Introduction to defense systems of the body

• Our defense against microbial infections are commonly divided into non-specific resistance and specific resistance, which employs the T-cells and B-cells of our immune system

• The 1st line of defense consists of more-or-less constant features of our make-up, and our constant microbial companions, our “normal flora”. We’ll discuss this aspect of defense first.

• The 2nd line of defense includes phagocytic WBC that respond to a infection. Significant infections will mobilize a class of these cells to activate the immune system.

The non-specific defenses of our normal flora

• Starting at birth, “harmless” and beneficial microorganisms colonize the surface of our skin, and portions of our respiratory and digestive passages

• Bacteria in colon include - acid-producing Lactobacilli that prevent proliferation of “opportunistic” microorganisms

• The distal ends of both the male and female urethra can be coated with normal flora (voiding urine helps prevent colonization of the bladder)

• The female genitalia are lined by normal microflora, also including the acid-producing Lactobacilli that prevent proliferation of “opportunistic” microorganisms - YEAST

• It is estimated that our mouths have over 6 billion microorganisms

• It is estimated that 400-500 different species make up the billions [1011] of microorganisms in our colons – and in total, they weigh ~4 lbs!

The non-specific defenses of our normal flora

• Just the fact that our normal non-pathogenic flora covers the surfaces of our skin and some mucous membranes prevents colonization by pathogenic organisms– The normal flora don’t want to “share” the niche they’ve found for

themselves, so they don’t provide a hospitable environment for pathogens to colonize these surfaces - Competition

• In addition, normal flora on our skin and in the colon and other areas produce acidic environments which are unfavorable to pathogens– The lactobacilli produce pathogen-inhibiting lactic acid

• Some opportunistic diseases occur when normal flora are depleted, as may happen when an individual takes antibiotics for an extended period of time

• Antibiotics are not specific for pathogens!

• Some normal flora are beneficial to our health– For example, some gut flora produce vitamin K which is absorbed into

our bodies– Calcium absorbance and blood clotting

Examples of interruption of normal flora• An opportunistic microorganism is one that takes advantage of a change in the

normal environment by proliferating– This can occur when normal flora are depleted, as may happen when an

otherwise healthy individual takes antibiotics for an unrelated infection for an extended period of time

– While the antibiotics may destroy the bacteria causing the infection, they also may wipe out normal flora, disrupting the normal “balance” of microorganisms

• One example of this is a sometimes fatal condition called antibiotic-associated colitis, also known as pseudomembranous colitis– A hospital patient on antibiotics may have normal colon flora disrupted by drugs. – This may allow the opportunistic Clostridium difficile (“C. diff”) to multiply in

the colon and release exotoxins which severely damage the colon.

• A less severe example is a woman taking antibiotics for strep throat (caused by Streptococcus pyogenes). Her strep throat clears up, but she develops a yeast infection. Why?– The antibiotics wiped out the acid-producing Lactobacilli in her vagina,

which caused the pH of her vagina to rise (become less acidic), which set up an opportunity for the small number of yeast, Candida albicans, there to proliferate

C. difficile and antibiotic-associated colitis

• A “hypervirulent” strain has emerged that releases HIGHER LEVELS of TOXINS

• An intestinal infection by Clostridium difficile – “antibiotic-associated colitis”, can result in diarrhea, blood poisoning and potentially, death - the death rate has risen from 2% to 6%

• Most cases occur in hospitals and long-term care facilities, and are associated with antibiotic use, although healthcare workers can spread the bacteria to patients or contaminate surfaces in the patient’s room by touch

• Annually there are 35,000 cases in hospitals and 10s of thousands in nursing homes. In the USA. the infection kills 15,000 to 20,000 people annually.

• Clostridium difficile is a normal inhabitant of the colon, but because of competition there, its numbers are relatively low– an endospore-forming bacterial species - is found in human feces

• Patients on long-term antibiotics that end up wiping out normal colon flora without affecting C. difficile, allows the bacteria to proliferate

• After proliferation, “C. diff” releases toxins that can severely damage the lining of the colon, leading to a condition called pseudomembranous colitis, which can be fatal

• Treatments include VANCOMYCIN, but in sever cases, the colon is removed

C diff proliferation

Acids as a barrier• The stomach has acid-producing (parietal) cells that create a pH of 1.5-3.5, which can destroy most microorganisms coming in during digestion (although Helicobacter pylori thrives in the stomach)• (This is NOT the only reason for the acid)

• Normal anal and vaginal flora, mainly the bacterial genus Lactobacilli, acidify the surface of these tissues

• In the vagina, Lactobacilli anaerobically metabolize glycogen supplied by vaginal cells and produce lactic acid - the acidic environment is inhibitory to the growth of other microorganisms

• Diet, stress, infections and other factors can disrupt the microbial balance

The epidermis as a mechanical barrier

• The epidermis of skin is a multi-layered mechanical barrier, with an outer layer of dead, keratinized cells - (STRATUM CORNEUM) – the top layer of which flakes off, removing millions of microorganisms with each flake

• Most viruses trouble infecting the skin because they cannot exist on the dead cells• The deeper epidermal layers prevent microbial penetration to the dermis

• Fatty acids, converted from sebum from sebaceous glands by surface bacteria, coat the surface and lower surface pH (to pH 3.5-5.8); this acidity is anti-microbial

• Normal flora covering the skin also is an important component of this barrier, preventing attachment by pathogenic organisms

The anti-microbial role of surface acid• Many pathogens live in physiological pH (~7.4) and cannot live or thrive in acidic environments

• The sebum of the sebaceous glands is converted into fatty acids by skin bacteria (Propionibacterium acnes)• The fatty acids lower skin pH to 3.5-5.8, and this acidity inhibits the growth of pathogens.

• This is an image of a hair follicle. The acidic sebum is released through the shaft of the follicle unto the surface of the skin.

• The shaft also is used by pathogens, like Staph aureus, to enter the skin and cause boils, sties, and other skin infections

Antibacterial “lysozyme”

• The lacrimal gland continually releases lysozyme in tears

• Lysozyme is an antibacterial enzyme (mostly effective against the cell wall of gram-positive bacteria) released in tears and saliva

• The salivary glands release lysozyme in saliva

The respiratory mucociliary escalator

• Mucus traps particles, that can include any breathed-in microbes and regularly-beating cilia move the mucus and microbes to the throat to be swallowed and destroyed by the stomach’s acid

• An individual with cystic fibrosis have ineffective ciliary escalator systems and are susceptible to chronic infections, most commonly by Pseudomonas aeruginosa

• A mucociliary escalator is found in the lining of the nasal respiratory epithelium and in the lining of the trachea of the respiratory tract

• It consists of mucus produced by goblet cells and cilia that “beat” in a steady and synchronized manner

Natural chemical protection against the spread of viral infections: interferon

• Interferon is produced and secreted by an infected cell to signal other cells to prepare for infection

• The cells that receive the interferon signal can then inhibit viral replication by degrading mRNA after they’re infected. The cell dies and the virus cannot replicate.

