αβ chains associated with one γε and one δε heterodimer and one ζζ homodimer. All three components of CD3 (γε, δε, and ζζ) contribute to TCR signal transduction via immunoreceptor tyrosine-based activation motifs (ITAM). On antigen engagement, TCR ITAMs bind cellular kinases that activate cell signalling. A minor subset of T-cells, γδ T-cells, have TCR containing γδ heterodimers (distinct from the γδ chains of CD3). TCR diversity Generated by having multiple alternative genes encoding separate segments of each chain of the TCR present in the germline DNA (the inherited chromosome)—same principle as immunoglobulin diversity. As lymphocytes differentiate, these genes are rearranged and an individual clone of lymphocytes contains DNA that encodes just one gene for each segment (Fig. 12.4) chain is made up of one copy of the possible V and J genes combined with a C gene. The β chain is made up of one copy of the possible V, D, and J genes combined with a C gene. This gene rearrangement requires the products of the recombination activation genes (RAG) -1 and -2 to allow enzymatic cleavage during recombination. TCR show allelic exclusion (only one parental chromosome is used), so all TCR in a clone have identical antigen specificity. TCR gene rearrangement allows for the generation of approximately 1015 separate TCR. T-cell selection T-cell maturation occurs in the thymus. The TCR expressed (αβ or γδ) is determined and TCR gene rearrangement occurs. T-cells at this stage are CD4+/CD8+ (dual positive). If TCR binds antigen, the cell survives (positive selection); if not, dies by apoptosis. The cells become educated to recognize the antigen only if presented by MHC (MHC restriction). Interdigitating DC present antigen to MHC-restricted T-cells and, if the reaction is of too high affinity, the cells die by apoptosis (negative selection or central tolerance). This prevents autoreactive T-cells. After negative selection, T-cells downregulate either CD4 or CD8. T-cells are now MHC-restricted and self-tolerant. Mature T-cells traffic to regional lymphoid organs or peripheral tissues. T-cells that have not previously encountered antigen are termed naïve and selectively home to regional lymphoid tissue, while activated or memory T-cells selectively migrate to peripheral tissue. Some autoreactive T-cells escape to the periphery but undergo activation-induced cell death— a form of apoptosis mediated by Fas ligand (peripheral tolerance).

Fig. 12.4 Organization of the genes encoding the α and β chains of the human T-cell receptor.

P.811
P.812
Antigen presentation to T-cells MHC-bound peptide interacts with the most variable region of the TCR (CDR3), while the CDR1 and CDR2 regions interact with conserved regions of the MHC. The interaction between the MHC-peptide complex and the TCR is low affinity and insufficient to activate the T-cell. In addition, CD4 or CD8 bind to conserved regions of MHC class II or I respectively, allowing adhesion and signal transduction via specific cellular kinases. Co-stimulatory pairs must also bind before T-cell activation (Fig. 12.5): CD40 on the APC with CD40 ligand (CD154) and B7.1/B7.2 (CD80/CD86) on the APC with CD28. Later in the activation process, B7.1/B7.2 may bind with CTLA-4 and inhibit further activation. The co-stimulatory molecule interactions help localize molecules involved in the signal transduction induced by TCR engagement. Adhesion molecules help stabilize the APC—T-cell interaction: CD58 with CD2 and CD54 with CD11a/CD18. TCR engagement results in signal transduction involving src family tyrosine kinases that phosphorylate ITAMs on the intra cytoplasmic tails of CD3. ZAP-70, another tyrosine kinase, is then activated, as are, ultimately, transcription factors, including NF-κB and nuclear factor of activated T-cells (NF-AT), resulting in gene transcription. Initially, cells are activated and proliferate. This is associated with cytokine production. T-cell effector functions Activation of T-cells results in the development of characteristic phenotypes:

  • T helper cells (Th). The effector function is influenced by the nature of antigen and APC. Th1 CD4 T-cells produce characteristic cytokines (IL-2, IFN-γ, TNF-β). Th1 responses are evoked by bacterial and viral antigens and activate CD8 T cells, NK cells, and macrophages to kill cells with intra-cellular micro-organisms. Th2 cells produce IL-4, IL-5, IL-10, and IL-13. This triggers IgE production by B-cells and activation of eosinophils in response to parasites and allergens
  • Cytotoxic T-cells (CTL). CTL are CD8 T-cells adapted to kill cells expressing specific antigen bound to MHC class I. Activation induces target (micro-organism infected, tumour or transplanted cells) killing. CTL can be activated by Th1 cells or by DC presentation of antigen. CTL kill using granules containing perforin and granzyme and, to a lesser extent, using Fas ligand. This results in lysis or apoptosis of the target, depending on the pathway. Some CD4 T-cells also function as CTL
  • Memory T-cells. Clonal downsizing follows initial proliferation of an antigen-specific clone. Activated cells are killed by apoptosis (activationinduced cell death). A proportion of the clone survives as memory T-cells. Memory T-cells (CD45RO+) express different antigens from naïve T-cells (CD45RA+, CD62L+). Memory T-cells are more easily activated by antigen and may not require co-stimulation by CD28. Professional suppressor T-cells are T-regulatory T-cells which retain a unique ability to suppress other immune responses
  • P.813

  • Suppressor T-cells. T-regulatory cells express immunomodulatory cytokines such as IL-10 and TGF-β CD4+, CD25+ regulatory T-cells prevent activation of autoreactive T-cells that have not been removed by other mechanisms of tolerance
  • γδ T-cells. Found in the skin, respiratory tract, and intestine and increase during infection, especially tuberculosis.
Fig. 12.5 Antigen presentation to a CD4-T cell involves presentation of the peptide bound to the MHC class II molecule of the antigen presenting cell (APC) to the T-cell receptor (TCR). The CD4 molecule and the TCR both bind to the MHC class II molecule. T-cell activation also requires co-stimulation via CD40 (binding to CD154) and B7 (binding CD28).

