Transcribed by Albert Cheng 9/8/12

[General Pathology] [Lccture 17-18: Inflammation III & IV by Dr. Craig]

Slide 41 – Mechanisms of inflammatory cell killing[Dr. Craig] Welcome back, so last Friday we started a discussion of inflammation. We had 2 hours on inflammation…covered a lot of material and what I’d like to do today is just review…or at least the beginning of today…review a little bit high points from those first 2 hours…and then we’ll finish up the inflammation lecture with how innate immune cells recognize pathogens or tissue damage. And then we’re going to talk a little bit about…very very briefly about chronic inflammation. We’re not really focused on chronic inflammation in this course per say. And then in the 2nd hour, we’ll address the question does infection or inflammation in a local site affect the body systematically and if it does, does it have any significance for dentistry. And we’ll talk a little bit about the relationship between periodontal disease and atherosclerosis. Let’s summarize some of the main points from last Friday.

First off the main event that occurs during an innate immune response is inflammation. Inflammation is the reaction of a tissue and its microvasculature to an insult. 3 things occur. First off is generation of inflammatory mediators. Inflammatory mediators affect the endothelial cells that line the microvasculature. So that’s the arterioles, capillaries, and post-capillary venules. Those little endothelial cells are real intelligent. They have on their basal surface, receptors for inflammatory mediators and then they do all sorts of neat things to generate edema. They contract…they expose the underlying endothelial basement membrane and then fluid from the vasculature leaks out…or you could say filters out the surrounding area. That’s an important event because in that serum is going to be complement. Complement activation is sort of a watershed event that occurs early on during an inflammatory response. It tends to focus and amplify the subsequent events of inflammation into that site. Later on, the same endothelial cells will start to express cell adhesion molecules that will target out cells that are appropriate for the challenge that tissue is undergoing whether it be monocytes, leukocytes…what have you. Once into that tissue, innate immune cells like this neutrophil or polymorphonuclear leukocytes are targeted to the site of activity through things like chemokines. IL-8 we talked about as being a major chemokine. Also products associated with arachidonic acid cascade. Leukotriene B4…very important or process associated with the complement activation or bacterial products themselves. We talked a lot of endotoxins. We talked about those 3 amino acids that start protein synthesis. fMLP (formulated Met-Leu-Phe) is a very strong chemotactic factor. Those things act in concert to get this little guy and his “brothers” to the site. These guys are just decorated with all sorts of receptors for pathogen associated molecular pattern or immobilized C3b…right…opsonins which coat the bacteria…covalently tagging it for phagocytosis. That occurs… and all sorts of neat things can occur in those vesicles in there. We talk a little bit about oxidative means of killing bacteria…generation of reactive oxygen species and reactive nitrogen species and also generation of killing events not using oxygen…a number of enzymes that these little guys have to kill. Also within these vesicles are things that destroy host extracellular matrix. At first that was kind of weird, when people started to characterize this, when I went to

dental school…I was taught tissue was destroyed because of the invading bacteria…bacteria have all sorts of collagenases and other terrible enzymes that will liquefy tissue and allow them to migrate into the host tissue AND THAT’S NOT TRUE. It’s the collateral damage if you will that these little guys have as they release these granules out into the extracellular matrix which contain matrixmetalloproteases to solubilize that extracellular matrix so that these relatively large cells in relation to bacteria can get in and dock with, phagocytose, and kill the bacteria. What else did we talk about [starts flipping through slides]