• (interferons also are produced by WBCs and fibroblasts)

Cellular response to viral infection: INTERFERON

Our 1st line of defense against invading bacteria: neutrophils

• Among our WBCs (also called leukocytes) are phagocytes like Neutrophils and Macrophages, cells that engulf (“phago” means “eat”) and internally destroy microbes

• The abundant NEUTROPHILS are typically the 1st line of defense against bacterial infections

– They engulf bacteria and die in the process, creating “pus”– Many neutrophils counted in a blood smear signals an acute bacterial infection

NEUTROPHILS are attracted to bacteria

• After bacteria BREECH a compromised barrier (like wounded skin), chemical signals stimulate capillary endothelial cells to express “docking stations” to which neutrophils attracted by other signals dock into – a process called margination• Next the neutrophil squeezes between endothelial cells - called diapedesis – and migrates to the bacteria to consume them

Infection- bacteria cause release of the chemical signals

Macrophages are “multi-taskers”• As their name implies, macrophages also are phagocytes

– The macrophages are abundant in tissues, but are derived from monocytes circulating in the bloodstream and originating from red bone marrow

• Macrophages are the cells that have 2 additional roles when “activated”:: When they engulf gram-negative bacteria, the endotoxin activate

them to release “pyrogens” that notify the hypothalamus to create a fever (and other “cytokines” that target other tissues)

: Macrophages also mobilize to activate the immune cells in a serious infection

• As antigen-presenting cells (APCs), where they “present” a part of the microbe (called the “antigen”) to the helper T-cells of the immune system, so that the immune cells can recognize the microbes in order to lethally attack them

• (Note: In latent re-activation of TB, it is macrophages that Mycobacterium tuberculosis are dormant within)

“Activated” macrophages and fever production• Fever is one of the strongest indicators of infectious disease, including colonization by gram-negative, endotoxin-containing, bacteria• In a Gram-negative infection, after a macrophage engulfs a bacterium, the endotoxin activates the macrophage

• Activated macrophages release chemicals called pyrogens (interleukin-1 [IL-1] and tissue necrosis factor [TNF])• Pyrogens stimulate the hypothalamus to raise body temperature, i.e., start a fever

• Pathogenic bacteria grow best at about 98.60F (370C) and fever is commonly defined as a core temperature above 1000F (37.80C)

• The fever kills pathogens and increases activity of the defenses to wipe out the rest

• The body’s “thermostat” is in a part of the brain called the hypothalamus

A macrophage has various microbe-specific receptors on its surface

• Flagellin is found on bacterial flagella

• Peptidoglycan is the major component of the bacterial cell wall (particularly gram-positive bacteria)

• LPS (lipopolysaccharide) is the outer coating of gram-negative bacteria

• Bacteria have their own DNA and RNA sequences

• A major function of macrophages is their role as Antigen-Presenting Cells (APCs), which “present” microbial antigens to helper T-cells of the immune system to initiate an immune response to an infection

Another chemical defense: the “defensins”

• Defensins are “ancient” short antimicrobial peptides produced by amphibian, reptiles, bird, mammals, and even plants

• In us, they have recently been found on mucous membranes and within certain phagocytes.

• 4 different types of defensins have been characterized.– For example, paneth cells located within the lining of the intestinal tract have

been shown to release defensins in response to bacterial infections of the intestinal lining

– These peptides are believed to insert themselves into bacterial plasma membranes, forming pores, or holes, that cause the bacterium to leak out its contents and die

– Defensins also have been shown to be effective against fungi and enveloped viruses

• Not too much is known about these molecules at this time. However, various defensins are being intensively studied for potential use an antimicrobial drugs.

Introduction to defense systems of the body

•specific resistance, which employs the T-cells and B-cells of our immune system

Overview of the lymphatic system• Most T-cells and B-cells reside in lymph nodes, located along the pathways of lymphatic vessels (which eventually drain “lymph” into venous blood)

• Antigen-presenting cells, or APCs, like macrophages and “dendritic cells” reside in the nodes• When a pathogen or exotoxin enters a node, APC phagocytosizes it and “presents” it as an antigen to a helper T-cell

• Digestive, respiratory and urogenital pathways all have lymphatic nodules under the epithelium• These nodules have T-cells, B-cells, and APCs

• Natural Killer (NK) cells are a class of lymphocytes that attack virally-infected cells and tumor cells, but don’t form a “memory”, so are not considered true immune cells

• The spleen has pockets of lymphoid tissue that monitors blood

• Loss of the spleen can make an individual more susceptible to infectious diseases

The specific defense system: the IMMUNE SYSTEM

• Our 3rd line of defense is the (healthy) immune system

– It consists of specialized defense cells (WBC) called lymphocytes: B-cells (B-lymphocytes) and T-cells (T-lymphocytes)

– Activation of lymphocytes requires an additional defense cell called an Antigen-Presenting Cell, or APC

• There are a number of T-cell subclasses:

– The helper T-cell is the lymphocyte that is initially activated by an APC after invasion by a pathogen

• There are subclasses of the T-cells

– The cytotoxic T-cell is a lymphocyte that responds to viral infections (and cancer, and tranplanted tissue)

• The B-cell usually requires that a foreign antigen be presented to them before it responds

– This requires an activated APC and an activated helper T-cell

– The B-cell will divide into memory B-cells and plasma cells

– A plasma cell has 1 job: release antibodies

The origins of lymphocytes (B-cell and T-cells)• All WBCs, including the precursors of APCs and the precursors of lymphocytes originate from “stem cells” in red bone marrow

• B-lymphocytes differentiate and “mature” within the red bone marrow before they are released into circulation

• The pre-cursors of T-lymphocytes migrate from red bone marrow to an organ called the thymus gland

• It is in the thymus that these precursors are “programmed” (or, “educated”) into mature T-cells

• The mature T-cells - including helper T-cells and cytotoxic T-cells – they permanently leave the thymus and enter circulation

The steps involved in activating the immune system

• When a pathogen enters the body, the 1st defense cells of the immune system it encounters (ignoring neutrophils, which are defensive “kamikazee cells”, but NOT cells of the immune system) are antigen-presenting cells, or APCs– Macrophage– “dendritic cell” in the epidermis of skin (where they are called “Langerhan cells”)

in body tissues and in lymph nodes

• The macrophage engulfs the pathogen and then inserts an “antigen” of the pathogen into its plasma membrane– The macrophage/APC then searches out the proper helper T-cell (may take

days)

• The macrophage/APC-helper T-cell complex then searches out a cytotoxic T-cell or a B-cell– If pathogen is bacterial, fungal, protozoan, or viral, a B-cell is activated

– If pathogen is a VIRUS, a cytotoxic T-cell ALSO is activated

The macrophage as an APC

• The macrophage is one of the links (there are other APCs) between non-specific defenses and the specific, immune response because it “presents” a surface feature of the pathogen to the helper T-cell of the immune system, a step usually required to activate an immune response

• An immune response activated early enough is typically lethal to the pathogen!

Activating a B-cell

• In most cases, to activate a B-cell it must be “presented” with a foreign antigen by an APC and a helper T-cell

• After activation, the B-cell will produce a clone of memory B-cells and a clone of plasma cells

• The plasma cells are the primary source of antibodies that are specific for that specific antigen

• Estimates are that a plasma cell can secrete about 2000 ab molecules per second• After abs bind to the foreign antigens at least 3 outcomes are possible: neutralization, complement activation, or opsonization

The 5 classes of antibodies/immunoglobins• Antibodies (abs) are also called immunoglobins, written as Ig

• There are 5 different classes:

• IgM - st to appear in response to an initial infection

– If high concentrations of IgM are detected it signals an initial infection

• IgG - account for 80% of circulating abs

– They are quickly produced when memory B-cells again contact a pathogen. Almost all circulating IgG indicate previous exposure to a pathogens (or vaccines).

– IgG immunoglobins also are important in passive immunity

• IgA antibodies are most common in mucous membranes and in body secretions (and thus is overall the most abundant antibody-class in the body)

• IgE abs attach to mast cells and basophil (for defense against parasites), but this arrangement can lead to allergies and anaphylaxis

• IgD abs are located on the surface of B-cells, presumably for B-cell activation, but their exact role is not well understood

The 5 antibody classes

• IgM antibodies are TOO LARGE to pass through the placenta

• IgG antibodies are the most common type in circulation and can pass through the placenta and into breast milk

• IgA antibodies are in secretions, including breast milk

• IgE antibodies form in response to an immune response to an “allergen”. Then they dock into the surface of mast cells.

