Defence Mechanismsã The Trustee of the Wellcome Trust 1998Reviewed by: Dr M S Murphy, Institute of Child Health, The University of Birmingham,Professor G Dougan, Department of Biochemistry, Imperial College of Science and Technology, London andDr W A M Cutting, Department of Child Life and Health, The University of Edinburgh, UK Picture: Invasion of a cultured epithelial cell by Shigella. Zambia Copyright Image from Sansonetti PJ.Image references ################ .\IMAGES\T45958.jpg Contents Click on the underlined text to jump tothat screen. Screen3Objectives 4Introduction 5 Overview of Defence Mechanisms 9Non-immunological Mechanisms 12 Assessment 13Humoral Immune Responses 25 Assessments 27Cell-mediated Immune Responses 30Innate Immune Responses 36 Assessment 37Mucosal Vaccines 41 Assessment 42Development of Defence Mechanisms 46Assessment47 Tutorial Assessment48Summary Underlined text is interactive. Click on underlined text to view extra information or to jump to another screen. Picture: Particles of rotavirus (EM). Vaccines against rotaviral diarrhoea are currently under development.Copyright Copyright Image from Centers for Disease Control and Prevention.Image references ################ .\IMAGES\T33755c.jpg ObjectivesAt the end of this tutorial you should be able to: 1. describe the roles of the bowel in balancing defence and tolerance 2. summarize non-immunological protective factors in the gastrointestinal tract 3. review innate, humoral and cell-mediated immune responses to enteric pathogens and how they are regulated 4. outline the rationale behind the development of mucosal vaccines against diarrhoea 5. describe the development of gastrointestinal defence mechanisms in the breast fed infant Image references ################ Introduction Diarrhoea Diarrhoea is: · an increase in the number, volume and water content of stools · a global cause of much illness and death· a major factor in childhood malnutrition This tutorial is about how the body defends itself against the pathogens that cause diarrhoea. Picture: The epithelium overlying Peyer’s patch in the mouse small bowel showing M cells (M) and enterocytes (E). The brush border of microvilli on the enterocytes can be seen. Both types of cell are central to gastrointestinal immunology. E M Copyright Image from McI Mowat A. Diarrhoea morbidity and mortalityEach year there are: · approximately 3.3 million deaths due to diarrhoea, 80% in children under 2 years of age · over 1 billion episodes of diarrhoea, most in the developing world · 5 - 10 million travellers affected by diarrhoea Image references ################ .\IMAGES\T45475.jpg Overview of Defence MechanismsOverview of Defence Mechanisms Image references ################ Overview of Defence Mechanisms - 1 The GI tract in health To maintain health the gastrointestinal (GI) tract has to: · defend the body against ingested pathogens and their toxins · tolerate a massive antigenic load from: - commensal bacteria- ingested food, ie. ‘oral tolerance’ (seepicture) Maintaining a balance between defence and tolerance requires tight regulation. Picture: The result of intolerance to dietary antigens. This is a jejunal biopsy from an adult with coeliac disease, which is an immunologically mediated reaction to dietary gluten, a protein in wheat. Note the subtotal villous atrophy.Copyright Image from The Wellcome Trust courtesy of Association of Clinical Pathologists. Commensal bacteriaThese are over 400 species of mostly anaerobic bacteria that: · live in the healthy GI tract as the normal bowel flora · ordinarily do not cause disease· often perform useful metabolic functionsMaintaining a balanceA shift in the balance towards tolerance might cause: · persistence of enteric pathogens in the bowel · small bowel overgrowth by commensal bacteria · reduced villus architecture A shift in the balance towards defence might cause: · food allergies, eg. coeliac disease · inflammatory bowel disease, which probably has an immunological pathology Diarrhoea is a common clinical feature in all these conditions. Image references ################ .\IMAGES\T14146.jpg Overview of Defence Mechanisms - 2Intestinal defence mechanisms Non-immunological mechanisms · Protective factors form a ‘first line of defence’. Immunological mechanisms (see table) · Innate immunity, including inflammatory reactions in the mucosa.· Humoral immunity - the major mechanism of defence. · Cell-mediated immunity. Where are these immuneresponses generated?Adaptive versus Innate Immunity Innate immuneAdaptive immune system system Key solubleComplement, Antibodyfactors acute phase(humoral proteins immunity) Effector cells Neutrophils,Cytotoxic T macrophages, lymphocytes natural killer(cell-mediated (NK) cells immunity) Specificity Low High Memory1 No Yes 1. Immunological ‘memory’ is characterized by a much greater immunological response on repeat exposure to an antigen.Protective factorsThese non-specific chemical and physical factors include: · mucus· epithelial cell turnover · peristalsis · gastric acid · bile · proteolytic enzymes · normal bowel flora See screens 10 - 11 for a more complete list and further details. Humoral and cell-mediated immunityThese immunological mechanisms developed later in vertebrate evolution than the innate mechanisms. They are:· highly specific, due to binding between: - antibody and antigen in humoral immunity - T cell receptor and antigen in cell-mediated immunity · adaptive - a stronger response develops after repeat exposure to the antigen (immunological ‘memory’)Humoral and cell-mediated immunityThese immunological mechanisms developed later in vertebrate evolution than the innate mechanisms. They are:· highly specific, due to binding between: - antibody and antigen in humoral immunity - T cell receptor and antigen in cell-mediated immunity · adaptive - a stronger response develops after repeat exposure to the antigen (immunological ‘memory’)Innate immunityInnate immunity includes such mechanisms as: · killing of pathogens by the complement pathway · engulfing of pathogens by phagocytic cells, eg. macrophages and polymorphonuclear neutrophils (PMNs) Innate immunity is the ‘primitive’ part of the immune system. It is not: · inherently specific against particular pathogens · adaptive - repeat exposure to the antigen does not generate a stronger response Generation of immune responsesPicture: Structure of the villus in the small bowel showing the epithelium (E) and lamina propria (LP). Image from The Wellcome Trust. E LP The immunological responses are produced by an interaction between the: · mucosal immune system in the intestinal lamina propria and epithelium · systemic immune system in the circulating blood Image references ################ .\IMAGES\Gvillus3.jpg Overview of Defence Mechanisms - 3V.cholerae O1 Immune responses to pathogens vary The nature of the immune response is determined in part by the pathogen. Important characteristics of the pathogen include: · surface antigenic structure· intracellular or extracellular location in the host · mechanism of pathogenicity (see picture) What else determines the immune response?Rotavirus Increasing degree of invasion Shigella Salmonella Picture: Structure of the small bowel showing the extent of invasion by four different pathogens. Shigella does infect the distal ileum but more typically infects the colon.Copyright Image from The Wellcome Trust. Surface antigensThe surface of a microbe can be its first point of contact with the host immune system. The diversity of surface antigens varies greatly between pathogens such as: · viruses, eg. rotavirus Diversity of· bacteria, eg. Vibrio cholerae O1 surface antigens · parasites, eg. Cryptosporidium increasing.Intracellular or extracellular locationThe immune responses to: · extracellular pathogens are generally mediated by antibody · intracellular pathogens such as viruses are generally cell-mediated This is because intracellular pathogens are usually inaccessible to antibodies. Note that the immune response to most pathogens is a combination of humoral and cell-mediated immunity.Mechanism of pathogenicityThe immune response to non-invasive pathogens that adhere to the epithelium and secrete toxins (eg. Vibrio cholerae O1): · is primarily an antibody response · has evolved to stop the pathogen adhering to the epithelium and neutralize the toxin The immune response to invasive pathogens that cross the epithelium and enter the mucosa (eg. Shigella): · involves both an inflammatory innate response and an antibody response · has evolved to kill the pathogen and degrade any toxic products What else determines the immune response?The other major determinant of the immune response is the immunological status of the host. This depends on factors such as: · previous exposure to the pathogen (acquired immunity) · malnutrition · micronutrient deficiency · human immunodeficiency virus (HIV) status Image references ################ .