Medical Microbiology and Immunology for Dental Students PDF

[Date] Medical Microbiology and Immunology For Dental students Microbiology I By Prof.Dr. Asmaa Mostafa El Gendy Professor of Microbiology & Immunology  ‫رؤية و رسالة الجامعة‬...

[Date] Medical Microbiology and Immunology For Dental students Microbiology I By Prof.Dr. Asmaa Mostafa El Gendy Professor of Microbiology & Immunology  ‫رؤية و رسالة الجامعة‬ ‫الرؤية‬ ‫تتطلع الكلية إلى أن تكون من أكثر الكليات تميزا على المستوى اإلقليمي والدولي‬ ‫في مجال طب الفم واألسنان والبحث العلمي بما يتناسب مع أخالقيات المهنه‬ ‫ومعايير الجودة‬ ‫الرسالة‬ ‫تهدف الكلية إلى إعداد أطباء أسنان يتميزون بالجدارة العلمية وقادرون علي مواكبة‬ ‫التطور العلمي واإلسهام فيه باالنشطة البحثية بما يلبي احتياجات المجتمع وسوق‬ ‫العمل المحلي والدولي‬ Vision The college aspires to be one of the most distinguished colleges at the regional and international levels in the field of oral and dental medicine and scientific research in line with professional ethics and quality standards. Mission The college aims to prepare dentists of professional merit who are able to keep pace with scientific development and contribute to it in research activities to meet the needs of society and the local and international labor market.  ‫الغايات واألهداف اإلستراتيجية‬ ‫الغاية األولى‪ :‬تحقيق قدرة تنافسية متميزة فى تعليم طب األسنان‬ ‫الهدف األول‪:‬‬ ‫تطوير إستراتيجيات التدريس والتعلم بما يتفق مع اتجاه الدولة المصرية لتطوير التعليم الجامعي‪.‬‬ ‫الهدف الثاني‪:‬‬ ‫تطوير المحتوى العلمي للبرنامج ونظم التقويم والكتاب الجامعي واألنشطة الطالبية لتنمية مهارات طالب وخريجي الكلية بما يتفق‬ ‫مع متغيرات سوق العمل‪.‬‬ ‫الهدف الثالث‪:‬‬ ‫استيفاء أعداد أعضاء هيئة التدريس والهيئة المعاونة بما يتناسب مع أعداد الطالب‪.‬‬ ‫الهدف الرابع‪:‬‬ ‫استخدام تكنولوجيا المعلومات وأساليب التعلم الحديثة‪.‬‬ ‫الغاية الثانية ‪ :‬التميز واإلبداع في مجال البحث العلمي‬ ‫الهدف األول‪:‬‬ ‫تحفيز منظومة البحث العلمي بما يدعم تقديم خدمات بحثية ذات تطبيقات عالجية تلبي حاجة المجتمع المحلي والدولي‪.‬‬ ‫الهدف الثاني‪:‬‬ ‫توسيع مجاالت التعاون والشراكة البحثية محليا واقليميا وعالميا‪.‬‬ ‫الهدف الثالث‪:‬‬ ‫تطوير البنية البحثية والتكنولوجية للكلية‪.‬‬ ‫الهدف الرابع‪:‬‬ ‫االلتزام بأخالقيات البحث العلمي وضمان حقوق الملكية الفكرية‬ ‫الهدف الخامس‪:‬‬ ‫تشجيع أعضاء هيئة التدريس والهيئة المعاونة على نشر األبحاث العلمية المحلية والدولية والحث على المشاركة العلمية في‬ ‫المؤتمرات‪.‬‬ ‫الهدف السادس‪:‬‬ ‫إنشاء برامج تعليمية لمرحلة الدراسات العليا تلبي احتياجات الخريجين في سوق العمل‪.‬‬ ‫الغاية الثالثة ‪ :‬التكامل مع المجتمع المدني لتقديم خدمات عالجية فى طب األسنان‬ ‫الهدف األول‪:‬‬ ‫التوسع في التعاون مع مؤسسات المجتمع المدني المحيط لتلبية احتياجات المجتمع‪.‬‬ ‫الهدف الثاني‪:‬‬ ‫التوعية التثقيفية المستمرة داخليا وخارجيا لتلبية احتياجات المجتمع المحيط بالرعاية الصحية لألسنان‪.‬‬ ‫الهدف الثالث‪:‬‬ ‫التطوير المستمر للخدمات العالجية بالعيادات الخارجية للكلية‪.‬‬ ‫الهدف الرابع‪:‬‬ ‫دعم برامج التواصل مع الخريجين‪.‬‬ ‫الغايــة الــرابعة‪ :‬التــ ُميز واإلبــداع الـمؤســسي‬ ‫الهدف األول‪:‬‬ ‫تطوير البنية التحتية والتكنولوجية للكلية‪.‬‬ ‫الهدف الثاني‪:‬‬ ‫تنمية قدرات القيادات االكاديمية واالدارية الحالية والمستقبلية‪.‬‬ ‫الهدف الثالث‪:‬‬ ‫تنمية قدرات اعضاء هيئة التدريس والهيئة المعاونة والجهاز اإلداري‪.‬‬  Strategic goals and objectives The first aim: achieving distinct competitiveness in dental education First goal: Developing teaching and learning strategies in line with the Egyptian state’s direction to develop university education. Second goal: Developing the program’s scientific content, evaluation systems, university book, and student activities to develop the skills of college students and graduates in accordance with labor market variables. Third goal: Fulfilling the numbers of faculty members and supporting staff in proportion to the numbers of students. Fourth goal: Using information technology and modern learning methods. The second aim: excellence and creativity in the field of scientific research First goal: Stimulating the scientific research system to support the provision of research services with therapeutic applications that meet the needs of the local and international community. Second goal: Expanding areas of cooperation and research partnerships locally, regionally and globally. Third goal: Developing the college’s research and technological infrastructure. Fourth goal: Commitment to scientific research ethics and ensuring intellectual property rights. Fifth goal: Encouraging faculty members and supporting staff to publish local and international scientific research and encouraging scientific participation in conferences. sixth goal: Establishing postgraduate educational programs that meet the needs of graduates in the labor market.  The third aim: Integration with civil society to provide therapeutic services in dentistry First goal: Expanding cooperation with surrounding civil society institutions to meet community needs. Second goal: Continuous educational awareness, internally and externally, to meet the needs of the community surrounding dental health care Third goal: Supporting alumni communication programs. Fourth goal: Commitment to scientific research ethics and ensuring intellectual property rights.. The fourth aim: Institutional excellence and creativity First goal: Developing the college’s infrastructure and technology. Second goal: Developing the capabilities of current and future academic and administrative leaders. Third goal: Developing the capabilities of faculty members, supporting staff, and the administrative staff. ‫عميد الكلية‬ ‫وحدة ضمان الجودة‬ ‫معيار التخطيط االستراتيجي‬  LIST OF CONTENTS Chapter 1: Introduction and bacterial structure………………………………......1 Chapter 2: Bacterial Growth and physiology ……………………………………11 Chapter 3: Bacterial genetics…………………………………………………….16 Chapter 4: Host parasite relationship……………………………………….........25 Chapter 5: Oral microflora and Dental plaque……………..……………33 Chapter 6: Introduction to immunology…………………………………38 Chapter 7: The acquired(specific) immune response…………………..45 Chapter 8: Immunogens and antigens………………………………………..47 Chapter 9: Lymphoid organs ………………………………….………..53 Chapter 10: Cells of the immune system………………………………. 55 Chapter 11: Antibodies……………………………………………….....68 Chapter12 :The Complement System……………………………………76 Chapter 13: Defense mechanisms of the mouth……………………........84 Chapter 14: Immunopathology…………….……………………………….......86 References……………………………………………………….….........91 General Bacteriology  Chapter 1 Introduction and bacterial structure Microbiology is the science that deals with living microorganisms or microbes that cannot be seen by the naked eye. In medical microbiology, we deal with microbes that are harmful and cause disease to man. The agent of human infectious diseases belongs to 5 major groups of organisms: Bacteria, fungi, protozoa, helminths and viruses. Viruses are quite distinct from other organisms as they are not cells but can replicate only within the host cells which provide the machinery for protein synthesis &energy generation Bacteria are prokaryotic cells. They are more simple and smaller than fungi and protozoa. Their genetic material is a single naked chromosome with no nuclear membrane, Surrounded by a rigid cell wall containing peptidoglycan. They contain 70S ribosome but no organelles and replicate by binary. Fungi & Protozoa are euokaryotoic cells contain a true nucleus with a nuclear membrane ,nucleoli,multiple chromosomes and organells such as mitochondria,lysosomes ,and 80S ribosomes Features Bacteria Fungi Diameter Approximately 1µm Approximately 4 µm Nucleus Prokaryotic (no nuclear membrane and no Eukaryotic (have nuclear nucleolus) membrane and nucleolus) Cytoplasm Mitochondria and endoplasmic reticulum are Mitochondria and endoplasmic absent reticulum are present Cell membrane Sterols are absent(except mycoplasma) Sterols are present Cell wall content Peptidoglycan Chitin Spores Endospore for survival not for reproduction Spores for reproduction 1  Bacteria are classified into orders each containing many families. Each family includes a variety of genera. Each genus comprises some species which may be further divided into Types. Individual organisms are recognized by their generic & species names. Example: Mycobacterium tuberculosis , Salmonella typhi Bacterial Morphology and Arrangement 1. Bacilli 2. Cocci 3- Spiral Bacterial Arrangement: Certain cocci occur in pairs (pneumococci),some in chains(streptococci) and others in grape-like clusters(staphylococci). Bacteria can be easily seen under the Microscope when stained by simple stains e.g.methylene blue or by differential stains e.g.Gram stain. 2  Bacterial cell structure Bacterial cell structure includes: The Cell wall Structures inside the cell wall: the cell membrane ,the cytoplasm, ribosomes ,and bacterial genome Structures outside the cell wall: capsules, fimbriae, flagella and spores. I-Bacterial Cell Wall It is rigid structure that maintains the shape of bacteria. It is the outermost component of bacterial cell It is common in all bacteria except mycoplasma It is osmotically insensitive , it protect the cell from bursting in hypotonic solutions General structure: The chemical composition of the cell wall differs considerably according to whether bacteria are gram positive or gram negative a) Gram positive bacteria: The cell wall of gram positive bacteria is formed of two layers : 1-Peptidoglycan : 3  It is a thick layer which constitutes up to 80% of the cell wall thickness It is made by N-acetylglucosamine and N-acetylmuramic acid. The entire cell wall structure is cross linked by covalent bonds. This provide the rigidity necessary to maintain the integrity of the cell. N-acetylmuramic acid is unique to prokaryotic cell. Components and structure of Gram positive bacteria 2-Teichoic acid(TA): Polymer of ribitol TA or glycerol TA Found in the cell wall and cytoplasmic membrane. It is the major surface antigen of gram positive bacteria Function of TA: * Antigenic determinant * Participate in the supply of Mg to the cell by binding Mg++ * regulate normal cell division. (b) Gram –ve bacteria: Components and structure of Gram negative bacteria 4  The cell wall of gram negative bacteria is composed of: 1- Thin peptidoglycan layer which constitutes up to 5-10% of Gram negative cell wall 2- Outer membrane : It is formed of two parts: -The inner part is bilayered phospholipids -The outer part contains distinctive component, the lipopolysaccharide. The lipopolysaccharide (LPS) consists of three parts: a-Inner lipid A (endotoxin of gram negative bacteria) b-Middle polysaccharide core