P.814
B-cells Produce antibody and can present antigen. Antibody structure Antibodies are proteins (immunoglobulins) that are secreted or membrane-bound. The basic units of immunoglobulin are two light chains and two heavy chains as shown in Fig. 12.6. Disulfide bonds hold these together. The light chains are identical and contain a variable domain and a constant domain. Each domain is stabilized by an intra-chain disulphide bond that creates a loop in the peptide chain that forms a compact globular structure. The heavy chains are also identical and contain a variable region and three constant domains. Each light chain associates with one heavy chain. A disulphide bond at the hinge region holds the two heavy chains together. The light chains of any given immunoglobulin are of one of two classes, κ or λ. The heavy chains are made of one of five classes (isotypes) separated into subclasses (both heavy chains are identical in a given molecule). The region containing the variable and first constant domains is termed the Fab fragment and the region containing the second and third constant domains is the Fc fragment. The heavy chain determines biologic activity: half-life in the circulation, Fc receptor binding, etc. Within each variable domain lie three hypervariable regions (complementarity determining region—CDR): cf TCR. The three adjacent CDR form the specific antigen binding site. Antigen antibody binding is non-covalent and requires a close fit. The strength of this association is the affinity. Flexibility at the hinge regions allows the two antigen binding sites to interact with antigens on a large structure (e.g. a bacterium). Multivalent antigens have numerous interactions of different affinity and the overall binding energy is the avidity. Genetics of antibody diversity Germline DNA encodes multiple genes for each segment of antibody light or heavy chain (cf TCR). During early B-cell maturation gene rearrangement occurs; selected genes for each variable segment are aligned and transcribed into RNA. Recombination requires RAG-1 and -2. For light chains, there are ~40V genes and 4 or 5J genes encoding the variable domain. For κ, there is one C gene, but for λ light chains there are four alternative C genes, each associated with one of the four J genes. For heavy chains, there are ~50V genes, 20D (diversity) genes and six J genes that encode the variable domain. The D and J genes together encode the CDR 3 region. Any B-cell clone makes antibody containing only one class of light chain of fixed antigen specificity. Genes from only one parental chromosome are used (allelic exclusion). There are multiple C genes, one for each isotype and subtype of immunoglobulin. Those closest to the J genes are Cµ and Cδ. P.815
During the early stages of development, B-cells undergo VDJ recombination and alternative splicing allows translation of protein with either a M or D immunoglobulin isotype heavy chain. At later stages of differentiation (requires cytokines and specific antigen), alternative isotypes such as IgG, IgE, or IgA are produced (class switching). Random association of V(D)J genes and heavy/light chains creates diversity. Junctional diversity (deletion or alterations in nucleotide sequence at the sites of gene rearrangement), insertional diversity (addition of nucleotides at V—D or D—J junctions), and somatic hypermutation (point mutations in the V(D)J genes occurring during secondary immune responses create increased antibody affinity) all enhance diversity. Biologic functions of antibody

  • Agglutination (clumping of insoluble antigen) and precipitation (clumping of soluble antigen) by IgG, IgM
  • Opsonization. Antibody is bound to micro-organisms and the Fc portion binds to the Fc receptors of phagocytes
  • Complement activation. Complement cleavage products enhance opsonization, induce chemotaxis of leukocytes to sites of infection, or directly lyse bacteria
  • Antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells also express Fc receptors but the engagement of these leads to killing of the target cell not phagocytosis
  • Neutralization of toxins or viruses
    Fig. 12.6 Antibody structure. Immunoglobulin consists of a light chain containing a variable domain VL and a constant domain CL. The heavy chain consists of one variable VH and three constant domains CH1–3. The segment containing the CH2–3 of both heavy chains is the Fc portion and the two segments containing VL, VH, CL, and CH1 are each termed Fab fragments.
  • P.816

  • Immobilization of motile bacteria. Antibodies may bind to structures such as flagella
  • Protection of the foetus and neonate. Maternal IgG crosses the placenta and protects the foetus and neonate, which is incapable of producing its own antibody.

IgM is the initial antibody produced during infection or immunization. IgG is produced later but constitutes the majority of serum immunoglobulin and has the longest half-life. IgA is the major immunoglobulin in secretions in the gut or respiratory tract and in combination with lysozyme is bactericidal, although it is unable to activate complement. IgM exists as a pentameric structure with five immunoglobulin units bound by a J chain, while secreted IgA consists of two units bound by a J chain. IgA also contains a secretory component when present in secretions. IgE is important in host defence against parasites such as worms and can also lead to allergic manifestations such as anaphylaxis and atopy (Table 12.6). B-cell differentiation Early differentiation in the bone marrow. Cells successfully rearranging antibody genes express pre-B-cell receptor (BCR) and undergo positive selection; the remainder die by apoptosis. Immature B-cells express monomeric IgM as a BCR. If these cells encounter self-antigen, they are deleted by apoptosis or become inactivated (anergy). This results in negative selection and self-tolerance. Mature B-cells express surface IgM and IgD and subsequent foreign antigen binding results in activation. This occurs in the germinal centres of secondary lymphoid organs. B-cells proliferate and differentiate. Mature antibody-producing B-cells are termed plasma cells and can demonstrate antibody class switching. B-cell effector populations and functions

  • Plasma cells. Differentiated B-cells; secrete high levels of antibody. Lack cell surface immunoglobulin.
  • Memory B-cells. Long-lived non-proliferating B-cells available for antigen rechallenge. Express surface immunoglobulins other than IgM or IgD. Somatic hypermutation allows affinity maturation.
  • B-1 B-cells. Minor subpopulation found in body cavities such as pleura and peritoneum. Produce low-affinity IgM in response to bacterial polysaccharide.
  • B-cell antigen presentation. Not a specific subpopulation. B-cells constitutively express MHC class II and possess co-stimulatory molecules such as B7, upregulated with activation and CD40 to allow antigen presentation to CD4 T-cells. Plasma cells do not express surface antibody and cannot present antigen. Adhesion molecules also aid interaction.

B-cell activation Antigens with multiple repeating epitopes (e.g. polysaccharides) directly activate B-cells (thymus-independent antigens). These antigens do not induce class switching or memory cells. Protein antigens have single epitopes and require additional signals from CD4 Th cells to activate B-cell P.817
(thymus-dependent antigens). The nature of the cytokines produced by Th determines the class of antibody produced. CD40/CD40 ligand interaction is critical for B-cell proliferation and antibody class switching. B-cell activation results in signal transduction (cf TCR signalling). Immunoglobulins activate src family kinases to phosphorylate ITAMs on signalling molecules associated with immunoglobulin in the BCR complex (Igα and Igβ). Other molecules, such as CD19 and CD21, also enhance BCR complex signal transduction. When phosphorylated, ITAMs activate Syk (a kinase) to activate pathways that allow transcription factors such as NF-κB and NF-AT to translocate to the nucleus and stimulate gene transcription (immunoglobulins and cytokine receptors). Th cytokines enhance B-cell proliferation and differentiation.

