ANFS332 Lecture Notes - Lecture 7: Strangles, Neutrophil, Macrophage
26 Feb 2018
School
Department
Course
Professor
•
Red text = important
•
Blue text = do not need to know
Chronic Inflammation - bad
A pathological condition
o
Chronically inflamed tissue is characterized by the infiltration of
macrophages, tissue destruction, and repeated attempts at healing
o
In chronically inflamed tissue the stimulus is persistent , and therefore
recruitment continues, existing macrophages are tethered in place, and
proliferation of macrophages is stimulated
o
•
Acute vs chronic inflammation
•
Acute
•
Chronic
•
Causative
Agent
•
Pathogens, injured tissues
•
Non-degradable pathogens, persistent
foreign bodies, autoimmune
reactions
•
Major Cells
Involved
•
Neutrophils, macrophages
•
Macrophages, lymphocytes,
fibroblasts
•
Primary
Mediators
•
Cytokines
•
Cytokines, growth factors, ROS,
hydrolytic enzymes
•
Onset
•
Immediate
•
Delayed
•
Duration
•
A few days
•
Up to many months or years
•
Outcomes
•
Healing, abscess
formation, chronic
inflammation
•
Tissue destruction
•
Termination of the Inflammatory Response
Resolution of an acute inflammatory response normally initiates in the first
few hours after an inflammatory response begins.
o
Neutrophils also initiate the termination sequence.
o
Neutrophil recruitment ceases and programmed death by apoptosis is engaged.
Neutrophils are starting to make other cells that will stop the
recruitment process
§
o
Consequently, apoptotic neutrophils undergo phagocytosis by macrophages,
leading to neutrophil clearance and release of anti-inflammatory and
reparative cytokines. The anti-inflammatory program ends with the departure
of macrophages
o
•
Mechanisms of Pathogenicity
How does
Streptococcus equis
cause the disease (strangles)?
•
Many virulence factors
•
1. Nonantigenic hyaluronic capsule
Virulent isolates (strains) have a capsule, while avirulent strains do not –
SOUND FAMILIAR?
o
Capsule inhibits the activity of neutrophils and is required for function of SeM
(see later slide)
Reduces the ability of the neutrophils to phagocytize and destroy the
cells
§
o
•
2. Β-hemolysis
Streptokinase S (enzyme)
o
Binds to erythrocytes and forms a transmembrane pore resulting in lysis of the
cell
Destroys membrane of cells
§
o
•
3. Pyrogenic toxins - SPE
Molecules that non-specifically stimulate immune cells to release
proinflammatory cytokines resulting in an uncontrolled acute inflammatory
response
o
Called pyrogenic exotoxins (SPE) or superantigenic toxins (does toxic shock
sound familiar?)
Analogous to toxic shock of horses
§
o
Pyro = fire (think inflammatory response)
o
•
4. Streptococcal M protein - SeM
Discovered by Rebecca Lancefield (remember her?)
o
Found on fibers on the bacterial cell surface
o
Blocks recognition of the bacteria by factors responsible for phagocytosis
(opsonization and the complement cascade –later)
Not only is the capsule making theses cells less able to be recognized by
neutrophils for phagocytosis, but the M protein also inhibits
phagocytosis
§
o
Loss of SeM expression results in loss of virulence but not infectivity.
Virulence does not mean infectivity!
§
Just because something infects, doesn’t mean it will cause disease
§
o
Antibodies to SEM can cross react with normal heart muscle proteins and is
the basis for rheumatic fever
o
•
•
Diagnosis of Strangles
Serotyping
Streptococci are divided into groups by antibodies that recognize surface
antigens (e.g. Group A)
o
•
Serology (ELISA)
Detect antibodies to SeM
o
•
Polymerase chain reaction (PCR)
Can we find the Dna fragment from streptococcus equis that codes for the SeM
gene?