c- outer polysaccharide side chain (somatic O antigen) -The LPS is the main inducer of septic shock 3- Periplasmic space : It is the space between the inner cytoplasmic membrane and phospholipids bilayerof the outer membrane. It contains 1-2 sheets of peptidoglycan layer and gell like solution of proteins. Function of the Cell Wall: a- Maintains the shape of bacterial cell b- Supports the weak cytoplasmic membrane c- Plays a role in the cell division d- Is responsible for passive diffusion of fluid and nutrients and for staining properties of the organisms. Cell wall-Deficient Bacteria: Naturally occurring: -Mycoplasma are the only bacterial species deficient in cell wall. Hence they are polymorphic (has no defined shape) and are not destroyed by penicillin -Protoplasts, Spheroplasts and L- form Bacterial cell wall may be removed by certain environmental conditions e.g. treatment with penicillin and lysozyme. If such treated cells are placed in osmotically protective 5 media, they liberate Protoplasts from gram positive cells and spheroblast from gram negative cells. If such cells are able to grow & divide they are called L-forms. Unlike mycoplasma They can revert to parental form on removal of the cell wall inhibitor. L form differ from parent bacteria in lacking rigid cell wall (vary in size and shape),but they are viable ,capable of growth and multiplication. L-forms may be formed during active infection under the effect of antibiotics and can be isolated from patients with chronic infection e.g. urinary tract infection Gram +ve Gram –ve Peptidoglycan layer Thick and outer Thin and inner Teichoic acid Present Absent Lipopolysaccharide Absent Present Toxins production Exotoxin Endotoxin Effect of antibiotics Protoplast Spheroplast II- Cytoplasmic Membrane -Just inside the peptidoglycan layer of the cell wall lies the cytoplasmic membrane. Structure: It is composed of a phospholipid bilayer similar in microscopic appearance to that in eukaryotic cells. -They are chemically similar but eukaryotic membranes contain sterols, whereas prokaryotes generally do not. -The only prokaryotes that have sterols in their membranes are members of the genus Mycoplasma Function: a. Selective permeability b. Transport electrons and protons for cellular metabolism c. Excretion of extracellular enzymes and toxins d. Secret hydrolytic enzymes 6  e. Plays an important role in cell division. f. Respiration :the cytochrome and respiratory enzymes are localized in the cytoplasmic membrane. It resembles mitochondrial membrane in euokaryotic cells. Mesosomes o A large invaginations of the cytoplasmic membrane, irregular in shape. o Increase in membrane surface, which may be useful as a site for enzyme activity in respiration and transport. o They function as the origin of the transverse septum that divides the cell in half and as the binding site of the cell DNA which duplicates to provide the genetic material of each daughter cell III- The Cytoplsm: The cytoplasm of the bacterial cell is a viscous watery solution or soft gel that contain a variety of organic and inorganic solutes Ribosome : Function :It is the site for protein synthesis They have sedimentation constant of 70S Intracytoplasmic granules o The cytoplasm contains granules which represent accumulation of food reserve. o They may be rich in lipids or carbohydrates. o The volutin granules rich in metaphosphate are found in the genus Corpebacterium. IV- The nuclear apparatus: o Bacterial nucleus is composed of a packed bundle of a double stranded DNA molecule (chromosome). o There is no nuclear membrane or nucleolus. 7  V- The capsule Present only in some bacteria and usually formed in vivo only. The capsule usually formed of polysaccharide or polypeptide or hyaluronic acid It has an antiphagocytic function so it determine the virulence of many bacteria It also plays an important role in attachment of bacteria to the mucous membrane The capsule can not be visualized by the ordinary stain and need a special capsular stain. VI- Flaglla: It is the organ of motility It is short and hair like projections It arises from the cytoplasm and extrude through the cell wall Structure of the flagellum: protein in nature: flagellin VII-Pili or Fimbriae Shorter than flagella and straighter , smaller. Only on some gram-ve bacteria. Function: adherence. One of the invasive mechanism on bacteria. Some pathogens cause diseases due to this. If a mutant has no fimbriae it is not virulent. Origin: Cell membrane Position: Common pili , numerous over the cell, usually called fimbriae Sex pili 1-4/cell Structure: composed of proteins which Pilin. 8 Sex pili : It is necessary for bacterial conjugation resulting in the transfer of DNA from one cell to another. VIII-Spores Some bacterial genera are capable of forming highly resistant resting phase or endospores, e.g. Bacillus group and clostridia group. The spore is formed outside the body and cannot be stained by ordinary stains. Spore is highly resistant to dryness, heat and disinfectants, but can be destroyed by certain methods of sterilization. Once formed, the spore has no metabolic activity &can remain dominant for years. Upon exposure to water &appropriate nutrients, germination to vegetative form occurs. Question I-MCQ: 1-Which of the following components is present in gram-positive bacteria but not in gram- negative bacteria? a. peptidoglycan b. capsule c. flagella 9 d. teichoic acid e. pili 2- Each of the following statements concerning Flagella is correct EXCEPT: a.Are the organs of motility in all motile bacteria. b.Are shorter and thinner than pili. c.Are composed of a single protein; flagellin. d.Specific antisera can be prepared, against flagella antigens. 3-Cell wall of Gram-negative bacteria have the following characters Except: a. Composed of inner layer of peptidoglycan. b. The peptidoglycan provides structural support. c. The peptidoglycan is less thicker than in Gram-positive. d. Some bacteria have layer of teichoic acid. 4. Which of the following components is present in gram-negative bacteria but not in gram-positive bacteria? a. peptidoglycan b. lipid A c. capsule d. flagella e. Pili II-Enumerate 4 differences between bacteria (prokaryotic cells) and fungi (euokaryotic cells). 10  Chapter 2 Bacterial Growth Growth involves both increase in size of the organism and increase in the number of individuals making up a population or a culture. Generation time (doubling time): Bacteria reproduce by binary fission, a process by which one parent cell divides to form two progeny cells. The doubling (generation) time of bacteria ranges from as little as 20 minutes for Escherichia coli to more than 24 hours for Mycobacterium tuberculosis.The doubling time varies not only with the species but also with the amount of nutrients, the temperature, the pH, and other environmental factors. Bacterial Reproduction Bacteria reproduce by binary Fission. Method of bacterial reproduction: cell divides exactly in half single cell division reproduction of the entire organism. 11  Bacterial chromosome is a double stranded DNA molecule which is circular and wound on itself to form the nuclear mass. Before cell division the 2 strands of the chromosome separate, each strand is attached to a mesosome and acts as a template on which a complementary strand is formed by the action of DNA polymerase enzyme. Cell division follows chromosomal replication by formation of a transverse septum between the attachment sites. Each daughter cell will contain an identical copy of the parent chromosome Bacterial Growth Curve If a small number of bacteria are inoculated into a liquid nutrient medium and the bacteria are counted at frequent intervals and the results plotted, a characteristic growth curve with four phases is obtained: 1-Lag phase: It is the first phase during which no cell division occurs. The bacteria adapt to the new environment by formation of new enzymes and macromolecules needed for replication Bacterial Growth Curve 12 2-Logarithmic phase: During this phase, rapid cell division occurs and the number of bacterial cells increases steadily by time. Many antibiotics are effective during this phase, e.g. beta lactam drugs, such as penicillin, which act when the cell is making peptidoglycan. 3-Stationary phase: As the nutrients in the medium are exhausted and toxic products accumulate, the rate of growth decreases. The number of dying cells equals that of newly formed cells. The number of living bacteria remains constant. 4- Decline phase: As the exhaustion of nutrients and accumulation of toxic products continue. The death rate exceeds the multiplication rate and the number of living bacteria decreases steadily. Growth requirements Bacteria –like all cells- require nutrients for maintenance of their metabolism and for cell division. Growth requirements include: I-Nutritional elements: 1- Carbon and Nitrogen: These substances are essential for synthesis and maintenance of bacterial growth. According to carbon requirement, bacteria are subdivided into two groups: a-Autotrophs: These are bacteria which can utilize simple inorganic substance, e.g. CO2 as a source of carbon and ammonium salts as a source of nitrogen; from which they synthesize organic substances, e.g. proteins, carbohydrates. 13  b- Heterotrophs: These bacteria require complex preformed organic substances, e.g. sugars, proteins etc..., which are derived from plant or animal sources. All bacteria of medical importance are heterotrophs. They live in or on the animal body and are called parasitic bacteria. Many of the heterotrophs can grow on simple media containing peptone or meat extract as in broth and nutrient agar. Others require complex organic material, e.g. blood, serum, etc... Media containing such substances are called enriched media. 2-Growth Factors or Essential Metabolites Many pathogenic species of bacteria require for growth essential substances –in small amounts- which they cannot synthesize, e.g. vitamins, amino acids, purines and pyrimidines. These have to be added to culture media. However, these substances are present in host tissues 3-Inorganic ions: Small amounts of inorganic ions are required by all bacteria e.g. sulpher, phosphorus ,potassium ,magnesium and calcium. II. Gaseous Requirements: On the basis of oxygen requirement ,bacteria are divided into: 1-Obligate aerobes: Grow only in the presence of oxygen e.g. M.Tuberculosis and pseudomonas aeruginosa. 