15/16: Inflammation I/II

7/21/2019 15/16: Inflammation I/II

http://slidepdf.com/reader/full/1516-inflammation-iii 1/26

Transcribed by Mandy Weil 5 September 2014

[General Pathology] – Inflammation by R. Craig, DMD, PhD

[Dr. Phelan] – Did you all get the message that the revised schedule is up for the

fall? The only difference in that schedule is that the exam is on Monday. And the

conferences for that week are cancelled. They will be on your schedule cause I

can’t fix that. But there are no conferences. Having conferences the week of anexam means that somebody has… a whole bunch of you will have conferences the

morning of an exam—you’d rather be anyplace else than in a conference on

another subject. Nicely, there was an extra conference that was scheduled, that I

hadn’t used and that’s the one that was on the 27th. So i was able to move that and

free up that week of the exam from conferences. But those are the only changes.

Everything else should be exactly the same.

[ Slide 1] – [Title Slide ]

[Dr. Craig] – Can everyone hear me? I guess so.. Hello? It works. It works.

Alright ….Okay. Good morning everyone. I’ve been asked to be with you for the

next 6 sessions. Talk a little bit about inflammation and wound healing. And Iknow you’ve already had your unit about inflammation with Dr. McCutcheon. Dr.

McCutcheon went over innate immunity and the prime effector of innate

immunity is the inflammatory response. So some of this going to be redundant.

Some of this might be a review. My intent is to try to present inflammation from

perhaps a bit of a clinical perspective because we’re going to deal with

inflammation on a daily basis in your practice. So with that, I should also tell you

that we have some learning aids—as you usually do with me—for these 6 hours. If

you get onto NYU courses you should be able to locate the power points and you

should be able to locate the lecture handouts. On the lecture handouts, as I usually

do, right in the front, there’s a reference section and I usually divide references

into required, which means fair game on educational assessments, otherwise

known as exams. And then, non-required. Non-required are for those folks who

want to see some of the primary reference materials that I use. And i will be using

that in wound healing and also in the systemic effect of inflammation lectures. So,

having said that, when you get to the Robbin’s textbook, they write a lot on

inflammation and actually it’s at a fairly good level for us. The Robbin’s textbook is

really written for medical students. And medical students have different needs

than dental students. And so there’s certain areas that I’m gonna kinda expand

that are not covered in the Robbin’s textbook, and certain areas that I’m not gonna

touch on at all. So when you read the Robbin’s textbook, and I highly suggest you

read the Robbin’s textbook, have the lecture handout by your side as a handyguide. If I’m not making a big deal about the 4 million cytokines that participate in

the inflammatory response, but only focus in on three, I would probably

concentrate on those three. Questions so far? I want you responsible for what the

resources are. Okay. So, um, lets talk a little bit about inflammation.

[Slide 2] – [Outline]

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[Dr. Craig] – I’m gonna try to set the stage a little by talking about the host

defense against infection and I know Dr. McCutcheon has prepared you well for

that. And then we’re gonna talk about the clinical signs of inflammation. Because

this is kind of historically how people came to grips with this reaction. And then

we’re gonna divide our comments today and then the beginning of next

week—we meet next Monday, is my understanding—into acute inflammation andchronic inflammation. And this is how classically the inflammatory response has

been divided. You’ll see how there is some artificiality based here. We’ll spend

most of our time talking about so called acute inflammation. And, um, we’ll talk a

little bit about how edema is generated. How the body, how your patient recruits

out of the vascular compartment specific and appropriate cells of the innate

immune response into the site to deal with the challenges being presented. We’re

also gonna talk a little bit about how phagocytes, things like macrophages,

monocytes and especially polymorphonuclear leukocytes—otherwise known as

neutrophils—how they target, how they snarf down bacteria. Cause most of our

diseases in dentistry are bacterial diseases. And how they kill them. This is of

importance to us because as we’ll see, as you’ll see in clinic too, the inflammatoryresponse comes at a really big price. There’s a lot of collateral damage. You know,

as I read the newspapers, I see this—collateral damage. Well it really kind of

applies to the inflammatory response because a lot of the mechanisms that the

body uses to uncover pathogen also has bad effects for the host, especially if that

reaction carries on for a long time. We’ll talk a little bit about how, um, innate

immune cells recognize pathogens. I know Dr. McCutcheon probably talked to you

about Pathogen Associated Molecular Patterns (PAMPs). We’ll also talk about

damage associated molecular patterns—so called inflammosome. And then we’ll

talk about ways that you can resolve inflammation and theres some really neat

things on the future that you’re gonna use clinically I predict that are gonna be

radically different than say what I’ve done in my practice.

[Slide 3] – [Host defenses against infection]

[Dr. Craig] – So, as Dr. McCutcheon probably told you: there’s three levels of host

response to infection and trauma. The first is physical barriers. The integument.

You know stratified squamous epithelium with keratin. Keratin’s really great.

Also, has acidic secretion, sebum, those sorts of things, which inhibit bacterial

colonization. Mucosal surfaces are those surfaces of the body that are specialized

for communication with the outside. Gastrointestinal tract for nutrition.

Respiratory for air exchange. So on and so forth. And those surfaces are really

susceptible to pathogenic challenge, to infection, because they don’t have keratin.They’re kinda easy to breach. Most of them, except our area, have mucous. And one

of the things that’s interesting about periodontitis is we have these teeth that

come from a calcified mesenchymal tissue—pierces through that barrier—and is

sitting out there in the gastrointestinal tract. And when we talk about

periodontitis in your next course, which is Diagnosis and Treatment of Oral

Diseases, i think in October, we’re gonna come back to that idea. So anyways, so,

the first layer is physical surfaces. You know in my practice, I shake the patients

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hand: “Hello Mrs. Jones, how are ya?” Sit her on a seat, and immediately I’m past

physical barriers because I give a local anesthetic. Probably pick up a Bard Parker

blade, start doing surgery. So immediately I’ve breached that. You know, I do clean

surgery, I don’t do aseptic surgery like they do at the Langone Medical Center.

Right. So as you put your Bard Parker blade in, you’re gonna drag in

bacteria—some really bad bacteria—into the site of the incision. So the patient’sresponsibility is to mount a really effective innate immune response. We’re just

gonna briefly go over the innate immune response so it’s kinda firm in your mind

what that response is. And the focus of our attention is going to be the

inflammatory response, which is the prime effector of innate immunity. And then

Dr. McCutcheon ably talked about adaptive immunity. I’m just gonna have a few

um comments to compare and contrast those two. And so this is kind of an

interesting idea. So innate immunity happens very, very quickly as you know. And

if it works, you don’t even see signs or symptoms of much disease. If it doesn’t

work—then you have pathology of quite often, quite significant pathology. The

medical profession has been very interested in those challenges that breach

innate immunity. So physicians in the medical profession have really focusd in onthis third level: adaptive immunity. Howe the patient mounts an adaptive

response. How you can help patients during that time. What are the mechanisms

involved. For years, folks didn’t really focus in on innate immunity until Charlie

Janeway—Charlie, like I know him—Charles Janeway, who is diseased. Past chair

of Microbiology at Yale. Came up with the idea that, gee, we’ve been studying all

this immunology, but what really paves the way for the adaptive immune

response is the so-called hard-wired innate immune response. So now people are

really interested in the inflammatory response. And in the last 10-15 years or so,

the knowledges gained has really been unbelievable with lots of payoff in our

profession. We’ll talk more about that on Monday. So, innate immunity: ancient

form of host defense. It’s not the most ancient, but it is an ancient form of host

defense. Um, and what characterizes the innate immune response is that your

successful—

[Slide 4] – [Innate immunity]

[Dr. Craig] –

—ancestors, the ones that lived long enough to actually contribute their genes to

the gene pool. Not the ones that didn’t. But, the successful ones, they over time,

evolutionarily, learned to recognize certain immutable characteristics of

pathogens. It can be a structure or it can be a metabolic product that that pathogen

can’t mutate away from. And so, you have a set, half from your mom, half from yourdad, of genes that encode for receptors that recognize various structures that

pathogens can’t mutate away from. Pathogen associated molecular patterns. So

since, this is dentistry, lets focus in on bacteria. Tell me something bacteria do or

have that you as an eukaryote don’t have…What do they do differently that you

can exploit …(student response, inaudible)…Hm? They have cell walls. Alright? Do

you have cell walls? No. You don’t have cell walls. You’re a multicellular organism.

You use your integument to keep a constant osmotic pressure. Keep a constant pH.

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So, you have a plasma membrane, you don’t have a cell wall. Bacteria don’t have

that. So they have this structurally complex—that you learned about in

Micro—cell wall hat buffers them form radical changes in osmotic pressure,

radical changes in pH, those sorts of things. And on that cell wall, as you know

from Micro, there’s a lot of interesting carbohydrates. One of which is mannose.

And I forget the derivative of mannose—but it’s mannose, right? When mannose ispresented on a surface within the body, there are receptors that recognize

mannose and they’ll say “Ack! That’s not self.” It has to be a pathogen…LPS,

endotoxin—especially in our field—endotoxin really drives um both innate and as

you know, adaptive immunity. Right? Second stimulus in adaptive immunity.