Table 12.6 Characteristic features of immunoglobulin isotypes
Characteristic IgG IgM IgA IgD IgE
% total Ig in plasma 80 6 13 <1 <1
Location Intra/extra vascular Intravascular Secretions/intravascular B-cell surface Mast cells, basophils, secretions
Structure Monomeric Pentameric Dimeric Monomeric Monomeric
Protein subunits None J J and S None None
Half-life (days) 23 5 6 3 2
Special features Placental passage Primary antibody produced Present in milk and secretions B-cell receptor Mediates atopy

P.818
Lymphatic organs Lymphocyte maturation occurs in the primary lymphoid organs: bone marrow for B-cells, thymus for T-cells. The thymus has an outer cortex with epithelial cells and thymocytes, where T-cells initially mature, and an inner medulla, where T-cell selection occurs in association with DC and macrophages. B-cell maturation occurs in the foetal liver but, after birth, occurs in the bone marrow. Mature lymphocytes are released into the circulation. Differentiation and proliferation occurs in secondary lymphoid organs: the lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT). The spleen contains red pulp and white pulp. Antigens in the blood are concentrated in the spleen and antigen presentation leads to lymphocyte proliferation and differentiation in the white pulp. The germinal centres are B-cell-containing regions, and T-cell-rich areas are peripheral in the white pulp. Lymph nodes receive antigen from peripheral tissues via afferent lymphatics. The cortex contains lymphoid follicles with germinal centres (B-cell-rich) and paracortical (T-cell-rich) areas. The medulla contains sinuses, and plasma cells migrate to the medulla to secrete antibodies. The efferent lymphatics carry antibody and primed T-cells from the lymph nodes. P.819
Immunization Passive (administering immunoglobulin) or active (vaccination). Vaccines may be administered to the whole population or specific groups at high risk of an infection. Types of vaccines include:

  • Toxoids. Produced by inactivation of toxins (e.g. tetanus). Highly immunogenic
  • Polysaccharide. Polysaccharide antigens are T-cell independent and fail to produce immunologic memory. Protein conjugate vaccines link polysaccharide to an immunogenic protein and allow immunologic memory and immune response in children <2 years old
  • Recombinant antigens. Recombinant DNA technology allows the expression of virus surface antigen in yeast cells and its purification for vaccination. Used in many viral vaccines, including hepatitis B
  • Live attenuated organisms. Administration of micro-organisms that express common antigens to the pathogen being immunized against but of attenuated virulence due to laboratory manipulation. Examples include the oral polio vaccine and bacille Calmette—Guérin (BCG) used to vaccinate against Mycobacterium tuberculosis (TB). Live attenuated vaccines should not be used in immunosuppressed individuals in whom disseminated infections may result
  • Whole inactivated micro-organisms. Some vaccines use whole micro-organisms that have been inactivated (e.g. influenza virus vaccine). Many of the older heat-killed vaccines are relatively ineffective and are rarely used (e.g. older cholera vaccines)
  • Newer strategies. These include using virus-carrier vaccines with a harmless virus (such as vaccinia virus or adenovirus) genetically engineered to express a gene encoding the immunizing antigen, synthetic peptide vaccines, or DNA vaccines in which DNA plasmids are injected. These strategies may be combined with DNA vaccination followed by virus carrier or peptide (prime-boost regimens) and are under investigation in infections for which immunization has been unsuccessful (e.g. HIV).

Immunity following vaccination wanes with time and revaccination (a booster) may be required after a set period of time. P.820
Cytokines Small molecular weight proteins that regulate cellular effector functions in the immune system (Table 12.7). Approximately 20 cytokines identified to date. Role in immune homeostasis but also contribute to disease pathology. Some have direct effector functions on micro-organisms (e.g. antiviral effects of IFNα). Cells usually respond to a particular pattern of multiple cytokines synergistically (the observed effect is geater than the sum of the individual effects) or antagonistically. Function is regulated by expression of the cytokine and its receptor. Chemokines are related chemotactic cytokines. They can be grouped into families depending on the arrangement of amino terminal cysteine residues (e.g. CC and CXC chemokines where C is a cysteine residue and X a non-conserved amino acid). IL-8 is a CXC chemokine. Chemokines enhance adherence of leukocytes to the endothelium, transmigration through blood vessels, and activation of the leucocyte in inflamed tissue. Cytokine effects include systemic effects (e.g. fever) and release of acute phase protein reactants including CRP and MBL. Cytokine responses to antimicrobial products (such as bacterial LPS and superantigens) mediate signs of sepsis (fever or low body temperature, increased heart rate, increased respiratory rate, increased or decreased numbers of PMN) and of septic shock (signs of sepsis with a low blood pressure that fails to correct despite fluid resuscitation).

TABLE 12.7 Cytokines and their functions
Cytokine Cell of origin Function
Interleukin-1 IL-1α, IL-1β) M/M, EN, B, o Lymphocyte activation, macrophage stimulation, fever and acute phase response
Interleukin-2 T Activation and proliferation of T-cells, co-factor in B-cell proliferation
Interleukin-4 T, MA, BA Growth factor for B-cells and Th2 T-cells; inhibits Th1 T-cells; stimulates IgG and IgE production
Interleukin-5 T, MA B-cell growth and antibody production; eosinophil differentiation
Interleukin-6 T, M/M, EN, o T-cell activation; B-cell antibody production; haematopoietic progenitor cell growth; acute phase response
Interleukin-8 M/M, EN, EP, o PMN chemoattraction
Interleukin-10 M/M, T, o Inhibits macrophage cytokine production; inhibits Th1 response, stimulates antibody production
Interleukin-12 M/M, B Activates Th1 T-cells and NK cells
Interferon-α M/M, T, o Inhibits RNA viruses; downregulates IL-12; expands memory T-cells; upregulates MHC class I molecules
Interferon-β EP, F Inhibits RNA viruses; upregulates MHC class I molecules
Interferon-γ T, NK Activates macrophages; activates Th1; inhibits Th2 responses
TNF-α M/M, T, NK, o Activation of macrophages, PMN, endothelial cells; co-factor for B-cell and T-cell proliferation; fever and septic shock
Transforming growth factor-β M/M, T, o Downregulates pro-inflammatory cytokines; wound healing
Granulocytemonocytestimulating factor T, M/M, EN, F, o Growth of PMN and macrophages; enhances function
Key: T (T-cell); B (B-cell); M/M (monocyte, macrophage); NK (natural killer cell); EN (endothelial cell); EP (epithelial call); F (fibroblast); MA (mast cell); BA (basophil); o (others).

P.821
P.822
Treatment The immune response to infections can be enhanced or, during transplantation/autoimmune disease, suppressed. Therapeutic interventions include:

  • Active immunization. Used for selected micro-organisms and being investigated in the therapy of other diseases such as cancer
  • Passive immunization. Transfer of specific factors such as immunoglobulin
  • Adoptive transfer. Cells can be transfused into individuals. Examples are transfusion of bone marrow stem cells or PMN to individuals after bone marrow transplantation or CTL, removed from individuals and primed in the laboratory by DC to respond to a specific virus (e.g. Epstein—Barr virus infection)
  • Cytokines. Specific cytokines (IFN-α, TNF-α, IL-2) are used therapeutically to enhance immune responses or may be blocked using inhibitors or soluble receptors (anti-TNF antibodies, soluble IL-2 receptors). Colony-stimulating factors such as GM-CSF or G-CSF may be used in neutropenic hosts
  • Steroids. Used to suppress inflammation (p.826) and lymphocyte activation. May modify inflammation in certain infections such as meningitis or be used to prevent autoimmune diseases or transplant rejection
  • Cytotoxic drugs. Azothioprine is a purine antagonist that blocks RNA and DNA synthesis. Mycophenolate mofetil selectively inhibits purine synthesis in lymphocytes and blocks T- and B-cell proliferation. Cyclophosphamide inhibits DNA metabolism by alkylating DNA. Methotrexate inhibits folic acid-dependent DNA biosynthesis. Both agents are cytotoxic for lymphocytes and used in immunosuppression.
  • Cyclosporine and tacrolimus. Inhibit IL-2 gene transcription by binding to a cytoplasmic receptor and inhibiting calcineurin—a cell signalling phosphatase. Sirolimus, a similar drug, inhibits IL-2 function by blocking IL-2 receptor signal transduction but has effects on the signalling of many other cytokines
  • Antibody therapy and inhibition of co-stimulation. Used in transplantation. Anti-lymphocyte globulin or anti-CD3 antibody (OKT3) inhibit T-cell activation by transplanted antigens. Antibodies to IL-2R e.g. daclizumat may represent a more selective approach to decreasing T-cell activation. Experimental approaches involve using CTLA-4 to bind B7 and block co-stimulation.
  • Miscellaneous therapies. Recombinant host defence molecules are being studied in investigational protocols (e.g. BPI). Abnormalities in clotting cascades are a feature of sepsis in association with certain patterns of cytokine production. Activated protein C is being used to treat certain kinds of bacterial sepsis.

P.823
P.824
Inflammation Overview If the human body was not exposed to infective agents, such as viruses, bacteria, and protozoa, or to the effects of trauma, then inflammation would be an unnecessary process. As it is, inflammation is a vital process which repels infections and initiates repair and regeneration after trauma. This should be borne in mind when studying the process of inflammation, since it is often seen as an entirely adverse process: patients regard inflammation as manifest by abscesses, redness, or pain—as the disease, rather than a reaction to it. Doctors aim many of their treatments at reducing the effects of inflammation. Acute and chronic inflammation Inflammation is traditionally classified into acute and chronic forms which would appear to relate to the time course of each type. This is not the case for all inflammatory processes. For example, infectious mononucleosis (glandular fever) is an acute illness caused by infection with the Epstein—Barr virus but is characterized by a purely ‘chronic’ inflammatory process from its onset. The classification into acute and chronic types does describe the predominant inflammatory cell type present in the process—neutrophils in acute inflammation, and macrophages and lymphocytes in chronic inflammation—which is a useful distinction. However, it might be better to label these types ‘neutrophil-predominant’ and ‘macrophage/lymphocytes-predominant’ rather than acute and chronic. P.825
P.826
Cellular mediators of inflammation Neutrophils (neutrophil polymorphonuclear leucocytes)

  • Short-lived cells, 10–20 hours in blood, which are produced in the bone marrow
  • Can be thought of as the first-wave, front-line rapid deployment defenders who appear rapidly at a site of inflammation and die for the overall ‘good’ of the body
  • Very effective killers of bacteria
  • Cell surface receptors for C3b component of complement, Fc portion of antibody tails—these facilitate phagocytosis of micro-organisms
  • Contain vacuoles with lysosomal enzymes and oxidating protein complexes which kill ingested bacteria
  • Release some chemical mediators of inflammation.

Eosinophils

  • Can be thought of as an analogue of neutrophils but with design favouring the killing of multicellular parasites, such as enteric worms, rather than bacteria
  • Longer-lived than neutrophils— 4 days in blood, weeks in tissue
  • Found in relatively large numbers in certain tissues, especially the lamina propria in the gastrointestinal system, even in the absence of disease
  • Cytoplasmic granules containing cationic proteins which can kill worms (and other cells) by binding to the cell surface
  • Have an apparently misdirected action in asthma where they accumulate in the bronchial mucosa and the cationic proteins damage the ciliated epithelium.

Mast cells

  • Have cytoplasmic granules containing sulphated glycosaminoglycan to which chemical mediators of inflammation, including histamine, are reversibly bound
  • Main reaction is degranulation which involves release of granule contents very rapidly into surrounding tissue
  • Key components of type I hypersensitivity (p.838).

Macrophages

  • Also known as monocytes when in the blood
  • Long-lived cells; months in tissues
  • Found in second wave of cells in acute inflammation, after neutrophils
  • Have phagocytic functions and vacuoles with digestive and free radical generating systems; less effective at killing common bacteria than neutrophils but more effective at killing atypical bacteria such as Mycobacteria
  • P.827

  • Some specialist macrophages e.g. dendritic macrophages in skin, present antigens to T cells
  • Activated macrophages produce many chemical mediators of inflammation and immune response including lymphokines, tumour necrosis factor
  • Can fuse together to form multinucleated giant cells, often in response to material that is too large for a single cell to phagocytose e.g. exogenous material such as silica particles
  • When activated, may take on an epithelioid appearance with abundant eosinophilic cytoplasm. A cluster of epithelioid macrophages surrounded by a rim of lymphocytes is called a granuloma and is characteristic of certain causes of inflammation, especially infective mycobacteria such as tuberculosis and leprosy.

P.828
Chemical mediators of inflammation There are a vast number of chemical mediators of inflammation, as the list below indicates, and the actions of these, and the cellular responses to them, regulate the inflammatory response. Because there are so many factors, it is very difficult to dissect out the role of a single mediator in any particular inflammatory situation. However, many therapeutic agents have been developed against specific mediators, so it is important to know something about their actions so that response to these agents can be predicted. With the range of mediators described, it is easy to lose sight of the basic functions of them:

  • To bring other cellular mediators of inflammation to the site in an appropriate number and at an appropriate time
  • To increase blood flow and blood vessel permeability so that cellular mediators can get to the site of inflammation and so material can be carried away.

Complement system Is a cascade system of proteins which may be activated in a number of different ways in acute inflammation:

  • By the Fc portion of antibodies which have combined with specific antigen—the classical pathway of activation
  • By the endotoxins of Gram-negative bacteria—the alternative pathway
  • By enzymes releases by dying cells in tissue necrosis
  • By some products of the kinin and fibrinolytic systems.

Once the complement system has been activated, it produces a number of proteins which are active in inflammation:

  • C3a and C5a—chemotactic for neutrophils, increases vascular permeability, causes release of histamine from mast cells
  • C567 complex—chemotactic for neutrophils
  • C56789 complex—the membrane-attack complex which punches holes in cell membranes leading to cell lysis
  • C4b, C2a, C3b—all opsonize bacteria by binding to them; macrophages have specific receptors for these proteins.