2-Obligate anaerobes: Cannot multiply in the presence of oxygen often killed by traces of oxygen in their environment 3-Facultative anaerobes: Grow best with oxygen, but can grow without oxygen. They use O² to generate energy by aerobic respiration if it present. They undergo aerobic respiration if oxygen is available and anaerobic respiration if no oxygen is present. 14  4- Micro-aerophilic bacteria: These require for growth a low oxygen tension, higher concentrations are inhibitory. III. Physical requirements 1-Temperature for Growth Most medically important species grow at a range of temperature between 25- 40°c. Optimum growth occurs at 37°c, i.e. the normal body temperature. Non-pathogenic bacteria may grow at temperature lower than 20°c or higher than 55°c. 2- Hydrogen Ion Concentration (pH) Most pathogenic species of bacteria can grow at a narrow range of pH7.2- 7.6.However, few species, e.g. V.cholerae grow at an alkaline pH 8 and lactobacilli prefer an acid pH 4 Questions 1-In which of the phases would growth not be detectable? a-lag phase b- death phase c- log phase d- stationary phase 2-In which stage of growth is there a slow loss of cells through death that is just balance the formation of new cells through growth and division. a-lag phase. b- logarithmic phase. c-stationary phase. d- decline phase. 3-In which of the phases a rapid cell division occurs and the number of bacterial cells increases steadily? a-lag phase. b- logarithmic phase. c-stationary phase. d- decline phase. 15  Chapter 3 Bacterial genetics Genetics is the study of the different characters from parents to offspring. Genes: are units of heredity. They are segments of DNA that carry information for specific functions or structures. Genome: is the sum of genes of an organism.The bacterial genes are carried on the chromosome, plasmids,transposons and prophages. I.Bacterial Chromosome It consists of a single molecule of double stranded DNA which is circular and wound on itself to form a double helix. Chemically, it is composed of a backbone of phosphate and sugar (deoxyribose) alternating with one another; to which are attached the purine and pyrimidine bases; guanine, adenine, cytosine and thymine. The two strands are not identical but they are complementary, they held together by hydrogen bonds between neighboring bases. The bonds are formed only between adenine and thymine (AT) or guanine and cytosine (G-C). 16  Thus, a sequence of bases along one strand must be matched on the opposite strand by a complementary sequence. The chromosome is functionally subdivided into segments or genes. The sequence of nucleotides in a gene determines the amino acid sequence and hence, the structure of a discrete protein, e.g. an enzyme, a cell wall component etc. Genes essential for bacterial growth are carried on the chromosome. Few genes associated with specialized functions are carried on plasmids. The bacterial chromosome replicates by semiconservative method of DNA replication in which the two strands are separated and each strand acts as a template to synthesize a complementary strand. II.Plasmids -These are extrachromosomal double stranded circular DNA molecules -That are capable of replicating independent of the bacterial chromosome -Some genetic properties in the bacterial cell are carried on plasmids. -These properties are not essential for growth. -Some plasmids can transfer to other bacteria of the same or different species. -Transfer takes place normally by conjugation. -Multiple copies of the same plasmid may be present in each bacterial cell. -Different plasmids may coexist within the same bacterium. -They are inherited by daughter cells. 17 Properties of plasmids: a-Self transfer: These are plasmids of gram negative bacteria. They can transfer themselves to other bacteria of the same or different species by production of sex pili through which genetic material can transfer,a process called conjugation. Plasmids of Gram positive bacteria are non transmissible plasmids. b-Recombination:plasmid DNA can be integrated into bacterial chromosome It is known as episome. Cell Properties Determined by plasmids: 1-Drug resistance: genes coding for drug resistance for many bacteria are carried on plasmid(R-plasmid) which code for enzymes that degrade antimicrobial drugs, e.g. β-lactamase enzyme that degrade penicillin and chloramphinicol acetyl transferase that inactivates chloramphinicol. 2-Virulance: some plasmids carry genes whose products are virulence factors of some bacteria ,e.g. Tox plasmid coding for enterotoxin production in entrotoxigenic E.coli. 3-Bacteriocin production: Bacteriocins are antibiotic-like substances produced by certain bacteria of the same or closely related species,e.g.col factor plasmid of E.coli that produces colicin substance. 4-Sex pilus formation:some plasmids carry genes (tra genes) that code for the formation of sex pilus which mediates the process of gene transfer between bacteria (conjugation). 5-Other functions :Antibiotics production (e.g. streptomycin produced by streptomyces),sugar fermentation, and resistance to heavy metals III.Transposons ‟jumping genes” ▪ These are non-replicating pieces of DNA that move readily from one site to another either within or between the DNAs of bacteria, or from plasmid to plasmid or from plasmid to bacterial chromosome ▪ Due to their unusual ability to move, they are called‟jumping genes”. ▪ Simple insertion sequences: They are simple type of transposon that have fewer bases (800-1500 base pair). They carry genes coding for enzymes responsible for its transposition 18 ▪ Complex transposons: They carry genes coding for enzymes responsible for its transposition plus antibiotic resistance or toxin production gene. Characters: ▪ In contrast to plasmids or bacteriophages, transposons are not capable of autonomous replication and they do not exist in the free state; hence they replicate as part of the recipient DNA. Bacteriophage Bacteriophages are viruses that can infect and destroy bacteria. Head formed of protein capsid which enclose phage DNA. Retractile body: that contracts to transport DNA to bacteria cytoplasm. Base Plate. Tail fibers: used for attachment of the phages to cell wall receptors. 19  Types of Bacteriophage ▪ Lytic or virulent phage: Phage that can only multiply within bacteria and kill the cell by lysis. ▪ Lysogenic or temperate phage: Some phages “temperate phage” fail to replicate or lyse the cell they infect. Their DNA is integrated with the bacterial chromosome and it passes to daughter cells as the bacteria divide. The part of the phage DNA integrated in the bacterial chromosome is called the “prophage”. The bacteria carrying the prophage are called“ lysogenic” bacteria Importance of Bacteriophages ▪ Gene transfer: transfer of genetic information from one bacterial strain to another by temperate phage (transduction). ▪ Lysogenic conversion: in which phage’s own DNA confer new properties to the cell, e.g. erythrogenic toxin production by Streptococcus pyogenes and toxigenicity of Corynebacterium diphtheria. ▪ Bacterial typing: phage typing is one of the method used to identify bacteria Bacterial Variation Bacterial variation may be phenotypic or genotypic variation. A genotype is the set of genetic determinants within the cell. A phenotype is the observable structural and physiologic properties of the cell. Phenotypic Variations Are changes in bacterial characters under the influence of the environment with no underlying genetic changes. Genotypic Variation Is a heritable irreversible variation due to changes in genetic constitution. 20  Genetic variation occurs through: ▪ 1- Mutation. 2- Gene transfere 1-Mutations: A mutation is a change in the base sequence of DNA that usually results in insertion of a different amino acid into a protein and the appearance of an altered phenotype. Mutations result from three types of molecular changes: Mutation may be: a-Spontaneous: occurs as a result of error of replication b-Induced by: physical agents ,e.g. visible light, ultraviolet rays or gamma rays, or by chemical agents as alkylating agents and nitroso compounds. N.B: Induced mutation is used to manipulate microbial genome to get mutant of low virulence that can be used as vaccine, or mutant producing large amount of antibiotics. 2- Gene transfer: There are 3 types of gene transfer: Transformation, Conjugation and Transduction a-Transformation (Uptake of pure naked DNA): This occurs when a recipient cell takes up a fragment of bacterial DNA, present free in the surrounding medium. There must be a homology between donor and recipient bacteria. If the DNA is completely unrelated, the absence of homology prevents recombination and the DNA is degraded. If the DNA fragment recombined with bacterial DNA can propagate new character,e.g. virulence factor. 21 b-Cojugation : It is the mating of 2 bacterial cells, of the same or different species during which DNA is transferred from the donor to the recipient cell. The transfer of DNA among bacteria by direct contact through sex pilus. This process is controlled by sex factor or F factor carried by plasmids. Bacteria cell that contain this plasmid is called F+ cell,and the other cell is called F- cell. Conjugation occurs when two cells come in contact. C- Transduction: Fragments of chromosomal DNA can be transferred or transduced into a second bacterium by bacteriophage Genetic engineering Genetic engineering means modification of genotypes of organisms by incorporation of new genes from entirely different species, allowing the manufacture of a genetic material in the laboratory. Gene cloning: Insertion of genes (DNA segments) coding for certain proteins into a vector and the subsequent propagation of the recombinant DNA molecule in a host organism by gene cloning to form new combinations. Cloning Vectors: These are vehicles used to carry and induce foreign DNA fragments into a host cell. They include: Plasmids, bacteriophages , viruses (e.g. retroviruses, adenoviruses). 22 Recombinant DNA Technique: Chromosomal DNA from any desired source is extracted and cleaved by a specific restriction endonuclease to produce the “insert“. 1. The vector, e.g. plasmid is cleaved by the same endonuclease, used in step 1. 2. The “insert” and the plasmid are mixed under conditions 3. The DNA ligase enzyme is added to seal the gaps. 4. This results in the recombinant plasmid (plasmid + insert). 5. By a process of transformation, the recombinant plasmid is introduced into a suitable host cell in which it can autonomously replicate. Application of genetic engineering: 1- Production of substances as hormones (insulin) and recombinant vaccines (hepatitis B vaccine). 