o
Detects the DNA sequence of the SeM gene
o
Can be completed in a few hours
o
Does not distinguish between living and dead organisms
o
•
Culture
Culture of nasal swabs, nasal washes, or pus on blood agar plates gram stain,
catalase, hemolysis
o
•
•
ELISA (Important)
General test; not just for strangles
•
Enzyme-Linked ImmunoSorbent Assay
•
Determines the existence of a particular protein in a sample (in this case specific
antibodies)
•
ELISA can also be used to detect antigen (see SNAP test later)
•
ELISAs are performed in 96-well plates
•
Steps;
The bottom of each well is coated with the protein (SeM) to which the
antibody that we want to measure (anti-SeM) will bind (1)
o
Whole cells are removed from a blood sample (Horse) by centrifugation
resulting in a clear serum which contains antibodies. This is diluted and added
to the plate (2).
o
After the antibody to SeM has a chance to bind all other proteins in the sample
are washed off the plate (3).
o
A second antibody (antibody to horse antibodies) is added (4).
o
Attached to this second antibody is an enzyme that converts a colorless
substrate to a colored product (chromagen)
o
•
•
ELISA Example
One way of determining sample titers. What are the titers of the three samples?
More antibodies, the more color
When you have to dilute it further it means you have more antibodies
§
o
•
•
SNAP Test
A SNAP test is an ELIZA test
•
This test can also be used to detect the presence of the “antigen”in the sample
Examples;
Idexx Pavovirus Elisa
§
Combo Tests
§
o
•
•
PCR
Polymerase Chain Reaction
•
A method for amplifying a piece of nucleic acid (DNA) up to a million times, in
vitro, within a few hours (30-40 cycles)
•
Taq DNA polymerase
•
I cycle = 3 temps
•
•
PCR –Important
•
5 min/cycle, 30 cycles = 1,000,000,000 copies
DNA –Primers - DNA polymerase –nucleotides
•
Denature (94) - Anneal (~60) –Extend –(72) - REPEAT
•
The products are visualized on an agarose gel
•
•
More Strangles, ELISA, PCR
Tuesday, October 25, 2016
8:23 AM
•
Red text = important
•
Blue text = do not need to know
Chronic Inflammation - bad
A pathological condition
o
Chronically inflamed tissue is characterized by the infiltration of
macrophages, tissue destruction, and repeated attempts at healing
o
In chronically inflamed tissue the stimulus is persistent , and therefore
recruitment continues, existing macrophages are tethered in place, and
proliferation of macrophages is stimulated
o
•
Acute vs chronic inflammation
•
Acute
•
Chronic
•
Causative
Agent
•
Pathogens, injured tissues
•
Non-degradable pathogens, persistent
foreign bodies, autoimmune
reactions
•
Major Cells
Involved
•
Neutrophils, macrophages
•
Macrophages, lymphocytes,
fibroblasts
•
Primary
Mediators
•
Cytokines
•
Cytokines, growth factors, ROS,
hydrolytic enzymes
•
Onset
•
Immediate
•
Delayed
•
Duration
•
A few days
•
Up to many months or years
•
Outcomes
•
Healing, abscess
formation, chronic
inflammation
•
Tissue destruction
•
Termination of the Inflammatory Response
Resolution of an acute inflammatory response normally initiates in the first
few hours after an inflammatory response begins.
o
Neutrophils also initiate the termination sequence.
o
Neutrophil recruitment ceases and programmed death by apoptosis is engaged.
Neutrophils are starting to make other cells that will stop the
recruitment process
§
o
Consequently, apoptotic neutrophils undergo phagocytosis by macrophages,
leading to neutrophil clearance and release of anti-inflammatory and
reparative cytokines. The anti-inflammatory program ends with the departure
of macrophages
o
•
Mechanisms of Pathogenicity
How does
Streptococcus equis
cause the disease (strangles)?
•
Many virulence factors
•
1. Nonantigenic hyaluronic capsule
Virulent isolates (strains) have a capsule, while avirulent strains do not –
SOUND FAMILIAR?
o
Capsule inhibits the activity of neutrophils and is required for function of SeM
(see later slide)
Reduces the ability of the neutrophils to phagocytize and destroy the
cells
§
o
•
2. Β-hemolysis
Streptokinase S (enzyme)
o
Binds to erythrocytes and forms a transmembrane pore resulting in lysis of the
cell
Destroys membrane of cells
§
o
•
3. Pyrogenic toxins - SPE
Molecules that non-specifically stimulate immune cells to release
proinflammatory cytokines resulting in an uncontrolled acute inflammatory
response
o
Called pyrogenic exotoxins (SPE) or superantigenic toxins (does toxic shock
sound familiar?)
