IMMUNITY = Ability of organism to resist infection --- by means of interactions of a variety of cells and cell products

1. Nonspecific (innate): Phagocytes: (1) engulf, (2) digest and (3) destroy bacteria in lysosomes, (4) present antigens and(5) attract cells of Specific immunity

2. Specific (adaptive) (1) Cell-mediated:

Cell TC degranulation apoptosis

APC TH1 cytokines inflamation

(2) Humoral (antigen-antibody mediated)

APC TH2 interleukins Bplasma Ig

memory cells

APC=Antigen Presenting Cell

• The innate immune response is mediated by phagocytes.

• Phagocytes recognize pathogen-associated molecular patterns (PAMPs) via a family of membrane-bound pattern-recognition molecules, or PRMs.

•Interaction of the PAMPs with PRMs activates phagocytes to produce metabolic products that kill the pathogen or limit its effects.

• Many pathogens have developed mechanisms to inhibit phagocytes.

1. Enzymes: Lysozyme Protease Phosphatase Lipase Nuclease

2. Bactericides: H-peroxide Superoxide Hydroxyl radical Singlet oxygen

3. Aerobic/Anaerobic switch

4. APC – system (antigen-presenting cells)

Phagocyte armor

• Nonspecific phagocytes present antigen to Specific T cells, triggering the production of effector T cells and antibodies.

• Immune T cells and antibodies react directly or indirectly to neutralize or destroy the antigen.

Inflammation

is characterized by pain, swelling (edema), redness (erythema), and heat. The inflammatory response is a normal and generally desirable outcome of an immune response. Uncontrolled systemic inflammation, called septic shock, can lead to serious illness and death.

• The adaptive immune response is characterized by specificity for the antigen,

• The ability to respond more vigorously when re-exposed to the same antigen i.e. memory, and

• The ability to discriminate self antigens from nonself antigens, i.e. tolerance.

•Specificity

•Memory

•Tolerance

• Immunogens are foreign macromolecules that induce an immune response. Molecular size, complexity, and physical form are intrinsic properties of immunogens. When foreign immunogens are introduced into a host in an appropriate dose and route, they initiate an immune response. • Antigens are molecules recognized by antibodies or T-Cell-Receptors or TCRs. Antibodies recognize conformational determinants; TCRs recognize linear peptide determinants.

T cells recognize antigens presented by APCs or by pathogen-infected cells. At the molecular level, TCRs bind peptide antigens presented by MHC proteins. These molecular interactions stimulate T cells to kill antigen-bearing cells or to produce cell-stimulating proteins known as cytokines.

IMMUNITY

1. Nonspecific: phagocytosis & destruction in lysosomes

PMN, Monocytes (circulate) Macrophages (fixed to tissue)

2. Specific a) Cell-mediated:

Cell TC degranulation apoptosisAPC TH1 cytokines inflamation

b) Humoral (antigen-antibody mediated)

APC TH2 interleukins Bplasma Ig

memoryc) APC cells are Macrophages, Dendritic cells, B-cells

TH1 and TH2

cells play pivotal roles in cell-mediated and antibody- mediated immune responses. TH1 inflammatory and TH2 helper cells each stimulate effector cells through the action of cytokines.

• T-cytotoxic (TC )cells recognize antigens on virus-infected host cells and tumor cells through antigen-specific TCRs.

• Antigen-specific recognition triggers killing via perforin and granzymes.

• Natural killer (NK) cells use the same effectors to kill virus-infected cells and tumors. However, NK cells do not require stimulation, nor do they exhibit memory. NK cells respond in the absence of MHC proteins.

Ig (antibody) proteins consist of four chains, two heavy and two light. The antigen-binding site is formed by the interaction of variable regions of heavy and light chains. Each class of Ig has different structural and functional characteristics.

The complement system catalyzes bacterial cell destruction and opsonization. Complement is triggered by antibody interactions or by interactions with nonspecific activators. Complement is a critical component of both innate and adaptive host defense.

Opsonization

The antigen-binding site of an Ig is composed of the V (variable) domains of one heavy chain and one light chain. Each heavy and light chain contains three complementarity-determining regions, or CDRs, that are folded together to form the antigen-binding site.