Antibody class-switching from IgM to IgG• In an initial infection, B-cells and plasma cells start producing IgM antibodies - a

fact that is diagnostically useful in determining that the patient is being exposed to the pathogen for the 1st time

• After helper T-cell signaling, plasma cells will “class-switch”:

• Plasma cells from lymph-node B-cells switch to production of IgG molecules– These antibodies circulate in lymph and blood and enter tissues

– IgG antibodies also can pass through the placenta, giving the embryo-fetus natural passive immunity

– Also, an injection of “gamma globulin” for exposure to pathogens like the hepatitis A virus consists of IgG antibodies, conferring artificial passive immunity

• (Class-switching does NOT occur in T-cell independent responses of B-cells)

How pathogens are destroyed by antibodies• An IgG antibody is a Y-shaped protein

• Each antibody is specifically designed to attach to a surface feature on the pathogen - called an antigen (antigen stands for an antibody-generating)

• When numerous abs attach to a pathogen, it becomes inactivated, referred to as neutralization, after which the pathogen effectively becomes disarmed and must just wait to be destroyed by phagocytes roaming through the body

• Additionally, when an antibody attaches, it changes confirmation and “sends up a flag” that is easily recognized by phagocytic cells like neutrophils and macrophages (This process of destroying the pathogen is called opsinization).

• In other cases, the “flag” activates a set of plasma proteins referred to as the complement proteins circulating in blood

• Activated complement proteins drill holes into membranes of pathogens, destroying it.

• A Y-shaped antibody

3 roles of antibodies• Antibodies are Y-shaped proteins produced by B-cells and their progeny, plasma

cells

– When an antibody is produced in response to a certain pathogen or toxin the antibody has a binding site that specifically fits onto an antigen on the surface of the pathogen or toxin

• Antibodies attach to antigens, forming an antigen-antibody complex

• When antibodies attach to the surface of a pathogen or toxin, the “invader” is neutralized - neither a pathogen coated with antibodies nor a toxin coated with antibodies is able to attach to cells

• When an antibody attaches to the surface it also “sends up a flag” to notify the 2 different defense systems:

– Complement proteins circulating in blood are activated by the “flagged” antibody

• Complement proteins then drill a hole into the pathogen, contents within the pathogen leak out and the pathogen is destroyed

– Neutrophils and macrophages are attracted to the “flagged” invader and will phagocytoze it and the antibody - a process called opsonization (as opposed to simple phagocytosis of a pathogen) – the “cheeseburger effect”

The multiple roles of pathogen-bound antibodies

• Opsonization

• (Cheeseburger effect)

The antibody-activation of complement

• Activated complement proteins, called “MAC”, will drill holes in the membrane of a pathogen, a “Big MAC attack”!

• Holes in the plasma membrane of the bacterium causes cytoplasmic contents to leak out and the cell dies

Cytotoxic T-cell response to viral infections• Besides B-cell activation and the production of antibodies against a VIRUS, cytotoxic (“killer”) T-cells ALSO are activated by helper T-cells

• After being activated by an APC - helper T-cell, a cytotoxic T-cell will search out virally-infected cells

• A cell infected by a virus will have viral antigens sticking out of its plasma membrane

• The cytotoxic T-cell will attach to the virally-infected cell and destroy it by releasing molecules called “perforins” that put holes in the infected cell - destroying it and the virus within it molecules

• The cytotoxic T-cell also will divide many times and form a clone of memory cytotoxic T-cells specific to the original viral “antigen”

• The memory cytotoxic T-cells respond rapidly to a subsequent infection

The cytotoxic T-cell and cell-mediated cytotoxicity

• Cytotoxic T-cells recognize and destroy cells infected by a virus, because the infected cells have new surface features (like the viral “spikes”)

• The cytotoxic T-cell binds to the cell and then release chemicals called perforins that put holes in the membrane of the infected cell, killing it

• Cancer cells and transplanted tissue also can be destroyed by cytotoxic T-cells when they express novel antigens on their surfaces

• An infected cell with viral antigens sticking out

• An activated cytotoxic T-cell

Perforin molecules put holes in the infected cell

The perforin molecules of cytotoxic T-cells• The perforin molecules embed in the plasma membrane of the virally-infected cell, putting holes in the membrane (i.e., “perforating” it)

• “Granzymes” are proteases that then enter through the perforations and degrade cellular contents

• Leakage of intracellular contents results in cell death, which prevents the spread of the virus

• Cancer cells (as shown in this illustration) and the cells of transplanted tissues and organs are destroyed in the same manner

• Natural Killer cells, or “NK cells”, are lymphocytes that work in the same manner as cytotoxic T-cells, but NK cells don’t require “assistance” from a helper T-cell and don’t form a “memory” of the virus that caused the infection

Antibody-dependent cell-mediated cytotoxicity: (ADCC)

• For large parasites that cannot be phagocytosed by a single phagocyte, antibodies will be produced that will coat the surface of the parasite

• The antibodies will send up their “flags” and attract a number of phagocytes to the parasite, including macrophages, eosinophils, and NK cells for a form of opsonization called ADCC

• The defense cells will release lytic enzymes and perforins in-mass to destroy the parasitic “cell” (thus, the “cytotoxicity”)

B-cell activation independent of helper T-cells• The lipopolysaccharide/endotoxin of gram-

negative bacteria and a bacterial capsule both can activate a B-cell independent of a helper T-cell

• However, the B-cell response is generally weaker than those that require helper T-cells and no memory B-cells are produced, so there is NO long-term immunity

• Kids < 2yrs are susceptible to Streptococcus pneumoniae and Hib infections, principally because both have a capsule (the “grease” of the “greased pig” strategy)

• Capsular vaccines – like the one against Strep. pneumoniae - also do NOT protect these kids, but now a vaccine called “Prevnar” is available

• The green Y-shaped “receptors” are IgM molecules projecting from the surface of the B-cell

The activated B-cell: memory B cell and plasma cell clones

Antibody class-switching from IgM to IgA

• In an initial infection, B-cells and plasma cells start producing IgM antibodies - a fact that is diagnostically useful in determining that the patient is being exposed to the pathogen for the 1st time

• Plasma cells in mucosal tissues switch to production of IgA antibodies– MALT mucosal associated lymphatic tissue) consists of lymphatic nodules lining

the respiratory, urinary, reproductive, and digestive tracts (the locations of “Peyer’s patches”)

– The plasma cells in these tissues produce IgA antibodies that “monitor” mucosal linings for the “antigen”, be it on the pathogen or part of an exotoxin

– IgA antibodies also are abundant in colostrum and breast milk supplied to a mother’s offspring, giving the infant additional natural passive immunity

• (Class-switching does NOT occur in T-cell independent responses)

Natural immunity• When one is infected by a pathogen or exotoxin, the response of the immune system

in producing memory cells and IgG antibodies results in a natural active (NA) immunity against that pathogen or toxin

• IgG antibodies circulating in a pregnant woman from previous infections (or vaccines) can pass through the placenta (the mom’s blood and the fetus’ blood does NOT mix) into the fetus’ circulation, giving the fetus natural passive (NP) immunity to those pathogens or toxins

• IgA antibodies passed in breast milk give the fetus additional natural, passive immunity

– These natural passive immunities are temporary. Months after breast-feeding, this passivity immunity is gone and the infant has to rely on her/his own defenses against pathogens and exotoxins.

• There also are artificial immunities - those supplied by the medical community:

– Artificial passive (AP) immunity is a temporary immunity against a pathogen or toxin one may have been exposed to. It consists of IgG antibodies, called a “gamma globulin” shot.

– Artificial active (AA) immunity against a pathogen or exotoxin results from a vaccine

Vaccines and immunization• A vaccine is a substance introduced into the body to activate an immune

response that results in memory lymphocytes and circulating antibodies

– In other words, an effective vaccine immunizes an individual against a disease, a process called immunization (actually, an artificial acquired immunity as opposed to natural immunity after recovering from a disease)

– Helper T-cells, anti-viral cytotoxic T-cells, and anti-pathogen B-cells all are capable of producing memory cells after being “activated”

• A vaccine can contain weakened (called “attenuated”) pathogens that typically cannot make the individual ill, fully-inactivated, or “dead” pathogens, a part of a pathogen (i.e., an “antigen” from the pathogen), or an inactivated exotoxin (called a “toxoid”)

• After the vaccine is introduced by injection (the “parenteral route”) or ingestion, memory cells will be produced and circulate for variable periods of time, conferring immunity to a disease

Vaccines: an artificial mechanism of active immunity

• A vaccine is a preparation of weakened (“attenuated”) living or dead microorganisms or viruses, or their components, used to immunize an individual against a specific disease.– The best “components” for an effective immune response are protein and/or

carbohydrate (CHO) antigens; fats are poorly immunogenic.