Slide 52: Cartoon of osteoblast/osteoclast resorbing boneWe also said that bone resorption can kind of evolutionarily be looked upon as sort of a continuation of this idea. Now the host has not only soft tissues that can be infected but has hard tissue (skeletal & dental tissue) that can be infected. It tends to use this kind of permutation of a system that it already has in order to resorb…in this cartoon…bone. Up on the top, there’s stromal cell/osteoblast, you could say well maybe that’s a PDL fibroblast and it’s expressing monocyte colony stimulating factor (M-CSF) which binds with a receptor on this recruited monocytes. When the monocyte is induced to express the RANK receptor…if that little regulatory cell up there is expressing RANK ligand…well that induces the monocyte to fuse with other monocytes to become a giant cell. So now it has a large surface area…it’s like a foreign body cell. Large surface area then it can start to acidify and remove the ECM (extracellular matrix) from this bone. And as it turns out, at least, in perio, a group of cell is responsible for RANK ligand or antigen specific B and T cells. Remember when we mentioned last week, there was this big confusion when…at least when I went to dental school I was taught periodontitis was a plasma cell rich lesion. What are all these plasma cells doing there? They’re doing several things…one of those things is orchestrating bone resorption. So I think we got up to where we left off

Slide 53: Innate immune recognitionSo I kind of told this story…not in a direct fashion. Really the first thing that happens during an innate immune reaction or an inflammatory response is that the host has to actually recognize that there is an insult whether that insult is of an infectious nature (bacterium, parasite etc) or from you (your Bard-Parker blade or surgery) or whether because a cell has died and that cell needs to be cleared from that site. How does this occur. The innate immune system is characterized by receptors that recognize PAMP and these are expressed on macrophages, dendritic cells (DCs) and all those kind of sentinel cells. Receptor activation of a PAMP first results through NFk-B transcription…notice that the inflammatory molecules a lot of time will use NF kappa B pathway…and it turns out that synthesis of these 3 guys (TNFa, IL-1B, IL-6). Another thing that occurs is synthesis of chemokines (IL-8). Lastly expression of co-stimulatory molecules in secondary lymphoid tissue. So selection and generation of appropriate B and T cells. One of the group or family of molecule…the idea of families of proteins/genes come back again…is something called the Toll-like receptors (TLRs). And the Toll proteins and genes were first characterized in the fruit fly. When a laboratory in Yale started looking at how was the innate immune system turned on…they stumbled across this family of proteins that had a lot of homology with these fruit fly proteins. So they named them Toll-like receptors after the fruit fly.

Slide 54: The Toll-like receptor familyAnd what did these guys look like? Well they’re a family of proteins and these are a cartoon of their body plan. They can have two proteins together or they can be monomers. If you look at the body plan, the receptor that actually binds to the ligand (PAMP) is sitting out here in this case the extracellular…they also can be docked and sitting inside a vesicle within the cell. A very interesting set of PAMPs are recognized. Here’s a receptor for bacterial flagellin. This is the one that we kind of have to think about…TLR-4…this cartoon is a little simplistic and that there should be another protein sitting here which is called CD14. This is important because this is going to come back when we discuss pathogenesis and periodontal disease in your next course DOD. CD14 which is made by the host binds with endotoxins LPS and then that complex binds with TLR-4. Then, inside these intracellular vesicles, you also have TLR that bind to nucleic acids that are not associated with the host…ssRNA/dsRNA…you never see that naked in the cytoplasm…bacterial DNA. The other part of their body plan. They’re always associated with plasma membrane. This portion sticks into the cytoplasm…whenever you see that, you think oh there’s going to be a phosphorylation/dephosphorylation signal transduction pathway linked up and you’d be absolutely right

Slide 55: The Toll-like receptor family (signal transduction cartoon)In the event of binding CD14 and that complex binds with TLR…is transduced into this nightmarish looking biochemistry. You don’t have know to…every issue of cell…these pathways change…so I don’t think it’s the pathway that’s important, it’s the end of the pathway that’s important. So there’s a number of phosphorylation/dephosphorylation of various substrates that occur finally climaxing with NFk-B which translocate from the cytoplasm to the nucleus and if a promoter region has a binding domain that’s complimentrary to NFk-B…that turns on transcription and the genes that we’re kind of interested in here would be IL-6, IL-1B and TNF-alpha. The 3 so called pro-inflammatory cytokines that are going to be expressed.