Recombination allows shuffling of various pieces of the final Ig genes. Random reassortment of the heavy- and light-chain genes maximizes genetically encoded diversity. Imprecise joining of VDJ and VJ segments as well as hypermutation and affinity maturation also contribute to virtually unlimited immunoglobulin diversity.

• Antibody production is initiated by antigen contact with an antigen-specific B cell that processes the antigen and presents it to an antigen-specific TH2 cell.

• The activated TH2 cell then signals the antigen-specific B cell to produce antibody.

• Activated B cells live for years as memory cells and can rapidly produce large quantities (high titers) of antibodies upon re-exposure to antigen.

• The thymus is a primary lymphoid organ that provides an environment for the maturation of antigen-reactive T cells.

• Immature T cells that do not interact with MHC protein (positive selection) or react strongly with self antigens (negative selection) are eliminated by clonal deletion in the thymus.

• T cells that survive positive and negative selection leave the thymus and can participate in an effective immune response.

• B cell reactivity to self antigens is controlled through clonal deletion, selection, and anergy.

• Cytokines are soluble mediators produced by leukocytes that regulate interactions between cells. Several cytokines such as IL-1, IL-2, and IL-4 affect leukocytes and are critical components in the generation of specific immune responses. Other cytokines such as IFN and TNF affect a wide variety of cell types. Chemokines are produced by a variety of cell types in response to injury and are potent attractants for nonspecific inflammatory cells and T cells. (IL stands for Interleukin)

Cytokines – small soluble regulatory proteins

Lymphokines – cytokines produced by lymphocytes

Interleukins – interleucocyte regulationIL1 Macrophages to TH activationIL2 TH2 to TH2 autocrene proliferationIL3 TH1 to stem cellsIL4 TH2 to B proliferation plasma cells IgG, EIL5 TH2 to B proliferation plasma cells lgAIL8 Leukocytes chemoattracts T-cells & PMNsIL10 TH2 to TH1 inhibitionIL12 Macrophages to TH1 activation

ChemokinesIFN – InterFeroNs leukocytes tissue cells – antiviralTGF – T-cell Growth FactorTNF – Tumor Necrosis FactorMCP – Macrophage Chemoattractant Protein

ImmunizationAgammaglobulinemia

Natural Artificial

Active Passive

Exposure

Receives immunogens

Immunization(vaccination)

Receives antibodies

Active: Immunogens Passive: antiserum (antitoxin)

Killed pathogens (formaldehyde) Antibodies in serumAttenuated strains of pathogens Hyperimmune personsToxoids Pooled Immunoglobulins1. Synthetic epitope peptide; 2. Recombinant vector vaccine: Viral genes cloned in E. coli3. Cloned genes injected and expressed (tumor-specific antigen)

Hypersensitivity - type I Immediate hypersensitivity Allergy, asthma, anaphylaxis

Hypersensitivity - type II Organ-specific Autoimmune diseases

Autoantibodies: Hashimoto’s disease against thyroid gland Juvenile diabetes against Langerhans islets

Hypersensitivity - type III Systemic Autoimmune diseases

Systemic lupus erythematosis (SLE)

Hypersensitivity - type IV Delayed hypersensitivity

Contact dermatitis, tuberculin test

• Immunity to infectious disease can be either passive or active, natural or artificial.

• Immunization, a form of artificial active immunity, is widely employed to prevent infectious diseases.

• Most agents used for immunization are either attenuated or inactivated pathogens or inactivated forms of natural microbial products.

Hypersensitivity results when foreign antigens induce cellular or antibody immune responses, leading to host tissue damage. Autoimmunity occurs when the immune response is directed against self-antigens, resulting in host tissue damage.

Hypersensitivity - type I Immediate hypersensitivity Allergy, asthma, anaphylaxis

Hypersensitivity - type II Organ-specific Autoimmune diseases

Autoantibodies: Hashimoto’s disease against thyroid gland Juvenile diabetes against Langerhans islets

Hypersensitivity - type III Systemic Autoimmune diseases

Systemic lupus erythematosis (SLE)

Hypersensitivity - type IV Delayed hypersensitivity

Contact dermatitis, tuberculin test

T-cell Cytotoxic Attack

T-cell

B-cell Antibody Attack

Complement alternatives

Mimicry(Rabies, Rhino, Vaccinia)

Hiding the binding site(Polyo, Influenza, HIV)