Vasoactive amines Histamine—causes vasodilatation and increased permeability of blood vessels. Nitric oxide Potent vasodilator, inhibitor of platelet and monocyte adhesion and, at high concentrations, a powerful cytoxic agent. Kinin system products Bradykinin—vasoactive and pain-stimulating functions. P.829
Clotting system products Factor XII (Hageman factor)—activates the coagulation, fibrinolytic, and kinin systems. Arachidonic acid metabolites

  • Prostaglandins—potentiate increased vascular permeability, inhibit or stimulate platelet aggregation
  • Leukotrienes—vasoactive properties.

Cytokines Interleukins. P.830
Patterns of inflammation Acute neutrophilic This is the prototypic pattern of acute inflammation which may be found in an abscess of a localized bacterial infection. It is characterized by masses of neutrophils which phagocytose and kill the bacteria. Many of these neutrophils will die during this process producing an amorphous mass of lysed nuclei and cytoplasm. Acute progressing to chronic This occurs in the later stages of neutrophil-rich inflammation, such as the abscess described immediately above. Macrophages arrive in great numbers and phagocytose the debris. Some fibroblasts may appear and produce fibrous scar tissue to ablate the abscess cavity. Lymphocytes and plasma cells will be less common, unless there is a persisting infection. Eosinophilic Acute inflammation in which eosinophils, rather than neutrophils, predominate occurs with parasitic, rather than bacterial, infections. Chronic granulomatous Granulomas occur in special types of chronic inflammation and this may give clues as to the cause of the inflammation:

  • Mycobacterial infections
    • Tuberculosis
    • Leprosy
  • Other specific infections
    • Syphilis
    • Invasive parasitic infections
  • Idiopathic inflammatory diseases
    • Sarcoidosis
    • Crohn’s disease
    • Wegener’s granulomatosis
  • Hepatic reaction to some drugs
    • Allopurinol
    • Phenylbutazone
    • Sulphonamides.

P.831
Systemic effects of inflammation Raised temperature Any substantial focus of acute inflammation causes a pyrexia due to the release of endogenous pyrogens (e.g. interleukin-2) from inflammatory cells which set the thermoregulatory area of the hypothalamus to a higher temperature. Cachexia There is often a negative nitrogen balance in substantial chronic inflammation with considerable weight loss, hence the historical term for tuberculosis—‘consumption’. P.832
Treatments The wide range of mediators involved in the inflammatory response represent the various targets of anti-inflammatory therapy. However, the highly complex and interactive nature of the inflammatory response limits the effectiveness of highly specific drugs that only target isolated inflammatory pathways. Steroidal anti-inflammatory drugs Glucocorticoids have an anti-inflammatory effect, primarily through inhibition of transcription of the gene for IL-2 required for cloning of Th2 cells crucial to the inflammatory response. In addition, they inhibit transcription of other inflammatory cytokines, including TNF-α, IL-1, and IFN-γ, as well as the expression of inducible inflammatory enzymes, like phospholipase A2 (and, consequently, PAF), COX-2, and inducible NO synthase. The broad impact that glucocorticoids have on different inflammatory pathways is instrumental in their effectiveness. Non-steroidal anti-inflammatory drugs Perhaps the best-known anti-inflammatories are the non-steroidal anti-inflammatory drugs (NSAIDs), most of which do not require a prescription. These drugs are usually taken to reduce the effects of minor aches or pains and for headaches, but they are also sometimes recommended for rheumatic pain. NSAIDs act by irreversibly inhibiting COX enzymes (COX-1 and COX-2), leading to reduced prostanoid synthesis. This inhibition has a number of anti-inflammatory effects, including:

  • Reduced vasodilatation in response to protaglandins PGE2 and PGI2 (prostacyclin). As a result, there is less oedema and swelling. This is probably the main effect in headache
  • Reduced sensitization of nociceptive nerve endings to 5-HT and bradykinin
  • Reduced fever.

Aspirin is the best-characterized NSAID and its action is known to be primarily mediated by acetylation of a specific serine residue in the COX enzyme to irreversibly block its action. Ibuprofen binds to a different site in the enzyme, to the same effect. Some of the actions of paracetamol might also be mediated via inhibition of COX, but it is not generally recognized to be an anti-inflammatory drug. The major side-effect of NSAIDs is gastric bleeding due to a loss of the protective effects of prostaglandins in the stomach, resulting in increased gastric secretion and reduced gastric blood flow. P.833
Antihistamines Calcium-mediated release of histamine from mast cells is partly responsible for the first two elements of the so-called ‘triple response’ to a mild insult to the skin:

  • The reddening due to arteriolar vasodilatation
  • The wheal due to increased permeability of venules.

(The ‘flare’ in surrounding tissue is due to release of vasodilators (e.g. CGRP) from nerve endings of sensory nerves in the vicinity of the insult.) Histamine also stimulates sensory nerve endings to cause itching and, at a systemic level, stimulates bronchoconstriction associated with the immediate phase of asthma (p.396). These actions of histamine are mediated via stimulation of H1 histamine receptors, and antagonists of this specific subclass of receptors are commonly referred to as antihistamines (not to be confused with H2 receptor antagonists that are used to treat and prevent peptic ulcers). Although H1 receptor antagonists should theoretically have an antiinflammatory effect, through their inhibition of the inflammatory responses outlined above, in reality, their use is limited to treatment of allergy (rhinitis), insect bites, and drug hypersensitivities. Many of the H1 receptor antagonists characterized to date have non-specific effects, through inhibition of muscarinic, 5-HT, and α-adrenoceptors. These nonspecific actions contribute to the drowsiness caused by some antihistamines and mediate some of the effects of antihistamines that are prescribed for motion sickness or even sedation. Bradykinin Bradykininin is an endothelium-dependent vasodilator that acts to relax vascular smooth muscle, primarily through G-protein-coupled B2 receptors on endothelial cells, stimulating the release of PGI2 and NO. However, it can also cause spasm in the bronchial tree and is associated with many of the symptoms experienced in allergic reactions (including vasodilatation, increased vascular permeability, and pain). Icatabant is a peptide antagonist for B2 receptors, but is not used therapeutically. A number of non-peptide antagonists are under investigation, which may be more suitable as orally active agents for use in allergy. P.834
Repair Cell involvement: regulation An infection causes some damage to tissues in the body; the amount of damage and the type of tissue determine what happens after the infection has been eradicated:

  • If the damage does not cause the death of cells, then the cells will recover and the tissue will return to normal
  • If there is death of cells in a tissue that is capable of regeneration (e.g. the liver), then the regenerative cells will divide to replace those that died and, after some remodelling, the tissue will return to normal
  • If there is death of cells in a tissue that is incapable of regeneration, then the tissue will heal by repair, which will lead to formation of scar tissue and some loss of function.

Cell involvement Fibroblasts

  • Primary cell involved in repair
  • Recruited by multiple growth factors (e.g. platelet derived growth factor, interleukin-1) from platelets, endothelial cells, macrophages, and neutrophils
  • Produce collagen and fibronectin to constitute fibrous connective tissue which changes with age to relatively acellular bands of collagen.