• Vaccines confer long-time immunity because of the formation of MEMORY CELLS during the immune response

• Recombinant vaccines are those where a specific pathogen’s protein is “inserted” into the genome of a yeast or bacterial cell. The cell then produces an uncontaminated “vaccine”.

• A number of serious to deadly diseases have decreased up to 100% due to immunizations!

Memory cells are essential for a 20 response

• This graph shows that when an individual is initially exposed to a pathogen’s antigen - which could be either by getting the disease or by getting a vaccine, first IgM antibodies are released, followed by “class-switching” to IgG antibodies

• Because of memory B-cells, when the same antigen is encountered again, they and their plasma cells flood the body with IgG antibodies, which neutralize and “eliminate” the pathogen• This is essentially why vaccines/immunizations work so well

Vaccines and “length” of immunity• A vaccine prepared with a “live” (viable), but weakened, i.e., “attenuated”,

virus preparation offers the longest immunity– The weakened virus is injected. It will stimulate the immune system, and long-

lived memory cells will be produced, maybe circulating for a lifetime– Many viral vaccines use this method. The danger is transmitting the virus to

others before it is eliminated.

• A vaccine prepared with “dead” (“inactivated”) organisms does elicit an immune response, but the immunity does not last as long– The annual flu shots contain “dead” flu viruses– Presently, the polio vaccine used in the USA contains “dead” polio virus, but the

vaccine is “enhanced” to stimulate a stronger immunity– For bacteria, the vaccine may contain surface protein and/or glycoprotein surface

antigens; because fats are poorly immunogenic it can be more challenging developing a vaccine against gram-negative bacteria

• Vaccines against exotoxins, like the diphtheria toxin and the tetanus toxin, give the shortest immunity - about 10 years– The exotoxin - basically a protein - is neutralized and then injected as a “toxoid”– Memory cells are produced, but they are effective for only a number of years– Thus for protection, “booster shots” are necessary. For diphtheria toxin and the

tetanus lasts only 10 years, and then a booster shot of the vaccine is required.

Long-term vaccines given to children in the USA

• MMR: measles (rubeola), mumps, and rubella; all viruses

– On March 21st, 2005, the U.S. government announced that thanks to the vaccine congenital rubella syndrome has been eliminated in the USA

• DTaP: diphtheria, tetanus, and “acellular” pertussis (“whooping cough”); all bacteria

– There now is a new adult vaccine - “Adacel” - to prevent the rise of adult pertussis, or the “100-day cough”

• Chickenpox (Varicella-zoster virus)

• Hib: a vaccine against the bacterium Haemophilus influenzae type b

• The vaccine against Hepatitis B virus (HBV) is called “Recombivax”– It is a vaccine against surface antigens of the virus, produced by a yeast plasmid

• A vaccine against rotovirus, a leading cause of childhood diarrhea, and in the undeveloped world, childhood death by dehydration, was approved in the USA in 2006, and may be added to the list of childhood vaccines

More long-term vaccines• The vaccine for children against strains of Streptococcus pneumoniae, the cause

of pneumococcal pneumonia and other diseases is called “Prevnar”, which starts at 2-months of age

– There also is an adult vaccine against the capsule of Streptococcus pneumoniae, given to the elderly (it doesn’t work effectively in young children)

• There is a vaccine available against the Hepatitis A virus (HAV), also called “infectious hepatitis” and passed via fecal-oral route

– Many non-vaccinated individuals in the USA exposed to HAV are given a shot of gamma globulin (IgG) - effective if given within 2 weeks of exposure

• There is a vaccine available against the cause of tuberculosis (Mycobacterium tuberculosis) - a live, attenuated bovine strain (BCG)

– This vaccine is NOT routinely used in the USA

• The rabies virus vaccine is given to high-risk individuals, and people bitten by a rabid animal

• Yellow fever, typhoid fever, and cholera vaccines are given to travelers

• Anthrax and Smallpox vaccines are “available” for terrorism threats

Need for other vaccines• HIV (Human Immunodeficiency Virus) – the cause of AIDS

– 40 million infected worldwide; 14,000 new cases/year

• Malaria (Plasmodium – a protozoan)– 300-500 million infected; 3 million deaths/year

• Influenza virus– 30-50 million cases/year, worldwide; 10-40,000 deaths/year in USA

• Strep throat (Streptococcus pyogenes)– 20 million cases/year in USA

• Genital herpes (Herpes simplex virus-2, or HSV-2)– 45 million infected worldwide; 500,000 new cases/year in USA

• Hepatitis C virus– 170 million infected worldwide

• Anti-cancer vaccines:– The vaccine against Hepatitis B virus (HBV) is, in effect, a vaccine against HBV-

caused liver cancer– The vaccine against the strains of Human Papilloma Virus (HPV) that cause

genital warts is a vaccine against cervical cancer

• IgG abs can cross the placenta and IgG and IgA abs are abundant in breast colostrum and milk to confer natural passive immunity from mother to newborn

• IgG, called “gamma globulin” is injected for artificial passive immunity as a prophylaxis or to prevent disease after infection

Immunological clinical tests for infections

• The basis for many of these tests is the specificity of antibodies for the antigens of certain pathogens, but note that individuals exposed to infectious agents for the 1st time do NOT have detectable specific antibodies in their blood serum until about 7-10 days after infection at the earliest

• Seroconversion is the clinical term used when certain antibodies are detected in a patient’s blood

– After the seroconversion, antibody titer (concentration in blood) of that specific antibody typically increases. This rise in titer indicates an active infection.

– After someone is infected with the human immunodeficiency virus (HIV), his/her blood will show a seroconversion. It had no antibodies against HIV before but “converts” to having anti-HIV antibodies in circulation. Looking for anti-HIV antibodies is the most common method for detecting an HIV infection.

• Immunological tests can also be performed on sputum, urine, and CSF samples

– Clinical immunological tests include latex bead-agglutination tests and ELISA tests

Latex bead-bound antibody tests for pathogens • These tests utilize the clumping of latex beads – called agglutination – when a test is

positive for a particular pathogen

• In a direct agglutination test the beads are chemically-bound to antibodies known to attach to antigens on a specific pathogen

• If a sample contains the pathogen, the bead-bound antibodies will bind to the antigens on the pathogens and direct agglutination will result, where you’ll see clumping in the solution– In lab, this type of test is used to confirm the presence of Staph Aureus– We also will test for Group B strep (Streptococcus agalactiae), which can cause

serious neonatal infections– A similar test can be used to detect Streptococcus pyogenes, (called the “ELISA

test) when strep throat is suspected. We do not perform this one in lab (we do a PYR disc test instead)

• An indirect agglutination test are those that use the same method in reverse, to detect antibodies against the pathogen in the patient’s sample– The most common test for HIV is an indirect agglutination test, called an “ELISA

test”

The ELISA tests

• ELISA stands for Enzyme-linked immunosorbent assay– These tests utilize the specificity of antibodies for particular antigens – for us,

particularly those on the surface of pathogen organisms– Either antibodies specific for a pathogen or the antigens of specific pathogens

are adsorbed (i.e., attached to) to the inner surface of a test tube well– In both a direct ELISA and an indirect ELISA, there is a certain kind of enzyme

attached, i.e., linked, to antibodies used in the test

• A direct ELISA test starts with pathogen-specific antibodies attached to the inner well of a test tube– It tests for the presence of the pathogen

• An example is the “quick test” for strep throat

• An indirect ELISA test starts with antigens from a pathogen attached to the inner well of a test tube– It tests for the presence of antibodies to the pathogen

• An example is the test for antibodies against HIV

The indirect ELISA test to detect antibodies• Antigens specific for an antibody are adsorbed/coated onto the surface of the test tube