2- Diagnosis of diseases through the use of single stranded DNA or RNA labeled with radioactive isotope or enzyme which used in hybridization experiment to detect the presence of a complementary sequence of microbial genes in clinical samples. 3- Chromosomal mapping and DNA sequencing this means the determination of location of genes in any organism ҆s genome i.e. to know the exact sequence of nucleotide bases in DNA. 4- Gene therapy which involves insertion of a normal gene into human cells to replace a specific genetic disorder. 23  Questions 1-Each of the following statements concerning types of gene transfer are correct EXCEPT: a. Transformation b. Mutation c. Conjugation d. Transduction 2-The mode of gene transfer in which purified DNA is directly taken up by the recipient cell is known as: a. Transformation. b. Transduction. c. Conjugation. d. Cell fusion. 3-Which of the following characterizes plasmid? a.Single-stranded DNA molecules b.Capable of replicating independently of the bacterial chromosome c.Are always found in linear form d.Cannot integrated into bacterial chromosomes 4-Each of the following statements concerning bacterial chromosome are correct EXCEPT: a. It is double stranded DNA molecule. b. Genes essential for bacterial growth are carried on the chromosome. c. Few genes associated with specialized functions are carried on plasmids. d. Chromosome is double stranded DNA molecule smaller than the plasmids 5-These properties concerning transposons Except: a. Move freely through plasmids & chromosomes. b. Exist in free state. c. Cannot replicate autonomously. d. Inserted randomly in different DNA. 24  Chapter 4 Host parasite relationship A microorganism is a pathogen if it is capable of causing disease, however ,microorganisms vary in their degree of pathogenicity Parasitic bacteria are those which live in or on a living host. It can multiply in the host tissues causing disturbance of physiologic functions , thus leading to disease and are called pathogenic bacteria Saprophytic bacteria are those which live freely in nature on decaying organic matter, in soil or water. Normal flora is the term used to describe the various bacteria and fungi that are permanent residents of certain body sites. Significance of normal flora -They can cause disease, especially in debilitated and immunocompromised individuals. They can be pathogens in other parts of the body. -The nonpathogenic resident bacteria occupy attachment sites on the skin and mucosa that can interfere with colonization by pathogenic bacteria (colonization resistance). -The intestinal bacteria produce several B vitamins and vitamin K. Commensal bacteria: Are parasitic bacteria live on the external or internal surfaces of the body without causing disease. They constitute the largest group of normal body flora. These organisms may even be beneficial to the host, e.g. commensals in the gut digest polysaccharides and are a source of certain vitamins. They also compete with pathogenic organisms for nutrition thus inhibit their growth. Under certain conditions, commensal bacteria may cause disease and should be considered as potential pathogens or opportunists. 25 Some of these conditions are: 1-Lowered host defense mechanisms e.g. immunosuppressed, diabetics or leukemic patients. 2-Alteration of the host tissues, e.g. strept viridans, a normal inhabitant of the mouth and throat may cause endocarditis after tooth extraction if the host has a predisposing heart lesion. 3-Change in the natural habitat of the organisms, e.g. E. Coli is a normal inhabitant of the intestine. If it reaches the renal system through blood or lymphatics, it causes urinary tract infection Infection and Disease Infection is the process by which the parasite enters into a relationship with the host. Its essential steps are the following: 1-Entrance of the parasite into the host. Portals of entry include: -Gastrointestinal tract. -Respiratory tract (mouth and nose). -Genitourinary tract. -Skin and mucous membranes; through abrasions, insect bites or injections. 2-Multiplication of the parasite within the host: The parasite may multiply locally at the portal of entry or it may spread through the tissues, blood or lymphatics to reach a target organ. For the organism to be propagated in nature, it should have: 3-Mode of transmission to a new host by insects bite, faecal contamination of food, inhalation of droplets, sexual contact, blood transfusion. 4-Portal of exit from the host, e.g. in urine, stools, respiratory or genital discharges or from the blood by insects or injections. 26 Carriers -A carrier is an apparently healthy individual who carries a pathogenic organism without showing clinical manifestations. -He can transmit this organism to other susceptible individuals. Carriers can be classified as follows: According to the site of carriage: 1-Urinary or intestinal salmonella carriers. 2-Nasopharyngeal meningococcal carriers. 3-Throat (oropharyngeal) diphtheria carriers. 4-Nasal, skin or throat staphylococcal carriers. B- According to the duration of carriage: 1-Transient carriers: e.g. during the incubation period (incubatory carriers) or early convalescence. 2-Permanent (chronic) carriers: e.g. hepatitis B.virus carrier. C- According to the mode of discharge of the organism: 1-Intermittent carriers. 2-Continuous carriers. Factors that Govern Disease Production: Only few infections end in clinically manifest disease. Most infections are abortive, silent or subclinical. The outcome of infection depends on interactions between: 1-Microbial factors (pathogenicity and virulence). 2-Host resistance factors (natural and acquired immunity). 27 Microbial Factors: Pathogenicity It is the ability of an organism to cause disease. Virulence -It is the degree of pathogenicity -In a pathogenic species of bacteria, e.g. C. diphtheria, some strains are highly virulent producing severe disease. Others are moderately virulent producing mild disease. While others are avirulent, i.e. unable to produce disease. This depends on the ability of different strains to produce a toxin and on its potency. Virulence Factors These are certain structures or products that help microorganisms to overcome body defense mechanisms and cause disease 1-Adherence factors: Certain bacteria have specialized structures or substances that help their adhesion to host cell mucous membranes thus allow them to start the disease process. Mutants that lack these structures are often avirulent. For example, the pili of N. gonorrhoeae and Esch. Coil mediate the attachment of the organisms to the urinary tract epithelium. Lipoteichoic acid found on the fimbriae causes adherence of the streptococci to buccal mucosa. Glycocalyx of Staph.epidermidis and some streptococci allows their adherence to the endothelium of heart valves. 2-Invasive factors This is the ability to invade tissues, multiply and spread rapidly causing the inflammatory process. This may be partly due to the antiphagocytic action of certain surface components that protect the bacteria from phagocytosis and destruction: 28  a-The capsule of many organisms e.g. pneumococci. b-The “M” proteins found on the fimbriae of Strept. Pyogenes. c-Protein A of Staph. Aureus 3-Ability to survive intracellular: This is a property of some organisms which use several different mechanisms to allow them to survive and grow intracellularly escaping extracellular killing by phagocytic cells. Examples are: M. tuberculosis , Legionella &Listeria 4-Extracellular enzymes: These are substances produced by some bacteria that help the spread, invasion and establishment of microorganisms into the tissues. These include: Collagenase is produced by Cl. Perfringens. It breaks down collagen fibers and promotes spread of infection. b- Lecithinase is produced by Cl.perfringens. It breaks down lecithin a constituent of cell membranes. c- Coagulase is produced by Staph. Aureus. It coagulates plasma with deposition of a fibrin wall around staphylococcal lesions which helps them to persist. It also causes deposition of fibrin on the surface of the individual organisms protecting them from phagocytosis. d-Fibrinolysin (streptokinase) is produced by Strept. Pyogenes. It causes lysis of fibrin clots and assists in spread of organisms in tissues. e- Hyaluronidase splits hyaluronic acid; a constituent of the cement substance of connective tissue. It helps spread of infection and is produced by streptococci and clostridia. f- Deoxyribonuclease (streptodornase) is produced by Strept. Pyogenes and clostridia. It breaks down DNA &facilitates spread of organisms. 29  g- Immunoglobulin A (IgA) protease: It is produced by N. gonorrhoeae, N.meningitis , H.influenzae and Strept. Pyogenes It degrades the secretary IgA on mucous surfaces and thus eliminates protection of the host by antibody 5- Toxin production: Toxins are bacterial products which have a direct harmful action on tissue cells. They fall into two groups: a- Exotoxins b- Endotoxins a-Exotoxins They are protein toxins. Secreted by living bacteria Diffuse freely into the surrounding medium i.e. extracellular toxins. The production of most exotoxins is controlled by genes in plasmids or bacteriophages rather than by chromosomal genes. b- Endotoxins These are integral part of the cell wall of gram negative bacteria from which they are liberated when the cell dies and disintegrates. The toxicity of the endotoxin is associated with the lipid A fraction of LPS. Organisms producing endotoxins include Salmonella, Shigella, E. Coil, and V. choierae. Endotoxins are the most important cause of endotoxic or septic shock. All endotoxins produce generalized non-specific toxic effect of fever, shock, hypotension, disseminated intravascular coagulation (DIC) and sometimes death due to massive organ failure. Endotoxins cause these effects through activation of: a- macrophages to produce IL-I, TNF-α and nitric oxide which cause the fever, tissue damage, hypotension and shock. b- complement which causes bradykinin-induced vasodilatation, increased vascular permeability, hypotension and shock. 