Analogous to toxic shock of horses
§
o
Pyro = fire (think inflammatory response)
o
•
4. Streptococcal M protein - SeM
Discovered by Rebecca Lancefield (remember her?)
o
Found on fibers on the bacterial cell surface
o
Blocks recognition of the bacteria by factors responsible for phagocytosis
(opsonization and the complement cascade –later)
Not only is the capsule making theses cells less able to be recognized by
neutrophils for phagocytosis, but the M protein also inhibits
phagocytosis
§
o
Loss of SeM expression results in loss of virulence but not infectivity.
Virulence does not mean infectivity!
§
Just because something infects, doesn’t mean it will cause disease
§
o
Antibodies to SEM can cross react with normal heart muscle proteins and is
the basis for rheumatic fever
o
•
•
Diagnosis of Strangles
Serotyping
Streptococci are divided into groups by antibodies that recognize surface
antigens (e.g. Group A)
o
•
Serology (ELISA)
Detect antibodies to SeM
o
•
Polymerase chain reaction (PCR)
Can we find the Dna fragment from streptococcus equis that codes for the SeM
gene?
o
Detects the DNA sequence of the SeM gene
o
Can be completed in a few hours
o
Does not distinguish between living and dead organisms
o
•
Culture
Culture of nasal swabs, nasal washes, or pus on blood agar plates gram stain,
catalase, hemolysis
o
•
•
ELISA (Important)
General test; not just for strangles
•
Enzyme-Linked ImmunoSorbent Assay
•
Determines the existence of a particular protein in a sample (in this case specific
antibodies)
•
ELISA can also be used to detect antigen (see SNAP test later)
•
ELISAs are performed in 96-well plates
•
Steps;
The bottom of each well is coated with the protein (SeM) to which the
antibody that we want to measure (anti-SeM) will bind (1)
o
Whole cells are removed from a blood sample (Horse) by centrifugation
resulting in a clear serum which contains antibodies. This is diluted and added
to the plate (2).
o
After the antibody to SeM has a chance to bind all other proteins in the sample
are washed off the plate (3).
o
A second antibody (antibody to horse antibodies) is added (4).
o
Attached to this second antibody is an enzyme that converts a colorless
substrate to a colored product (chromagen)
o
•
•
ELISA Example
One way of determining sample titers. What are the titers of the three samples?
More antibodies, the more color
When you have to dilute it further it means you have more antibodies
§
o
•
•
SNAP Test
A SNAP test is an ELIZA test
•
This test can also be used to detect the presence of the “antigen”in the sample
Examples;
Idexx Pavovirus Elisa
§
Combo Tests
§
o
•
•
PCR
Polymerase Chain Reaction
•
A method for amplifying a piece of nucleic acid (DNA) up to a million times, in
vitro, within a few hours (30-40 cycles)
•
Taq DNA polymerase
•
I cycle = 3 temps
•
•
PCR –Important
•
5 min/cycle, 30 cycles = 1,000,000,000 copies
DNA –Primers - DNA polymerase –nucleotides
•
Denature (94) - Anneal (~60) –Extend –(72) - REPEAT
•
The products are visualized on an agarose gel
•
•
More Strangles, ELISA, PCR
Tuesday, October 25, 2016 8:23 AM
Document Summary
8:23 am: red text = important, blue text = do not need to know. Chronically inflamed tissue is characterized by the infiltration of macrophages, tissue destruction, and repeated attempts at healing. In chronically inflamed tissue the stimulus is persistent , and therefore recruitment continues, existing macrophages are tethered in place, and proliferation of macrophages is stimulated. Neutrophil recruitment ceases and programmed death by apoptosis is engaged. Resolution of an acute inflammatory response normally initiates in the first few hours after an inflammatory response begins. Neutrophil recruitment ceases and programmed death by apoptosis is engaged. o o o. Neutrophils are starting to make other cells that will stop the recruitment process. Consequently, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines. The anti-inflammatory program ends with the departure of macrophages. Many virulence factors: nonantigenic hyaluronic capsule o o. Virulent isolates (strains) have a capsule, while avirulent strains do not .
