Download BIO 580 - Medical Microbiology - Unit One Part II

Objectives:
1. understand the multiple lines of defenses against microbial infection
2. recognize the signs of acute inflammation when presented in a clinical context and describe
how each sign of acute inflammation is generated at a cellular/tissue level
3. know why/how the alternate complement cascade is activated, what the important molecules
formed are, what their function is, and what the consequences of complement activation are
4. understand how complement and phagocytosis are integrated processes
5. compare and contrast PMNs and macrophages
6. understand the importance of direct cell-cell contact in phagocytosis and the relationships
between capsules, opsonins, and phagocytosis
II. HOST DEFENSES
The immune system is composed of 2 arms
 innate = nonspecific – already in place, response is rapid, not as efficient
 adaptive = specific – must be induced, response is slower, highly efficient, enhances
nonspecific. Has “memory”
A. Nonspecific Defenses
1. Defenses against entry into the host (1st line defenses)
a. Physical defenses (examples)
1. epithelial cells
2. turbulence
3. shedding, scraping, flushing (saliva, urine)
4. muco-ciliary clearance (1-3 cm/hr)
b. Chemical defenses (exs)
1. acids (e.g. gastric, fatty acids)
2. enzymes (e.g. lysozyme in saliva, tears, perspiration, urine)
3. other microbicidal chemicals (e.g. zinc, dermicidin)
c. Biological defenses (exs)
1. normal microbiota – physical, competition, inhibitory substances
2. immune defense cells and molecules
2. Defenses of the interior of the host (2nd line defenses)
Inflammation
a. phagocytic cells
b. nonspecific cytolytic cells
c. complement cascade
d. acute phase proteins
e. interferons
Inflammation - a process that coordinates and regulates all aspects of non-specific interior
defense.
Acute inflammation is characterized by:
1. increased blood supply to the area
2. increased capillary permeability
3. accumulation of neutrophils
Signs of acute inflammation – 4 signs
1.
2.
3.
4.
Triggers of acute inflammation – 2 triggers
1. cell/tissue damage/injury  chemical “alarms”
2. cell wall components of bacteria (peptidoglycan, LTA, LPS)
How Signs of Acute Inflammation are Produced
1. release of Inflammatory Mediators (=IM; see table 9.3)
2. vaso-dilation &  blood flow 
2. endothelial cells of vessels contract 
3. plasma leaks out of vessels & into tissues = exudation 
4. swelling  pressure on nerve endings 
bradykinin 
***Increased blood flow & capillary permeability - a mechanism for white blood cells and
critical soluble factors to enter the tissues to combat microbial invaders.
The Critical White Blood Cells (WBC = leukocytes)
a. Phagocytes – professional engulfing cells
2 main roles for phagocytes
1. engulf and destroy foreign matter
2. secrete chemicals (esp. cytokines)
2 main kinds of professional phagocytic cells
1. polymorphonuclear leukocytes (= PMN = polymorph = neutrophils) – are granulocytes 2. mononuclear leukocytes - monocytes & macrophages – are agranulocytes 1. polymorphonuclear leukocyte
 dominant cell type in early stages acute inflammatory response
 made in the bone marrow - 80 mil/min
 dominant WBC, ~5,000/ul of blood - ↑15,000-20,000/ul
 live for 2-3 days - function in anaerobic environments
 abundant cytoplasmic granules contain loads of antimicrobial enzymes and chemicals
(acid hydrolases, myeloperoxidase, lysozyme, lactoferrin)
 best with extracellular pathogens, esp. bacteria
2. mononuclear leukocyte
 majors players later in inflammatory process
 made in bone marrow
 in the blood - monocytes (~600/ul); in tissues - macrophages (~60,000)
 conc. in lung, liver, lymph nodes, spleen
 live for weeks or months
 fewer granules (acid hydrolases, peroxidase)
 one of the antigen presenting cells
 secrete lots of different proteins (incl. PG, IL-1, TNF)
 best with intracellular pathogens
Process of Phagocytosis – 6 steps
1. Activation a. margination
b. pavementing
c. diapedesis
2. Migration via chemotaxis
3-6. – illustrated in diagram
3. Attachment
Pathogen-Associated Molecular Patterns
Microbe - PAMP – PRR – phagocyte
Pattern Recognition Receptors
PAMP incl. LTA & LPS (table 9.2)
After PAMP-PRR interaction, macrophages secrete pro-inflammatory cytokines (TNF, IL-1)
 enhance antigen-presentation  leads to activation of Th1. We’ll talk about this later.