30  c- Hageman factor; an early component of the coagulation cascade causing DIC, resulting in thrombosis, purpuric rash, and tissue ischemia Exotoxins Endotoxin -Protein in nature -Lipo-polysacharide -Heat Labile -Heat stable -Secreted by cells -Part of the outer membrane (Lipid A) -Separable by filtration -Obtained by cell lysis -Specific effect -Effect non-specific -Highly antigenic -Weakly antigenic -Mainly produced by Gram positive -Produced only by Gram negative bacteria bacteria Questions 1-Enumerate 5 differences between endotoxin and exotoxin 2-Mention 3 virulence factors of bacteria 31  Chapter 5 The Oral Microflora and Dental plaque The oral flora is one of the most ecologically diverse microbial populations known to man. This flora exists, normally, in harmonious relationship with the host. This relationship can be distributed by any changes to the habitat, e.g. xerostomia or the use of broad spectrum antibiotics, and may lead to mucosal infections and increase the prevalence of both dental caries and periodontal disease. Composition and acquisition of the oral microflora -Although during birth the neonate comes into contact with the microflora of the mother ҆s vagina, these organisms do not usually become established and the oral cavity of the fetus is sterile. -From the first feeding, microorganisms are transferred from the surrounding environment such as maternal saliva or the skin flora of the mother and nursing staff. -By 24 hours after birth ,the first species have become established. Stereptococci , particularly S.salivarius which bind to modify their environment by producing extracellular products , which enhance conditions for growth of other species. -By one year of age teeth have erupted , the predominant species isolated are streptococcus species , Neisseria spp., Veillonella spp. and Staphylococcus species. Less frequently isolated species include, Lactobacillus, Actinomyces,Prevonella and fusobacterium. -Tooth surface and gingival tissues provide new habitats for colonization, with resultant formation of dental plaque. -Other shifts in the microbial flora take place during the lifetime of an individual for example ,only 18-40% of 5 year olds have Spirochaetes and black pigmenting anaerobes, compared with 90% of 13-16 year olds. From approx.70 years age there is an increased proportion of lactobacillus and staphylococcus species in saliva of non denture wearers , -After 80 years of age the number of yeast increases. 32  Dental plaque Definition: Dental plaque is a general term for the complex microbial community found on the tooth surface, embedded in the matrix of polymers derived from bacteria and saliva. Formation: жThe formation of pellicle is the first step in the plaque formation.The pellicle comprises mucins, salivary glycoproteins , minerals and immunoglobulins. -Following pellicle formation , there is passive transport of oral bacteria to the tooth surface. Microorganisms are generally transported passively to the tooth surface by the flow of saliva ; few oral bacterial species are motile (e.g. posses flagella), and these are mainly located subgingivally. -Initial adhesion: At distances of 10-100 nm , weak reversible forces come into effect.As the bacterium approaches closer(2nm) ,strong forces come into play. -Attachment: Following initial adhesion, a more permanent attachment can occur by bond formation between specific receptors on the host surface (ligands ) and components on the bacterium (adhesins). -Colonization and biofilm formation: once bound to a surface ,the bacterium can divide and remain attached.Extra-cellular products are formed and daughter cells repeat the process so that microcolonies coalesce, producing a more complex three dimensional arrangement.This is due to coaggregation between similar species and between different bacterial species. -Plaque maturation is characterized by increasing quantity and diversity of microorganisms on the tooth surface. After 7 days Streptococci are still the main organism present ,but after 14 days there is a shift to anaerobic rods and filaments , with Streptococci comprising only 15% of the cultivable flora. Significance of plaque as a biofilm: Oral bacteria growing as biofilm such as dental plaque display a markedly reduced sensitivity to antibiotics and antimicrobial agents, including those used in tooth pastes and mouthwashes. 33  -The mechanisms that cause the increased tolerance of biofilm cells to antimicrobial agents include: a)Limited penetration , b) inactivation (e.g. by neutralizing enzyme ) c)Unfavorable environmental conditions, d) slow microbial growth rates, and (e) expression of a novel microbial phenotype. Rough surfaces allow more plaque to adhere because of the increase surface area and protection from the forces of removal. Water repelling surfaces e.g. teflon binds fewer microorganisms than water attracting e.g. enamel. In order to minimize plaque accumulation, intra oral devices should be designed with water repelling surface. Anti-plaque agents: Three main approaches have been utilized for chemically interfering with the formation of dental plaque. 1-Antimicrobial agents -Currently the most effective antiplaque agent is chlorhexidine. -The molecule has a positive charge at either end and binds to negatively charged sites on the enamel pellicle, mucosal cells and bacterial cell wall structures. -It can exert its antimicrobial effect by damaging the microbial cell membrane and precipitating the cell contents. -Chlorhexidine also inhibits microbial adherence since it is able adsorb onto a surface and is slowly released ,maintaining its antimicrobial activity. 2-Plaque disruption agents: -Sodium lauryl sulphate is used commonly in tooth paste and mouth rinses as an anionic detergent with solubilizes plaque to reduce its accumulation. -In addition, it has a moderate degree of substantivity and antimicrobial activity ,although much less than chlorhexidine. 3-Anti-adhesive compounds: -These compounds are designed to alter the surface binding within the oral cavity, thus preventing interactions between bacteria and the oral environment. -Fluoride containing compounds are reported to have antiadherent properties, although the mechanism of action is uncertain. 34 Ж Factors affecting the growth of oral microorganisms: -The oral flora is influenced by a wider range of factors which may be associated with the diet, saliva gingival, cervical fluid ,microbial products and host factors. 1-Diet: The presence of fermentable carbohydrate e.g. sucrose, glucose and lactose will lead to increased plaque formation and the accumulation of microbial products. Thus , frequent eating of sucrose is associated with high caries activity. The physical consistency of large molecules e.g. proteins restrict availability to bacteria, since they may be removed from the mouth before they have been degraded. 2-Saliva: -The mean PH of unstimulated saliva is 6.75-7.25. -A regular decrease in PH by frequent sugar intake will lead to the proliferation of Strept.mutans and Lactobacillus species and increase in dental caries. -Changes in the flow rate will affect the concentrations of the ions bicarbonate, calcium and phosphate, which are of great importance to the balance of mineralization and demineralization. -Saliva also contains numerous antimicrobial factors such as lysozyme (lyses some bacterial cell walls,lactoferrin (binds iron which bacteria need for growth ), salivary peroxidase system (inhibit bacterial cell metabolism) and immunoglobulins. 3-Microbial interactions: -Extracellular end products produced by one species may be essential for the growth of another species. Conversely ,metabolic products e.g. hydrogen peroxide produced by strept.oralis group may inhibit other bacteria. 4-Gingival cervical fluid This is a serum transudate which contains proteins e.g. albumin and immunoglobulins as well as amino acids ,minerals,vitamins and glucose. It has protective functions for the host by its flushing effect and high numbers of viable polymorphs, antibodies and complement proteins. 5-Gaseous environment -Most oral microorganisms are facultative anaerobes, however ,some organisms are obligate anaerobes and are killed by oxygen. 35  -Early colonizers of plaque utilize O2 and produce Co2 ,thus allowing bacteria e.g. anginosus group of streptococci, to become established. -late colonizes of plaque will produce H2 and other reducing agents which will gradually lower the oxygen tension, allowing more anaerobic bacteria ,e.g. Prevtella spp., to colonize. 6-The host -Host factors such as presence of systemic disease, broad spectrum antibiotic usage, or chemotherapy for cancer, may disturb the host / microbial flora interactions. -One of the easiest ways the host can influence the oral flora is the use of oral hygiene methods, e.g. tooth brushing. -This produces a persistently young plaque containing many facultative anaerobic Gram positive bacteria with a limited numbers of obligate anaerobes and flora which is compatible with oral health. Questions 1-Define dental plaque and describe its formation 2-Enumerate 5 factors that affect the growth of oral microorganism 36 Immunology 37  Chapter 6 Introduction to immunology The main function of the immune system is to prevent or limit infections by microorganisms such as bacteria, viruses, fungi and parasites. This defensive function is performed by various cellular & humoral components which interact with each other producing a coordinated immune response directed towards eliminating the pathogen or minimizing the damage it causes. The immune system is divided into 2 functional divisions, namely the innate immune system and the acquired immune system. Innate immunity acts as a first line of defense against infectious agents which are checked before they cause overt infection. If these innate defenses fail, the acquired immune system is called upon. Definition of the innate immunity Non-specific immune system and first line of defense. It comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. Innate Immunity is the 1st line of defense, It includes external and internal barriers. The external defenses (barriers) ( physical, chemical, physiological and biological). Components of the innate immune response: Cells: phagocytes, natural killer cells, mast cells. Soluble molecules: mainly proteins present in serum and extracellular fluids e.g. complement components and cytokines 38  Physical and chemical barriers of the innate immunity: A-Physical barriers: 1-The skin: The epidermis consists of several tightly packed epithelial cells with the outermost layer consisting of dead cells filled with waterproof protein (keratin). Sweat and sebaceous glands are present in the dermis. This architecture prevents penetration of microorganisms into deeper tissues while release of dead skin cells removes them from the skin surface. Sweat and sebum secretions render skin pH unfavorable for pathogens. 