\IMAGES\Fourpath.jpg Non-immunological MechanismsNon-immunological Mechanisms Image references ################ Non-immunological Mechanisms - 1Protective factors Protective factors have evolved to maintain the integrity of the GI mucosa. They: · physically limit contact between the pathogen and host epithelium · chemically inactivate pathogens or their productsPicture: Protective factors in the GI tract. Click on each label in the picture for details. Saliva Mucus Peristalsis Gastric acid Proteolytic enzymes Bile Cryptdins Epithelial turnover Commensal bacteria Copyright Image from The Wellcome Trust. SalivaSaliva contains the following proteins: · peroxidase, which produces free radicals to attack pathogens · lactoferrin, which binds to iron required for bacterial growth · lysozyme, which digests the coat of Gram-negative bacteriaMucusMucus: · contains a complex mix of glycoproteins · acts as a mechanical and chemical barrier against pathogens and toxinsGastric acidThe pH in the stomach is 2 - 3. This is due to the secretion of hydrochloric acid by the gastric glands. This highly acidic environment: · degrades toxins · kills many bacterial pathogens, eg. V. cholerae O1 is killed at a pH of less than 2.4 · does not kill the cysts of some parasites, eg. Entamoeba histolytica and Giardia lambliaCommensal bacteria The normal bowel flora prevent colonization of the intestine by pathogens. For details see screen 11.BileBile contains acids that inhibit the growth of bacteria, eg. Clostridium and enterococci. These bile acids include: · cholic acid · chenodeoxycholic acidCryptdinsThe Paneth cells in the small bowel crypts of Lieberkühn secrete several peptides called cryptdins. These are thought to kill Salmonella, E. coli and other bacteria by: 1. increasing the permeability of the bacterial membrane 2. lysing the target cell Epithelial turnover Epithelial cells in healthy bowel: 1. arise continuously from stem cells in the crypt region of the mucosa 2. migrate towards the luminal surface over 4 - 5 days 3. are shed from the surface into the bowel lumen Shedding of epithelial cells from the tips of the small bowel villi helps to eliminate pathogens (eg. rotavirus) or toxins within epithelial cells (enterocytes).PeristalsisThe normal movement of bowel contents along the GI tract: · limits the time that pathogens are in contact with the epithelium · ‘flushes out’ pathogenic material from the bowel lumen Diarrhoea itself can be seen as a defence mechanism to expel pathogens. This is one reason why antimotility drugs such as loperamide are not generally recommended in its treatment. Proteolytic enzymesThe numbers of viable pathogens are reduced by: · pepsin in the stomach · pancreatic proteases and lipases in the small bowel Image references ################ .\IMAGES\Gitract4.gif Non-immunological Mechanisms - 2 Numbers of bacteria (per ml) withinthe healthy adult GI tract.Commensal bacteria The bowel flora within the healthy GI tract (see picture): · compete with enteric pathogens for nutrients and sites of adhesion · help to maintain normal bowel structure· secrete antimicrobial factors, eg: - colicins - short chain fatty acids What happens if the normal flora are disturbed? Gastric acid Evidence that gastric acid protects against enteric pathogens comes from studies of: · experimental volunteers· patients with hypochlorhydria Click on the labels for details of the bacterial species. Stomach 0 - 103 Duodenum 0 - 105 Ileum 103 - 107 Large bowel 1010 - 1012 Data from Simon and Gorbach 1984. Copyright Image from The Wellcome Trust. Bowel structureExperimental animals bred since birth in a sterile environment: · do not develop commensal bacteria in the bowel · have an abnormal thin mucosa, irregular villi and reduced lymphoid tissue These changes are reversible if the animals are exposed to bacteria. Disturbance of bacteriaReduction of the number of commensals, eg. by antibiotic treatment, increases the risk of: · infection with enteric pathogens· diarrhoea Volunteer studiesIn experimental volunteers, temporary neutralization of gastric acid with oral sodium bicarbonate: · reduces the infective dose of V. cholerae O1 by a factor of 10,000 · increases the multiplication rate of S. flexneri by a factor of 3 Patients with hypochlorhydriaPatients with reduced secretion of gastric acid (hypochlorhydria) are at increased risk of infection with pathogens such as: · non-typhoid Salmonella · Shigella · V. cholerae O1 Common causes of hypochlorhydria include: · malnutrition · old age · infection · gastrectomy · H2 receptor antagonists, eg. given for peptic ulcer StomachThe stomach contains: · very few bacteria, because of its acidity · mostly Gram-positive aerobes such as: - lactobacilli - streptococci - staphylococci · H. pylori in over half of many studied populations DuodenumThe proximal small bowel contains mostly Gram-positive aerobes such as: · lactobacilli · streptococciIleumThe distal small bowel: · includes more Gram-negative than Gram-positive bacteria · contains significant numbers of: - Bacteroides - Bifidobacterium - Fusobacterium - Clostridium Large bowelThe ileocaecal valve marks a sharp increase in the concentration of bacteria. In the colon: · nearly one-third of the faecal dry weight is viable bacteria · anaerobes dominate, notably: - Bacteroides - Bifidobacterium - Eubacterium - peptostreptococci - Clostridium Image references ################ .\IMAGES\Gitract5.gif Where does each non-immunological defence mechanism act in the GI tract? Click your mouse on a box below.Hold the mouse down and drag the box to the correct site in the GI tract. To return to the start of the section. Non-immunological Mechanisms: AssessmentLysozyme pH < 3 Cryptdins Largest number of commensals Yes. That's right. Yes. That's right. Yes. That's right. Yes. That's right. No. That's wrong. Try again. Well done. You have now finished this assessment.Image references ################ .\IMAGES\Gitract4.gif Humoral Immune ResponsesHumoral Immune Responses Image references ################ Humoral Immune Responses - 1Pathogens LUMEN Secretory IgA and IgM The most important humoral response to enteric pathogens is secretion into the bowel lumen (see picture) of: · large quantities of secretory IgA (sIgA) · lesser amounts of secretory IgM (sIgM) These antibodies bind to pathogens and toxins in the lumen and inactivate them. Do any other humoral immune responses occur? Picture: The humoral response of the mucosal immune system. sIgA sIgM Secretion of antibodies Plasma cell(B lymphocyte) sIgA sIgM LAMINA PROPRIA Copyright Image from The Wellcome Trust. Large quantities of sIgAThe mucosal immune system in adults secretes over 3 g of IgA into the intestinal lumen each day.A Summary of Humoral Immune Responses Mucosal (bowel) Systemic (blood) immune systemimmune system sIgA sIgM IgGIgE2 1. IgG and IgE are important in inflammatory reactions to invasive pathogens such as Shigella and Salmonella. Some IgG also leaks into the bowel lumen. 2. IgE is also important in the response to intestinal helminths. Secreted into lumen Smaller amounts Active in lamina propria1Image references ################ .\IMAGES\Sigasecr.gif Humoral Immune Responses - 2sIgA IgG sIgA has a unique structure The pictures compare the structures of the major antibodies in:· mucosal secretions - sIgA · serum - IgG Click on the picture labels for details. The unusual dimeric structure of sIgA is important for its function in the bowel lumen. Fab Fc Fc Fab Copyright Images from The Wellcome Trust modified from Roitt IM, Brostoff J, Male DK, eds. Immunology. 4th ed. London: Mosby, 1996.Major antibodies· 90% of all antibody in the intestinal lumen is sIgA, in two subclasses (sIgA1, sIgA2) · 70% of all antibody in the blood is IgG, in four subclasses (IgG1 - IgG4)sIgAEach molecule of IgA has a ‘standard’ antibody structure of: · two identical heavy chains · two identical light chains Secretory IgA is a dimer, ie. is made up of two molecules of IgA. Each dimer is held together by: · a small J (‘joining’) protein chain · one molecule of secretory componentSecretory component J chain Fab sites of sIgAEach sIgA dimer has four identical antibody binding (Fab) sites. These each bind to antigen in a highly specific interaction.Fc portion of sIgAEach sIgA dimer has two identical Fc portions. These bind: · to a receptor that helps secrete sIgA into the lumen · poorly with Fc receptors on macrophages and neutrophils In ‘standard’ antibodies such as IgG, binding of Fc to receptors on these cells promotes killing of pathogens by: · phagocytosis (engulfing) · cytotoxic (cell killing) chemical reactions The poor binding of the Fc portion of sIgA to macrophages and neutrophils means that sIgA does not help to kill pathogens by the above mechanisms.IgGIgG is a monomer, in contrast to dimeric sIgA. Each molecule of IgG has a ‘standard’ antibody structure of: · two identical heavy chains · two identical light chains Fab sites of IgGEach IgG molecule has two identical antibody binding (Fab) sites. These each bind to antigen in a highly specific interaction.Fc portion of IgGEach IgG molecule has one Fc portion. This binds to receptors on macrophages and neutrophils to promote killing of pathogens by: · phagocytosis (engulfing) · cytotoxic (cell killing) chemical reactions Image references ################ .