Many points in adaptive immunity. So we got cell wall. What else do bacteria do

differently than you do? (silence) —Not all at once!….

Haha—Dr. Craig! You’re so early and I”m still you know, thinking about the

weekend…How do they do protein synthesis? What are the first 3 amino acids that

they synthesize with any protein that’s gonna be exported? —(student response)

— Okay! So f-Met-Leu-Phe, so formulated Methionine, Leucine, Phenylalanine.

Bacteria, prokaryotes, because of their ribosomes, start everything that way. Youdon’t start your protein synthesis that way. You wouldn’t be caught dead—well

actually that’s a pun—synthesizing that way. So, if there are any proteins or that

3-amino acid peptide present in the extracellular matrix, the immune system

knows. F-Met-Leu-Phe—Oh! It has to be a prokaryote in the area. So that’s another

pathogen associated molecular pattern. You also know that bacteria have different

adjuncts onto their DNA than you have. Right? Naked DNA itself is a bad thing.

Right? Should be in the nucleus. But especially when it has these different

adjuncts on it. Ugh..It has to be coming from a prokaryote. So you kinda get the

idea. That characterizes the innate immune response. Second characteristic of the

innate immune response is: once the host recognizes that Pathogen Associate

Molecular Pattern, it either tries to destroy the source and/or it starts to signal to

the outside that it needs recruits. Alright? So, the containment of that infection in

an innate immune response happens in the site of infection. Right? Happens in the

site of infection. And once recognized, theres a set of host effector mechanisms

that hopefully we’ll get to at the end of today, that contain, or hopefully destroy

that pathogen. As opposed to—Do i have a slide on this? Actually I don’t.— As

opposed to adaptive immunity.

What characterizes adaptive immunity? —Right? The immune response is not

assembled at the site of infection. But it’s assembled at deep holes ((hard to

decipher this phrase; said when there is 1:42 of podcast remaining)) where the

body has anatomically set up to increase the concentration of antigen drainingfrom an area. Lymph nodes. Secondary lymphoid tissue. Right? Theres a set of

antigen presenting cells. What are those antigen presenting cells in secondary

lymphoid tissue? —Dendritic cells. We got one. A person..is it Steinman? Just up

the street. Got the Nobel Prize two years ago for his work on Dendritic Cells.

Unfortunately passed away. What’s next? —Macrophages. What’s the third? — B

cells. Excellent. So Dr. McCutcheon did a good job.

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So being presented in various forms in secondary lymphoid tissue on the antigen

presenting cells are these wonderful bits of the pathogen. And then B and T cells

come in, right? Via the arterial circulation. Circulate through the secondary

lymphoid tissue and test the goodness of fit of their B cell receptor or T cell

receptor to bind with that piece of the pathogen. Those T cell receptors and B cell

receptors are generated by very unique genes. RAG 1 and RAG 2. Actually takespieces of genes in B cells and T Cells during development and cuts them and

splices them together. Almost in a Montecarlo stochastic—by chance—function to

generate genes that will encode for proteins that become the B cell and T cell

receptor that may or may not have affinity during that cells life for anything

outside of nature. So what characterizes the adaptive immune response is you

sequester all of the antigen in one place and you try to sort out from all of those B

and T cells that you have circulating in your body the few, the very few, the

select—right? it’s like the marines—the very few that actually can bind with that

pathogen. And then you take those cells and you actually teach them to bind with

higher and higher affinity to make either effector T cells or plasma cells, which

makes antibody.What blows my mind, well lots of things do as I get older, but, what really blows

my mind is this strategy that vertebrates have come up with, because adaptive

immunity is a characteristic of vertebrates, right? Helminth worms, those guys

don’t have it. But vertebrates, backbone, we’ve got it. This strategy that’s been

adapted will recognize pathogens that haven’t even evolved yet. That’s amazing!

That’s really powerful, you know. And that was all done by evolution, I guess. So,

anyways, that’s the difference between innate and adaptive immunity. So let’s

focus in on innate immunity. So The primary effector of innate immunity is

inflammation. Okay? The inflammatory response. Let’s talk a little bit about the

inflammatory response. So the inflammatory response is the reaction of a tissue.

And it’s microcirculation to a pathogenic insult. And you’ll see that the insult can

be very broad.

[Slide 5] – [The inflammatory response]

[Dr. Craig] – So this is a very generalized reaction that the body has. Inflammation.

The primary objective of the inflammatory response is elimination or containment

of the insult and removal of damaged tissues. That’s what it says in the textbook.

But there’s a number of complex diseases that we’ll talk about when we get into

Diagnosis and Treatment of Oral Diseases class that are so called complex diseases.

And there are some diseases, like type 2 diabetes. where inflammation is a major

component, but it appears to be a derangement in lipid biosynthesis. And a few ofthe things called the metabolic syndrome..So even, theres no real, insult there,

other than being overweight, that incites the inflammatory response. So the

inflammatory response is really kind of universal throughout vertebrate biology.

And as you know, you need to have inflammation in order to get an adaptive

immune response. So this is my…I don’t know why…I always get the most difficult

lectures to give. I wish I could just get—I don’t know, like taking alginate

impressions or something that was like, very simple and straightforward. I always

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get these complex things. So anyways, you can imagine that if inflammation is so

important for the survival of the species and there’s been this complex interplay

over evolution between pathogens who want--

[Slide 6] – [The inflammatory response]

[Dr. Craig] – --to garner all the energy and resources that are in our bodies. And

our successful ancestors who are successful at thwarting those infections, so, you

know bacteria and viruses and other pathogens have sort of mutated and evolved

to get into us, and we’ve kinda mutated and evolved to come up with defenses

against it. And this has been going on for eons. You can imagine the complexity

that must be there. It is complex. So what I’m gonna try to do is, I’m gonna try to

distill out, in my best attempt, to give you the details of the inflammatory

response that are important to us at this moment as clinicians. But I want to give

you the idea that we’re entering into very complex ground. And our

understanding of inflammation is still developing. As I said, it’s an intense area ofinvestigation for the last 12-15 years or so. So during your clinical lifetimes, you

have to kinda keep up with this area because there’s gonna be a lot of applications

for clinical dentistry and you don’t want to be left out. So what’s the clinical

significance? Inflammation is encountered daily in dental practice. Caries. Um,

pulpal disease. Periodontal disease. Inflammatory diseases, right? Many chronic

diseases have inflammatory components. As we’ll talk about on Monday:

atherosclerosis, type 2 diabetes, Alzheimer’s disease, rheumatoid arthritis,

osteoarthritis. They all have a major component of an inflammatory response that

perhaps is not appropriate.

[Slide 7] – [Clinical significance]

[Dr. Craig] – Inflammation is an essential first step in wound healing…so this is

gonna kinda like pave the way for our discussion next Friday on wound healing,

and uh finally, this has been the golden fleece in my little area: chronic

inflammation, perhaps from oral sources, may contribute to systemic diseases,

such as heart attack and stroke. Okay. So the Egyptians actually wrote about

inflammation. But the first person who is actually credited with it is the Roman

Celsius. My reference says..uh..second century source says first century. I don’t

read Latin, so I really have a hard time with that. But anyways, he’s usually

credited with classifying the four classic signs of an inflammatory response:

redness, heat, swelling and pain.

[Slide 8] – [Classical (cardinal) hallmarks of inflammation]

[Dr. Craig] –And this is uh, this periodontal patient—whoops, there goes…—he

already lost a tooth to periodontitis, right? And if you look at this patients

tissues, you kinda see this redness here, right? And if you had an instrument, and

there is an instrument called the periotron, which you put, uh, thermal ____

((cannot decipher this word, 1:33:30 of podcast left)) on the core temperature of

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the patient and have like a little hand piece measuring device. And you can

actually put it into the sulci and you’ll actually measure the differences between

basal core temperature and peripheral temperature. You’ll actually pick up sites

that have increased, you know, uh temperature. So you have Calor—heat. You’ve

got swelling in this area. This is not normal gingival architecture. The only thing

that you don’t normally have, or usually have, is pain. Because most of thisinflammatory exudate, swelling and stuff, things that cause pain, have egress into

the oral cavity. But if this same response was further down—say in the apex, you’d

have extreme pain. So um, periodontal inflammation has the 4 characteristics,

right. So what are the characteristics of an inflammatory response. The first thing

is to identify that there is an insult, whether it be pathogenic, whether it be you.

Whether it be something else that is considered an insult, and there are many

things. And the first thing that happens is generation of various soluble

inflammatory mediators. And we’re gonna talk quite a bit about what they are

because they’re wonderful targets for you in clinic, uh, to help control

inflammation for your patient.