• The patient’s sample is added to the tube• IF the sample has the antibodies, they will bind to the antigens

• The rest of the sample is washed away – the antibodies are not

• A 2nd set of antibodies specific for the 1st antibodies - and with an enzyme attached to them – are added to the tube. They attach to the 1st antibodies (forming a “sandwich”)

• A second wash clears away unbound second-antibodies

• A chromogen that will change color when the enzyme is present is added

• IF a color-change is seen, then the patient’s sample had the antibodies

• This test is used to detect anti-HIV antibodies in a patient’s sample• If done too soon after infection, a false-negative result is likely

The direct ELISA test to detect antigen• Antibodies specific for a pathogen’s surface antigens are adsorbed/coated onto the surface of the test tube

• The patient’s sample is added to the tube• IF the sample has the pathogen, it will bind to the antibodies

• The sample is washed away – the antigens are not

• A 2nd set of antibodies also specific for the pathogen – but with an enzyme attached to them – are added to the tube, “sandwiching” the pathogen

• A second wash clears away unbound second-antibodies

• A substrate, called a chromogen that will change color ONLY when the enzyme is present is added

• IF a color-change is seen, then patient’s sample had the antigen

• This test is used for detecting Streptococcus pyogenes when strep throat is suspected

• It also is used for detecting drugs or HCG (pregnancy test) in urine

The Western blot test

• The Western blot, also referred to as the immunoblot test, combines electrophoresis (a technique to isolate proteins/antigens that electrically-migrate within a gel) and ELISA to separate and then immunologically determine the identity of antigens of a certain size in a sample

• Its main clinical use is the confirm positive ELISA screening tests for antibodies against HIV (i.e., the indirect ELISA test) because there is a small percentage of false positives with the ELISA test alone– The “test kit” has HIV antigens “blotted” from the gel onto a strip of filter paper– The patients serum is added and then enzyme-labeled anti-HGG antibodies are

added• Development of a color on the strip change indicates infection

Florescent-linked antibody tests for pathogens

• Antibodies against specific pathogens (I.e., against their antigens) are generated in a lab

• The antibodies then have a fluorescent dye chemically-linked to each antibody

• When the antibody binds to the pathogen, fluorescence is seen with a fluorescent microscope

Immune system disorders• In unforeseen circumstances, the immune system can respond inappropriately, called immune

system disorders

• Here we will briefly examine “hypersensitivity” of the immune system. Immune cells become inappropriately sensitive, or “responsive” We briefly will examine 4 hypersensitivities:

– A Type I hypersensitivity is an allergic reaction

• A severe allergic reaction is called anaphylaxis, which can lead to anaphylactic shock

– A Type II hypersensitivity can destroy cells, called “cytotoxicity”

• An example is Hemolytic Disease of the NewBorn (HDNB)

– A Type III hypersensitivity can result when antibody-antigen complexes activate defense cells, damaging healthy tissue

– A Type IV hypersensitivity generates memory helper T-cells that will activate a “delayed hypersensitivity”

• One example is the reaction to poison ivy

• The tuberculin skin test to determine if an individual has ever been exposed to Mycobacterium tuberculosis, the cause of tuberculosis, applies type IV hypersensitivity

An allergy is a type I hypersensitivity

• A type I sensitivity develops when the immune system responds to a harmless antigen, resulting in a allergy, or in severe cases, potentially-deadly anaphylaxis

• A type I hypersensitivity develops when B-cells (and plasma cells) produce a class of antibodies called IgE antibodies, in response to initial exposure to the “allergen”

• The IgE antibodies - which are “keyed” specifically for the allergen - attach to receptors in the plasma membrane of mast cells

• When the individual is exposed to the allergen molecules again, the molecules bind to the IgE antibodies

• This binding causes the mast cell to release its store of chemicals, including histamine (an “inflammatory” vasodilator) and leukotrienes, causing a local inflammatory response (also called localized anaphylaxis)

Anaphylactic shock is a severe type I hypersensitivity

• Hives (urticaria) is a localized skin reaction characterized by the formation of an edematous itchy swelling called a “wheal”, surrounded by a red rim, called a “flare”

– Mast cells in the skin with IgE antibodies embedded in their plasma membranes respond to an antigen and release their chemicals, causing the hives

• Asthma also is a localized anaphylaxis: IgE-bound mast cells in the lining of the respiratory tree release their chemicals

• Generalized systemic anaphylaxis is a rare but serious form of IgE-mediated allergy

– Both mast cells and their sisters, basophils circulating in the bloodstream, can have IgE antibodies embedded in their plasma membranes

– When the antigen is introduced, there will be a massive release of chemicals like histamine, that will cause a loss of fluid from the blood and tissue edema

– This can lead to anaphylactic shock – a dramatic loss of blood pressure that can lead to death

• To prevent suffocation, an injection of adrenaline, which relaxes the smooth muscles of the airway passages, is necessary

HDNB is a Type II hypersensitivity

• Hemolytic Disease of the NewBorn (HDNB), also known as erythroblastosis fetalis causes the rupture of fetal RBCS after antibody-fixation activates complement to destroy the cells (i.e., “cytotoxicity”)

• It occurs if an Rh- female produces anti-Rh factor antibodies after exposure to the blood of her Rh+ newborn• This risk can be prevented if the Rh- mother is passively immunized with anti-Rh+ antibodies in a medicine called “RhoGam”

Drug-induced thrombocytopenic purpura is a Type II hypersensitivity

• In “drug-induced thrombocytopenic pupura”, a drug attaches to the surface of platelets (thrombocytes), initiating an immune response that produces abs that bind to the drug

• Complement binds to the abs, and the platelets lyse

• This results in a loss of platelets (i.e., “thrombocytopenia”) and their loss results in internal hemorrhaging that appear on the skin as purple (“pupura”) spots

Post-streptococcal acute glomerulonephritis is a type III hypersensitivity

• Type III reactions involve antibodies that bind to soluble antigens circulating in the serum, with a certain ratio of these antigen-antibody complexes escaping opsonization

• The antigen-antibody complex can deposit in the lining of capillaries and activate circulating complement

• Certain complement factors attract neutrophils that release enzymes which damage the tissue

• This can occur after complexes of Streptococcus pyogenes and attached antibodies lodge in kidney glomerular capillaries, causing an inflammation called glomerulonephritis which can damage the filtering system

A poison ivy rash is a type IV hypersensitivity

• Chemicals on the leaves of the poison ivy initiate the production of memory T-cells• There is NO skin reaction the 1st time of exposure

• Any time the skin is exposed to the poison ivy after this sensitization, the activated memory T-cells cause a skin inflammation - a dermatitis, or rash commonly called “poison ivy”

The tuberculin skin test utilizes type IV hypersensitivity • Exposure to Mycobacterium tuberculosis, the causative agent of tuberculosis also will generate memory T-cells

• A positive test – seen as a localized rash (deramtitis) of certain dimensions – is evidence that the individual was exposed to the pathogen• It does NOT mean the person has TB

• The reaction results from the “delayed” congregation at the site of memory T-cells which were already exposed to the organism• The T-cells release factors that attract macrophages and the local release of chemicals causes a characteristic inflamed mound of a certain diameter.