\IMAGES\Sigaieg.gif Humoral Immune Responses - 3Picture: Gut associated lymphoid tissue in the small bowel. The intestinal mucosa is a major site of immunological activity in the body. Gut associated lymphoid tissue The central component of the mucosal immune system is the gut associated lymphoid tissue (GALT). This comprises: · Peyer’s patches Sites of the immune · solitary lymphoid folliclesinductive phase. · scattered lymphoid cells in the: - lamina propria Sites of the immune - epithelium effector phase.Does mucosa elsewhere have associated lymphoid tissue? To recap about B and T cells.Peyer's patch Villus Epithelial cell M cell Lamina propria lymphocytes Intraepithelial lymphocyte Follicle Copyright Image from The Wellcome Trust. Other mucosa associated lymphoid tissueGALT is only one part, but the major part, of the mucosa associated lymphoid tissue (MALT). Other lymphoid tissues include: · bronchus associated lymphoid tissue (BALT) in the respiratory tract · nasopharyngeal associated lymphoid tissue (NALT) · genitourinary tract associated lymphoid tissue (GURALT) Recapping B and T cellsLymphocytes in the immune system are of three basic types. These are: · B cells, which differentiate to produce antibody · T cells, with two broad types: - helper T cells (mostly ‘CD4+’), which regulate humoral and cell-mediated responses - cytotoxic T cells (mostly ‘CD8+’), which kill pathogens and infected cells in cell-mediated immunity · cytotoxic NK cells Peyer’s patchesPeyer’s patches: · are most common in the distal ileum · are collections of up to 300 lymphoid follicles, each with a core of B cells and peripheral CD4+ helper T cells (see picture) · include macrophages and other antigen presenting cells (APCs) · are the best characterized sites of immune induction Solitary lymphoid folliclesSolitary lymphoid follicles: · are most common in the appendix and rectum · have a core of B cells with peripheral CD4+ helper T cells · include macrophages and other antigen presenting cells (APCs) Inductive phaseThe inductive phase is when the immune system recognizes antigen and becomes primed to produce an immune response. During induction each antigen stimulates the proliferation of a clone of identical B or T cells specific to that antigen (clonal selection). Scattered lymphoid cellsScattered lymphocytes are concentrated in the small bowel in the: · lamina propria (lamina propria lymphocytes) · epithelium (intraepithelial lymphocytes) Effector phaseThe effector phase is when the products of the immune system (eg. antibodies) act to neutralize, eliminate or kill the infecting pathogen. The effector mechanisms of sIgA and sIgM are described on screens 21 and 22. Image references ################ .\IMAGES\Galt.gif Humoral Immune Responses - 4The mucosal humoralresponse Secretion of sIgA and sIgM by the mucosal immune system can be divided into three steps (see picture): 1. induction 2. lymphocyte migration 3. antibody secretion Picture: The three stages of the mucosal humoral immune response.Antibody secretion 3. Antigen 1. B and T cells B and T cells 2. Thoracic duct Lymph nodes Copyright Image from The Wellcome Trust modified from Brandtzaeg P. APMIS 1995;103:1-19. 1. InductionDuring the inductive phase, B and T lymphocytes respond to antigens, cytokines and cell-cell interactions by: 1. activation 2. proliferation 3. partial differentiation Induction happens at specific sites in the GALT, ie. at Peyer’s patches and the solitary lymphoid follicles. 2. Lymphocyte migrationB and T lymphocytes primed at one site within the GALT: · undergo a complex migration around the body · finally localize to: - distant sites in the intestinal lamina propria, the major site for migration - other mucosal sites in the body, eg. in the breast or lung By this mechanism an immune response induced at one mucosal site is disseminated to multiple sites. This is the concept of a common mucosal immune system. Image references ################ .\IMAGES\Mucusumm.gif Humoral Immune Responses - 5 Induction B cells become committed to make sIgA specific to luminal antigen (see picture) by four steps. These are: 1. antigen ‘sampling’ 2. antigen processing and presentation 3. priming of helper T cells 4. priming of B cells Picture: Induction of the humoral response at Peyer’s patches. The picture shows the best characterized mechanism of induction. However, other means of antigen presentation probably exist. Click on the numbers in the picture for details. Pathogen 1. M cell 2. APC 3. 4. T B Pathogen Processed antigen MHC-II T cell receptor Copyright Image from The Wellcome Trust. 1. Antigen ‘sampling’The M (‘microfold’) cell in the epithelium overlying Peyer’s patch is specialized to take up: · intact luminal antigen, eg. cholera toxin· whole viruses, bacteria or small parasites The M cell takes up antigenic material into a small vacuole by a non-specific process called pinocytosis. 2. Antigen processing Antigen is delivered intact from an M cell to an antigen presenting cell (APC) which expresses major histocompatibility complex (MHC) class II. APCs include: · dendritic cells· macrophages APCs in the basolateral pocket of the M cell: 1. kill whole pathogens and process antigen into small peptide fragments 2. present these fragments at the cell surface bound to MHC-II3. Priming of helper T cellsAntigen specific CD4+ (helper) T cells in Peyer’s patch: 1. bind to the antigen-MHC-II complex on the APC 2. become activated and secrete cytokines, initially interleukin 2 (IL-2) and interferon g (IFN-g) Activated helper T cells in Peyer’s patch then undergo: 3. proliferation 4. partial differentiation 4. Priming of B cellsImmature B cells in Peyer’s patch respond to antigens and activated helper T cells by: 1. activation 2. proliferation 3. partial differentiation Immature B cells ordinarily make IgM, but during activation most undergo ‘isotype switching’ to make IgA instead. Image references ################ .\IMAGES\Peyers.gif Humoral Immune Responses - 6Lymphocyte migration Primed B and T lymphocytes leave Peyer’s patches and migrate (see picture) to the: 1. lymphatic system 2. mesenteric lymph nodes 3. thoracic duct4. systemic circulation 5. intestinal lamina propria and other mucosal effector sitesPicture: Migration of primed B cells and helper T cells. The intestinal lamina propria contains large numbers of memory helper T cells and significant numbers of antibody secreting plasma B cells. Peyer's patch Lamina propria 5. 1. Breast Mesenteric lymph node 4. 5. 2. 3. Thoracic duct Copyright Image from The Wellcome Trust modified from Brandtzaeg P. APMIS 1995;103:1-19. Thoracic ductThe thoracic duct is the main collecting duct of the lymphatic system. It: · receives lymph from lymphatic vessels in the GI tract, lower body and elsewhere · empties into the left subclavian vein, which feeds into the superior vena cava Effector sitesMigration of lymphocytes from the circulation to effector sites is controlled by specific interactions between: · ‘homing’ receptors on lymphocytes · addressin molecules on mucosal tissues Lymphocytes primed in Peyer’s patches migrate to: · distant intestinal lamina propria - the major site for homing · the mammary glands - during pregnancy and lactation · the upper respiratory tract Image references ################ .\IMAGES\Lympmigr.gif Humoral Immune Responses - 7 Secretion of IgA and IgM 1. Plasma cells in the lamina propriaproduce IgA. 2. IgA binds to an epithelial cell receptor. 3. Receptor-IgA is taken up and transported across the cell. 4. Secretory IgA is released into the bowel lumen. How does secretion of IgM differfrom this? Animation: The mechanism of secretion of sIgA.To run the animation. pIgR sIgA B Copyright Image and animation from The Wellcome Trust modified from Brandtzaeg P. APMIS 1995;103:1-19. Plasma cellsPlasma cells are: · antibody producing cells · formed when B lymphocytes homing to the lamina propria complete their differentiationIgAPicture: IgA dimers as released by plasma cells. Image from the Wellcome Trust.Plasma cells release IgA as: · mostly dimers · some polymers Dimers and polymers of IgA (see picture) are held together at this stage by a J protein chain.J ReceptorIgA binds by its Fc portion to a specific receptor on the basolateral surface of epithelial enterocytes. This receptor: · is called the polymeric immunoglobulin receptor (pIgR) · also binds pentameric IgM TranscytosisThe transport of receptor-IgA across the enterocyte (transcytosis) is: · mediated by intracellular vesicles · accompanied by: 1. enzymatic cleavage of the receptor to produce the secretory component (SC) 2. covalent linkage of the IgA dimer to SC by two disulphide bridges Secretion of IgAThe epithelial cell secretes sIgA into the lumen as a covalently bound complex that contains: · two molecules of IgA · one J chain molecule · one SC molecule sIgA is different in structure from serum IgA, which is mostly monomeric. Secretion of IgMSecretion of sIgM is similar to that of sIgA and uses the same receptor (pIgR). The main differences are that sIgM is: · secreted at only 10% of the levels of sIgA· pentameric, whereas sIgA is dimeric · not a covalently bound complex, and hence is less stable in the lumen Image references ################ .\IMAGES\Siga0001.bmp .