Macrophages and neutrophils

  • Present from the initiating infection
  • Not directly involved in repair but secrete chemical mediators which attract fibroblasts
  • Macrophages may continue with debris-removing activities.

Endothelial cells

  • Present in granulation tissue at the start of the repair process
  • Growth is stimulated by angiogenic factors such as vascular endothelial growth factor.

Regulation Repair, like many processes in the body that occur in response to injury, is a double-edged sword:

  • Production of fibrous tissue to replace cells that have died allows retention of the structural integrity of the organ (e.g. a fibrous scar at the site of a myocardial infarct prevents the heart wall from rupturing at that point)
  • Production of fibrous tissue can cause loss of function in the tissue surrounding it (e.g. fibrosis in the lungs can cause physical constriction of blood vessels leading to pulmonary hypertension and right-sided heart failure)
  • Overproduction of fibrous tissue can cause a much larger fibrous scar than was necessary to repair the immediate damage with distortion and loss of function in the surrounding tissue (e.g. a keloid scar on the skin).

P.835
Regulatory processes

  • Positive factors for formation of fibrous tissue
    • Production of growth factors that recruit fibroblasts
    • Production of factors that increase vascular permeability (e.g. vascular endothelial growth factor) and so lead to increased deposition of plasma proteins such as fibrinogen and fibronectin
  • Factors which inhibit formation of fibrous tissue
    • Matrix metalloproteinases which break down proteins in the extracellular matrix, such as collagen, laminin, and fibronectin.

P.836
Examples of repair and its consequences Lung—after slowly resolving bacterial pneumonia (p.399):

  • If the elastic walls of the alveoli are damaged, then the lung cannot regenerate
  • Fibrin and inflammatory cells fill the alveoli
  • Fibroblasts are recruited
  • Fibroblasts synthesize collagen
  • Fibrin and inflammatory debris is removed by macrophages
  • Final state = fibrous scarring in lung with reduced expansion of lungs (thus reduced vital capacity on respiratory function tests) and reduced area for gas exchange (so reduced VO2max).

Liver—after years of hepatitis C infection:

  • Liver cells have the ability to regenerate so a single episode of damage does not usually intiate the repair process, however continuing damage whilst the cells are attempting to regenerate does lead to repair
  • Fibrous tissue forms between liver cells
  • Bands of fibrous tissue form between portal tracts with intervening regenerative nodules (cirrhosis)
  • The fibrous tissue obstructs flow through the portal venous system leading to portal hypertension
  • Replacement of most hepatic tissue by fibrous tissue leads to liver failure.

Skin—after recurrent bacterial abscesses:

  • Skin is relatively resistant to damage and will heal without much repair
  • However, an abscess destroys many cells and leaves a cavity
  • The cavity is lined by a wall of fibrous tissue after repair
  • The fibrous tissue may contract to obliterate the cavity which will just leave a scar
  • If the cavity is not obliterated, then recurrent bacterial infections will occur, leading to greater scarring (e.g. hidradenitis suppurativa = recurrent bacterial infections in the apocrine skin glands in the axilla and groin).

P.837
P.838
Immunopathology Hypersensitivity Overview The body has a number of immune systems which function to prevent or limit infections from viral, bacterial, or parasitic organisms. These systems may produce some adverse effects within the body, but this is more than balanced by the prevention of damage that would occur if the infective organism established an infection within the body. However, sometimes these immune systems react against objects that are not infective organisms (e.g. pollen grains in hayfever) and then the damage caused by the immune system itself is disadvantageous and inappropriate. This is termed a hypersensitivity reaction. It should always be borne in mind that hypersensitivity reactions are caused by systems that normally prevent infections, since any therapy that is used to reduce the effects of a hypersensitivity reaction may lead to a vulnerability to infection. Hypersensitivity reactions are classified into different types according to the specific system which is mediating the reaction. Type I hypersensitivity (Fig. 12.7) (OHCM6 p.780) This is mediated by mast cells and is typified by hayfever. Mast cells have IgE antibodies on their surface which are directed against a specific antigen, particular types of pollen in the case of hayfever. When an antigen binds to the antigen-specific site on these antibodies, a signal is transmitted through the mast cell which causes it to degranulate, releasing the contents of granules in its cytoplasm into the immediate surrounding environment. These granules contain many chemical mediators of acute inflammation, such as histamine, which lead to vasodilation and oedema in the surrounding tissues—manifest as nasal obstruction and watery secretion in hayfever. Severe type-I hypersensitivity reactions manifest as anaphylaxis (OHCM6 p.780). There are a number of steps in this pathway which can be blocked to prevent this hypersensitivity reaction:

  • Affected individuals can avoid the precipitating antigen (but this can severely limit their lifestyle in the case of ubiquitous environmental antigens such as pollen)
  • The number of mast cells in the affected organ can be reduced by long-term administration of immunosuppressants such as steroids
  • The cell membrane of the mast cells can be stabilized, so it is less likely to degranulate, by administration of disodium chromoglycate
  • If all these fail, then the effects of the chemical mediators of acute inflammation can be blocked using drugs such as antihistamines.

Type II hypersensitivity (Figs. 12.8, 12.9) This is mediated by specific antibodies directed against and by both cells and activation of the complement system. An example is the breakdown of red blood cells in a Rhesus blood antigen-positive foetus of a mother who is Rhesus antigen negative and has been exposed to Rhesus antigen positive blood in the past (usually by a previous pregnancy) (p.405). The maternal antibodies which are directed against the Rhesus antigen cross P.839
P.840
the placenta into the foetal circulation, where they bind to the Rhesus antigens on the surface of the foetal red blood cells. The binding of the antibodies to the antigen causes a conformational change in the structure of the antibody tail region which then binds the first component (Clq) of the complement protein cascade. This leads to production of activated complement components (e.g. C3b) which form a ‘membrane attack complex’ which attaches to the membrane of the red blood cells, forming a hole in the membrane which causes cell lysis by uncontrolled influx of water into the cell. This process leads to a haemolytic anaemia in the foetus which may be so severe that it requires intrauterine blood transfusions. In this instance, the best method of treatment is prevention by ensuring that all Rhesus negative mothers receive injections of antibodies directed against the Rhesus antigen during pregnancy and immediately after birth. Thus, any foetal red blood cells that do get into the maternal circulation are immediately lysed by these antibodies before the maternal immune system has time to react against them.

Fig. 12.7 Type I hypersensitivity. The binding of the antigen to IgE molecules on the surface of mast cells leads to degranulation of those cells with relase of chemical mediators of acute inflammation.
Fig. 12.8 Type II hypersensitivity. Mediated by macrophages binding to specific antibodies which have bound to antigens on a host cell. The macrophage could phagocytose the antigen-covered cell or a natural killer cell could induce its death.
Fig. 12.9 Type II hypersensitivity. Mediated by complement which binds to the tails of the specific antibody and either leads to formation of the membrane attack complex and lysis of the cell or attraction of neutrophils by the C3b component.