2-The mucus membranes: Cover surfaces of many areas exposed to the environment. Several protective mechanisms exist: The thin layer of the sticky mucus entraps microorganisms. The cilia in respiratory epithelium propel mucus entrapped organisms towards the throat where it is swallowed or cleared by sneezing or coughing. 3-The secretions e.g. saliva, tears: wash away potential invaders and contain antimicrobial substances e.g. lysozyme. 4-Nasal hairs: add to the mechanical defense. 5-The urine flow: reduces microbial populations of the urethra B – Physiological barriers 1-Temperature: many species are not susceptible to certain infections simply because their body temperatures inhibit pathogen growth. 2- PH: gastric acidity, high acidity of vagina is inhibitory to microbial growth. 3-Bile from gallbladder (alkaline) together with the enzymes in the small intestine can kill different microorganisms. 39  C – Biological barriers The normal flora that tends to colonize the skin and some areas of the mucus membranes competes with pathogens for attachment sites and for essential nutrients, some produce inhibitory substances e.g. colicins. Suppression of normal flora by antibiotics may lead to superinfection with potential pathogens. Breaching surface barriers e.g. cuts, wounds, smoking, exposure to air pollutants, alcohols, narcotics etc. can suppress this line of defense and allow entry of organisms into the tissues. Innate Immune Response Cells of the innate Immune response: a-Macrophages are large cells with specialized granules (lysosomes) and organelles. The monocytes rapidly emigrate from blood to healthy tissues and differentiate into long-lived mature resident tissue macrophages. They remain in a resting state performing a scavenger job unless stimulated. Resident macrophages detect infection in tissues and can recruit neutrophils from circulation using signals in the form of cytokines. Functions of macrophages 1.Phagocytosis 2. Antigen processing n presentation to T cell 3- Secretion of cytokines ( IL-1, IL-6, IL-8,TNF, G-CSF and IL-12 4. cytotoxicity 1-Phagocytosis A metabolically active process, triggered by target binding through one of the receptors. 40 It is a multi-step process involving: 1– Chemotaxis: The phagocyte moves to the site of infection/inflammation in response to a chemotactic agent. 2– Binding: The particle (pathogen) to be phagocytosed is attached to the phagocyte surface. 3– Internalization (endocytosis). The pathogen is internalized to the inside of the phagocyte in a vesicle called phagosome that moves towards lysosomes. 4– Fusion of phagosomes and lysosomes, contents are mixed. 5- Killing and degradation of the pathogen. The pathogen is degraded into smaller and simpler components e.g. amino acids, sugars, etc that are disposed outside the cell or reutilized by the cell. Steps of phagocytosis 41  b – Natural killer cells (NK cells) Definition: Large granular lymphocytes, Innate cytotoxic lymphocytes Source : Bone marrow precursors Location : 10% or 15% of lymphocytes in peripheral blood and 1% or 2% of lymphocytes in spleen Function : Cytotoxic for tumor cells, viral infected cells, bacterial, fungal, and parasitic infection Responsible for antibody–dependent cell mediated cytotoxicity (ADCC). Destroy infected or mutated cell by removing them from the site of infection. Serve as early line of defense Secretion of cytokines (IFN-γ) Their cytoplasmic granules contain killing proteins e.g. perforins that are responsible for removing foreign matter present in the intracellular compartment e.g. virus infected cells and tumor cells They are very effective killers. Mechanisms of killing Of natural killer cells: 1–Direct killing: this requires close contact with the target cell. a -cytoplasmic granule contents will be released on the target cell surface causing damage. b-Activation of apoptosis. 2–Antibody dependent cell mediated cytotoxicity ADCC. When target cells are bound by IgG Abs. The Fc part of IgG binds also to Fc receptors on the NK cell and directs it to kill the target cell. Mast cells ◦ These cells are present in very low concentrations in blood. ◦ Their cytoplasmic granules contain pharmacologically active compounds e.g. histamine. Its cell surface carries receptors for IgE of any antigen specificity. 42  It is responsible for allergic reactions through the action of IgE fixed to its surface receptors by the Fc part. It is an inflammatory cell similar to basophils. EOSINOPHILS They account for 1 – 6% of total leukocytes. Function: Phagocytosis like neutrophils, however they are more specialized against helminth parasites BASOPHILS These cells are present in very low concentrations in blood. Their cytoplasmic granules contain pharmacologically active compounds e.g. histamine. Cell surface carry receptors for IgE of any antigen specificity. Responsible for allergic reactions through the action of IgE fixed to its surface receptors by the Fc part. Soluble mediators of the innate Immune Response: They are families of proteins in blood and tissue fluids, expressed throughout life. At times of need, their levels can be increased. They recognize carbohydrates, lipids, nucleic acid. Examples: 1-Complement: The complement system is one of the major defense systems of the body. It is always ready to act fast against invading pathogens. Its activation leads to lysis of bacteria or other foreign cells, split products enhance inflammation and opsonise organisms enhancing their removal by phagocytes (phagocytosis). 2-Lysozyme: An enzyme that attacks cell wall of bacteria leading to its lysis. 43 3-Acute phase proteins: Through binding to bacteria, they can activate complement and promote phagocytosis. They also reduce bacterial toxicity and limits tissue damage. 4-Interferons: It is a family of proteins (cytokines) made by various cells following viral infections, produced locally, has the ability to stop virus growth in other uninfected cells. -IFNs (interferons α & β): cover the period required to initiate innate immune response. Interferons inhibit intracellular replication of viruses in neighbouring cells by inducing proteins that prevent the translation of viral mRNA, i.e. they block viral protein synthesis. -Interferons have no direct effect on extracellular virus. Remeber: The innate immune response is a series of nonspecific resistance mechanisms that: - The individual is born with (present since birth). - Does not change greatly with age. -Is fast, act within minutes – hours of infection. -Leaves NO MEMORY after the encounter with the target. - Is not enhanced by repeated exposure. - Alerts the adaptive immune 44  Questions I-Choose the correct answer: 1- Which of the following best describes Natural killer cells? a) Can kill virus infected cells by specific receptors recognition b) Can kill virus infected cells by nonspecific recognition c) Have memory cells d) Recognize the antigen in association with MHC class I molecule 2– All of the following are professional antigen presenting cells except… a – Mast cells b – B cells c – Macrophages d – Dendritic cells 3-Which of the following is not a correct statement concerning neutrophiles? a- They constitute the first line of non-specific cellular defence for the host. b- They, together with monocytes/MØs are the principle phagocytic cells. c- They cannot present antigen. d- They can differentiate into B cells on activation. II-Enumerate soluble mediators of the innate Immune Response. III-Discuss phagocytosis. IV-List three functions of macrophages. 45  Chapter 7 The Acquired (specific) Immune response The acquired immune response is more specialized than the innate immunity. It has several generalized features that characterize it: 1-It is highly specific. 2-It discriminates between “self” and “non-self” 3-Diversity: It exhibits remarkable diversity and can respond to millions of different antigens. 4-Development of immunologic memory after first exposure to a foreign antigen. So that on second exposure to the s `ame antigen a more rapid and longer response is evoked. This explains the long lasting and sometimes lifelong immunity to many infections. The innate immune system activates Acquired immunity Cells of the innate immune system activate the specific immune response. Antigen presenting cells (APC) activate the acquired immune system. Macrophages, dendritic cells and B-cells are examples of types of APCs. APCs turn on the acquired immune system by activating T-Helper cells (TH-cells). TH-cells in turn activate either the cell mediated or the humoral immune system. 46 NB. The innate and adaptive immune systems operate in concert: ☼ Phagocytic cells process and display antigens, facilitate activation of T cells ☼ Macrophages secrete cytokines which help trigger initiation of specific immune response. ☼ T cells produce cytokines, enhance activities of Phagocytes and NK cells ☼ Antibodies produced by plasma cells bind to (opsonize) target and assist phagocytosis Questions 1-Compare between innate and acquired immune response. 2-Mention 5 differences between innate and acquired immune response. 47  Chapter 8 Immunogens and antigens Immunogens It is any foreign substance which is capable of generating a specific immune response and binding to immune components (antibodies and cell mediated immunity) Antigen is a substance capable of binding specifically to immune components.It may or may not induce an immune response. The two words (antigen and immunogen) are used interchangeably. Most antigens are: (a)Components of invading microbes or their products: protein, nucleoprotein, lipoprotein, glycoprotein, large polysaccharides They are found in cell wall , capsule, flagella, viral capsid or envelop and toxins (b) Allergens (dust, pollen, hair, food). (c) Foreign tissues or cells (transplant, transfusion) (d)Body own cells that are recognized as non-self (cancer, infected cells). Haptens are Low molecular weight molecules which ,by themselves are not capable of inducing an immune response. However , when coupled or conjugated to a larger carrier protein become capable of inducing an immune response. Examples of carrier molecules are: Antibiotics, drugs e.g penicillin,asprin ,neomycin ,cosmetics and soap. Adjuvants: are substances that, when mixed with an antigen before its administration will increase the immune response to that antigen. Examples of adjuvants: Incomplete Freund’s adjuvant-oil-in- water emulsion Complete Freund’s adjuvant-oil-in-water emulsion plus dead Mycobacteria Aluminum hydroxide gel 48 Antigenic determinant: Only small part of the molecule can stimulate the production or bind to antibody. These parts are called (epitopes) or antigenic determinants. Each may be composed of 4-7 amino acids or monosaccharide residues The binding of antigen to antibody binding site can be likened to a “lock and key” situation. The most efficient immunological responses occur when the antigen and antibody fit exactly (high affinity antibody) Types of Antigens 1-Bacterial antigens: These may be:- a-Soluble antigens: They are products excreted into the environment, e.g. exotoxins, enzymes, hemolysins, etc... b-Cellular antigens: They are different structures of the cell, e.g. capsular antigens in capsulated organisms, they are often polysaceharides as in Strept. Pneumonia 2-Viral antigens: These may be: a-Protein coat viral antigens b-Soluble antigens which diffuse in the surrounding fluids during virus growth, e.g. soluble nucleoproteins as in influenza and mumps viruses 3-Human tissue antigens: a-Blood group antigens A and B as well as Rh antigens on red cells. These are of importance in blood transfusion reactions. Rh Incompatibility can cause erythroblastosis foetalis. b-Histocompatibility antigens: These are glycoprotein molecules present on membranes of tissue cells.  