\IMAGES\Siga1.gif Humoral Immune Responses - 8Effector mechanisms of sIgA Secretory antibodies in the mucus layer and lumen prevent pathogens and toxins from attaching to and invading the epithelium by: · agglutination of pathogens in the lumen · formation of immune complexes with luminal antigens · neutralization of toxins, viruses and bacteria sIgA does not activate complement and so the mucosal humoral response is not inflammatory. How do sIgA and sIgM avoid proteolysis by digestive enzymes? Animation: Agglutination. The high number of Fab sites on sIgA (four) and sIgM (ten) promotes cross-linking of antigenic sites.To run the animation. Pathogen sIgA sIgM Copyright Image and animation from The Wellcome Trust. NeutralizationsIgA and sIgM bind to toxins, viruses and bacteria to prevent them from: · functioning - eg. antibody inactivates bacterial flagella · binding to epithelial cells - many pathogens and toxins attach to enterocytes and colonocytes by a specific mechanism Not an inflammatory responsePathogens which do not invade the mucosa (eg. V. cholerae O1, enterotoxigenic E. coli): · induce a large sIgA response · do not cause gross damage to the mucosa, which appears histologically normal and uninflamed on light microscopy Avoiding proteolysisThe small bowel lumen contains many digestive enzymes, including several that degrade proteins. Molecules of sIgA and sIgM: · are protected from these proteases by their J chain and SC components · have a half-life in the lumen of 5 - 6 days for sIgA (less for sIgM) Some pathogens secrete specific proteases that target sIgA. Image references ################ .\IMAGES\Aggl0001.bmp .\IMAGES\Aggl0001.bmp Humoral Immune Responses - 9Other effector mechanisms of IgA There is evidence that IgA within the lamina propria may: · inhibit the replication of viruses in epithelial cells (see picture) · help to excrete antigen from the lamina propria back into the lumen Does IgA in the lamina propria take part in more ‘standard’ mechanisms? Blocked viral asembly Synthesis of virus Secretion of sIgA Golgi complex pIgR sIgA Virus Picture: Inhibition of viral replication by intracellular IgA.Copyright Image from The Wellcome Trust modified from Lamm ME, Nedrud JG, Kaetzel CS, Mazanec MB. APMIS 1995;103:241-6. Inhibition of viral replicationDuring secretion of sIgA, the vesicle which contains IgA crossing the enterocyte may: 1. fuse with vesicles containing intracellular viruses 2. inhibit viral assembly or release This is an unusual effector mechanism because intracellular pathogens are usually considered to be:· inaccessible to antibody · targets for cell-mediated rather than humoral responses Excretion of antigensPicture: Excretion of antigens by sIgA. Image from The Wellcome Trust. IgA in the lamina propria may: 1. bind to antigen that has penetrated the epithelium 2. carry the antigen out into the lumen when sIgA is secreted pIgR sIgA Antigen More ‘standard’ mechanismsIgA in the lamina propria may help to promote: · phagocytosis by coating (opsonization) of pathogens · antibody-dependent cell-mediated cytotoxicity However, these mechanisms depend on sIgA binding to an Fc receptor on a macrophage or neutrophil (see screen 15), and are therefore unlikely to be very important. Image references ################ .\IMAGES\Sigavir.gif .\IMAGES\Psigaant.gif Humoral Immune Responses - 10sIgA Regulation of the mucosal humoral response Cytokines that are probably important at various stages of mucosal sIgA production (see picture) include: · transforming growth factor b (TGF-b) · IL-2 and IL-4 · IL-10 · IL-5 · IL-6 These are mostly produced by type 2 helper T (Th2) cells. Pathogen APC Activation Differentiation B B Plasma cell T TGF-b IL-2/IL-4 IL-5 IL-6 IL-10 Picture: A model for the cytokines involved in the mucosal humoral response. The evidence for this comes mainly from studies in mice. Copyright Image from The Wellcome Trust modified from Brandtzaeg P. APMIS 1995;103:1-19. Th2 cellsCD4+ helper T cells exist in two distinct populations. · Type 1 helper T cells (Th1 cells) secrete cytokines associated with cell-mediated and inflammatory responses, eg. IL-2 and IFN-g. · Type 2 helper T cells (Th2 cells) secrete cytokines associated with humoral (antibody) responses, eg. IL-4, IL-5 and IL-10. Image references ################ .\IMAGES\Humcks.gif Humoral Immune Responses - 11Serum derived IgG Small amounts of IgG enter the lamina propria from the serum (see picture) by two routes: · leakage of IgG · extravasation of B lymphocytes IgG activates the complement pathway, which promotes an inflammatory reaction. IgG LUMEN Transudation LAMINA PROPRIA IgG Extravasation Leakage IgG B Picture: Routes by which IgG enters the lamina propria. Note also that a small fraction of the IgG leaks into the intestinal lumen. Blood vessel Copyright Image from The Wellcome Trust modified from Brandtzaeg P. APMIS 1995;103:1-19. B lymphocytesThese cells: 1. leave the circulation by squeezing between cells in the vascular endothelium (extravasation) 2. enter the lamina propria and produce IgG Leakage of IgG into the lumenThe IgG: 1. enters the lumen by passing between intestinal epithelial cells (transudation) 2. is rapidly degraded by proteolytic enzymes (it is not protected in the way that sIgA is) 3. has only a limited role in immunity against luminal pathogens such as V. cholerae O1Inflammatory reaction Significant leakage of IgG and extravasation of B lymphocytes occur only as part of an acute inflammatory reaction (see screen 31) in which: 1. the vascular endothelium becomes more permeable 2. products of the systemic immune system leave the circulation and enter the lamina propria Image references ################ .\IMAGES\Serumigg.gif Humoral Immune Responses: Assessment - 1Are the following statements about the mucosal humoral response true or false?To return to the start of the section. Click on the True or False button for each statement. 1. sIgA contains two IgA molecules, one J chain and one molecule of secretory component.2. The best characterized sites of induction of immunity are the tips of small bowel villi.3. Primed lymphocytes migrate in turn to the mesenteric lymph nodes, thoracic duct, circulation and lamina propria.4. Secretion of sIgA and sIgM occurs by leakage between intestinal epithelial cells.Correct This structure: · is different from that of a ‘standard’ antibody such as IgG · protects sIgA against proteolysis by digestive enzymes Incorrect Text 11 pt Arial dark blue goes here Incorrect This structure: · is different from that of a ‘standard’ antibody such as IgG · protects sIgA against proteolysis by digestive enzymes Correct Text 11 pt Arial dark blue goes here Correct Text 11 pt Arial dark blue goes here Incorrect Induction of the humoral response occurs in the GALT at: · Peyer’s patches in the small bowel · solitary lymphoid follicles in the large bowelIncorrect Text 11 pt Arial dark blue goes here Correct Induction of the humoral response occurs in the GALT at: · Peyer’s patches in the small bowel · solitary lymphoid follicles in the large bowelCorrect Migration of primed lymphocytes around the body: · is controlled by specific interactions with homing receptors · disseminates immunity from the gut to distant mucosal sitesIncorrect Text explaining the answer (11-pt plain blue) Incorrect Migration of primed lymphocytes around the body: · is controlled by specific interactions with homing receptors · disseminates immunity from the gut to distant mucosal sitesCorrect Text explaining the answer (11-pt plain blue) Correct Incorrect Secretion of sIgA and sIgM occurs by a specific mechanism in which: 1. plasma cells in the lamina propria produce IgA or IgM 2. antibody binds to a receptor on an enterocyte 3. receptor-antibody is taken up and transported across the cell 4. sIgA or sIgM is released into the bowel lumen bound to a secretory component Incorrect Text explaining the answer (11-pt plain blue) Correct Secretion of sIgA and sIgM occurs by a specific mechanism in which: 1. plasma cells in the lamina propria produce IgA or IgM 2. antibody binds to a receptor on an enterocyte 3. receptor-antibody is taken up and transported across the cell 4. sIgA or sIgM is released into the bowel lumen bound to a secretory component Image references ################ Secretory IgA has effector mechanisms at several different sites within the mucosa and lumen. Click your mouse on each of the boxes.Hold the mouse down and drag the box to label each of the mechanisms. To return to the start of the section. Humoral Immune Responses: Assessment - 2Inhibition of intracellularviral replication Agglutination, neutralization & formation of immune complexes Excretion of antigens boundto sIgA Yes. That's right. Yes. That's right. Yes. That's right. No. That's wrong. Try again. Well done. You have now finished this assessment.Image references ################ .\IMAGES\Sigasite.gif Cell-mediated Immune ResponsesCell-mediated Immune Responses Image references ################ Cell-mediated Immune Responses - 1Intraepithelial lymphocytes Cell-mediated responses are possibly due in part to intraepithelial lymphocytes. These are: · a major population of lymphocytes · concentrated in the small bowel villi (see picture) · nearly all T cells Intraepithelial lymphocytes are a major population of lymphocytes in the mucosal immune system, yet their function is largely unknown. Picture: Intraepithelial lymphocytes (boxed) in the mouse small bowel villus (light micrograph). These lymphocytes undergo induction and migration in broadly similar ways to lamina propria lymphocytes. Copyright Image from Mowatt AMcI.Major populationIntraepithelial lymphocytes represent 60% of all the T cells in the body. Image references ################ .