Epitope cover-up (wolf in sheep’s skin – E. coli)

Hiding in the police Department

(Shigella, Mycobacterium, Chlamydia)

Clinical methods- Culture diagnostic:

- Blood , Bacteremia -> Septicemia -> Septic shock

- Urine , Bacteriuria – bacteria in urin

- Feces, Salmonella, Shigella

- Antibiogram

- Serodiagnostic- Antibody titer- Polyclonal antibodies- Monoclonal antibodies

Safety in the clinical laboratory requires effective training, planning, and care to prevent the infection of laboratory workers with pathogens. Materials such as live cultures, inoculated culture media, used hypodermic needles, and patient specimens require specific precautions for safe handling.

Proper sampling and culture of the suspected pathogen is the most reliable way to identify an organism that causes a disease. The selection of appropriate sampling and culture conditions requires knowledge of bacterial ecology, physiology, and nutrition.

Antimicrobial drugs are widely used for the treatment of infectious diseases. Pathogens should be tested for susceptibility to individual antibiotics to ensure appropriate chemotherapy. This rigorous approach to antimicrobial drug treatment is usually applied only in health care settings.

An immune response is a natural outcome of infection. Specific immune responses, particularly antibody titers and skin tests, can be monitored to provide information concerning past infections, current infections, and convalescence.

Polyclonal and Monoclonal antibodies are used for research and clinical applications. Hybridoma technology provides reproducible, monospecific antibodies for a wide range of clinical, diagnostic, and research purposes.

Antigen–antibody reactions require that antibody bind to antigen. Specificity and sensitivity define the accuracy of individual serological tests. Neutralization and precipitation reactions are examples of antigen-binding tests that produce visible results involving antigen–antibody interactions.

• In serological reactions, high specificity prevents false-positive reactions. High sensitivity prevents false-negative reactions.

Specificity: binding with a single antigen(positive + negative controls no false +)

Sensitivity: lowest amount of antigen dedectable(no false -)

______________________________________Precipitation

soluble - solubleAgglutination (100 x precipitation sensitive)

surface bound - soluble

Direct agglutination tests are widely used for determination of blood types. A number of passive agglutination tests are available for identification of a variety of pathogens and pathogen-related products. Agglutination tests are rapid, relatively sensitive, highly specific, simple to perform, and inexpensive.

Fluorescent antibodies are used for quick, accurate identification of pathogens and other antigenic substances in tissue samples and other complex environments. Fluorescent antibody-based methods can be used for identification, quantitative enumeration, and sorting of a variety of cell types.

EIA (ELISA) and RIA methods are the most sensitive immunoassay techniques. Both involve linking a detection system, either an enzyme or a radioactive molecule, to an antibody or antigen, significantly enhancing sensitivity. ELISA and RIA are used for clinical and research work; tests have been designed to detect either antibody or antigen in many applications. For more specificity apply IMMUNOBLOT

Immunoblot procedures are used to detect antibodies to specific antigens or to detect the presence of the antigens themselves. The antigens are separated by electrophoresis, transferred (blotted) to a membrane, and exposed to antibody. Immune complexes are visualized with enzyme-labeled or radioactive secondary antibodies. Immunoblots are extremely specific, but procedures are complex and time-consuming.

Nucleic acid hybridization is a powerful laboratory tool used for identification of microorganisms. A nucleic acid sequence specific for the microorganism of interest must be available in order to design a probe. Perhaps the most widespread use of probe-based technology is in the application of gene amplification (PCR) methods. Various DNA-based methodologies are currently used in clinical, food, and research laboratories.

Virus propagation in vitro can be accomplished only in tissue culture. Therefore, most diagnostic techniques for viral identification are not growth-dependent, but routinely rely on immunoassays and nucleic acid-based techniques. Electron microscopy techniques are useful for direct observation of viruses in host samples.

Epidemiology

The study of disease in populations. To understand infectious disease, the epidemiologist studies the interactions of the pathogen with the host population. It is a population level ecology

Concepts of Epidemiology

Prevalence: % of diseased

Incidence: # of diseased

Endemic - Epidemic - PandemicMortality - Morbidity

Infection – Incubation - Acute period – Decline - Convalescence

Emerging Infectious Diseases

Transmissions:

• Reservoirs - Vectors - Carriers

Public Health Measures• Surveillance - Reservoir control -

Transmission control -

• Immunization - Quarantine - Pathogen eradication

To understand how diseases spread, the pathogen reservoir must be known. Some pathogens exist in soil, water, or animals. Other pathogens exist only inhumans and are maintained solely by person-to-person contact. An understanding of disease carriers and pathogen life cycles is critical for controlling disease.