4. Engulfment
5. Phagosome-lysosome fusion and intracellular killing
6. Expulsion of debris
Phagocytosis - Diagram
Role of Opsonins in Process of Phagocytosis - Diagram
Microbe
+phagocyte
Opsonin
none
present
Rate of Phagocytosis
Phagocyte Intracellular Killing Mechanisms
1. Oxygen-dependent killing (see box 9.2) – PMN and macrophages
“oxidative burst”
Reduction
O2
NADPH
NADP
superoxide anion
hydrogen peroxide
singlet oxygen
hydroxyl radicals
O2H2O2
1
O2
OH
Reactive Oxygen
Intermediates (ROI)
Other reactive intermediates
 reactive nitrogen (nitric oxide = NO)
(macrophages)
 reactive
chlorine
(OCl)
+
myeloperoxidase (PMN)
phagosome
membrane
2. Oxygen-independent killing compounds – PMN and eosinophils
 acid hydrolases (PMN)
 cathepsin G (PMN
 cationic proteins (PMN, eosinophils)
 defensins (PMN)
 lactoferrin (PMN)  lysozyme (PMN, also macrophage) –
 peroxidase (eosinophils)
b. Nonspecific Cytolytic Cells
1. natural killer cells (NK)
use - perforins + granzyme  apoptosis
also secrete TNF
target - intracellular pathogens, primarily viruses
also – secrete -IFN that can activate macs to intracellular killing
2. basophils and mast cells
can be triggered to discharge cytoplasmic granules
release – histamine, heparin, anaphylactic factors
target – parasites
3. eosinophils
can be triggered to discharge cytoplasmic granules
use - basic proteins, perforins, ROI  chemical burns
target - large parasites (e.g. helminthes)
The Critical Soluble Factors
c. Complement - activation of the alternate cascade (= properdin pathway)
A group of 20 serum proteins – form an enzymatic cascade
C3 (most abundant)
C3a
C3b
Factor B
C3bB
Factor D
C3bBb =
C3
C3a
C3
C3b
C3a
C3b
C5
C5a
C5b
C5b678 multiple C9
cell lysis
d. Acute Phase Proteins
 Plasma proteins – proteins that increase in concentration 2-100X during the acute phase of
an infection.
Ex. Many of the complement proteins
Ex. C-reactive protein (CRP) – produced by liver in response to pro-inflammatory cytokines
(exs. IL-1, TNF)
 uses pattern recognition to bind to bacteria
 promotes binding of C3b (opsonization)
 activates complement cascade
Microbe
+phagocyte
Opsonin
none
C3b
CRP
Rate of Phagocytosis
e. Interferons – one of many kinds of “cytokines”, chemicals used in cell to cell communication
Cytokines: (see table 11.2)
 small secreted proteins that mediate and regulate inflammation, immunity, and
hematopoiesis
 act over short distances, short duration, and low conc.
 receptor binding  signal transduction
Interferons - interfere w/ viral replication
3 classes of Interferons:
1.  - produced by leukocytes
2.  - produced by fibroblasts and other cells
3.  - produced by T lymphocytes and NK (does NOT interfere with viral replication)
Integration of Nonspecific Defenses
1. Stimulus
a. cell/tissue injury
inflammatory mediators released
b. microbial surface polysaccharides
2. Within seconds to minutes
a. acute inflammation begins
 vaso-dilation of capillaries  increases blood flow to/volume at the site
 increased vascular permeability  exudation of plasma, proteins, and cells
b. complement activation
 C3a and C5a  mast cell degranulation
maintains vaso-dilation/vascular permeability
 C5a attracts phagocytes from vasculature
 C3b and C5b bind to cell surfaces
c. Acute phase proteins increase in concentration – CRP, complement, and IFN
3. Minutes to hours
a. PMNs arrive in huge number and encounter cells opsonized by C3b and CRP 
phagocytosis is enhanced.
4. Hours to days
a.
Macrophages arrive
b. NK arrive
SUMMARIZE – Nonspecific Defenses
List the Nonspecific Interior Defenses important against Bacteria
List the Nonspecific Interior Defenses important against Viruses
CONCEPT CHECK – Nonspecific defenses
Bubble Map
Characteristics unique to PMN
PMN
Shared characteristics
Characteristics unique to macrophages
Macrophage
CONCEPT CHECK – Nonspecific defenses
Draw a concept map that illustrates the integration of complement and phagocytosis
CONCEPT CHECK - Nonspecific Host Defenses
Steve, a college student, was backpacking in a remote wilderness region with some friends.
While pitching a tent, he tripped and fell. In an attempt to break his fall, he extended his arms
and sustained a puncture wound to his right palm. Although the wound was painful and bled for
a short time, it didn’t appear to be serious, and Steve fell asleep that night unconcerned.
By the next morning, however, Steve noticed that the tissues immediately surrounding his wound
were red, swollen, and warm. A round area about 1 inch in diameter really looked abnormal
compared to the rest of his hand. The affected area was also painful, especially when he touched
it or bumped it. After hiking all day, the sore hand was even more painful, and a thick yellow
discharge oozed from the open wound. Steve felt unusually tired, his body ached, and a brief
chill made him aware that he was getting a fever. His friends helped him elevate his arm and
applied warm compresses to his palm, hoping that he would feel better in the morning.
1. List the nonspecific defenses against entry that are relevant to skin.
2. How were Steve’s skins defenses against entry overcome?
3. List the nonspecific interior defenses that would become activated in this case of bacterial
invasion.
4. What are the signs in the case history that Steve is experiencing an inflammatory process? List
them.