They are called the major histocompatibility complex (MHC) antigens or human leucocyte antigen (HLA).  It encodes for proteins called MHC molecules that are expressed on the cell surface, with significant function in recognition of antigens. 49  Factors influencing antigenicity a)Factors related to the host e.g. genetic constitution, age, nutritional state, etc. b) Factors related to the molecule include: 1-Foreigness For a substance to be antigenic it must be foreign to the animal in which it introduced.The immune system of an individual can normally distinguish between body components (self )and foreign substances (non self). The body is tolerant to its own components and does not initiate an immune response against them. However Under certain circumstances this auto –tolerance may be disturbed permitting the individual to react against himself leading to auto immune diseases. 2-Chemical nature The most potent antigens are proteins. Some polysaccharides of high MW are antigenic. The more complex structure of proteins the more antigenic ( globular proteins are more antigenic than fibrillar molecules 3-Molecular size Small molecules such as amino acid or monosaccharides are usually not antigenic As a rule molecule which a molecular weight of less than 10000 have no or only weak antigenicity Heterophile antigens and Cross reacting antigens: Antigens that share one or more similar epitope are called heterophil antigens. They are capable of binding to low affinity antibodies that were produced (poor or partial fitting) and result in cross reaction phenomenon. 50 Heterophile antigens and Cross reacting antigens Importance of cross reaction: In vivo: this phenomenon occurs in acute rheumatic fever, where antibody formed against Strept. Pyogenes antigen in the throat cross react with the host heart tissue antigens leading to myocarditis and valvular damage. Human tissue antigens (Isoantigens): a) Blood group antigens: -A, B, AB, Rh on red blood cells. These antigens are of special significance in: Blood transfusion and Erythroblastosis foetalis b)Major Histocompatability antigens (MHC): Human Leukocyte Antigens (HLA), class I and II, have significance in tissue transplantation. c)Immunoglobulin can act as an antigens. 51  Superantigens They are potent antigens that activate many T cells specially CD4 T cells. It results in release of excessive amount of cytokines that can cause harmful immune responses. They are active at very low concentrations. They are Not processed or presented by antigen presenting cells (APC), but bind MHC outside the peptide cleft of APC. They also bind to TCR of T lymphocytes outside the variable β chain (V β). Super antigen Antigen Superantigen 1-Processing Processed Not processed 2-MHC presentation -Inside the antigen -Outside the antigen binding binding cleft cleft 3-Site of binding to T cell -Binds to both α and β -Binds to the β chain only receptors chains of T cell receptors 4-Induced response Protective Harmful 5-Example -Bacterial - Toxic shock syndrome toxin antigen(e.g.Capsular antigen) -Viral antigen(envelop antigen) 52  Questions 1-A hapten is a nonimmunogenic small protein. Which of the following statements best describes haptens? a. Haptens activate T cells b. Penicillin is a hapten c. Haptens do not react with specific antibody d. Haptens bind the major histocompatibility complex (MHC) e. Poison ivy is caused by a small protein that is not a hapten 2-Good immunogen should be which of the following? a. Foreign and chemically simple b. Complex foreign material c. Chemically complex, non-degradable d. Non of the above 3 – Which of the following is the site of the antigen that interacts with antibody? a.Epitope b. Idiotope c. Isotope d. Paratope 4 – Which of the following describes a hapten ? a – Can stimulate response alone b – Enhance response to other antigens c – Induce response when mixed with a carrier protein d – Stimulate response by nonspecific manner 53  Chapter 9 Primary and secondary lymphoid organs A – Primary lymphoid organs Primary lymphoid organs are sites for development and maturation of immune competent cells. They include the bone marrow and the thymus. 1-The Bone marrow The soft tissue located in the center of most bones. Its main function is the production of all types (lineages) of blood cells: red blood cells (erythrocytes), white blood cells (leukocytes: myeloid lineage and lymphoid lineage) and platelets. The Bone marrow is the site of development and maturation of B lymphocytes. Mature B cells leave the bone marrow through blood circulation to reach secondary lymphoid organs. 2 – The thymus 54 A large organ located in the thoracic cavity near the heart. It is fully developed at birth and continues to grow in size until puberty then shrinks and becomes replaced by adipose tissues. The remnant is thought to be still active. Mature T cells leave the thymus through blood circulation to reach secondary lymphoid organs. Children with defective thymus suffer from immunodeficiency disorders B – Secondary lymphoid organs These are sites of immune reactivity They provide the microenvironment necessary for the interaction between foreign antigens (collected from tissues or blood or mucosal surfaces) with re-circulatory lymphocytes. They are situated in strategic positions where infectious organisms are most likely to be found. Secondary lymphoid organs include: 1 – Lymph nodes: A network of structures, located throughout the body. They filter foreign material from lymph passing through it before lymph goes to the thoracic duct. The tonsils, adenoids and Peyer’s patches are specialized lymph nodes. In the event of serious infections, the lymph node becomes swollen. 2 – Spleen: located in the upper left quadrant of the abdomen. It filters blood. 3 – Mucosa – associated lymphoid tissues: these are aggregates of lymphoid cells located under all mucosal surfaces. They trap antigens entering into the body through the mucosa. 55  Chapter 10 CELLS OF THE IMMUNE SYSTEM Mature cells whose receptors can recognize self antigens are considered harmful and die by apoptosis before they are allowed to leave the organ. This phenomenon is called clone deletion and through it the body is sure that his immune cells will not attack his own tissues = central tolerance. Development of T cells. Note the positive and negative selection that occur in the thymus 56 Different immune cells can be identified by certain surface markers called cluster of differentiation molecules (CD system). The presence of such markers can be detected by specific monoclonal antibodies. Cells involved in innate immunity are : Neutrophils, monocytes/macrophages, eosinophils, natural killer lymphocytes (NK cells), mast cells. Cells involved in adaptive immunity: Lymphocytes and antigen presenting cells Lymphocytes These are mononuclear cells. Each lymphocyte has individual receptors enabling it to recognize a particular antigen (confers specificity on the lymphocyte). On activation, they undergo clonal proliferation. Lymphocytes may be T, B or NK cells. I-T lymphocytes and their maturation T cells learn to recognize what is self and those T cells with‘ TCR that do not react to self proteins and recognize MHC molecules, are “positively selected” allowed to proliferate and mature in the thymic medulla CD4&CD8 Types of cells: T-cell progenitors differentiate under the influence of thymic hormones into T-cell subpopulations. These cells are characterized by certain surface glycoproteins, e.g., CD3, CD4, and CD8. All T cells have CD3 proteins on their surface in association with antigen receptors (T-cell receptor, TCR). CD3 is required for signal transduction to the inside of the cell. 57  T cells are subdivided into two major categories on the basis of whether they have CD4 or CD8 proteins on their surface. Mature T cells have either CD4 or CD8 proteins but not both. T lymphocytes and their maturation T-cell Receptor (TCR): The TCR for antigen consists of two polypeptide chains α and ß, linked by disulfide bonds. It is similar to a Fab fragment of an antibody molecule having variable and constant regions. The variable regions are highly polymorphic which gives T cells a remarkable ability to recognize millions of different antigens They recognize and bind specific protein antigens. TCRs do not recognize free soluble or surface bound antigens, but recognize antigens that are processed and presented on the surface of another cells; antigen presenting cell (APC) in the form of short peptides, associated with a cell membrane protein molecule called major histocompatibility complex (MHC) molecule. An individual T cell contains only a single type of α ß dimer and recognizes and responds only to a specific combination of antigen and MHC. Besides the most common isotype, the αß TCR, there is a second isotype composed of γ andδ chains. The γδ T cells are found mainly in the skin and at the mucosal surfaces. Activation of T Cells The activation of helper T cells requires that their TCR recognize a complex on the surface of antigen-presenting cells (APCs), e.g., macrophages and dendretic cells, consisting of both the antigen and a class II MHC protein. 