Reservoires

• An endemic disease is constantly present at low incidence in a specific population. • In epidemics, an unusually high incidence of disease occurs in a specific population. • A pandemic disease is global

Infectious diseases cause morbidity (illness) and may cause mortality (death). An infectious disease follows a predictable clinical pattern in the host and its population.

A pathogen can be transmitted from ….

• host-to-host, … directly or

indirectly by:• vectors …... live intermediates, or• fomites … ..Inanimate objects

• vehicles ….use of food and water.

Epidemics origin: may be (A) common-source or (B) host-to-host

Transmission

Common source waterborn

Host-to-host1. Airborn2. Direct contact3. Vectors - animals

Mosquito – transferred deseases

• Malaria

• Eastern equine encephalitis

• Western equine encephalitis

• St. Louis encephalitis

• West Nile virus

Water-transmitted diseases

Salmonella typhi Vibrio cholerae Escherichia coliLegionella pneumophilaEntamoeba histolithica, Giardia

Water treatment 1. Primary2. Secondary3. Tertiary

Parasite – Host Co-Evolution

• Hosts and pathogens co-evolve with time and arrive at a steady state that favors the continued survival of both. • With Herd immunity, a large fraction of a population is immune to a given disease, and it is difficult for the disease to spread. • Disease cycles occur when a large, recurring, non-immune population such as children entering school is exposed to a pathogen.

Many Respiratory Pathogens are gram-positive Bacteria. Because gram-positive Bacteria are resistant to drying, they are easily transmitted in air. Most respiratory pathogens are transferred from person to person via respiratory tract. Transfer is generated by coughing, sneezing, talking, or simply breathing.

1. Air-born transmissions – Respiratory diseases

Bacterial1. Streptococcus pyogenes, S. pneumoniae2. Corynebacterium diphtheriae3. Bordetella pertussis4. Mycobacterium tuberculosis, M. leprae, M. bovis5. Neisseria meningitidis

Viral

1. Measles, Mumps, Rubella, Varicella, 2. Common cold, Influenza

Diseases caused by streptococci include streptococcalsore throat and pneumococcal pneumonia. Occasionally, Streptococcus pyogenes infections develop from pharyngitis into serious conditions such as scarlet fever and rheumatic fever. Pneumonia caused by Streptococcus pneumoniae is a serious disease with high mortality. Definitive diagnosis for both pathogens is by culture. Infections with both pathogens are treatable with antimicrobial drugs, but drug-resistant strains are known, especially for Streptococcus pneumoniae

Tuberculosis is one of the most prevalent and dangerous single diseases in the world. Its incidence is on the increase in developed countries, in part because of the emergence of drug-resistant strains. The pathology of tuberculosis and leprosy is influenced by the cellular immune response.

Colds and influenza, or flu, are the most common infectious diseases. While they are not usually life-threatening diseases by themselves, they can lower resistance and allow serious secondary bacterial infections. Influenza outbreaks occur annually and more serious epidemics and pandemics occur periodically.

Neisseria meningitidis is a common cause of meningococcemia and meningitis in young adults and occasionally occurs in epidemics in closed populations. Bacterial meningitis and meningococcemia are serious diseases with very high mortality rates. Treatment and prevention strategies are in place to deal with epidemic outbreaks, but an effective universal vaccine is not yet available.

Infection with a novel zoonotic virus, SARS-CoV (ssRNA), causes severe acute respiratory syndrome (SARS). Person-to-person spread is by respiratory means. Control of this high mortality virus is through rapid diagnosis and isolation of victims (source: civets, food-grown in Guandong Province, China – Nov. 2002).