Type II hypersensitivity can also be mediated by cells, such as macrophages or natural killer cells, which bind to the tails of antibodies that have bound antigen and then phagocytose the cell or release factors which kill it. Type III hypersensitivity (Fig. 12.10) If the ratio of antigen to antibody occurs within a certain range, then the antigens will crosslink antibodies to form large complexes which will lodge in membranous filtration systems in the body (e.g. the glomerulus or synovial membrane). Components of the complement system will bind to the antibody tails and damage will occur from the activated complement components themselves and from the inflammatory cells which they attract. Examples of immune complex disease include the glomerulonephritis, arthritis, and endocarditis that occur in rheumatic fever after a Streptococcal throat infection. Type IV hypersensitivity (Fig. 12.11) This is mediated by cells rather than antibodies. A common example is nickel allergy which may occur from exposure to this metal in wrist watches or metal components of clothing. Nickel, by itself, is too small to induce an immune response (hypersensitivity would ensure extinction of the species if reactions were elicited to pure elements), but if it binds to proteins in the body, such as keratin in the skin, then it can produce an allergenic complex. The antigenic complex is presented to T-cells by specific types of macrophages. The T-cells which are specifically reactive with the antigen divide to produce an expanded clone of cells. When the antigen is encountered again, it is recognized by these T-cells (again, usually when presented on the surface of specific types of macrophages) and they secrete a number of cytokines, including interleukin 12 (IL-12) and interferon gamma (IFN-γ), which produce an inflammatory reaction. These cytokines cause an accumulation of activated macrophages at the site of inflammation. Neutrophils are not usually involved in this type of hypersensitivity reaction.

Fig. 12.10 Type III hypersensitivity. Large complexes of antigen and antibody lodge in membranous filtration systems in the body (e.g. the glomerulus in the kidney) where they activate the complement cascade.
Fig. 12.11 Type IV hypersensitivity. Cell-mediated hypersensitivity caused by sensitized T-cells releasing cytokines after binding to the antigen.

P.841
P.842
Autoimmunity General principles An autoimmune reaction occurs when the body mounts an immune reaction to some intrinsic component within it (e.g. thyroid epithelial cells) rather than against some extrinsic object (e.g. bacteria, pollen, nickel). There are many regulatory processes in the body which are designed to prevent autoimmune reactions but there are some mechanisms which can breach these:

  • Exposure of a ‘hidden’ antigen to the immune system
    • Some tissues in the body are not patrolled by cells from the immune system
    • Some tissues in the body are in sealed compartments which are not exposed to cells from the immune system (e.g. proteins within myocardial cells)
    • If a tissue is damaged and proteins that immune cells have not been exposed to during the period of immune tolerance in utero and early childhood are released, then an immune reaction will be mounted against them (e.g. Dressler’s syndrome (OHCM6 p.722) following myocardial infarction)
  • Cross-reaction between an extrinsic and intrinsic antigen
    • Some infective organisms possess antigens that stimulate an immune reaction, but these antigens are similar to antigens in intrinsic body components so the antibodies or activated lymphocytes that are formed against the infective organism cross-react with these intrinsic components to produce an autoimmune reaction
    • An example is rheumatic fever (OHCM6 p.144), where the immune reaction directed against the cell wall of Streptococcal bacteria cross-reacts with heart valve tissue.

Autoimmune reactions may be mediated by any of the body’s immunologic mechanisms (p.838). Autoimmune disease is often classified into:

  • Single organ—where a single area in the body is affected, presumably due to the antigen only being present in that organ. Examples include:
    • Hashimoto’s thyroiditis (OHCM6 p.306)—where the epithelial cells in the thyroid are destroyed by activated lymphocytes leading to hypothyroidism, when there are insufficient numbers of cells left to produce normal amounts of thyroxine
    • Grave’s disease (OHCM6 p.304)—where, unusually, a stimulatory auto-antibody is produced which binds to the thyroid-stimulating hormone receptor on thyroid epithelial cells, causing excess production of thyroxine and, thus, hyperthyroidism
  • Systemic—where the whole body is affected due to widespread presence of the antigen. Examples include:
    • Systemic lupus erythematosus (OHCM6 p.422)—where there are many auto-antibodies present which are mainly directed against targets in the cell nucleus. The skin, joints, and kidney are the most commonly affected sites
    • Sjögren’s syndrome (OHCM6 p.734)—with autoimmunity directed against the salivary and lacrimal glands, with activated T-lymphocytes the effector cells.

P.843
Transplantation Transplantation of organs is now a common treatment (at least as far as the supply of donor organs allows) for failure of individual organs such as kidney, liver, heart, and lungs. The major difficulty with transplantation is preventing the new host from rejecting the organ by an immune process. Every individual has a large number of different antigens expressed on their cells which are highly variable. These include blood group antigens and the HLA (major histocompatibility loci antigens). The only organs that would not be rejected without immunosuppression would be those in a site in the body that does not have immune surveillance (e.g. the cornea) or those from an identical twin. For all other transplants, a number of methods of preventing rejection have to be employed:

  • Matching of donor and recipient
    • Match blood group type
    • Match HLA types as far as is possible
  • Immunosuppressive therapy
    • Corticosteroids
    • Azothioprine
    • Ciclosporin
    • Anti-lymphocyte antibodies
  • Monitoring of graft function and rejection
    • Functional measurements (e.g. glomerular filtration rate in kidneys)
    • Biopsy and histological examination
    • Adjustment of immunosuppressive therapy on the basis of these results.

Transplantation of bone marrow (commonly used to increase the dosage of chemotherapy that can be given in the treatment of malignancy) is an interesting reversal of the usual problems of transplant rejection since, in this case, it is the transplanted cells that are the immune system and can thus mount an immune attack on the host—graft versus host disease. P.844
Immunodeficiency (Primary/Secondary to Other Diseases) Deficiency of humoral immunity Most common inherited immunodeficiency. Results in frequent infections— otitis media, sinusitis, pneumonia due to Streptococcus pneumoniae or Haemophilus influenzae, and recurrent gastroenteristis. Treated by immunoglobulin infusion.