58 This is followed by the ingestion of the foreign protein (or microbe) into the APC. Within the cytoplasm of the macrophage, the foreign protein is cleaved into small peptides that associate with the class II MHC proteins. The complex is transported to the surface of the macrophage, where the antigen, in association with a class II MHC protein, is presented to the receptor on the CD4-positive helper cell. Similar events occur within a virus-infected cell, except that the cleaved viral peptide associates with a class I rather than a class II MHC protein. The complex is transported to the surface, where the viral antigen is presented to the receptor on a CD8-positive cytotoxic cell. Remember the rule of eight: CD4 cells interact with class II (4 x 2 = 8), and CD8 cells interact with class I (8 x 1 = 8). Costimulatory molecules:  Two signals are required to activate T cells.  The first signal is the interaction of the antigen and the MHC molecule with the T-cell receptors specific with the antigen.  The second signal is provided by costimulatory molecules which interact to help stabilize the contact between the T cells and the APCs. Examples are: LFA-1 protein on T cells binds to ICAM-1 on APC B7 protein on APC interact with CD28 on helper T cells Cell surface interactions between T helper cells and antigen presenting cells 59 CD (cluster determinant) antigens Is a uniform system of nomenclature for all cell surface markers. All T cells express transmembrane CD3 molecule associated with TCR which is necessary for a signal to be transduced to the cytoplasm after the TCR binds antigen. CD4&CD8 Types of cells Mature T cells have either CD4 molecule; that is expressed on helper T cells and binds to MHC class II to enhance the response of specific T cells or CD8 molecule; that is expressed on cytotoxic T cells and binds to MHC class I. T cells also express other molecules which are important in cell to cell contact and activation such as CD28, CD40L, adhesion molecules (CD2) and cytokines. T cell subpopulations T helper cells, T cytotoxic cells and T regulatory cells I-CD4 T-helper lymphocytes (TH): These constitute 65%-80% of peripheral T cells and predominate in the thymic medulla, tonsils and blood. TH cells are the principal orchestrators of the immune response as they are needed for the activation of the major effector cells in the response, i.e. cytotoxic T (Tc) cells and antibody producing B cells. CD4 have a long life span, responsible for cell-mediated immunity. Have a central role in generation and regulation of immune response When naïve T cell meets its specific antigen, it becomes stimulated, proliferates and differentiates into effector cells and memory cells. 60 TH1 cells : Produce interleukin -2(IL-2), INF-γ and TNF-β in response to viral and bacterial infections. These cytokines: Enhance cytotoxicity of Tc cells Activate macrophages and NK cells Recruit inflammatory cells to the infected site Enhance production of selected classes of antibody that participates in opsonization and ADCC. IL-12 and INF- γ increase the production of TH1. TH2 cells: Produce IL-4, IL-5, IL-6, IL-10 and IL-13. They are mainly concerned in B cell activation and differentiation and enhance production of all classes of antibodies including IgE and stimulation of eosinophils and mast cells. They respond mainly to allergic reactions and parasitic infections. IL-4 enhances the production of TH2. INF-γ inhibits production of TH2, whereas, IL-4 and IL-10 inhibit production of TH1. All subsets secrete IL-3 and GM-CSF. 61 Cytotoxic T lymphocytes These cells constitute 35% of peripheral T cells. They express CD8 molecule on their surface and recognize only those peptides associated with class-I MHC molecules. Remember the rule of 8: CD4cells interact with classII (4x2=8),and CD8 cells interact with classI (8xI=8) Functions of Tc: These cells are active against tumors, viruses, and allogeneic grafts. They kill the target cells by different mechanisms: -Granule exocytosis and release of cytotoxic proteins such as perforin and granzymes that cause pores in the cell membrane, leakage of cell components and lysis. -Programmed cell death or apoptosis that causes fragmentation of target DNA, shrinking of the cell & death through interaction of FAS ligand on Tc and FAS receptor on target cells. -Secretion of IFN-γ and TNF-β that activate macrophages and kills some tumor cells. 62 T suppressor cells Like T helper cells they have regulatory function. They suppress the immune responses after elimination of the pathogens through production of inhibitory cytokines such as transforming growth factor (TGF-β) and IL-10. They may express CD8 molecules on their surface. II-B –Lymphocytes The lymphoid stem cells differentiate into B cells in bone marrow where B cell precursors differentiate into immunocompetent B cells (they do not pass through the thymus). They undergo clonal deletion of those cells bearing antigen receptors for self proteins, a process that induces tolerance and reduces the occurrence of autoimmune diseases. They bear receptors for Fc portion of IgG, component of the complement and Epstein Barr virus. They all express an array of molecules on their surface that are important in B-cell interactions with other cells such as MHC II, B7 and CD40 which important for immunoglobulin class switching. B cell surface molecules and their functions 63  When naïve B cells meet their specific antigen, they become stimulated through this receptor and develop into effector plasma cells and memory cells. Function of B cells The function of B cells is secretion of immunoglobulins (Igs) following antigen activation. These antibodies play a central role in humoral immunity. B cells can also act as antigen presenting cells. Clonal Selection How do antibodies arise? Does the antigen “instruct” the B cell to make an antibody, or does the antigen “select” a B cell endowed with the preexisting capacity to make the antibody? It appears that clonal selection, accounts for antibody formation. Each individual has a large pool of B lymphocytes (about 107). Each immunologically responsive B cell bears a surface receptor (either IgM ) that can react with one antigen (or closely related group of antigens); i.e., there are about 107 different specificities. An antigen interacts with the B lymphocyte that shows the best “fit” with its immunoglobulin surface receptor. After the antigen binds, the B cell is stimulated to proliferate and form a clone of cells. 64  These selected B cells soon become plasma cells and secrete antibody specific for the antigen. Effector Functions of B Cells/Plasma Cells The result of the activation process is the production of many plasma cells that produce large amounts of immunoglobulins specific for the epitope. Plasma cells secrete thousands of antibody molecules per second for a few days and then die. Some activated B cells form memory cells, which can remain quiescent for long periods but are capable of being activated rapidly upon reexposure to antigen. Most memory B cells have surface IgG that serves as the antigen receptor, but some have IgM. Memory T cells secrete interleukins that enhance antibody production by the memory B cells. The presence of these cells explains the rapid appearance of antibody in the secondary response. The secondary immune response that is carried out by memory cells is different in 3 ways: 1-Memory cells produce antibodies that bind with greater affinity to their antigens than the antibodies produced in the initial response 2-The response time is much faster than the primary response. 3-A greater number of antibodies are produced. Primary Immune response Secondary Immune response Exposure to antigen First exposure Repeated exposure Time of onset About one week delay Within hours Lag phase Long Short Antibody concentration Lower level, decline rapidly Higher level, decline slowly Antibody type Mainly IgM Mainly IgG Class switch No Yes Memory No Yes Primary and Secondary Immune Response 65  Primary and Secondary Immune Response Questions 1-The major role of T cells in the immune response includes which one of the following? a.Recognition of epitopes presented with major histocompatibility complex molecules on all surfaces b. Complement fixation c. Phagocytosis d. Production of antibodies 2-It appears that HIV binds selectively to CD4 glycoproteins. Thus, HIV shows a selective infection with the destruction of helper T cells. Which of the following cells exhibit CD4 glycoprotein on their cell surface? a. Macrophages b. Polymorphonuclear leukocytes c. Suppressor lymphocytes d. Columnar epithelial cells e. Squamous epithelial cells 3-Which of the following statements about T cells is NOT true? a-The majority of circulating lymphocytes are T cells b-T cells have far fewer surface immunoglobulins than do B cells c-T cells are distributed in interfollicular areas of lymph nodes d-T cells proliferate in response to an antigen to which they have been primed e-T cells differentiate into antibody-secreting plasma cells 4-Most of the white blood cells de found in the peripheral blood of adults are a-granulocytes b- macrophages c- null lymphocytes d-B lymphocytes e-T lymphocytes 5-Which of the following is the site of B cell development in mammals? a – The bone marrow b – The bursa of fabricus 66 c– The thymus d – The lymph nodes e – The spleen 6-The major role of T cells in the immune response includes which one of the following? a-Recognition of epitopes presented with major histocompatibility complex molecules on all surfaces b. Complement fixation c. Phagocytosis d. Production of antibodies 7-CD8 is a marker of which of the following cells? a. B cells b. helper T cells c. cytotoxic T cells d. an activated macrophages 8-Immunity may be natural or acquired. Which of the following best describes acquired immunity? a. Increase in C-reactive protein (CRP) b. Presence of natural killer (NK) cells c. Complement cascade d. Maternal transfer of antibody e. Inflammatory response II-Each group of questions below consists of five lettered headings followed by a list of numbered words or statements. For each numbered word or statement, select the ONE lettered heading that is most closely associated with it. Each lettered heading may be selected once, more than once, or not at all. Questions10through 14 a. plasma cells b. T cells c. basophils d. resting B cells e. macrophages 1. Large phagocytic cells 2. Cells activated in anaphylaxis 3. Principal antibody-producing cells 4. Bursa of Fabricius in birds 5. Thymus-processed 67