\IMAGES\T45477.jpg Cell-mediated Immune Responses - 2Functions of intraepithelial lymphocytes Intraepithelial lymphocytes are: · 80 - 90% CD8+ (cytotoxic/suppressor) cells · different from T cells in the lamina propria and peripheral blood · large, motile and contain cytoplasmic granules (see picture) The number of intraepithelial lymphocytes is increased in: · giardiasis and other parasitic infections · active coeliac disease · Crohn’s disease What roles might these cells perform?Picture: Granules within the cytoplasm of a mouse intraepithelial lymphocyte (TEM).Copyright Image from Mowatt AMcI.Lamina propria T cellsMost of the T cells in the intestinal lamina propria are helper T cells (CD4+ phenotype). Cytoplasmic granulesThe granules in the cytoplasm of intraepithelial lymphocytes contain: · perforin · granzyme A These molecules are cytotoxic. Possible roles of intraepithelial lymphocytesVarious roles have been proposed. Intraepithelial T cells may: · be cytotoxic towards virally infected epithelial cells (cell-mediated immunity) · secrete a range of cytokines, including Th1 and Th2 types · help to regulate tolerance to dietary antigens by suppressing immune responses · promote epithelial growth by secreting soluble mediators It is likely that different subpopulations of intraepithelial lymphocytes have different functions. Image references ################ .\IMAGES\T45478.jpg Innate Immune ResponsesInnate Immune Responses Image references ################ Innate Immune Responses - 1Innate responses are inflammatory The gross physiological responses to a pathogen invading the mucosa are: · increased blood supply · increased vascular permeability · extravasation of immune cells from venules These events define an acute inflammatory reaction (see picture). Picture: The effects of Shigella on the colon of the rhesus monkey. Shigellosis causes mucosal inflammation and ulceration, with production of an exudate containing blood, mucus and pus.Copyright Image from Centers for Disease Control and Prevention. Increased vascular permeabilityThere are increases in the permeability of: · capillaries - allowing complement and serum IgG to leave the circulation and enter the lamina propria · venules - allowing cells of the immune system to extravasate Inflammatory reactionCollectively these processes help to concentrate the soluble and cellular products of the immune system at the site of infection. The inflammatory reaction involves both the mucosal and systemic immune systems. Extravasation of immune cellsPicture: Stages in extravasation.1, adhesion of the cell to the vascular endothelium;2, crossing the vascular endothelium. Image from The Wellcome Trust.Blood vessel 2. 1. LAMINA PROPRIA Increased permeability allows cells in the blood to cross the vascular endothelium and enter the lamina propria. These cells include: · polymorphonuclear neutrophils (PMNs) · macrophages · lymphocytes · eosinophils Image references ################ .\IMAGES\T33790.jpg .\IMAGES\Extravas.gif Innate Immune Responses - 2Innate immune responses These include: · activation of the alternative complement pathway· phagocytic (see animation) and cytotoxic activities of macrophages, PMNs and NK cells · infiltration of inflammatory cells into the lamina propria· degranulation of mast cells and eosinophils How specific are these mechanisms? Animation: Phagocytosis by macrophages of bacteria in the lamina propria. To run the animation. Copyright Image and animation from The Wellcome Trust. ComplementThe complement pathway: · is an enzymatic cascade of proteins · triggers release of inflammatory mediators, eg. histamine by mast cells · lyses bacteria and viruses · opsonizes pathogens for enhanced phagocytosis The complement cascade can be activated by: · antigen alone (eg. in the bacterial cell wall)- the alternative complement pathway · antigen bound to IgG and other antibodies - the classical complement pathway Macrophages and PMNsMacrophages and PMNs kill pathogens by two types of mechanism: · phagocytosis and killing of intracellular pathogens (see animation) · secretion of cytotoxic factors to kill extracellular pathogens The products used to kill pathogens in both cases include: · reactive oxygen intermediates (eg. superoxide, hydrogen peroxide) · reactive nitrogen intermediates (eg. nitric oxide) Inflammatory cellsExtravasated inflammatory cells (eg. PMNs, macrophages) in the lamina propria are attracted to the site of infection (chemotaxis) by soluble mediators. As inflammation develops, some PMNs may cross the intestinal epithelium and enter the bowel lumen. Mast cell degranulationThe molecules released by degranulation of mast cells include histamine, which: · activates PMNs· increases vascular permeability Activated mast cells also release lipid mediators such as leukotriene B4 (LTB4), which attracts PMNs bychemotaxis. EosinophilsThe molecules released by degranulation of eosinophils include:· major basic protein - toxic to many parasites· histamine - activates PMNs and increases vascular permeability Specificity of innate responsesThe innate immune responses characteristic of acute inflammation: ·have inherently low specificity·are capable of acting without antibody or T cells However, the innate responses are amplified and made more specific (see screen 33) by products of the adaptive immune system. Image references ################ .\IMAGES\Bacteat2.gif Innate Immune Responses - 3Interaction of the innate and adaptive immune responses Most invasive pathogens evoke both innate and adaptive immune responses. Innate responses interact with products of the adaptive immune system (see picture) to become:· amplified · highly specific These products of the adaptive system include: · cytokines· antibodiesPathogen B Macrophage Enhanced phagocytosis IgG, IgE Classical Complement Degranulation Mast cell Alternative Picture: Interaction between the innate immune system and antibody.Copyright Image from The Wellcome Trust. CytokinesCytokines produced by T cells help regulate the innate response. For example, IFN-g from Th1 cells activates: · macrophages· PMNs A wide range of other cells also produce cytokines and other soluble mediators that regulate the innate response. These include: · macrophages · intestinal epithelial cells · mast cells AntibodiesAntibodies from the humoral response have several roles. For example: · complement reactions in the lamina propria are made more specific (the ‘classical’ pathway) by binding of IgG to the antigen · phagocytosis in the lamina propria is made more specific by antibody coating the antigen (opsonization) · degranulation of mast cells is triggered by antigen cross-linking IgE on the surface of the mast cell Innate and adaptive responsesInvasive bacteria such as Shigella induce: · inflammatory innate responses, as described in this section · humoral immunity, by the mechanisms described on screens 17 - 20 Image references ################ .\IMAGES\Humoinna.gif Innate Immune Responses - 4Acute inflammation Initial events in the inflammatory response to invasive pathogens such as Shigella are: 1. invasion of M cells by bacteria 2. delivery of intact Shigella to macrophages for phagocytosis 3. death of macrophages, which release live bacteria and: · IL-1· IL-6 · TNF-a 4. infection of epithelial colonocytes 5. secretion by infected epithelial cells of IL-8 How is the humoral response toShigella induced? Shigella 1. M cell 2. 3. These cytokines are known as the proinflammatory triad. 5. 4. 3. Picture: Key events in the initial stages of the acute inflammatory response to Shigella.IL-1, IL-6, TNF-a IL-8 Copyright Image from The Wellcome Trust modified from Bloom PD, Boedeker EC. Semin Gastrointest Dis 1996;7:151-66.Death of macrophagesShigella is not killed after phagocytosis by the macrophage. Instead the bacterium: · survives within the macrophage · rapidly induces the cell to undergo programmed cell death (apoptosis) Shigella thus subverts the usual mechanism by which a protective immune response is induced. Proinflammatory triadIL-1, IL-6 and tumour necrosis factor a (TNF-a) trigger release of a network of regulatory molecules that cause an acute inflammatory reaction. The molecules in this network include: · vasoactive mediators - increase vascular permeability · chemotactic cytokines (‘chemokines’) - attract inflammatory cells to the site of infection · activating cytokines - increase the activity of immune effector cells Humoral response to ShigellaShigella also induces an antibody response. The mechanism is that described earlier (screens 17 - 20). 1. M cells take up whole bacteria and deliver them to APCs. 2. APCs present processed antigen to helper T cells. 3. Helper T cells prime B cells. 4. Lymphocytes migrate and home to the intestinal lamina propria. 5. Plasma B cells secrete anti-Shigella sIgA. This response is delayed because of the disruption caused by death of macrophages and colonocytes, and by the inflammatory reaction. Image references ################ .