[Slide 9] – [Components of the inflammatory response]

[Dr. Craig] – And then those inflammatory mediators work on the endothelial cells

of the microvasculature within that site. Right? So especially the arteriole,

capillary and post-capillary endothelium are gonna be major players in regulation

of inflammation. And then finally, under the control of these inflammatory

mediators released into the local environment, there’s gonna be first movement of

fluid from the vascular space to the extravascular space generating edema,

generating lymph. Increase lymph flow. And then finally those endothelial cells

are also gonna have a specialized mechanism for picking up the appropriate

leukocytes, white blood cells, that are passing through in the circulation and

recruiting them out of the vascular compartment into the extravascular

compartment to deal with the challenge. So those are the three components:

generation of the inflammatory mediators, change in those endothelial

cells—those endothelial cells are gonna become really important. And then,

finally, the movement of fluid and cells, regulated by those endothelial cells, from

the vascular to the extravascular space.

Okay so…how many people went to Parochial school? Just one? —Okay so I

remember when I was maybe about, I don’t know..fifth grade or something like

that …and I wasn’t a very good student. And I remember we were having this

lecture or something, some class thing and the girls gym class was outside, and it

was kind of a nice day. So, I was kinda like watching the girls gym class and notreally paying attention to what I was supposed to be paying attention to—

[Slide 10] – [Acute inflammation: physical signs]

[Dr. Craig] – —and I had my hands out and all of a sudden this yardstick came out

of the heavens, and Sister Brigid brought me back into the classroom with this

WHACK. And I remember kinda sitting there. At first it was painful. But I could see

the indentations of the yardstick. And the first thing that happened was the entire

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area across my hands became kind of white. So, the first thing that happens during

an inflammatory response—and this is under control of innervation of the small

arterioles—is just a transient vasoconstriction. Perhaps it’s there to try to control

hemorrhage in really severe lacerations. And then, as I’m watching and the

pain—it was a great rush, I remember the pain was just an unbelievable rush. I’m

looking and all of a sudden that area starts to get red again or hyperemic, right?Hyperemia. So both the arterioles and the venues in the area start to relax and

open up, so there’s more blood going into the area. That gives you that red

appearance and also in time the increase flow of blood to the area increases the

temperature of the area. And then, finally, after about, I don’t know, 5-10 minutes,

something like that …I remember because the girls gym class was going back

in…there was a lot of swelling that occurred in the area. So, there’s a number of

people whose names are kinda classically associated with—so this is the so-called

triple response first described by Lewis is 1924, that’s not important for us.

[Slide 11] – [Gingival health]

[Dr. Craig] – Alright. So here’s, just to drive it home, so here’s a picture of socalled gingival health. The gingiva mimics the cement enamel junction. If you push

on this tissue, it doesn’t blanch. It’s really nicely, um, conforms to the underlying

alveolar bone.

[Slide 12] – [Gingival inflammation]

[Dr. Craig] – As opposed to this poor gentleman, everything is red, everything’s

inflamed. This is a case of gingivitis, so you can kinda see.

[Slide 13] – [Cellular events and inflammation]

[Dr. Craig] – So anyways, now we’re beginning to focus down and we’re going to

leave the clinical, right. What you see chair side. And now we’re gonna begin to

focus down first at the cellular level, and then we’re gonna go into the molecular

level because there’s a lot of therapeutic targets here. So, classically, inflammation

has been divided basically by the histopathology of these two readtions into

something called acute inflammation and chronic inflammation. Again, as we begin

to understand more about inflammation, the demarcation between acute and

chronic is becoming less and less clear.

[Slide 14] – [Cellular events in inflammation: acute inflammation]

[Dr. Craig] – So, here we have a little diagram lifted from your text. So up here,

this is a normal capillary bed, arteriole, venule draining, a nice capillomeric plexushere. You may have some sentinel (?) cells in this connective tissue, dendritic

cells, right? And some macrophages kinda sitting there waiting for, um, to detect

if theres any pathogenic challenge in the area. And if there is a pathogenic

challenge then, especially the macrophages start to release vasoactive mediators.

And it goes to the arterioles and the venules and it tells those smooth muscles to

start to relax. Alright? So this increases blood flow in this area. But, because of

fluid dynamics, if you take a pipe and you keep the flow constant, or the pressure

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going in there constant, and all of a sudden you dilate it: now you’ve got a bigger

volume. So now the flow starts to go slower. So even though you have more blood

in the area, flow starts to decrease. And sometimes this flow is so slow that the

erythrocytes begin to lose their oxygen content. You’ll learn in clinic that

inflamed gingiva is sorta cyanotic. It turns, like, this bluish red color and thats

because of the changes in vascularity during inflammation. The next thing thatoccurs, is there’s increased vascularity because the endothelia’s cells that are

lining this microvasculature begin to contract and expose the underlying

basement membrane. And basement membrane works like an ultrafilter. So

anything in blood, which is below the molecular weight cutoff, will percolate out

of the vascular into the extravascular space. Another thing that happens during

inflammation is that endothelial cells start to release mediators to platelets and

platelets begin to clump together and degranulate. So intravascular platelet

activation. And then, these endothelial cells, later on will start to express adhesion

molecules that will select out appropriate cells of the innate immune system. So

early on, say its a bacterial challenge, it will be polymorphonuclear leukocytes,

like in this little cartoon. Later on it may need monocytes or other cells such as Tand B cells into the area.

[Slide 15] – [Cellular events in inflammation: acute inflammation]

[Dr. Craig] – So this is sort of a schematic of inflammation. What does this actually

look like? So here’s a slide that was generously donated by the Dr. Phelan

Research Foundation, I think, years ago. So anyways, this is what

histopathologically acute inflammation looks like. And what you see here—you

know, you guys don’t have to look at micrograph slides anymore so what you guys

are probably seeing is a bunch of dots, huh? But if you look at the dots real

carefully, you’ll see that they’re like individual cells and this dark stuff is the

nuclei. And you’ll notice that these nuclei are kinda like multi-lobed. Alright?

They’re polymorphonuclear leukocytes. So this whole field is almost completely

populated by polymorphonuclear leukocytes. This is the classic presentation of

acute inflammation.

[Slide 16] – [Cellular events in inflammation: Chronic inflammation]

[Dr. Craig] –Chronic inflammation. So chronic inflammation—according to the

textbook—usually follows acute inflammation. If acute inflammation hasn’t able to

resolve the challenge. But acute inflammation by insidiously develop in areas of

malignancy. Viral infections, parasitic infections—those sorts of stuff. So, you

don’t have to start an inflammatory response with a so called chronicinflammatory response. And what characterizes chronic inflammation is the

cellular infiltrate. The inflammatory cellular infiltrate. So in acute inflammation its

polymorphonuclear leukocytes. In chronic inflammation, it’s much more divers. So

you’ll see mononuclear cells and their derivatives: macrophages. You’ll also see

small lymphocytes. Right? And you may even see plasma cells. In fact, when we

talk about periodontitis, the characteristic lesion of established periodontitis are

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plasma cells. Actually antigen-specific-antibody-secreting plasma cells, which was

always a bit of a question mark in folks minds, up until recently.

[Slide 17] – [Cellular events in inflammation: Chronic inflammation]

[Dr. Craig] – So here’s the histopathology of a chronic inflammatory lesion. And

again, if you look at the pepper grains (chuckle), alright—the individual cells. Allof the cells that have that multilobular—the nuclei, rather, don’t have that multi

lobular appearance any longer. They look more like small lymphocytes that have

been populating this area.

[Slide 18] – [ Possible outcomes of the inflammatory response]

[Dr. Craig] – Okay, so what are the possible outcomes of the inflammatory

response? And so this is a classic term from pathology: so-called resolution. So if

everything works well, right, the innate immune response works well, identifies

the pathogen. The correct innate immune cells are recruited into the area, they

destroy the pathogen. Everything goes back to where it was before: you get

resolution. Right? And that’s always been, at least is perio, that’s been kind of likethe golden fleece. We wanna be able to get regeneration and you kinda know from

Craniofacial Biology course, that’s indeed possible. Unfortunately a lot of times

what happens is scarring. So, perhaps, resolution of the inflammatory response is

not 100%. And, you have inappropriate expression of type I collagen and other

genes associated with the extracellular matrix. So the anatomy is not truly

reinstated. Some of the function may be, but perhaps the anatomy is not.You end

up with something called fibrosis which can have very serious clinical sequelae. So

much so, that the NIH floated a very big program project last year looking for

grants that will study in depth fibrosis after wounding. And then what also can

happen is that that lesion can progress from an acute inflammatory response into a

chronic inflammatory response. In fact, there are diseases where components of an

acute inflammatory response coexist with chronic inflammatory responses and

they’re associated with excessive extracellular matrix degradation. And

periodontitis fits into that very, very well.

[Slide 19] – [Stimuli for acute inflammation]

[Dr. Craig] – Okay. What are the stimuli for an acute inflammatory response? So,

from Dr. McCutcheon, you probably learned that you can kinda loosely divide

pathogenic challenges into four different types, right? Um, bacteria, prokaryotes,

viruses, fungi and then this grab-bag of protozoans and worm-type parasites.