  • X-linked agammaglobulinaemia
    • Mutation in a B-cell cytoplasmic tyrosine kinase; maturation failure
    • Absence or profound decrease in B-cells and antibody
  • Common variable immunodeficiency
    • Not hereditary and exact mechanism unclear
    • B-cell maturation abnormal and numbers normal or low
    • IgA and IgG (±IgM) low
    • May not present until third decade
    • Autoimmunity and cancers associated
  • Transient hypogammaglobulinaemia
    • Low levels of IgG after 6 months of age but normal B-cell numbers
    • Self-resolving
  • Selective immunoglobulin deficiency
    • Normal B-cell numbers
    • IgA deficiency most common
    • Autoimmune manifestations
    • If associated IgG2 deficiency, frequent infections
  • Hyper-IgM syndrome
    • Majority X-linked (XL)
    • Lack CD40 ligand
    • IgM high; IgG, IgA, and IgE low
    • B-cell numbers normal
  • Secondary defects in humoral immunity: occur with malignancy (lymphoreticular malignancies e.g. multiple myeloma OHCM6 p.666), protein malnutrition, splenectomy (OHCM6 p.671), sickle cell disease (OHCM6 p.640), bone marrow transplantation (OHCM6 p.658), and AIDS (OHCM6 p.578) (especially in children).

P.845
Deficiency of cell-mediated immunity T-cell defects. Susceptible to opportunistic infections: cytomegalovirus (CMV), Candida sp., Pneumocystis jirovecii (carinii) pneumonia (PCP), mycobacterial infections, and Toxoplasma gondii.

  • Congenital thymic aplasia (DiGeorge syndrome)
    • Autosomal dominant (AD)
    • Thymic aplasia
    • Absent or few mature T-cells
    • B-cell numbers normal but defective responses to T-cell-dependent antigens
    • Absence of hypoparathyroid glands causes low serum calcium
  • Functional T-cell defects: normal T-cell numbers but mutations involving the TCR complex or ZAP 70 tyrosine kinase
  • Chronic mucocutaneous candidiasis
    • Selective defect involving response to Candida sp.
    • Chronic skin and mucocutaneous candidiasis only manifestation
  • Secondary defects in T-cell function
    • HIV (OHCM6 p.578) infection most common acquired cause
    • Also, transplantation, immunosuppressant drugs, malignancies (e.g. Hodgkin’s lymphoma OHCM6 p.658)
    • Pregnancy and advanced age decrease T-cell function.

P.846
Combined humoral and cell-mediated deficiency Severe combined immunodeficiency disease (SCID)

  • Most die within 2 years unless receive successful bone marrow transplantation
  • Heterogenous disorders
  • X-linked SCID
    • Associated with mutations in the γ-chain of the IL-2 receptor
    • T-cells low, B-cells present but can’t produce antibody
  • Other forms AR
    • SCID associated with mutation in JAK3 tyrosine kinase, similar to X-linked form
  • Adenosine deaminase deficiency (ADA)
    • Leads to a defect in purine metabolism of lymphocytes
    • Low T- and B-cells
    • Treatment options include gene therapy
  • Recombinase deficiency
    • Mutations in the RAG genes
    • Absent T- and B-cells

Bare lymphocyte syndrome • T- and B-cell numbers normal but antigen-presenting cells lack MHC class II molecules, so fail to present antigen. Wiskott—Aldrich syndrome

  • XL mutations in a specific protein cause combined immunodeficiency
  • Normal T- and B-cell numbers
  • Functional defects; low IgM and IgG, and high IgA and IgE levels
  • Low platelets; allergic reactions, including severe eczema; and increased incidence of cancer.

Ataxia—telangiectasia

  • AR defect in a tyrosine kinase required for DNA repair
  • Neurologic (unsteady gait) and ocular features associated with lymphopenia and low IgA and IgE (± low IgG)
  • Increased malignancies.

P.847
Defects in phagocyte function Susceptibility to pyogenic bacteria and fungal infections. Chronic granulomatous disease (CGD)

  • XL and AR defects in components of NADPH oxidase which catalyses: NADPH +2O2 → NADP+ + 2•O2- + H+
  • Generation of 2•O2- (superoxide) is defective
  • Infections with catalase-positive organisms; Staphylococcus aureus and the fungus Aspergillus sp. most frequent
  • Recurrent pulmonary infections and abscesses of skin, bone, and liver
  • Obstructive complications can involve gut or genito-urinary tracts due to formation of granulomatous lesions
  • Diagnosis by detecting normal PMN numbers but decreased superoxide production (nitroblue tetrazolium test or measurement of dihydrorhodamine 123 fluorescence by flow cytometry)
  • Treatment of infections prophylatic antibiotics and IFN-γ injections.

Leukocyte adhesion deficiency

  • AR defects in leukocyte adherence of variable severity
  • Most common form due to failure to make certain integrins that share a common polypeptide
  • High PMN numbers but no abscesses and defective complement-dependent phagocytosis (lack CR3)
  • Diagnosis: absence of specific integrins on leukocytes
  • Recurrent respiratory, gastrointestinal, and skin infections due to Staphylococcus aureus and gram-negative bacteria.

Myeloperoxidase deficiency

  • AR—most common defect of PMN, but usually clinically silent
  • Fail to convert hydrogen peroxide to the more potent microbial molecule, hypochlorous acid.

Neutropenia (OHCM6 p.662)

  • Congenital forms rare
  • Cyclic neutropenia characterized by decreased counts and infections every 3 weeks
  • Acquired due to chemotherapy or, occasionally, other drugs
  • Bacterial and fungal infections
  • Granulocyte colony stimulating factor (G-CSF) used to boost counts
  • Prophylactic antimicrobials when pyrexial (OHCM6 p.650) (neutropenic regimen).

Chédak—Higashi syndrome

  • AR condition characterized by large lysosomes in PMN
  • Recurrent bacterial infections and ophthalmologic and neurologic complications.

Job’s syndrome

  • AD, recurrent infections, and severe eczema
  • High levels of IgE and chemotactic defects.

P.848
Other defects Mononuclear cell defects

  • Defective production of IFN-γ/IL-12 or receptor mutations effect monocyte/macrophage function
  • Infections include atypical mycobacterial infections such as Mycobacterium avium complex and Salmonella sp.
  • Treatment includes IFN-γ

Anti-TNG-α theraphy • Mycobacterial infections or other infections with intracellular pathogens. TNF-α receptor-associated periodic syndromes (TRAPS)

  • Gain-of-function mutations in TNF-α receptor
  • Recurrent fever in absence of infections.

Complement deficiency

  • Most are AR defects in individual complement components
  • Deficiency of early classic or alternative pathway components leads to recurrent bacterial pneumonia and autoimmune conditions (e.g. systemic lupus erythematosis (SLE OHCM6 p.422))
  • Late complement deficiencies associated with recurrent and atypical Neisseria sp. infections, including meningitis
  • Diagnosed by complement component assays
  • MBL deficiency associated with recurrent respiratory and gastrointestinal infections.

Splenic dysfunction

  • Usually acquired; occasionally congenital
  • Sickle cell disease leads to autosplenectomy
  • Increased frequency of infections with encapsulated bacteria; Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitides, and parasites; Babesia microti, Plasmodium sp.
  • Immunization against encapsulated bacteria essential.

Leave a Reply


Time limit is exhausted. Please reload the CAPTCHA.

Categories

apply_now Pepperstone Group Limited