• The tuberculin skin test screens for previous exposure to M. tuberculosis, by inoculating tuberculin protein into the skin

• This image shows a positive test – a redness and a firm swelling in the skin at the injection site• This is the definitive test for determining exposure to Mycobacterium tuberculosis

Auto-immune diseases• Autoimmune diseases are those in which one’s immune system attacks one’s own cells

• A major culprit are the antibodies directed against certain surface “markers” on one’s cells

• The fact that the different classes of our cells have surface features of their own is the determining factor in autoimmune disease specificity

• In most cases, antibody attachment “signals” destruction of the cell

• The cause of most of these diseases is unknown, but it is suspected that an initial immune response to a foreign antigen (perhaps like one on a microorganism or an exotoxin) results in the autoimmune response

• For example, the cause of the autoimmune disease multiple sclerosis is unknown, but some suspect an initial viral infection

• 90% of the victims of autoimmune diseases are females• There is no known cure for autoimmune diseases

Examples of autoimmune diseases

• Myasthenia gravis (neuromuscular junctions)

• Rheumatoid arthritis (synovial joints)

• Multiple sclerosis (myelin/oligodendrocytes in CNS)

• Grave’s disease and Hashimoto’s disease (thyroid)

• Addison’s disease and Cushing’s disease (adrenals)

• Type I diabetes mellitus (beta cells in the pancreatic islets)

• Lupus Erythematosis (DNA)

Transplants and immunosuppression

• Immunosuppression is NOT truly a disorder of the immune system

• Other than autografts or transplants from identical twins, a transplanted tissue always is at risk of rejection

• After transplantation surgery the drug cyclosporin (from a mold) is prescribed – usually for life.Cyclosporin suppresses the secretion of IL-2, disrupting cell-mediated immunity by cytotoxic T-cells and NK cells

• A newer immunosupressive drug, sirolimus (rapamycin) inhibits both cell-mediated (cytotoxic T-cell) and humoral (B-cell) responses

• In this example the “yellow” cell is not infected – it is a transplanted cell

• On its surface are major histocompatibility complex (MHC I) markers that identify the individual the cell came from• When the cytokine IL-2 (from helper T-cells) activates cytotoxic T-cells, they bind to the transplanted cells and release perforin, which puts holes in the cells and kills them, destroying the transplanted tissue or organ

Immune deficiencies• There are 2 categories: congenital (like SCID) and acquired

• Drugs, cancer, and infections can result in acquired immune deficiencies

• The best known is Acquired Immunodefiency Syndrome, or AIDS, that results from an infection with HIV, the Human Immunodeficiency Virus

Latent and active HIV infection

• AIDS is an immunodeficiency disease because the helper T-cell (CD4 cell) population steadily declines typically over the decade after infection

• Helper T-cells are also called CD4 cells because CD4 is the name of the receptor in the helper T-cell plasma membrane that the HIV virus uses to gain entry into the cell• The loss of helper T-cells leads to other infections because there are no helper T-cells to assist in “presenting” antigen to B-cells, so the B-cells remain inactive. These infections are what eventually kills the individual.

The progression of an HIV infection

HIV antiviral drugs• There is NO CURE for HIV infections, although some individuals appear to be naturally

immune to developing AIDS

• Presently, a “cocktail” of drugs is used to keep the HIV infection in check, i.e., decrease the viral “load” in blood and keep helper T-cell numbers up

– AZT blocks reverse transcriptase, the enzyme required for the RNA of HIV to synthesize DNA

– Protease inhibitors prevent the cleaving of long protein chains into active proteins by the viral enzyme protease

• These drugs include indinavir and saquinavir

• Currently there is NO vaccine available against the HIV virus, although not for lack of trying

• The virus regularly mutates (there is no “proofreading” for RNA retroviruses); in fact, an infected individual may have a number of mutated viruses in his/her system

Introduction to antimicrobial drugs• Antimicrobial drugs can be classified by the class of microorganisms the drus are directed

against

• Anti-viral drugs (for example, “Acyclovir” for the herpesviruses) typically target viral enzymes necessary for viral replication within host cells– Also, recall that our “host” cells produce an anti-viral chemical called interferon that

notifies other cells of a viral infection. Those cells then take steps to prevent the spread of the virus by self-destructing after infection, which eliminates the virus’ ability to replicate and spread.

– In the developed world, 60% of infectious illnesses are estimated to be viral– Every year, 90% of the U.S. population suffer viral diseases

• Anti-fungal drugs like amphotericin B or the azoles target ergosterol in the plasma membrane of fungal cells (our cells have cholesterol)

• Drugs against eukaryotic parasites, like protozoa and worms, are among the most toxic to us because of the cellular structural and functional similarity

• Antibiotics are drugs directed against bacteria

Introduction to antibiotics• Antibiotics are drugs used against bacteria (15% of diseases in the developed world are

bacterial)– Antibiotics are NOT effective against viruses

• There are 2 methods that antibiotics help one overcome a bacterial infection:– Bacteriostatic drugs are antibiotics that prevent multiplication of the pathogen. The

remaining bacteria are then destroyed by the defense system.– Bactericidal drugs are antibiotics that directly kill bacteria

• There are 2 broad categories of antibiotics:– Narrow-spectrum antibiotics are drugs that disrupt the synthesis of new cell walls of

multiplying bacteria, making these drugs bacteriostatic• Because gram-positive bacteria rely on a thick cell wall, narrow-spectrum drugs

are generally most effective against Gram-positives. (However, there are some gram-negative bacteria that are susceptible to narrow-spectrum antibiotics).

– Broad-spectrum antibiotics are drugs that disrupt cellular mechanisms required for survival of the bacteria, making these drugs bacteriocidal

• Broad-spectrum drugs work against both gram-positive and gram-negative bacteria, but are more commonly used for Gram-negative infections

Narrow-spectrum antibiotics

• The narrow-spectrum antibiotics are those that attack the cell wall of newly-forming bacteria

• The narrow-spectrum drugs bind to the peptidoglycan of the cell wall during its synthesis

• Because the cell wall is thicker and more accessible on gram-positive bacteria, narrow-spectrum drugs are generally more effective against the gram-positive bacteria– The narrow-spectrum drugs do NOT readily get through the LPS of many gram-negative

bacteria and these species have less of a cell wall to disrupt, so the narrow-spectrum drugs, like penicillin, are not usually effective against most gram-negative bacteria

• Erythromycin is an antibiotic that inhibits bacterial protein synthesis by interfering with the bacterial 70s ribosomes– However, unlike broad-spectrum protein synthesis inhibitors, erythromycin is water-

soluble and cannot penetrate the LPS of Gram-negative bacteria, making it narrow-spectrum

• Narrow-spectrum drug don’t harm us (except for allergies) because our cells do NOT have a cell wall

The penicillin tree

• There are over 50 penicillin-based antibacterial drugs

• Natural penicillins are purified from the mold, Penicillium notatum

• They are narrow-spectrum and most effective against gram-positive bacteria

• Semi-synthetic penicillins are penicillins that are chemically-altered to enhance their effective

• Some semi-synthetic penicillins are fat-soluble and broad-spectrum, making them effective against some gram-negative bacteria

Beta-lactam ring narrow-spectrum antibiotics

Penicillin drugs inhibit bacterial cell wall synthesis

• Penicillins also are called beta-lactam drugs, because they have a beta-lactam ring that is the component of the drug that attaches to peptidoglycan penicillin-binding proteins (PBPs), disrupt peptidoglycan synthesis

• Mutations in bacterial PBPs that prevent attachment of the beta-lactam ring can result in bacterial resistance to the penicillin drugs

• The penicillin drugs target and disrupt peptidoglycan synthesis in the cell wall of growing bacteria

• This weakens the wall and kills growing cells (making penicillins bactericidal)

• Penicillins are generally more effective against gram-positive bacteria that have not acquired resistance to the drugs

• This reason for the effectiveness is that the gram-positive bacteria rely on a thick, peptidoglycan-rich, cell wall for survival

MRSA Staphylococcus aureus

• The semi-synthetic penicillin drugs, starting with methicillin, and followed by others like oxacillin and nafcillin, were designed to be resistant to inactivation by penicillinase– However, newer strains of penicillinase-producing Stapylococcus aureus mutated with

altered penicillin-binding proteins (or “PBPs”) that the drugs CANNOT attach, creating a rsistsance to these drugs referred to as Methicillin-Resistant Staphylococcus aureus, or MRSA strains of Staph aureus

• MRSA was once confined to hospital (“nocosomial”) settings. But it has spread into “communities”, resulting in 2 MRSA designations:

– HA-MRSA is “hospital-acquired (i.e., “nosocomial”)– CA-MRSA is “community-acquired”

• Because of the resistance of the MRSA strains to these drugs, more aggressive treatment is required for a MRSA infection:– This can include a cell-wall disrupting drug called vancomycin

• Vancomycin is an intravenous drug given for systemic MRSA infections. It must be administered carefully because it is toxic to our cells.