These guys are all, the individual entities are smaller than a human cell. Theseguys are bigger than a human cell. And you should know by now that certain arms

of the adaptive immune response have evolved to deal with these components.

Alright? But however, the inflammatory response is more broad than just dealing

with infections. It also can sense if there is tissue that has died, become necrotic or

tissue that is on its way to dying. And we’ll talk a little about that. Uh, foreign

bodies, sutures, are really notorious for setting up inflammatory responses. Um,

talc, from surgical gloves, really good at setting up inflammatory responses.

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Immune reactions, that you’ll learn about more when this course talks about auto

inflammatory disease, autoimmune diseases. And then of course, trauma. And most

of us in this room will inflict trauma everyday on our patients in the name of

surgery, alright? So surgery is nothing really more than, hopefully, controlled

trauma.

[Slide 20] – [Tissue injury or infection]

[Dr. Craig] – Okay, so this is sort of like a forest, not a trees slide here. This is

gonna be kinda like our outline for the next few minutes. So up here, we have

tissue injury or infection—it’s very, very wide category of things that can uh

precipitate an inflammatory response. And then we said: the first thing that

happens in an inflammatory response is the production of inflammatory

mediators. And those mediators come in 2 flavors. They can either act on

endothelial cells to increase the edema, increase dilation of the arterioles and the

venues in the area and also increase the permeability of that microvasculature,

which allows fluid to move from the vascular to extravascular space: producing

edema. And we’ll probably end up talking about this in this first hour.And then the second hour: the second form of inflammatory mediators—and some

of these will actually do both things, as we’ll see—are chemotactic factors. So

they’re factors that are released locally in the challenged environment, which tell

or signal the endothelial cell, “gee, I really need to have some neutrophils into this

area” if it’s a bacterial infection. And we’ll talk a little bit about these and these

should be kind of familiar to you from Dr. McCutcheon’s lecture. This results in

inflammatory cell recruitment and activation. And depending upon the type of cell

that’s recruited, you’ll either end up with acute inflammation or chronic

inflammation.

So, um, the list of vasoactive mediators goes from here approximately down to the

center of the earth. So I’ve only highlighted the ones that are targets for the drug

industry, that you will be using to help manage your patients. Or, ones that have

kind of interesting nuances that will probably be targets in the future. So

everything here is clinically relevant. And I’ve done kind of the same thing over

here. Chemotactic factors don’t go to the center of the Earth. They probably only

go 100 miles down or so. But, there’s an enormous amount of chemotactic factors

and we’re just gonna talk about the ones that are perhaps more clinically

significant to us. So just get a sense that you’re getting a view, sort of a

tip-of-the-iceberg kinda thing.

[Slide 21] – [Images][Dr. Craig] –So! What does this actually looks like? So here’s a little cartoon.

Here’s a normal venule or capillary cross-section, right? You got some

erythrocytes, they’re forming rouleaux. They form, because of fluid dynamics.

Here, this area will have some friction as fluid flows through it. And so, the fastest

flow is gonna be in the center of the capillary. In addition, endothelial cells have a

net negative charge and erythrocytes have a net negative charge. These’s

electrostatic repulsion. Sot his kind of keeps everything in the center of the flow.

11




And it’s the fastest. Anyways, if you look, this little venule here is lined by living

endothelial cells and where they touch one another, there’s a tight junction. So

here’s a transmission electron micrograph. So here’s the cytoplasm of one

endothelial cell. Here’s the cytoplasm of a second endothelial cell. And here’s this

little tight junction uniting the two of them. And all of a sudden if there is a

vasoactive amines that are being released into the area. There’s receptors for thevasoactive amines on the basal surface of those endothelial cells.Those endothelial

cells recognize that. They activate their cytoskeletal network and they begin to

contract. Um, we had a researcher that would culture endothelial cells in culture

plates. And if you added stuff to it, you could actually see them contract in culture.

It was kinda neat. But that’s what they do in situ here. And what’s happening in

this situation is that as their tight junctions disperse, and they expose the

underlying basement membrane, right? Now fluid from the vasculature can leak

out into the surrounding area, causing edema. So since the basement membrane is

in tact, and you can kinda think of it—What kind of collagen is there? In basement

membranes? student: four …Four! Excellent. Remember, when you see me, it’s

gonna be a collagen question. Alright. So anyways, type IV collagen forms amesh-like array, right? It has a molecular weight cutoff, just like an ultrafiltration

membrane. So, low molecular weight molecules are gonna get out. High molecular

weight molecules are not gonna get out. So early on, during an acute inflammatory

response, the edema is called either an ultrafiltrate of blood or a transudate.

Alright? And, if that’s enough to take care of the response if this is a very mine

response indeed, then these cells will reestablish their connections and the edema

generation will stop. So, heres an electron micrograph of one endothelial cell.

Here’s another endothelial cell. Here, the gap junctions have been opened up

exposing this underlying, um, basement membrane. If, uh, you have say a heat

injury, and you actually damage the endothelial cells, this extravasation of fluid

into the extravascular space will continue. And so you get continued generation of

inflammation.

[Slide 22] – [Inflammatory vasoactive mediators]

[Dr. Craig] – Ao let’s talk a little bit more about the actually mediators. So you can

kind of divide the mediators into two forms, right. This is the innate immune

response, so these are kinda generated very quickly, if not already pre-formed. So

those in the green box are either already pre-formed in cells or are made very

quickly de novo in cells. And these are the guys we’re gonna talk about.

And then there’s another group of vasoactive mediators that are pre-formed or

very quickly synthesized in the vascular compartment, and we’ll talk a little bitabout those too. So, um, your book starts off with histamine and serotonin. I’m

gonna start off with prostaglandin. So we’re gonna start off with these guys cause

they’re real important in our profession. The generation of any or all of these

components is going to act to increase permeability through contraction of those

endothelial cells, resulting in edema.

[Slide 23] – [Cell-derived mediators: arachidonic acid]

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[Dr. Craig] – So, let’s talk a little bit about arachidonic acid. So, this is a real

ancient signaling pathway based on membrane lipids. And it’s not just for

inflammation, unfortunately. It’s used throughout the body for various things.

Controlling permeability to kidneys and things like that. So, under the

appropriate stimulus, many, many cells, if not all cells, in the extracellular matrix,

by activating a protein called phospholipase A2 can takephosphatidylcholine—Where’s phosphatidylcholine found? -Cell membranes.

Excellent. There’s a lot of it right. — You can take phosphatidylcholine and make

arachidonic acid. Another enzyme, phospholipase C can take phosphatidyl inositol

and also make arachidonic acid. So once the cell pool of arachidonic acid begins to

increase, it can either go in one of two pathways. Or it can go in both, depending

upon the inciting molecules or depending upon the cell type.

The cycloxygenase pathway takes arachidonic acid, and this is the black box, so

there are several things in here that we don’t need to know, okay. And it can

generate 2 different classes of molecules. One class of molecules, derived form

lipid, are called prostaglandins and literally, the list of prostaglandins that have

been isolated, really goes way, way down off this screen. We’re only gonna ask youto remember two—only two. But realize, the list is long. Alright? Prostaglandin

E2 is synthesize early in an inflammatory response. It is used as a mediator for

many different things. One of the things it does is it increases edema in the area. It

also sensitizes nerve endings, pain, okay. And then, it’s synthesized macrophages,

monocytes, resident cells that have been damaged. Endothelial cells will also make

prostaglandins. And then, later on in the inflammatory response, during

resolution, endothelial cells will shift their synthesis and start making another

prostaglandin—that I’m gonna hold you to—Prostaglandin I2. And this one is sort

of—It’s not totally true, but for our discussions, we’re going to discuss it as sort of

being the antagonist of Prostaglandin E2. Okay? We’re gonna come back to

Prostaglandin E2 many times during our discussions. Especially during

periodontitis. The cycloxygenase pathway can also take a arachidonic acid and

make a whole class of lipid derived mediators called thromboxanes. The only one

I’m gonna ask you to remember isThromboxane A2. Everything in these red boxes,

by the way, are gonna increase edema. Increase endothelial permeability. But

Thromboxane A2 also has another function. It’s synthesized by platelets and is

very important in platelet aggregation and establishing the platelet plug, one of

the first steps in hemostasis. And it’s 8:52. At this point in time, perhaps this is a

good time to stop, and we’ll come back and finish up in the next hour. I’ll take

questions…my throat is beginning to go so… Have an 8 minute break.