2. Direct Contact Transmission – Sexual transmission

Bacterial1. Staphylococcus epidermis, S. aureus – toxic shock2. Helicobacter pylori3. Neisseria gonorrhoeae4. N. meningitidis5. Treponema pallidum6. Chlamydia trachomatis

Viral1. Hepatitis A-G, 2. Herpes simplex – 1, 2, 3. HIV

Gonorrhea and syphilis, caused by Neisseria gonorrhoeae and Treponema pallidum, respectively, are STIs with potential serious consequences if not treated. Although the incidence of these diseases has generally declined in recent years, there are still over 350,000 cases of gonorrhea and 6000 cases of syphilis annually in the United States.

Helicobacter pylori infection appears to be the most common cause of gastric ulcers. Treatment of gastric ulcers now involves antibiotics, which seem to promote a permanent cure.

Hepatitis caused by viruses can cause cirrhosis, an acute liver disease. HBV and HCV can cause chronic infections leading to liver cancer. Vaccines are available for HAV and HBV.

• The overall prevalence of hepatitis has decreased significantly in the last 20 years in the United States, but viral hepatitis is still a major public health problem because of the high infectivity of the viruses.

• Postexposure treatment: γ-globulin, HBV-specific

HIV

AIDS

Opportunistic diseases

AIDS is now one of the most prevalent infectious diseases in the human population. HIV destroys the immune system, and opportunistic pathogens then kill the host. There is still no effective vaccine for HIV. However, several antiviral drugs slow the progress of AIDS. The only prevention for the spread of HIV infection is through avoidance of behavior such as intravenous drug use (needle sharing) and unsafe sexual practices.

Nosocomial infections Many common microorganisms have the potential to be pathogens in a hospital environment. Hospital patients are unusually susceptible to infectious disease and are exposed to a variety of infectious agents, including opportunistic pathogens, in the hospital environment. Treatment of these infections is complicated by antibiotic resistance.

Food and water purity regulations, vector control, immunization, quarantine, disease surveillance, and pathogen eradication are public health measures that play a major role in reduction of disease incidence.

Infectious diseases account for nearly 30% of all worldwide mortality. Most infectious diseases occur in developing countries. Travelers to endemic disease areas should be immunized when possible and should take appropriate precautions to prevent infection.

Bacillus anthracis has emerged as an important pathogen because of its use as a bioweapon. Highly infective weaponized endospore preparations have been used as bioterror agents. Inhalation anthrax has a fatality rate of about 90% in untreated individuals. Effective treatment relies on timely observation and diagnosis of symptoms. Treatment does not guarantee survival for inhalation anthrax.

Changes in host, vector, or pathogen conditions, whether natural or artificial, can result in conditions that encourage the explosive emergence or reemergence of certain infectious diseases. Global surveillance and intervention programs must be developed to prevent new epidemics and pandemics.

Concepts of Epidemiology

Prevalence: % of diseasedIncidence: # of diseased

Endemic - Epidemic - PandemicMortality - Morbidity

Infection-Incubation-Acute period-Decline-Convalescence

Transmissions:Reservoirs - Vectors - Carriers

Public Health MeasuresSurveillance - Reservoir control - Transmission control - Immunization - Quarantine - Pathogen eradication

Emerging Infectious Diseases

Biological Weapons

INDUSTRIAL MICROOGANISMS AND

PRODUCTS

Industrial Microorganisms and Their Products

Properties of a Useful Industrial Microorganism Examples of Industrial Products

Primary and Secondary Metabolites Primary and Secondary Metabolism Pathways

Characteristics of Large-Scale Fermentations

Construction of an Aerobic Fermentor Fermentation Control and Monitoring

Fermentation Scale-Up The Scale-Up Process

• An industrial microorganism must produce the product of interest in high yield, grow rapidly on inexpensive culture media available in bulk quantities, be amenable to genetic manipulation, and, if possible, be nonpathogenic.

• Industrial products are many and include both cells and substances made by cells.

• Primary and secondary metabolites are produced during active cell growth or near the onset of stationary phase, respectively.

• Many economically valuable microbial products are secondary metabolites.

Large-scale industrial fermentations present several engineering problems: • Fermentation Scale-Up• Aerobic processes require mechanisms for stirring and aeration. • The microbial process must be continuously monitored to ensure satisfactory yields of the desired product.

Scale-up process is:

• Gradually converting a useful industrial fermentation from laboratory scale to• Production scale. • Aeration is a particularly critical aspect to monitor during scale-up studies.