5. Describe the mechanisms at the cellular/tissue level that cause each of the signs of
inflammation listed in 4 above?
Supplemental information FYI
Saliva (modified from Wikipedia) - Produced in salivary glands, human saliva is 98% water, but
it contains many important substances, including electrolytes, mucus, antibacterial compounds
and various enzymes
It is a fluid containing:
 Water
 Electrolytes: (sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate,
iodine)
 Mucus. Mucus in saliva mainly consists of mucopolysaccharides and glycoproteins;
 Antibacterial compounds (thiocyanate, hydrogen peroxide, and secretory IgA)
 Epidermal growth factor or EGF
 Various enzymes. There are three major enzymes found in saliva.
o α-amylase - starts the digestion of starch and lipase fat.
o lingual lipase
o Antimicrobials that kill bacteria:
 Lysozyme
 Salivary lactoperoxidase
 Lactoferrin
o Proline-rich proteins (function in enamel formation, Ca2+-binding, microbe killing and
lubrication)
o Minor enzymes
 Cells: Possibly as much as 8 million human and 500 million bacterial cells per mL. The
presence of bacterial products (small organic acids, amines, and thiols) causes saliva to
sometimes exhibit foul odor.
 Opiorphin, a newly researched pain-killing substance found in human saliva.
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are a family of
enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between
N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between Nacetyl-D-glucosamine residues in chitodextrins. Lysozyme is abundant in a number of
secretions, such as tears, saliva, human milk, and mucus. It is also present in cytoplasmic
granules of the polymorphonuclear neutrophils
(PMN).
Urine is approximately 95% water. The other components of normal urine are the solutes that
are dissolved in the water component of the urine. These solutes can be divided into two
categories according to their chemical structure (e.g. size and electrical charge).
Organic molecules These include:
 Urea – Makes up 2% of urine. Urea is an organic compound derived from ammonia and
produced by the deamination of amino acids. The amount of urea in urine is related to
quantity of dietary protein.
 Creatinine - Creatinine is a normal constituent of blood. It is produced mainly as a result of
the breakdown of creatine phosphate in muscle tissue. It is usually produced by the body at a
fairly constant rate (which depends on the muscle mass of the body).
 Uric acid - Due to its insolubility, uric acid has a tendency to crystallize, and is a common
part of kidney stones.
 Other substances/molecules - Example of other substances that may be found in small
amounts in normal urine include carbohydrates, enzymes, fatty acids, hormones, pigments,
and mucins (a group of large, heavily glycosylated proteins found in the body).
Ions These include:
 Sodium
 Potassium
 Chloride
 Magnesium
 Calcium
 Ammonium
 Sulfates
 Phosphates
Blood (modified from Wikipedia)
Blood accounts for 8% of the human body weight. The average adult has a blood volume of
roughly 5 liters (1.3 gal), composed of plasma and several kinds of cells; these formed elements
of the blood are erythrocytes (red blood cells; RBC), leukoytes (white blood cells; WBC), and
thrombocytes (platelets). By volume, the red blood cells constitute about 45% of whole blood,
the plasma about 54.3%, and white cells about 0.7%.
Cells:
One microliter of blood contains:
 4.7 to 6.1 million (male), 4.2 to 5.4 million (female) erythrocytes. The proportion of blood
occupied by red blood cells is referred to as the hematocrit, and is normally about 45%.
 4,000–11,000 leukocytes. White blood cells are part of the immune system; they destroy and
remove old or aberrant cells and cellular debris, as well as attack infectious agents and
foreign substances.
 200,000–500,000 thrombocytes: Platelets are responsible for blood clotting (coagulation).
They change fibrinogen into fibrin. This fibrin creates a mesh onto which red blood cells
collect and clot, which then stops more blood from leaving the body and also helps to prevent
bacteria from entering the body.
Plasma:
About 55% of whole blood is blood plasma, a fluid that is the blood's liquid medium, which by
itself is straw-yellow in color. The blood plasma volume totals of 2.7–3.0 liters (2.8–3.2 quarts)
in an average human. It is an aqueous solution containing 92% water, 8% blood plasma proteins,
and trace amounts of other materials. Plasma circulates dissolved nutrients, such as glucose,
amino acids, and fatty acids (dissolved in the blood or bound to plasma proteins), and removes
waste products, such as carbon dioxide, urea, and lactic acid.
Other important components include:
 Serum albumin
 Blood-clotting factors (to facilitate coagulation)
 Immunoglobulins (antibodies)
 lipoprotein particles
 Various other proteins
 Various electrolytes (mainly sodium and chloride)
The term serum refers to plasma from which the clotting proteins have been removed. Most of
the proteins remaining are albumin and immunoglobulins.
Constitution of normal blood
Parameter
Value
hematocrit
45 ± 7 (38–52%) for males
42 ± 5 (37–47%) for females
pH
7.35–7.45
base excess
−3 to +3
PO2
10–13 kPa (80–100 mm Hg)
PCO2
4.8–5.8 kPa (35–45 mm Hg)
HCO3
21–27 mM
oxygen saturation Oxygenated: 98–99%
Deoxygenated: 75%