[Same slide after the break]Okay everyone…so…um, what we need to remember or what I’m asking you to

remember, out of the enormous shopping list of arachidonic acid metabolites: for

at least for Cycloxygenase is that Cycloxygense makes both prostaglandins and

thromboxanes depending on the situation and the cell type. Uh, PGE2: early on,

big player in periodontitis. Sensitizes nerve endings, painful, that sort of stuff. Uh,

PGI2 synthesized mainly by vascular endothelial cells. Later on in the

inflammatory response promotes resolution. So it’s sort of like the antagonist in a

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way to PGE2. Thromboxanes made my platelets. Right. Not only are these

vasoactive mediators but they also participate in the reactions that allows

platelets to adhere. Right. Form platelet plug. Arachodonic acid can also be taken

down the Lipoxygenase pathway into a series of compounds that I can’t even

pronounce. So HETES is one of the end products and it’s very active vasoactive

mediator, promotes edema. Uh, it can also be made into something calledLeukotriene A4 (LTA4). And one of the products of Leukotriene A4 is Leukotriene

B4 (LTB4). And not only is this a vasoactive mediator, this also functions as a very

powerful chemoattractant, especially for neutrophils. And LTA4 can also be

metabolized into Leukotriene C4, D4 and E4. And these 3 together are classically

called “the slow reacting substances of anaphylaxis.” Cause they’re elaborated, um,

they were first described as being elaborated later in anaphylactic reactions, um,

through mast cells. But they’re also important. And these guys also play a role in

diseases such as asthmas and those sorts of things. So, um, this is sort of a scaled

down pathway chart of arachidonic acid metabolism. But, if you’re really

interested in controlling inflammation and controlling pain and you’re in the

pharmaceutical industry, you’d look at this as sort of like the happy huntingground. It’s fairly easy to synthesize some of the inhibitors. In fact, some of the

inhibitors were known hundreds of years before they were actually synthesized.

[Slide 24] – [Cell-derived mediators: arachidonic acid metabolite inhibition]

[Dr. Craig] – So, um, lets take the cycloxygenase pathway first. As it turns out,

there are two forms of cycloxygenase, the enzyme. COX1 and COX-2. And it was

sorta thought, up until a few years ago, that COX-1 was constitutively expressed in

many parts of the body. And that COX-2 was only associated with inflammatory

cells and it was only induced, its synthesis was only induced during times of

inflammation. So that’s kinda an interesting insight. And it has turned out that

there are a class of drugs called COX-2 inhibitors that came out of this finding. But

preceding COX-2 inhibitors were aspirins. So, Native Americans used to take

willow bark and chew on it if they had pain, either headaches, or arthritis, or what

have you. And, something in willow bark was very effective at inhibiting

inflammation and inhibiting pain. And it took folks at the Bayer company in

Germany, during the height of organic chemistry explosion, in the late 1800’s to

actually synthesize aspirin, the active ingredient in willow bark. And what aspirin

does is it places an acetyl group and irreversibly acetylates both COX-1 and

COX-2. And so this is a non-competitive irreversible inhibition. And as soon as that

acetyl group is placed on COX-1 or COX-2, that knocks out this pathway and then

everything downstream is inhibited. Alright? So aspirin, a wonderful drug. I wishI had that patent. I would not be here before you if I had that patent! Cause not

only does it do this, it does other things quite well, and we’ll come back to aspirin

several times.

So, consequently, If you’re knocking out cycloxygenase 1 and 2, right? And, you

happen to be a platelet, this knocks out thromboxane A2. Why? Well the COX-1

and COX-2 that you have has been irreversibly inhibited. And you don’t have a

nucleus, cause you’re a platelet. So, you can’t synthesize more, right? So patients

14




who are chronically taking aspirin are at a risk for hemorrhage. And so you wanna

determine that in your dental patients. And we’ll talk about low dose aspirin on

Monday, but there’s a lot of folks out there taking low dose aspirins. Uh, aspirin

has other problems though, because it’s knocking out COX-1 that has a lot of good

effects. It regulates renal flow, uh blood flow, to the kidneys. It’s used as a

signaling system in many parts of the body. Folks are really interested inknocking out COX-2 because this is the one that’s induced and it was thought it

was only being expressed in inflammatory cells. So, wouldn’t it be wonderful if we

could knock out COX-2 and leave COX-1 in tact? And the Merck Sharpe & Dohme

folks were actually the first ones to crystallize the enzyme COX-2. I remember

sitting at a research seminary and, you know, computer visualization at the time

was just kinda new and there was the active site. And then they synthesized this

molecule that fit into the active site. It was really kinda neat to see. And that was

developed into a drug called Vioxx. I loved Vioxx when it was available. There’s

another one called Celebrex out there also. And Vioxx, the story of Vioxx is kinda

neat. So anyways, you know, to get a drug through they have to go through Phase

1, 2 and 3 trials by the FDA. And you have to satisfy the Federal DrugAdministration that you have an effective, safe drug before you can market it. And

Merck Sharpe & Dohme did diligence. Their phase 3 trials had, I believe, over

9.000 patients. And it was considered to be a safe, and very effective drug. The

problem is phase 3 trials were, at that time, pure randomized control clinical

trials, where you take a defined patient population with a defined randomization

schedule. Give them half the drug, half something else. Look at the outcome. It was

effective. But folks that we kinda see in our practices, are not medically healthy or

not as medically simplistic as the ones in these randomly controlled clinical trials.

Right? We see folks who are at risk for heart attack and stroke. We see folks who

have type 2 diabetes. We see patients who have Alzheimer’s disease, all of which

were excluded from the phase 3 trials from Merck Sharpe & Dohme. So after a few

years, people at the CDC, Center for Disease Control, started to realize that “Gee,

there was this increase in heart attacks from people taking Vioxx.” So,

consequently, Vioxx now, I believe it’s been taken off the market. And, it’s kinda

sad because Vioxx was really really neat. It was very, very specific for

inflammation associated with inflammatory disease. Um, okay….Because of the

problems with aspirin, because it irreversibly acetylates both COX-1 and COX-2,

the pharmaceutical industry has been really, very diligent in developing

Non-Steroidal Anti-Inflammatory Drugs—NSAIDS. And also there’s been a big

move now to get away from narcotics, especially in the dental profession. And so

Non-Steroidal Anti-Inflammatories are really big now. They, as a class,non-competitively inhibit COX-1 and COX-2 by various methods. One that I really

like is Ibuprofen. So the majority of my surgical patients get preloaded with 600

mg of Ibuprofen before their surgery. And it’s really neat because I can knock

down these pro-inflammatory mediators. These vasoactive mediators, so I can

decrease inflammation. I can also use them as analgesics. So, it’s kinda a powerful

drug to use. On the lipoxygenase side, there’s several drugs that are being

developed. One that you might come into contact with in the clinic is Zileutin. It’s

15




a principal used for asthma patients. But it knocks down lipoxygenase, which

knocks down the incidence of these slow reacting substances of anaphylaxis. If

you’re anticipating a very large surgery, say, multiple dental implants or

craniofacial surgery, those sorts of things, um, there are folks in our profession,

oral surgeons, periodontists, those types, who may pre-medicate a day before,

patients with corticosteroids. Corticosteroids inhibit phosholipase A2. So if you dothat, you knock down the pool of arachidonic acid and you knock everything down.

Right. The problem with doing that is that a lot of these mediators are real

important for stimulating an inflammatory response. And, you know, the

inflammatory response is there for a good reason, right? To control pathogen, and

help in its elimination. So, um, use of things like corticosteroids, which knock

everything down, you have to be very careful in that now you have a patient who

may be very susceptible to post-surgical infections.

[Slide 25 and 26] – [Cell derived mediators: Platelet activating factor and Cell

derived mediators: Platelets]

[Dr. Craig] – Okay. A few more. Platelet activating factors. So it’s synthesized frommembrane phospholipids by our friend Phospholipase A2. You put corticosterioids

in, on a patient, you’re gonna knock this down also. And, um, what does it do? It

stimulates platelets to aggregate and release their granules of pre made factors;

one of which is serotonin, which is really important. And it also primes innate

immune cells for activity, a function that Dr. McCutcheon probably touched on.

And it’s synthesized by endothelial cells, inflammatory cells and cells that have

become injured in the area.

Platelets…real important..and there are a number of strategies to try to increase

the number of platelets and their products in surgical areas. You’ll learn about

platelet rich plasma. In fact, we have recombinant-human-platelet-derived growth

factor for use. Tends to be a little expensive. But, uh, they’re being used in the

clinic. So, platelets have several different vesicles and they have different names.

You don’t have to remember what’s in the vesicles. Just that dense granules

release serotonin, a very reactive vasoactive mediator. Calcium, to help with blood

clotting. ADP. Um, alpha granules release cationic proteins that begins to

neutralize that negative charge between cells that are in the vasculature. And the

endothelial cells release fibrinogen, coagulation proteins and we’ll be talking a lot

about platelet derived growth factor. Even in this early stage of inflammation, the

groundwork is already being set for wound healing. Platelet derived growth

factor is one of the factors that is very active in the early stages of the wound

healing cascade.They also have lysosomal vesicles that release acid hydrolyases that are

antimicrobial. And then we talked about thromboxane A2, promotes platelet

adhesion and aggregation.

[Slide 27] – [Cell derived mediators: Mast cells and baseophils]

[Dr. Craig] – Mast cells and basophils. So Mast cells have wonderful receptors for

the constant domain of IgE. And it’s thought that mast cells probably evolved, or

16




one of their functions was to evolve to take care of pathogens that were larger

than a single cell. So, things like parasites, helminth worms, those sorts of things.