\IMAGES\Shigecks.gif Innate Immune Responses - 5For picture legend. Development of the inflammatory reaction As the inflammatory reaction develops, two processes take place(see picture). 1. Increased vascular permeability. This is due to vasoactive mediators such as: · IL-1 · histamine 2. Chemotaxis of extravasated PMNs and macrophages. This is regulated by molecules such as: · IL-8 · MCP-1 · LTB4 · C5a What are the clinical consequencesof these processes? Infected epithelial cell IL-8 Chemotaxis PMN or macro-phage Mast cell LTB4 MCP-1 Histamine IL-1 Infected macrophage Vascular permeability Extravasation Blood vessel Copyright Image from The Wellcome Trust. IL-1IL-1 is secreted primarily by activated macrophages, such as those infected with Shigella.HistamineHistamine is released by degranulation of: · mast cells - in response to antigen, IgE and complement · eosinophils - in response to lipopolysaccharide in bacterial cell walls, IgE and IgG IL-8IL-8: · is secreted by intestinal epithelial cells infected with invasive bacteria (Shigella, Salmonella) · attracts PMNs and macrophages by chemotaxis MCP-1Monocyte chemoattractant protein 1 (MCP-1): · is secreted by infected intestinal epithelial cells · attracts macrophages by chemotaxisLTB4LTB4: · is secreted by mast cells · attracts PMNs by chemotaxis C5aC5a: · is a product of the complement cascade · attracts PMNs and macrophages by chemotaxis Clinical consequencesThe consequences of infection by invasive bacteria are due to: · direct pathogenicity, eg. pathogens releasing cytotoxins, or signalling molecules that disrupt host tissue integrity and immune responses · the secondary effects of inflammatory mediators released by the host Clinical effects include: · fever - due to TNF-a · PMNs in the stool - due to leakage across a damaged intestinal epithelium · blood, pus and mucus in the stool - due to inflammatory breakdown of the epithelium Picture legendPicture: Mediators of the inflammatory reaction. The picture shows examples of cells that produce inflammatory mediators (a macrophage, mast cell and intestinal epithelial cell) and examples of immune effector cells (a macrophage and PMN). The scheme is simplified and shows only selected interactions. Image references ################ .\IMAGES\Airncks.gif Innate Immune Responses: AssessmentAre the following statements true or false?To return to the start of the section. Click on the True or False button for each statement. 1. Innate responses to enteric pathogens are made more specific by interaction with antibodies.2. Key sources of inflammatory mediators are macrophages, mast cells and the intestinal epithelial cell.3. The ‘proinflammatory triad’ of cytokines is IL-1, IL-6 and TNF-a.4. Histamine is an important chemotactic agent for PMNs and macrophages.Correct Innate responses such as phagocytosis and complement interact with antibodies to become: · amplified · highly specific Incorrect Text 11 pt Arial dark blue goes here Incorrect Innate responses such as phagocytosis and complement interact with antibodies to become: · amplified · highly specific Correct Text 11 pt Arial dark blue goes here Correct These cells are important sources of cytokines (eg. interleukins, TNF-a) andother inflammatory mediatiors such as lipids (eg. LTB4).Incorrect Text explaining the answer (11-pt plain blue) Incorrect These cells are important sources of cytokines (eg. interleukins, TNF-a) andother inflammatory mediatiors such as lipids (eg. LTB4).Correct Text explaining the answer (11-pt plain blue) Correct This group of cytokines: · is characteristic of the acute inflammatory reaction · is secreted primarily by macrophages · triggers a network of inflammatory mediatorsIncorrect Text explaining the answer (11-pt plain blue) Incorrect This group of cytokines: · is characteristic of the acute inflammatory reaction · is secreted primarily by macrophages · triggers a network of inflammatory mediatorsCorrect Text explaining the answer (11-pt plain blue) Correct Incorrect Histamine: · is released by degranulation of mast cells · activates PMNs and increases the permeability of the vascular endotheliumIncorrect Text explaining the answer (11-pt plain blue) Correct Histamine: · is released by degranulation of mast cells · activates PMNs and increases the permeability of the vascular endothelium Image references ################ Mucosal VaccinesMucosal Vaccines Image references ################ Mucosal Vaccines - 1Mucosal vaccines For pathogens that adhere to or invade mucosal surfaces, such as pathogens that cause diarrhoea, vaccination by mucosal rather than parenteral routes is an attractive possibility. The idea is to: 1. deliver a vaccine to a mucosal surface2. induce mucosal and systemic immune responses3. elicit immunity at other sites within the mucosal immune system Picture: Immunization against measles. Most childhoood vaccines except oral poliomyelitis vaccine are delivered by injection and induce systemic (serum IgG) protective immune responses. What are the limitations of parenteralvaccine delivery? Copyright Copyright holder unknown. Image supplied by MERLIN picture library. Limitations of parenteral deliveryParenteral delivery of vaccines (eg. measles, tetanus): · is expensive · requires attendance by a trained health worker · can cause adverse effects of injection · carries a risk of transmitting blood borne diseases in contaminated needles and syringes, eg: - HIV - hepatitis B- hepatitis C Measles vaccination Note that the recommended site for intramuscular injection of the vaccine is the lateral thigh. Mucosal surfacesMucosal vaccines could theoretically be delivered by the: · gastrointestinal tract (oral vaccines)· nasopharynx · rectum · vagina · conjunctivaLess practical options. Immune responsesThe response to a vaccine delivered to a mucosal site (eg. Peyer’s patches) could include all or any of the immunological responses described in this tutorial, ie: · sIgA from the mucosal immune system · serum IgG from the systemic immune system· cell-mediated responses from the mucosal or systemic immune system Other sitesRemember how induction by antigen at one site within the MALT (eg. Peyer’s patches) is followed by migration of lymphocytes to: · distant sites in the intestinal lamina propria· other mucosal surfaces in the body An oral vaccine might exploit this common mucosal immune system by inducing immunity at multiple mucosal sites. Image references ################ .\IMAGES\T45823.jpg Mucosal Vaccines - 2Delivery of oral vaccines What are the barriers to development of an oral vaccine? The range of antigen delivery systems includes:· coadministration of antigen with immunogenic toxins· killed whole cells· live attenuated organisms · particulate carriers· recombinant ‘edible vaccines’ (see picture) Which diarrhoea vaccines are most advanced?Picture: Bananas have been proposed as a potential vehicle for an ‘edible vaccine’.Copyright Image from Peters W. Live attenuated organismsSome bacteria and viruses(eg. V. cholerae O1, Salmonella typhi, adenovirus) bind specifically to M cells. Attenuated live strains of these pathogens have been used as carriers of: · endogenous antigens (vaccines for cholera and typhoid) · foreign antigens from other pathogens (eg. Shigella O antigen expressed in Salmonella) Immunogenic toxinsAt least two bacterial enterotoxins bind preferentially to M cells and also act as adjuvants to stimulate sIgA, serum IgG and cell-mediated responses. The toxins are: · cholera toxin · enterotoxigenic E. coli heat labile toxin Oral and intranasal vaccines are being developed in which avirulent forms of these toxins are administered with the immunizing antigen. Killed whole cellsFor example, one of the two types of licensed oral cholera vaccines is based on whole cells of V. cholerae O1 inactivated by: · heat · formalin treatment Particulate carriersParticulate delivery systems which incorporate immunizing antigens include: · copolymer microspheres · proteosomes · liposomes · immunostimulating complexes · virus like particles Recombinant ‘edible vaccines’Edible transgenic plants (eg. potatoes, bananas) are under development, in which the plant is genetically engineered to express the foreign antigen. Bananas seem promising because they are: · grown in many tropical and subtropical areas · palatable to young children · edible without cooking, which might otherwise destroy the antigen Barriers to an oral vaccineAn effective oral vaccine must overcome several potential barriers. These include: · structural alterations while in the GI tract, eg: - hydrolysis by digestive enzymes - loss of critical epitopes by enzymatic digestion - conformational changes due to acidic or alkaline conditions · low contact time between the antigen and Peyer’s patches · poor binding or uptake of antigen by M cells Delivery systems that have been developed partly reflect attempts to overcome these problems. Diarrhoea vaccinesDiarrhoeal diseases for which oral vaccines are most developed include: · rotavirus · cholera (see screen 40) · shigellosis · enterotoxigenic E. coli · salmonellosis Image references ################ .