– A topical ointment of bacitracin, called “Bactriband”, also is effective

– A newer broad-spectrum drug, called “Zyvox”, also is effective against MRSA

Vancomycin and Bacitracin inhibit bacterial cell wall synthesis

• MRSA infections require alternate antibiotics to the penicillins

• Vancomycin interferes with peptidoglycan synthesis, but NOT at penicillin-binding proteins (PBPs), so its mechanism is NOT inactivated by the newer strains of MRSA

• Bacitracin is a topical ointment that disrupts peptidoglycan synthesis by interfering with the transport of newly-synthesized peptidoglycan precursors into the cell wall• As “Bactriband”, it can be an effective ointment against nasal carriers of MRSA

• “Zyvox” is the newest antibiotic (available since 2000). It is a broad-spectrum antibiotic, whose mechanism is to interfere with bacterial protein synthesis.

• Zyvox also is effective against MRSA

Penicillinase is an enzyme of bacterial drug resistance

• The enzyme penicillinase may have 1st been produced by the gram-positive bacillus, Clostridium difficile, which has pathogenic potential under certain circumstances

• The resistant gene with the blueprint for penicillinase was transferred in a plasmid to Staphylococcus aureus, creating Staph aureus strains that are resistant to penicillin

• A new series of semi-synthetic penicillinase-resistant penicillins were developed, including one called methicillin, with side-chains that prevented penicillinase from inactivating the drug

• Certain bacteria have undergone genetic mutations that have resulted in the production of an enzyme called penicillinase that inactivates penicillin• (The penicillinase cleaves the beta-lactam ring of penicillin. Thus, penicillinases are sometimes called beta-lactamases)

Semi-synthetic penicillins have a “broader” affect• To combat penicillin-resistant strains of Staph aureus (and other bacteria) that produce the

enzyme penicillinase, which inactivates penicillin before it can damge cell walls, semi-synthetic penicillins, like “Augmentin”, have been created– Augmentin has a chemical (called “clavulanic acid”) added onto the antibiotic

(“amoxicillin”, one of the penicillins) – The clavulanic acid blocks the enzyme activity of penicillinase released by the bacteria– With the penicillinase blocked, the penicillin can reach the cell wall (and bind to the

“penicillin-binding proteins, or PBPs) to disrupt peptidoglycan synthesis– New clones cannot survive, helping to control the bacteria while the defenses wipe out

the rest

• Some semi-synthetic penicillins have enhanced capability in penetrating the LPS of gram-negative bacteria because of side-chain additions– Because these penicillins can pass through the LPS, they are classified as broad-spectrum

antibiotics• The mechanism of action, however, remains the same: disruption of peptidoglycan

synthesis by attaching to PBPs– AMPICILLIN and AMOXICILLIN are acid-resistant lipid-soluble broad-spectrum antibiotics

that can be taken orally.• They are effective against Gram-positive bacteria and the following gram-negative bacteria:

Hib, E. coli, Salmonella, Shigella, and Proteus– TICARCILLIN or CARBENICILLIN are broad-spectrum semi-synthetic penicillins active against

the Gram-negative rod Pseudomonas aeruginosa, which is a major problem in nosocomial infections

Other beta-lactam drugs that disrupt peptidoglycan synthesis• Any drug with a beta-lactam ring that that can interfere with peptidoglycan synthesis is called

a beta-lactam drug

– As we’ve seen, this includes the penicillins

• Cephalosporins are beta-lactam drugs that are less susceptible to destruction by penicillinase (also called “beta-lactamase”)

– There are more than 70 different cephalosporins

– Cephalosporins can be used by individuals allergic to penicillin

– Cephalosporins are generally more effective against certain gram-negative bacteria

• The carbapenems are broad-spectrum beta-lactam antibiotics

– “Primaxin” appears to be active against 98% of all organisms isolated from hospital patients (nosocomial infections)

• The monobactams is an unusual class of beta-lactam drugs that is only effective against certain gram-negative bacteria, including E. coli and Pseudomonas aeruginosa

Erythromycin• Erythromycin (a “macrolides”) is a bacteriostatic, effective against many Gram-positive

bacteria and Mycoplasma pneumoniae, a tiny bacterium without a cell wall, that causes “walking pneumonia” – Erythromycin is an antibiotic that inhibits bacterial protein synthesis by interfering with

the bacterial 70s ribosomes– However, unlike broad-spectrum protein synthesis inhibitors, erythromycin is water-

soluble and cannot penetrate the LPS of Gram-negative bacteria, making it narrow-spectrum

– is generally used as an alternative for those allergic to penicillin– It also is a popular “pink”/red drug with children (“erythro” means red)

• Most other protein synthesis-inhibiting antibiotics and fat-soluble and broad-spectrum

• Lincosamides, which include “Zyvox”, are narrow-spectrum antibioticsA 70S bacterial

ribosome

“Zyvox”

• In 2000, the 1st new antibiotic in 35 years, “Zyvox” (linezolid) was approved by the FDA for vancomycin-resistant bacterial infections and particularly nocosomial infections (including nocosomial Gram-positive pneumonia) caused by these Gram-positive bacteria:

– Staphylococcus aureus

– Streptococcus pyogenes

– Group B strep (S. agalactiae)

– Streptococcus pneumoniae

– Enterococcus faecalis

• Linezolid inhibits bacterial protein synthesis by interfering with the 70s bacterial ribosome

Antibiotics and “the pill”

• Some antibiotics decrease the ability of the body to absorb the hormones estrogen and progesterone in birth control pills

– This, obviously, can increase the risk of conception and pregnancy

• The antibiotics listed as increasing this risk include:

– Penicillin, Amoxicillin, Ampicillin, and Augmentin

– The tetracyclines

– Sulfa drugs, like Septra, used for urinary tract infections

– Rifampin

– Others

• Additional birth control protection is advised during the antibiotic course of treatment up until at least one week after the woman is finished taking the antibiotics

The natural penicillin drugs

• Natural penicillins are narrow-spectrum antibiotics effective against non-resistant gram-positive spheres - the staphylococci and streptococci - Treponema pallidum, the syphilis-causing spirochete, and some gram-negative cocci, like the gram-negative diplococcal genus Neisseria

• Penicillin G (the 1st antibiotic) is usually given intramuscularly

• Penicillin V is a gastric-acid resistant modification that can be taken orally

Broad spectrum drugs

• The broad-spectrum antibiotics are effective against both gram-positive and gram-negative bacteria but they are primarily used against gram-negative bacteria– The broad-spectrum antibiotics are lipid-soluble drugs that can diffuse through the

LPS of gram-negative bacteria

• Certain broad-spectrum antibiotics disrupt bacterial protein synthesis– The mechanisms of action of these drugs are to target and disrupt some aspect of protein

synthesis occurring at the 70s bacterial ribosome– These drugs include the tetracyclines, the aminoglycosides, chloramphenicol, “Zyvox”

(based on a chemical called a “linezolid”) and others–

• The fluoroquinolones, including ciprofloxacin (“Cipro”), prevents the supercoiled, circular strand of bacterial DNA from “nicking open”– These drugs interfere with the bacterial enzyme which “nicks” the DNA open, a step

required for DNA replication and the “transcription” step of protein synthesis

• The sulfa drugs interfere with the conversion of PABA to folic acid, a vitamin required for survival of the bacterium

The 70s RIBOSOME is a major target

• All proteins are synthesized in RIBOSOMES - the “protein-manufacturing plants of a living cell

• Many antibiotics target the 70s ribosomes of bacteria, sparing the 80s ribosomes that carry out protein synthesis

Broad-spectrums that interfere with bacterial protein synthesis

• In bacteria, all proteins are assembled within organelles called 70s ribosomes

• Certain broad-spectrum antibiotics - including tetracycline - prevent protein synthesis by interfering with the 70s ribosomes

• Erythromycin also disrupts protein synthesis but is water-soluble and narrow-spectrum (it can’t penetrate the LPS of Gram-negative bacteria)