And, if the IgE is bound to its antigen, then the mast cells release their granules.

And inside those granules is histamine. Right? Serine proteases, chemotactic

factors for neutrophils and eosinophils. Usually will result in extreme contraction,

violent contraction to try to dislodge the pathogen from the mucosal surface. Butmast cells also function in other reactions against non-parasitic infections. Also, if

the receptors are bound, then the slow reacting substances of anaphylaxis are

synthesized and released into the area. Okay?

[Slide 28] – [Image]

[Dr. Craig] – Here’s a little diagram of the mast cell being stimulated. So here’s the

EM. And here’s a little cartoon. Here are the receptors for the constant domain of

the IgE antibody. If a ligand is bound on IgE antibody, the granules are released.

So you can kinda see all these dense granules being released into the area. Sort of

like a claymore mine for those of you who have been in the military.

[Slide 29] – [Cell derived mediators: Endothelial cells]

[Dr. Craig] – And endothelial cells. So, they’re the regulators of local tissue

perfusion, late in the inflammatory response, for our discussion. They synthesize

Prostaglandin A2, I2 rather (said with 41:19 remaining in podcast). Um, they also

have nitric oxide, a product of which is a very, very potent vasodilator. It’s also

used in bactericidal reactions that we’ll talk about more, probably on Monday.

They also synthesize a protein called Endothelin, which is another vasodilator.

Kinda getting the idea that there’s multiple overlapping and redundant mediators

that are being released. And they also release something called Procoagulation

tissue factor in response to endotoxin, LPS or interleukin 1, or TNF-alpha. Um,

so-called pro-inflammatory cytokines that we’ll talk more about very, very soon.

[Slide 30] – [Cell derived mediators: Macrophages, inflammatory cells]

[Dr. Craig] – Okay so macrophages and inflammatory cells, when their receptors

for PAMPs become bound, they can do several things. One of the most important

things is that genes—um, and we’re gonna talk more about something called the

NF-kappa-B pathway (?)—genes associated with the pathway are activated. And

one of the product is something called Tumor Necrosis Factor alpha. Kinda of an

unfortunate name for this cytokine. Uh, it not only increases vascular

permeability, but it also induces expression of endothelial cell adhesion molecules

and also has systemic effects. It usually is found in association with 2 othercytokines: Interleukin 1, which does similar things to TNF-alpha, and IL-6, which

is similar, it also activates something called the acute phase response, that we’ll

talk about more on Monday.

[Slide 31] – [Inflammatory vasoactive mediators]

[Dr. Craig] – Okay, so now, we’ve kinda gotten through the green box. These are

all cell-associated. You’re either pre-packaged or it needs to be synthesized de

17




novo during an acute inflammatory response. What about factors that are present

in the circulation? Present in blood. And there’s gonna be three of them. We have

the clotting factors…so, clotting and inflammation are very, very tightly linked

because evolutionarily, if one had a laceration, it was usually not done in an

operating room situation. But was done outdoors with lots of pathogenic

challenge. That first layer of defense has been breached and so there’s usuallygonna be a pathogenic challenge associated with a laceration. So it makes sense

that inflammation and uh, clotting, are linked. Um, and you’ve already talked about

complement, I hope, with Dr. McCutcheon. Complement. We’ll talk a little bit

about complement in something called the Kallikren-kinin System.

[Slide 32] – [Blood-derived vasoactive mediators]

[Dr. Craig] – Okay. So clotting/fibrin formation, kinin and complement are the

three interrelated cascade systems that are present in blood. So, uh, as you will

learn in this course, if you haven’t learned already: clotting is a complex cascade of

reactions. But it’s initiated by many, many different things. One of the best things

to initiate the clotting cascade is exposed naked type I collagen. Right. And thatsort of makes sense. Collagen is found extracellularly, but not within the

vasculature. And, if all of a sudden components of the blood are starting to see

exposed type I collagen, with its very unique structure, things aren’t good.

Alright. So it’s probably a good idea to institute the clotting cascade. Large

negatively charged surfaces: endotoxin/LPS can activate the clotting cascade. And

other endogenous expressed enzymes, such as plasmin and trypsin can also

activate the clotting cascade. We will learn that there’s two arms: intrinsic and

extrinsic to clotting. That’s not important to our discussion at this point in time.

What’s important is: activation is something called Factor XII, otherwise called the

Hageman factor. And once that factor is activated, many things can occur within

that site. Kinda linking inflammation and blood clotting cascade. So one of the

things that can happen is the clotting cascade, you get, um, the conversion of

fibrinogen to fibrin. And that stabilizes that initial platelet plug in the

area—which is a good thing. Um, Factor XII can also activate the Kallikren Kinin

Cascade, ending up with Brady Kinin and other kinds of molecules derived from

that cascade. These guys are also very active vasoactive mediators. They increase

inflammation. They also increase pain. And you’ll learn more about that in your

endodontics lectures. Uh, Factor XII takes plasminogen and makes plasmin, an

enzyme. And plasmin can activate complement through the alternate pathway and

generate anaphylotoxins—that we’ll talk about in the next slide—increasing

edema in the area. It can also, later on in the cascade, begin to degrade that initialfibrin clot. As it turns out, fibrin degradation products themselves also tend to

stimulate inflammation in the area. So the idea that you should be getting at this

point in time is that there’s this complex interwoven series of factors all

converging on endothelial cells to increase edema into the area.

[Slide 33] – [Blood-derived vasoactive mediators: Hageman factor activation]

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[Dr. Craig] – And last, but not least, for blood products, complement—I’m not

gonna spend a lot of time on complement because I assume that Dr. McCutcheon

has done that before me. I just want to point out several things that are important

to inflammation. So complement is a group of over 20 proteins that are

synthesized in the liver. In a cascade, oops..get outta here…that are synthesized in

a cascade fashion. And there are four functions that complement activationaccomplishes. The first is targeted cell opsonization. And that’s like, real

important. Why is that real important? Well. Most receptors for PAMPs bind

non-covalently with their ligands. Alright? They bind non-covalently with their

ligands. In fact antibody binds non-covalently with its antigen. Right? So, if it’s

non-covalent, that means there’s an equilibrium. There’s an on/off. Right? Very

few places during an inflammatory response do you get a covalent tagging of a

structure during these reactions. And this one is the exception. So, through the

generation of a phosphodiester bond, complement component C3b is covalently

attached, hopefully to the bacterial cell surface. And now you have a permanent

covalently bound tag to identify that cell for phagocytosis. Alright? That’s a really

important function of complement. Something called the MAC Attack. MembraneAttack Complex effects, hopefully, bacterial cell lysis. Some components diffuse

away from the complex that’s build up during complement activation. And one of

those are C5a which acts as a chemoattractant for neutrophils. And then, C5a, 3a

and 4a together work on endothelial cells to increase, um, the amount of edema in

the area. And as you know, complement activation can occur through three

pathways: classical, lectin and alternative.

[Slide 34] – [Plasma-derived vasoactive mediators: complement activation]

[Dr. Craig] – Did Dr. McCutcheon tell you why they call it complement?…Anyone

know? So, the story goes…up the street somewhere, I think it was at Cornell. This

was like, before WWII. They were immunizing, I forget the experimental animal,

and I don’t even remember the bug. But they would take heat-killed

whatever-the-bug-was, immunize the bunny rabbit. Make believe it’s a bunny

rabbit. And they would take the blood from the immunized bunny rabbits and put

it in with a live bacteria. And the bacteria would be lysed. And, if they took the

blood from a non-immunized bunny rabbit, against this bacterium, the bacterium

would not be lysed. Ah! So this is an immunology lab. So what we’re gonna do is

we’re going to do is purify all the antibody. As it turns out, it’s frickenly easy to

purify antibody from blood. So they did that. And when they reacted from the

immunized bunny rabbit, um, the antibody, purified antibody, with a bacteria, it

didn’t lyse the cells. Only if you added back in components from the blood backinto the antibody/bug mixture did you get cell lysis. Hence, there was something

in the blood that complemented the binding of the antibody to allow lysis to

occur. Hence, that’s why it’s called complement. Right? And, unfortunately,

because that was the first series of experiments that were done, they perhaps, the

latest evolved pathway—so called “classical pathway,” which you would think

would be the earliest pathway evolutionarily, based on the nomenclature—uh,

that’s how it kinda got its name. Okay, so binding of the antibody to the cell

19




surface generates a series of reactions that result in the generation of a C3

convertase. Perhaps the earliest is the so-called Alternate pathway. And I’m sure

Dr. McCutcheon went over the various ways that the Alternate pathway can be

activated, but that also results in a C3 convertase. And then, finally, there’s a

Lectin Binding Pathway. And, remember we said that mannose is expressed on

bacterial cell membranes? And it’s a PAMP. Well, we make a protein called amannose-binding protein in the liver—part of the acute phase response. It’s also

present. And binding of the mannose binding protein with its lectin will also

generate the C3 convertase. Everyone know what a lectin is, by the way? A lectin

is a protein that’s specific for a 3-dimensional structure on a carbohydrate.