\IMAGES\T37313.jpg Mucosal Vaccines - 3Live oral cholera vaccine The live attenuated oral cholera vaccine CVD 103-HgR:· is one of two types of licensed oral cholera vaccine· has been made by deleting the cholera toxin A subunit gene from wild type V. cholerae O1 · has a high efficacy in adult volunteers· induces mucosal and systemic humoral responses Picture: Severe dehydration due to cholera (as shown) requires expression of the cholera toxin. However, naturally acquired immunity to cholera is due mainly to protective antibodies against V. cholerae O1 surface antigens rather than against cholera toxin.Copyright Image from Bryceson ADM. CVD 103-HgR· CVD stands for Center for Vaccine Development, University of Maryland, where the vaccine was produced. · 103 is an arbitrary strain designation. · HgR indicates that the strain has been made resistant to mercury (chemical symbol Hg), making it easy to differentiate from wild type V. cholerae O1. Types of cholera vaccineThe other types of licensed oral cholera vaccine: · are killed whole cell preparations, with (WC-BS) or without (WC) cholera toxin B subunit · have been extensively tested in field trials in endemic areas, eg. Bangladesh · have an efficacy of about 50% over 3 years in endemic populations Adult volunteersExperiments with CVD 103-HgR in adult volunteers: · show that the vaccine has an efficacy of 76 - 100% in these subjects · are the basis for licensing of the vaccine for use in travellers to cholera areas The efficacy of CVD 103-HgR in subjects exposed to endemic cholera is currently being assessed in a large randomized trial in Indonesia. Humoral responsesCVD 103-HgR induces: · local sIgA in the gut · systemic IgG Image references ################ .\IMAGES\T39750.jpg Mucosal Vaccines: AssessmentAre the following statements about mucosal vaccines true or false? Click on the True or False button for each statement. Mucosal vaccines:To return to the start of the section. 1. are mostly delivered by the oral route2. induce only a sIgA response3. can be introduced by living or non-living delivery systems4. are most advanced for parasites such as Giardia and CryptosporidiumCorrect Mucosal vaccines are delivered by the: · oral route in most cases · nasal route in a few casesIncorrect Text 11 pt Arial dark blue goes here Incorrect Mucosal vaccines are delivered by the: · oral route in most cases · nasal route in a few casesCorrect Text 11 pt Arial dark blue goes here Correct Text 11 pt Arial dark blue goes here Incorrect Mucosal vaccines are most effective if they induce: · mucosal sIgA · serum IgGIncorrect Text 11 pt Arial dark blue goes here Correct Mucosal vaccines are most effective if they induce: · mucosal sIgA · serum IgGCorrect Live delivery systems include: · live attenuated organisms · recombinant ‘edible vaccines’ Non-living delivery systems include: · killed whole cells · particulate carriers. eg. liposomesIncorrect Text explaining the answer (11-pt plain blue) Incorrect Live delivery systems include: · live attenuated organisms · recombinant ‘edible vaccines’ Non-living delivery systems include: · killed whole cells · particulate carriers. eg. liposomesCorrect Text explaining the answer (11-pt plain blue) Correct Incorrect The most developed mucosal vaccines against diarrhoeal diseases target: · rotavirus · cholera · shigellosis Incorrect Text explaining the answer (11-pt plain blue) Correct The most developed mucosal vaccines against diarrhoeal diseases target: · rotavirus · cholera · shigellosis Image references ################ Development of Defence MechanismsDevelopment of Defence Mechanisms Image references ################ Development of Defence Mechanisms - 1Developing defence The development of GI defence mechanisms reflects genetic, maternal and environmental factors. Important aspects are: · transfer of maternal IgG across the placenta · transfer of maternal factors (eg. sIgA) in breast milk · development of the bowel flora Why does the incidence of diarrhoea peak in young infants? Picture: An Indonesian child being weaned. Weaning is important in establishing the bowel flora because it introduces different species of bacteria into the intestine. Passive immunity. Copyright Image from WHO Photo Library courtesy of Sprague S. Maternal IgGTransfer of IgG across the placenta from the maternal to fetal circulation: · begins in the third trimester from around 24 weeks of gestation · is complete by around 40 weeks of gestation Maternal IgG is: · present in the neonate at levels similar to those in adult blood · particularly important in: - infection by invasive pathogens, eg. Shigella - infants who are not breast fed Passive immunityPassive immunity due to uptake of maternal antibodies (serum IgG, breast milk sIgA) helps to protect the infant while its own immune system is developing. Bowel floraThe GI tract of a breast fed child: 1. is sterile in the fetus 2. becomes colonized with anaerobes from the vagina at birth 3. includes Lactobacillus bifidus within the first few days of life 4. contains more Bacteroides when weaning foods are introduced 5. has an adult type flora by the time weaning is completed Peak in young infantsThe peak incidence of diarrhoea in developing countries is in children aged 6 - 12 months. This peak reflects: · introduction of contaminated foods and liquids at weaning · increased exposure to faeces in the environment · poorly developed defence mechanisms such as: - limited production of gastric acid - a leaky mucosa - immature immunological responses Image references ################ .\IMAGES\T34755.jpg Development of Defence Mechanisms - 2Breast milk Breast feeding (see picture) protects against diarrhoea because breast milk contains: · large quantities of sIgA · lesser amounts of sIgM · immune cells · non-specific protective factors · bifidus factor Picture: Breast feeding, especially exclusive breast feeding, strongly protects against diarrhoea in developing countries. Copyright Image from John & Penny Hubley. Protective effects of breast feedingBreast feeding in developing countries strongly protects against: · morbidity due to diarrhoea · severe diarrhoea · mortality due to diarrhoea For details refer to the tutorial Diarrhoeal Diseases: Prevention and Control. Breast feeding prevents diarrhoea by: · transfer of maternal antibodies and other factors (see this screen) · shielding the infant from diarrhoea pathogens · favouring good nutritional statussIgAThe first breast milk (colostrum) contains about: · 12 g/l of sIgA - an extremely high concentration · 0.6 g/l of sIgM Levels of these secretory antibodies in breast milk: 1. fall by a factor of 4 over the first 2 weeks of breast feeding 2. remain fairly stable at this lower concentration throughout lactation 3. are countered by the larger volumes of milk that older babies take Immune cellsColostrum and, to a lesser extent, later breast milk contain: · macrophages (55 - 60% of leucocytes) · PMNs (30 - 40%) · lymphocytes (5 - 10%), mainly T cells At least some of these cells cross the intestinal epithelium of the infant and enter the lamina propria. Protective factorsNon-immunological protective factors in breast milk include: · lysozyme · lactoferrin · peroxidase · mucins For details of how these protect from diarrhoea see screen 10. Bifidus factorBreast milk contains bifidus factor, which promotes the growth of Lactobacillus bifidus in the breast fed baby’s bowel. This bacterium is: · an important component of the normal bacterial flora · responsible for the ‘sweet’ smell of the stool of a breast fed baby Image references ################ .\IMAGES\T26832.jpg Development of Defence Mechanisms - 3 Basis of protection by breast feeding Steps in the passive transfer of immunity through breast feeding (see picture) are: 1. induction of a humoral response in the mother’s bowel mucosa 2. migration of lymphocytes from GALT to the breast 3. secretion of sIgA and sIgM in the lactating mammary gland 4. ingestion of antibody by the breast feeding infant The antibodies transferred are specific to antigens that the mother has previously encountered and which dominate the infant’s environment.The basis of protection from diarrhoea in breast fed infants. Click on the numbers for details. Antigen 1. 3. Other sites Maternal bowel 4. Infant bowel Migration 2. Copyright Image from The Wellcome Trust modified from Brandtzaeg P. Development of the mucosal immune system in humans. In: Bindels JG, Goedhart AC, Visser HKA, eds. Recent developments in infant nutrition. London: Kluwer Academic, 1996:349-76.2. Lymphocyte migrationPrimed B and T lymphocytes leave the sites of induction and migrate to the: 1. mesenteric lymph nodes 2. thoracic duct 3. systemic circulation 4. breast in pregnancy and lactation3. Antibody secretion1. Plasma cells homing to the lactating mammary gland produce IgA. 2. IgA binds to a polymeric immunoglobulin receptor on the basolateral side of an epithelial secretory cell. 3. Receptor-IgA is taken up and transported across the secretory cell. 4. sIgA is released into the milk. This process is similar to that described for the intestine on screen 20. 