MAJOR INDUSTRIAL PRODUCTS FOR THE FOOD AND BEVERAGE INDUSTRIES

Alcohol and Alcoholic Beverages

Wine Varieties and ProductionBrewing: Making the WortBrewing: The Fermentation ProcessDistilled Alcoholic BeveragesCommodity Ethanol

MAJOR INDUSTRIAL PRODUCTS FOR THE

HEALTH INDUSTRY

Antibiotics: Isolation and Characterization

New Antibiotics, Purification, Incresed Yield

Industrial Production: Penicillins and Tetracyclines

b-Lactam Antibiotics: Penicillin and Its Relatives

Production of Tetracyclines

Vitamins and Amino Acids

Vitamins

Amino Acids

• Beer

• Ale

• Porter

• Stout

Brewing: Making the Wort

1. Mashed grains of barley (wheat, rice, corn): Malt2. Boiling: Natural enzymes convert starch to sugar, protein coagulates and is removed3. Aqueous extract removed by filtering: Wort4. Hops is added boiled for several hours

Brewing: Making the Wort

1. Mashed grains of barley (wheat, rice, corn): Malt2. Boiling: Natural enzymes convert starch to sugar, protein coagulates and is removed3. Aqueous extract removed by filtering: Wort4. Hops is added boiled for several hours5. Wort is sterilized, filtered, cooled & transferred to6. Fermentation vessels

Brewing:The Fermentation Process

Top fermenting (obergaering): Ales, Stout, English style, (Altbier) Sacharomyces cerevisiae 14 - 23°C ; 5 – 7 days – stored at 4-8°C

Bottom fermenting (untergaerig): Lager (Pilsener, Pils, Spezial, Bavarian lager), Sacharomyces carlsbergensis 6 - 12°C ; 8 -14 days – stored weeks at -1°C

Wart Alcohol• American light beer: ?

3.50%• Munich beer: 12%

4.25%• Oktoberfest beer >12% 5.00%• Maibock beer >12%

5.00%• Belgian beer ? ?

• Alcoholic beverages are produced by yeast from the fermentation of sugar to ethyl alcohol and CO2. • Wine is produced from grape juice, • Beer from malted grain, and • Distilled spirits from the distillation of fermented solutions. • Commodity alcohol is used as a gasoline additive and industrial solvent.

Vinegar Production Vinegar Production

Citric AcidCitric Acid Production: The Link to IronCitric Acid Production: Growth Media,

Conditions, and Purification

Yeast as a Food and Food Supplement Yeast Cell Production

Mushrooms as a Food Source Commercial Growth of Mushrooms

The industrial production of antibiotics begins with screening for antibiotic producers. Once new producers are identified, purification and chemical analyses of the antimicrobial agent are performed. If the new antibiotic is biologically active in vivo, the industrial microbiologist may genetically modify the producing strain to increase yields to levels acceptable for commercial development.

• Major antibiotics of clinical significance include the β-lactam antibiotics penicillin and cephalosporin and the tetracyclines. • All of these antibiotics are typical secondary metabolites, and their industrial production is well worked out . . . • despite the fact that the biochemistry and genetics of their biosynthesis are only partially understood.

Vitamins produced microbially include vitamin B12 and riboflavin, whereas the most important amino acids produced commercially are glutamic acid, aspartic acid, phenylalanine, and lysine. High yields of amino acids are obtained by modifying regulatory signals that control synthesis of the particular amino acid such that overproduction occurs.

Microbial biotransformation employs microorganisms to biocatalyze a specific step or steps in an otherwise strictly chemical synthesis.

Microorganisms are ideal for the large-scale production of enzymes. Many enzymes are used in the laundry industry to remove stains from clothing, and thermostable and alkalistable enzymes have many advantages in these markets. Enzymes from extremophiles are desirable for biocatalyses under extreme conditions. When an enzyme is used in a large-scale process, it may be desirable to immobilize it by bonding it to an inert substrate.

The active ingredient in vinegar is acetic acid, which is produced by acetic acid bacteria oxidizing an alcohol-containing fruit juice. Adequate aeration is the most important consideration in ensuring a successful vinegar process.

A number of organic chemicals are produced commercially by use of microorganisms, of which the most important economically is citric acid, produced by Aspergillus niger.

Yeast cells are grown for use in the baking and food industries. Commercial yeast is produced in large-scale aerated fermentors using molasses as the main carbon and energy source.