Alright? And you’ll see lectins over and over again. But anyways, I wanted to make

sure you had that. So, no matter which of the three pathways, as soon as you

generate a C3 convertase, then it’s off to the races, Alright? So, the convertase

takes complement component 3, splits it into two pieces. C3b gets a thiodiester

bond covalently bound to the surface, hopefully of the bacterium. C3a diffuses

away, goes to endothelial cells, tells them let’s get more edema into the area. That

complex then takes C5, okay C4 has been used up in there—they kinda got mixedup in the order—takes C5. The same thing happens. C5b stays with the complex

on the cell surface. C5a diffuses away. This is really good for neutrophil

chemotaxis. So on and so forth, on the surface of the bacterial cell to assemble a so

called Membrane Attack Complex and lysis of the cell by establishing a pore

through the bacterial cell membrane. So activation of complement is sort of like a

watershed event during inflammation. Cause, not only do you detect and tag that

pathogen, you’re also amplifying the acute inflammatory response to that area.

Now you’ve set up a gradient that cells can follow to get into the site of activity.

You’ve also generated uh inflammation, edema, and not only help clear pathogen,

but also, as the lymph is drained from that area to begin to set up an adaptive

immune response. You’ve helped identify cells to be phagocytosed by the cells

that being called through this covalent tag. There are receptors for immobilized

C3b on phagocytic cells. And also, this is probably the least important, you’ve also

begun to destroy the bacteria that’s present in the area through generation of the

Membrane Attack Complex.

[Slide 35] – [Diagram]

[Dr. Craig] – And so here’s kinda like a diagram that just illustrates that. Alternate

pathway: here’s the bacteria convergence on that C3 convertase, right? Generation

of C3b. Covalent tag tagging that bacteria for phagocytosis. Classical pathway uses

antibody. As we’ll see on Monday, there are other things that can stimulate theClassical pathway other than antibody. Um, that also generates a C3 convertase.

Lectin pathway: mannose binding protein activates a pathway again, culminating

in the C3 convertase. And it’s off to the races from there. Inflammation.

Chemotaxis. Cell-directed phagocytosis, generation of the Membrane Attack

Complex and lysis of the bacterium.

[Slide 36] – [Plasma-derived vasoactive mediators: complement effector functions]

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[Dr. Craig] –So, we’ve gotten through this side: so, now we have kind of an idea of

how is the inflammation generated. And how do we get important things that are

in the blood, like pre-form antibody if this person’s actually seen the challenge

before. But more importantly, getting complement from the vascular compartment

into the extravascular compartment cause activation of complement is really a

watershed event. How do we get specific cells into that site? So now we’re goingto go to this side of this diagram and we’re only gonna talk about a few

chemotactic factors. Uh, C5a, which you’ve actually already talked about. We’ve

also talked about arachidonic acid metabolism. Leukotriene B4. Uh,

f-Met-Leu-Phe—really important. And we’ve touched already on chemokines and

let’s kinda talk about how these interact

[Slide 37 and 38] – [Tissue injury or infection and Inflammatory cell recruitment:

Endothelial/leukocyte cell interactions]

[Dr. Craig] – Okay, so if you actually look at an area that’s undergoing

inflammation, if you look at the microvasculature under a microscope… and when I

was a dental student, we actually had something called lab, where we actuallykinda did this with a frog. It was kinda neat, you know? Well, it wasn’t too neat for

the frog. But the frog lived. So, it was nice. But, you’d anesthetize a frog and put

him under a microscope. And as it turns out for this species of frog, and I forget

which one it was, but the skin in between the individual digits was really thin,

and you could kinda like thin it out and you could see the blood going through the

individual small vessels. And then you could put various drugs on the area. And,

because frog skin doesn’t have a lot of, uh is very permeable, doesn’t have a lot of

keratin, you get very quick absorption, so you can see changes in blood flow. And

one of the things you can actually see, if you use vasoactive mediators, such as

Prostaglandin E2, those sorts of things. You can sorta see is not only is the area

becoming hyperemic, filled with blood, and because of the increased vascular

caliber, right, blood is flowing through slower, cause it’s under constant pressure.

But you also start to see: some cells start to become more located along the

endothelial cells and these are polymorphonuclear leukocytes for the most part.

And histologists call that margination. Another synonym that you’ll see in books

is ‘pavementation’ also. So as these polymorphonuclear leukocytes are kind of

rolling along in the circulation, the endothelial cells start to express receptors

that are specific for sugars. Right, they’re lectins…they’re called selectins, right. P

and E selectins are being expressed by the endothelial cell and L-selectin is

expressed by the polymorphonuclear leukocyte. And these selectins are specific

for the Sialyl-x-Lewis carbohydrates that are on the polymorphonuclearleukocytes. They used to be called addressins. Selectins and addressins. Every cell

has an address and it’s on its Sialyl-X-Lewis carbohydrate. So anyways, this allows

the endothelial cells to begin to select out of the cells that are passing by in the

blood flow, the ones they want. And they want these polymorphonuclear

leukocytes. By these interaction with selectins. Next, if you’re looking at the frog,

you can see further down in the stream, all of a sudden cells are becoming very,

very closely associated and very tight. They’re not rolling around the endothelial

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cell tube any longer. And that’s done by expression of integrins by the

polymorphonuclear leukocyte, which bind to cell adhesion molecule 1, that’s

being expressed by the endothelial cells. So now, you have tight adherence. And

then, finally, these endothelial cells kinda migrate to the gaps between the—the

polymorphonuclear leukocytes, rather—migrate to the gaps that have formed

between the endothelial cells. And they start to transmigrate across the basementmembrane into the extracellular matrix. So as they’re trying to do that, right, they

can get past the cells alright, cause the cells have moved off to the side. But, they

come up against the basement membrane. And basement membrane, being tight

for collagen and other basement membrane associated proteins, laminin, those

sorts of things, alright? Uh, this is associated with matrix metalloproteinase

(MMP) gene expression. Especially ones that are associated with the membrane.

So they chew their way through the basement membrane and out into the

extracellular matrix. And I think I have a little cartoon of that. Yes, we have a little

cartoon.

[Slide 39] – [Inflammatory cell recruitment: Endothelial/leukocyte cellinteractions]

[Dr. Craig] – So here’s the inside of a small vessel, and here’s those endothelial

cells have been induced because of pro-inflammatory cytokine release and also

pro-inflammatory mediators, such as PGE2. And these little guys sense that, and

they start to express selectins. These selectins are specific for Sialyl-X modified

carbohydrates on the leukocytes, in case monocyte (? mumbles at 19:55 left on

podcast) And, then, expression of integrins, which bind the intercellular adhesion

molecule 1 (ICAM-1) that’s being expressed on these little guys…Increasing the

tethering of this neutrophil. And now the neutrophil comes through and it has to

degrade it’s way through the underlying basement membrane and then it goes

out into the extracellular matrix. And starts to follow the pathway to the pathogen

using diffusion gradients of things like f-Met-Leu-Phe or Interleukin-8, those

sorts of things. Now, in doing this, it takes awhile for these endothelial cells to

resynthesize the basement membrane. So now you don’t have an ultrafiltrate in

this area any longer. You actually have a hole in the filter. So now, larger molecular

weight proteins can come into this area. So, later on during inflammation, the

edema that was originally characterized as a transudate or an ultrafiltrate of blood

now becomes an exudate. Has higher molecular weight proteins associated with it,

has polymorphonuclear leukocytes associated with it. Changes character from a

clear straw fluid to a milky, thick, sometimes almost caseous kinda exudate.

[Slide 40] – [Inflammatory cell recruitment: Chemotactic factors]

[Dr. Craig] – And, this is just a photomicrograph of what my frog looked like. So,

here’s some vascular channels, right? And erythrocytes inside the vasculature

being lined with polymorpholucular leukocytes. And all these little guys out here

are all PMNs that have been recruited to the extravascular space by things like

C5a, uh, bacterial products, leukotriene B4 and chemokines, such as interleukin 8.

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[Slide 41] – [Mechanisms of inflammatory cell killing]

[Dr. Craig] –Okay, so, now we’ve got the polymorphonuclear leukocyte out into

the extravascular space. And now we kinda have an idea, through receptors for

PAMPs and also receptors for things like C5a and receptors for leukotriene B4 that

start to migrate up this concentration gradient towards where the infection

actually is. And so what do they do when they get there? So here’s apolymorphonuclear leukocyte. Umm, it’s a transmission electron micrograph and

you can kinda see one of its little podocytes is beginning to to lift up and trying to

get down on the basement membrane between these two endothelial cells. And

this is the cell nucleus. It’s cut in cross section. It’s a lobated nucleus so that’s why

it looks like two, it’s really just one. But inside the polymorphonuclear leukocyte

are all these little vesicles. These dense vesicles. And they all contain interesting

substances that are gonna be released into the area of or fuse with phagosomes to

destroy bacteria. Okay? So, on the outside of a neutrophil, you can kinda think of a

neutrophil as sort of like, I don’t know, a policeman in a way or..some kinda dooms

day machine for bacteria. And they are studded with receptors for all sorts of

things. Uh, bound immunoglobulin, C5a, C3b, especially that, C3b, uh arachidonicacid, metabolites, pro-inflammotry cytokines.