4. Antibody uptakesIgA and sIgM taken up by breast feeding are stable in the infant’s intestine. Remember that sIgA: · includes covalent bonds between the IgA dimer and SC · is more stable than sIgM Specific antibodiesIn cholera endemic areas the sIgA in breast milk is specific to antigens in: · lipopolysaccharide in the outer coat of V. cholerae O1 · cholera toxin These antibodies protect the baby against cholera by the effector mechanisms described (screen 21). Pathogens for which specific antibodies have been demonstrated in breast milk include: · V. cholerae O1 · enterotoxigenic E. coli · Campylobacter · enteropathogenic E. coli 1. InductionAntigens in the mother’s bowel: · are sampled in Peyer’s patches and solitary lymphoid follicles · are presented to helper T cells by APCs · cause specific B and T lymphocytes to become primed For details refer to screen 18. Image references ################ .\IMAGES\Entomamm.gif Development of Defence Mechanisms: AssessmentAre the following statements true or false? Click on the True or False button for each statement. Breast milk contains: To return to the start of the section. 1. a high concentration of IgG2. macrophages and PMNs3. antibodies to antigens in the mother’s environment4. a range of non-immunological protective factorsCorrect Text explaining the answer (11-pt plain blue) Incorrect Breast milk contains a high concentration of sIgA and some sIgM. IgG in the baby results primarily from transfer of maternal IgG across the placenta during pregnancy.Incorrect Text explaining the answer (11-pt plain blue) Correct Breast milk contains a high concentration of sIgA and some sIgM. IgG in the baby results primarily from transfer of maternal IgG across the placenta during pregnancy.Correct Colostrum and, to a lesser extent, later breast milk contain: · macrophages · PMNs · lymphocytes, mainly T cells Incorrect Text explaining the answer (11-pt plain blue) Incorrect Colostrum and, to a lesser extent, later breast milk contain: · macrophages · PMNs · lymphocytes, mainly T cells Correct Text explaining the answer (11-pt plain blue) Correct Breast milk contains antibodies produced by plasma cells from the maternal mucosal immune system. These plasma cells: 1. are primed in the mother’s bowel 2. migrate to the breast 3. secrete antibody in the mammary gland Incorrect Text explaining the answer (11-pt plain blue) Incorrect Breast milk contains antibodies produced by plasma cells from the maternal mucosal immune system. These plasma cells: 1. are primed in the mother’s bowel 2. migrate to the breast 3. secrete antibody in the mammary gland Correct Text explaining the answer (11-pt plain blue) Correct These protective factors include: · lysozyme · lactoferrin · peroxidase · mucins Incorrect Text 11 pt Arial dark blue goes here Incorrect These protective factors include: · lysozyme · lactoferrin · peroxidase · mucins Correct Text 11 pt Arial dark blue goes hereImage references ################ The diagram summarizes the humoral response to non-invasive pathogens and the innate inflammatory reaction to invasive pathogens. Correctly identify the components labelled 1 - 6 in the picture. Click your mouse on a box below.Hold the mouse down and drag the box to the correct number. LUMEN Non-invasive pathogen To return to the start of the section LAMINA PROPRIA 3. 2. Invasive pathogen 1. 6. Mast cell 1. 2. 3. 4. 5. 6.PMN 5. Macrophage 4. Blood vessel To return to the start of the tutorial. Tutorial AssessmentsIgA IgG Plasma cell Macrophage Vasoactive mediators Chemotactic molecules Yes. That's right. Yes. That's right. Yes. That's right. Yes. That's right. Yes. That's right Yes. That's right No. That's wrong. Try again. Well done. You have now finished this assessment.Image references ################ .\IMAGES\Immsumm.gif Summary Click on the buttons below for summary information. sIgASecretory component J chain Overview of Defence Mechanisms Non-immunological Mechanisms Humoral Immune Responses Cell-mediated Immune Responses Innate Immune ResponsesMucosal VaccinesDevelopment of Defence Mechanisms Copyright Image from The Wellcome Trust. Overview of Defence MechanismsIntestinal defence mechanisms can be classified as: · non-immunological protective factors · immunological mechanisms: - humoral immunity, eg. sIgA- cell-mediated immunity- innate immunity, including inflammatory reactions Immune responses to pathogens vary with the: · pathogen, eg. toxins, degree of invasion · host immunity, eg. previous exposure, malnutrition Specific adaptive responses. Non-immunological MechanismsProtective factors: · physically limit contact between the pathogen and bowel epithelium · chemically inactivate pathogens or their products Protective factors include: · saliva · mucus · gastric acid · commensal bacteria, ie. the normal bowel flora · bile · cryptdins · proteolytic enzymes · epithelial cell turnover · peristalsis Humoral Immune ResponsesSecretion into the lumen of sIgA and sIgM occurs by: 1. induction in Peyer’s patches and solitary lymphoid follicles 2. migration of primed B and T lymphocytes around the body 3. antibody secretion by a specific mechanism sIgA and sIgM work mainly by: · agglutination of luminal pathogens · formation of immune complexes with luminal antigens · neutralization of toxins, viruses and bacteria Serum derived IgG: · enters the lamina propria in small amounts · is proinflammatory, ie. activates complement, in contrast to sIgA Cell-mediated Immune ResponsesCell-mediated responses are possibly due to intraepithelial lymphocytes. These are: · a major population of lymphocytes · concentrated in small bowel villi · nearly all T cells · primed by antigen presentation from intestinal epithelial cells Innate Immune ResponsesInvasive pathogens evoke an acute inflammatory reaction with: · increased blood supply· increased vascular permeability · extravasation of PMNs and macrophages Innate immune responses include: · complement · activation of macrophages, PMNs and NK cells · degranulation of mast cells and eosinophils The innate inflammatory response: · interacts with the humoral immune system · is regulated by cytokines, chemotactic molecules and vasoactive mediators Mucosal VaccinesMucosal vaccines: · are being developed for many enteric pathogens, eg. rotavirus, V. cholerae O1, Shigella · aim to avoid the limitations of parenteral immunization · are introduced by a range of oral delivery systems · induce mucosal (eg. sIgA) and systemic (eg. serum IgG) immune responses Development of Defence Mechanisms Important aspects of the development of GI defence are: · transfer of maternal IgG across the placenta · transfer of maternal factors (eg. sIgA) in breast milk· development of the normal bowel flora Breast milk contains: · large quantities of sIgA· lesser amounts of sIgM· immune cells· non-specific protective factors · bifidus factor Section 1 Section 2 Section 3 Section 4 jpg image goes here Copyright Copyright Image from ....... (copyright info) (10 point Arial, blue) Section 1 Pop-up text is in blue with a yellow title in 10 pt bold left aligned.The pop up is a display icon within the library, and if the text is too long then a scroll bar should be used, pop-up boxes can be larger. Section 2 Pop-up text is in blue with a yellow title in 10 pt bold left aligned.The pop up is a display icon within the library, and if the text is too long then a scroll bar should be used, pop-up boxes can be larger. Section 3 Pop-up text is in blue with a yellow title in 10 pt bold left aligned.The pop up is a display icon within the library, and if the text is too long then a scroll bar should be used, pop-up boxes can be larger. Section 4 Pop-up text is in blue with a yellow title in 10 pt bold left aligned.The pop up is a display icon within the library, and if the text is too long then a scroll bar should be used, pop-up boxes can be larger.Image references ################ .\images\Lapaz.jpg .\IMAGES\Sigas.gif You have now finished the tutorial Defence Mechanismsã The Trustee of the Wellcome Trust, 1998 Further reading Further activities Restart tutorial Picture: Follicle associated epithelium in the mouse small bowel showing M cells (M) and enterocytes (E) with microvilli.M E Copyright Image from McI Mowat A. Further reading Brandtzaeg P. Molecular and cellular aspects of the secretory immunoglobulin system. APMIS 1995;103:1-19. Brandtzaeg P. The human intestinal immune system: basic cellular and humoral mechanisms. Baillieres Clin Rheumatol 1996;10:1-24. Brandtzaeg P, Berstad AE, Farstad IN et al. Mucosal immunity - a major adaptive defence mechanism. Behring Inst Mitt 1997;98:1-23. Bloom PD, Boedeker EC. Mucosal immune responses to intestinal bacterial pathogens. Semin Gastrointest Dis 1996;7:151-66. Lamm ME. Interactions of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 1997;51:311-40. Langermann S. New approaches to mucosal immunization. Semin Gastrointest Dis 1996;7:12-8. Shalaby WSW. Development of oral vaccines to stimulate mucosal and systemic immunity: barriers and novel strategies. Clin Immunol Immunopathol 1995;74:127-34. Toy LS, Mayer L. Basic and clinical overview of the mucosal immune system. Semin Gastrointest Dis 1996;7:2-11. Further activities To look at pictures related to this tutorial, search the image collection using the following keywords: · pathology · organismsImage references ################ .\IMAGES\T45476.jpg