• These drugs do NOT disrupt protein synthesis in human cells because they have 80s ribosomes and the drugs do NOT attach to these

• Proteins are built within 70s ribosomes, organelles located within the cytoplasm of bacterial cells

Broad-spectrum drugs that inhibit bacterial protein synthesis

• The tetracyclines include doxycycline (a tetracycline that is retained in the body longer), “Terramycin” and “Aureomycin”.– Tetracycline use has risks: tetracyclines can stain the growing teeth of children and

can be toxic to the kidneys if given after the expiration date

• Chloramphenicol is effective against a wide range of bacteria and can cross the blood-brain-barrier– Chloramphenicol can cause a rare, but lethal, condition called aplastic anemia, where the

body cannot manufacture red or white blood cells, so alternates to it are commonly advised

• The aminoglycosides are a class of drugs that include streptomycin (used for tuberculosis), gentamicin (effective against Pseudomonas aeruginosa) and neomycin, which is topical only– All the aminoglycosides can cause hearing loss and kidney damage, and so are used

when alternatives are not available

Antimicrobials that inhibit bacterial nucleic acid synthesis

• These broad-spectrum drugs target the enzymes required for the “nicking open” of the bacterial DNA, which is in a continuous loop in its inactive form– The loop of DNA must be nicked open before a bacterial cell can replicate its DNA for

multiplication or before a bacterial cell can carry out “transcription” - the production of mRNA, with the instructions for protein synthesis

• Fluoroquinolones include ciprofloxacin, or “Cipro”, which became popular in the USA as a prophylactic defense against inhalation anthrax (Bacillus anthracis) during the bioterrorism attack in 2001

• Rifamycins, which include “Rifampin” block prokaryotic RNA polymerase– Rifampin also is effective against the acid-fast positive Mycobacterium tuberculosis –

the cause of tuberculosis, and Mycobacterium leprosae - the cause of Hansen’s disease (leprosy)

Sulfa drugs• Folic acid synthesis, from a chemical designated “PABA”, is required for bacterial survival because of its role in converting DNA and RNA precursors into DNA and/or RNA

• Sulfa drugs inhibit the synthesis of folic acid from PABA

• Because the drugs are chemically-similar to PABA, the PABA-enzyme locks onto the drug, preventing the necessary conversion of PABA

• Sulfa drugs sometimes are given in combination requiring only 10% of the normal dosage – called drug “synergism”

• An example is combining “sulfamethorazole”, which inhibits an early step in the PABA-to-folic acid pathway, with “trimethoprim” - as in the urinary tract drug, “Septra” - which inhibits a later step in the pathway, effectively disrupting the synthesis of folic acid at a much lower dose than would be used for either drug individually

• Most “UTIs” are caused by the facultative anaerobe, E. coli

Summary of antibiotic modes of action

Drug resistance

Targets of antibacterial drugs

Drug resistance

The disk-diffusion test to assess drug effectiveness

• The Kirby-Bauer disk-diffusion test is a method used to determine the effectiveness of a number of drugs – each infiltrated into a absorbent disk – against a single species

• The area of inhibition around the disk is measured to estimate the minimum inhibitory concentration (MIC) of each drug

• The LARGER the zone of inhibition, the more effective the drug

• The clear zones around each white disk (impregnated with a certain antibiotic) is the zone of inhibition• Here, the upper and lower disks are more effective

Drug treatment for TUBERCULOSIS

• Tuberculosis (TB) is an increasing health problem

– An estimated 10 million Americans are infected by Mycobacterium tuberculosis. The ID50 for TB is 10 bacteria!

– It is spreading among victims of AIDS (about 50% of who die of TB) and a risk to their health-care providers

• TB is the leading cause of death among AIDS vixtims

• Drug-resistant strains of TB has led to treatment with a “drug cocktail”

– The 1st line involve 5 drugs that are most effective and least toxic. These include:

• Streptomycin

• Isoniazid (INH) inhibits the synthesis of mycolic acids

• Etambutol inhibits other cell wall synthesis enzymes

• Rifampin blocks prokaryotic RNA polymerase

Some bacterial strategies against drugs• Penicillinase is an enzyme that interferes with the peptidoglycan-disrupting activity of

penicillin drugs. It was originally encoded on a plasmid in gut enterococci

– This R-plasmid was transferred to Staphylococcus aureus and other bacteria

• Chloramphenicol acetyltransferase interferes with the activity of chloramphenicol

• Bacteria can alter (mutate) the target molecule of the drug: some penicillin-resistance results from a change in the structure of the bacterium’s PBPs; a change in ribosomal RNA has made macrolides (erythromycin) ineffective against some bacteria

• Some Gram-negative bacteria are resistant to broad-spectrum anti-bacterials because they have changed the porin proteins (normal “channels” in the plasma membrane), preventing entry of the drugs

• Some bacteria are resistant to broad-spectrum anti-bacterials because they have altered their efflux pumps to eliminate a drug that has entered the cytoplasm

An antibiotic that damages the plasma membrane of bacteria

• Polymycin B is a broad-spectrum drug that damages the bacterial plasma membrane, leading to leakage of cellular contents– It only is topical because it also attaches to the plasma membrane of eukaryotic cells

- although to a lesser extent

Anti-viral herpes drugs: ACYCLOVIR

• Acyclovir is used against a number of the herpesviruses• Acyclovir resembles a viral nucleoside-enzyme and will “create” a “false nucleotide” that blocks the DNA synthesis of the viral genome

• Famciclovir and ganciclovir are acyclovir derivatives• The drugs can limit the duration of subsequent outbreaks of “cold sores” and “genital herpes”

• Trifluridine is used for acyclovir-resistant herpes keratinitis (an eye infection)

Cellular response to viral infection: INTERFERON

Other antiviral drugs

• RIBAVIRIN, which resembles guanine and interferes with viral replication, is used against hepatitis B and hepatitis C. Recombinant INTERFERON also is being used for hepatitis C infections.

• The enzyme neuraminidase, produced by influenza viruses, is inhibited by RELENZA and TAMIFLU

• Amantadine and Rimantidine are anti-FLU drugs that prevent the “uncoating of the envelope and capsid, trapping the RNA inside

HIV antivirals

• AZT blocks reverse transcriptase, the enzyme required for the RNA of HIV to synthesize DNA that is only found in retroviruses

• Protease inhibitors prevent the cleaving of long protein chains into active proteins by the viral enzyme protease. These drugs include indinavir and saquinavir.

Anti-fungal, -protozoan, and –helminth drugs• Antifungals, like amphotericin B (produced by Streptomyces), permealize the ergosterol-rich

plasma membrane – i.e., put holes in the membrane, killing the cells– Azoles interfere with sterol synthesis– Griseofulvin (a Penicillium antifungal) – given orally - binds to keratin and acts against

dermatophytes

• Flagyl is anti-protozoan drug, but it also works against obligate-anaerobic bacteria, like Trichomonas vaginalis

• Quinine and its derivatives (like “Larium”) are used against the malaria plasmodia, although resistant is becoming widespread

• Mebendazole disrupts the microtubules of worms, diethylcarbamazine alters the worm plasma membrane so the immune system can attack it, and other drugs paralyze worms (ivermectin, oxantel, piperzines, praziquantel)

A graph of anti-septic effectiveness

• Tincture of iodine is the most effective antiseptic, killing 90% of bacteria within 1-and-a-half minutes• Anti-septic effectiveness is followed by 70% ethanol

• Soap-and-water does NOT kill bacteria, but is effective because it washes microbes off the surface of the skin!• Hand-washing is one of the MOST EFFECTIVE personal strategies to prevent transferring pathogens – including in nosocomial settings

Sterilization: the AUTOCLAVE and filtering

• Moist, high heat (steam) at pressure kills microorganisms primarily by the coagulation of their proteins

• At 15 psi and 1210C, steam will also kills endospores in 15 minutes

• However, autoclaving does NOT destroy endotoxin• Thus, IV fluids are sterilized by filtration to remove any endotoxin