[Dr. Craig] – So they can recognize and dock with their targets. And once they

dock with their targets, histopathologically, there are several classes of vesicles

that are present in PMNs. I don’t want you to remember the various classes. All I

want you to remember is what’s present. So they contain things like acid

hydroxylases. Alright. So that would be good to kill bacteria. But they also…and

here’s some phosphorylase A2, which we’ve already talked about …and here’s some

myloperoxidase, and we’re gonna talk about how it generates reactive oxygen

species to blow holes in bacteria. But also we find things like MMP2 and 9, alright?

So, gelatinases. They’re not there kill bacteria. And they also contain lysozyme,

and that’s good, that gets rid of bacteria. Lactoferrin. That’s good. It chelates iron

so that bacteria can’t replicate as fast. But also has collagenase MMP-8. Alright?

That destroys the host. And tertiary granules: cathepsin and more gelatinase.

Alright? So, it seems like these bacteria come in, or these polymorphonuclear

leukocytes come in armed to get rid of bacteria by both enzymatic and oxidative

means. But, they also come armed with stuff to get rid of extracellular matrix.

—Yes?

Student question (can’t hear) —I believe so. Yea, I’ll have to check … Um..where

were we?So what’s the reason for having matrix metalloproteinases present in these cells?

(inaudible student response) —Yea… so, if you can kind of envision what’s

happening is that you’ve got a relatively large cell that’s climbing on the

extracellular matrix that’s made of collagen and other proteins and embedded in

this matrix are relatively small cells, such as bacteria. And so that extracellular

matrix has to be resorbed in order to uncover the pathogen. Alright? And hence,

that’s why there are matrix metalloproteinases present. So, while phagocytosis

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and engulfment and killing, bacterial cell killing is occurring, there’s also release

of matrix metalloproteinases to liquify that extracellular matrix and liberate the

pathogens that may be incorporated. So in order to actually get to the bacteria,

there’s some collateral damage. Okay. Polymorphonuclear leukocytes are

damaging their own tissue. So there’s two mechanisms…


[Dr. Craig] – Two generalized mechanisms for killing of bacteria by

polymorphonuclear leukocytes. One includes oxygen, called oxidative

mechanisms. And one does not, so non-oxidative mechanisms. So let’s talk a little

bit about oxidative. And you can actually measure this. Something called

respiratory burst. PMNs actually use molecular oxygen. Not for respiration, but

they actually generate reactive oxygen species and you can actually measure this.

So one of the things that happens is that inside the phagosome of a PMN, there’s

an enzyme called NADP oxidase (12:20 remaining—slide says NADPH oxidase),

and what that enzyme does is it takes molecular oxygen and converts it into a

superoxide ion. And then, there’s a second enzyme called superoxide dismutase,which takes the superoxide anion and forms hydrogen peroxide …gee, I use

hydrogen peroxide, alright? Bust holes in membranes. And then theres another

enzyme, myleoperoxidase, which takes the hydrogen peroxide and makes

something called hyperchlorous acid, which is really effective at blowing holes in

membranes and denaturing proteins…all sorts of things it can do against

biomolecules. And if that’s not enough, in the presence of iron, hydrogen peroxide

can also form hydroxyl radicals. Alright. All of which act to destroy the bacteria.

[Slide 44] – [Mechanisms of inflammatory cell killing Reactive oxygen (ROS) and

nitrogen (iNOS) species]

[Dr. Craig] –And here’s a little cartoon of that. So here’s a tagged, uh, covalently

tagged, in this case, bacterium. Here’s a receptor for that tag. Immobilized C3b. Uh,

this becomes… uh, podia begin to move around that docked, bound molecule, uh,

becomes a phagosome… a phagosome can be bound with other vesicles that contain

enzymes, which will non-oxidatively degrade the bacterium. On this end, the use

of oxygen. So, here’s the generation of the active oxygen species. Also, nitric oxide

can also be generated into a peroxide, nitrous peroxide molecule, which is also

very effective at destroying bacteria.


[Dr. Craig] – So, there are also NON-oxidative methods of killing bacteria byneutrophils. Not using oxygen, per se. Um, lysosomal hydroxylases have a broad

profile of specificities against both gram positive and gram negative. There’s

something called bactericidal permeability increasing protein, which

permeablizes gram negative bacteria. Defensins, that we talked a little bit about

with mucosal immunity, PMNs also have that. Lyses cell membranes. Lysozyme

attacks gram positive cell wall. And lactoferrin chelates iron that they can’t

replicate. So, we have both oxidating and non-oxidative means of killing. Um, but,

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there’s some collateral damage involved, right? So in order to uncover the

pathogen, you have matrix metalloproteinases that are liquefying the area so that

PMNs can dock with and destroy the bacteria.

[Slide 46] – [Mechanisms of host tissue destruction in inflammation]

[Dr. Craig] – You also have relatively non-specific ways of killing bacteria.Generating reactive nitrogen species, generating reactive oxygen species; are not

really specific for bacteria, but they’ll also kill host cells very easily. So there are

several different ways of generating collateral damage. So the release of reactive

oxygen and nitrogen species decrease tissue perfusion because there tends to be

blood stasis in the area if the inflammatory response takes a long time. Expression

of MMPs and local bone resorption.

[Slide 47 and 48] – [Matrix metalloproteinase family]

[Dr. Craig] –And let’s just finish up, I guess, with the matrix metalloproteinses. So

just to remind you, there’s a whole family of matrix metalloproteinases that are

out there. And, as it turns out, it’s really difficult to cleave type I collagen—thatrope like molecule, only has that one hinge region. And it turns out theres two

MMPs: MMP 1 and MMP 8 that invertebrates have the ability to cleave that hinge

region. Collagenase 1 is constitutively expressed in all cells. MMP 8 is induced in

inflammatory cells. There are gelatinases that take denatured collagen and

degrade them. As it turns out, there are two of them also. MMP9 tends to be

associated with connective tissue cells, but also tends to be associated with

inflammatory cells.

[Slide 49] – [Periodontitis is associated with MMP-8 expression and localized

bone resorption]

[Dr. Craig] – And…

as it turns out, periodontitis is associated with an inflammatory

lesion. So what has increased expression of MMP 8. And MMP 8, MMP 9, um, the

MMPs associated with inflammatory lesions are the ones associated with

extracellular matrix loss in periodontitis.

[Slide 50 and 51] – [Localized bone resorption: “Osteoimmunology”]

[Dr. Craig] – And, let’s just finish up with bone resorption and then we can start

on targeting next week. So, there’s a couple things that are kinda odd that people

couldn’t explain up until the last few years or so. So when I was a dental student,

and you will learn this too, there are several stages of the periodontal lesion

described by Page and Schroeder. And the one that’s actually associated with boneloss was something called the established lesion and if you look at the

inflammatory cell infiltrate in the established lesion, it’s mostly plasma cells.

What are plasma cells doing there? Right? Then, there’s a group that showed that

bone formation is always coupled with bone resorption. So you have to have bone

respiration before you have bone formation. So what’s happening here? So, there’s

this idea of osteoimmunology that we touched on in Craniofacial Biology. And

perhaps the easiest way to review this is to go right to a cartoon…

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[Slide 52] – [Picture ]

[Dr. Craig] –…So, in this cartoon, the cell is regulating this bone resorption in this

area. This is an osteoblast stromal cell. But for our purposes, make believe this is

an alveolar bone cell, or perhaps, even better still, periodontal fibroblasts. And it’s

an area of inflammation. So in comes mononuclear cells that have been recruited.And this periodontal cell expresses something called monocyte colony stimulating

factor. And theres a receptor for it on the monocyte. Once that ligand is bound,

then this monocyte starts to express RANK receptor. Receptor associated with

NF-kappa-B. Once that occurs, then this regulatory cell starts to express

RANK-ligand, which binds with RANK. And tells this mononuclear cell to begin to

fuse to become an activated osteoclast and begin to resorb this area of bone

matrix. You’ll also remember that there’s another factor that can be expressed.

OPG, Osteoprotegrin, which is a decoy receptor. So it binds RANK-ligand so that

the ligand can’t bind to the receptor. So, it’s really, bone resorption is a mix of how

much OPG is being expressed and how much RANK-ligand is being expressed. And

as it turns out, those plasma cells, and also T cells that are part of the establishedlesion of periodontitis,they’re a really good source of RANK-ligand. So that’s

probably one of the reasons why those antigen specific cells are present.

So at this point, we’ve gotten through the collateral damage that’s associated with

the inflammatory response. Next week, were gonna finish up with how cells are

targeted to the area. How they detect, rather, the pathogen. We’ll talk a little bit

about the inflammatory response, and then we’ll talk about systemic effects of

inflammation next Monday.

Have a good weekend.

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15/16: Inflammation I/II

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