Slide 56: Actions of pro-inflammatory cytokinesSo if you have really severe inflammation and a lot of this is going on…there’s going to be so much pro-inflammatory cytokines in that peripheral site…it’s going to back up into the circulation and serum levels are going to rise on those 3 pro-inflammatory cytokines. And these guys can get access globally or at least within the body. One of the things that these guys do will be the subject of our next talk. It’s going to go into the liver and regulate the synthesis of cassette of genes that are called the acute phase proteins and the mannose binding protein (MBP) that we already talked about in the lectin binding pathway of the complement activation. This is going to stimulate more complement activation and also decorating the pathogen with opsonins making it fit to eat. These pro-inflammatory cytokines can also go to the bone marrow…so called hemapoietic stem cell niche…which you will hear about if you haven’t heard about it already. This will allow leukocytes to be mobilized. These 3 guys can also go centrally to the hypothalamus and increase body temperature. That’s kind of a weird thing to do. It turns out a lot of pathogens have streamline their metabolism to have optimal rate at our body temperature. So if you increase the temperature, all of a sudden, it puts them at a disadvantages.

That’s one kind of permanent way of dealing with an infection. Also goes to fat and muscle to increase gluconeogenesis. So blood glucose levels start to go up. It can also go to DCs and other sentinel cells in the area telling them to take up the pathogen or fragments of the pathogen…leave the extracellular connective tissue…go into lymphatics…into the secondary lymphoid tissue…and begin to display their antigen in the context of MHC molecules. So these guys can do a lot if you have severe inflammation

Slide 57: Innate immune recognitionDid Dr. McCutcheon talk about the inflamazone???? to you guys? No? Ok, so you notice that the TLR and most receptors are tethered to the plasma membrane. So what happens if you have self damage…what if there were molecules that were indicative of damage that are within the cytoplasm. Just want to introduce this idea to you because I think it’s going to be big in the near future. So there’s another group of receptors called the Nod-like receptors (NLRs). They’re a family of receptor and in many ways they’re analogous to the TLRs and there’s some overalap in the ligands that they can recognize. Some of the NLRs will recognize PAMPs but most of them are really good at recognizing damaged associated molecular patterns (DAMPs). You can see there’s a lot of effort here put in to make these 2 systems parallel. Sometimes when you’re reading the literature, you’re goin to see “danger associated molecular patterns”. What these receptors will do…they tend to be monomers in the cytoplasm but if they bind with their ligand, they’ll form these very large complexes…and I have a little cartoon on the next slide…they’re called inflammasomes and these inflammasomes can do 1 of 2 things depending upon the milieu you find them in. They can either induce synthesis of pro-inflammatory cytokines via the NFk-B pathway…if you’re in the pharm industry, you’re beginning to look at that NFk-B pathway as a potential for drug developing…OR if you’re not gonna sound an alarm, then you want that cell to die so you’re going to activate the apoptosis pathway.

Slide 58: This is a terrible slide. All I want you to do is focus in on the inflammasomes. Here’s an individual with 8 NLRs that have bound to their ligands…perhaps a component of a damaged cell and sitting out here is Caspase-1 that becomes activated so called inflammasomes.

Slide 59: There’s another signaling system that doesn’t depend on cell membrane of the inflammasomes and I just wanted to introduce that topic to you because I think it’s going to be very important in the future. So we talked a little bit about how the inflammatory response is initiated. Now you should have some understanding of how those molecules are recognized and how effector mechanisms come into play during inflammation. Clearly, you don’t want inflammation going on forever. The objectives of inflammation is to wall off that agent, deal with it, get rid of it and go for resolution…go back to homeostasis. So when I took this course, up until recently what was thought was most of these pro-inflammatory cytokines (IL-1, IL-6, TNF-alpha) have very short half-life…the protein and mRNA. And a lot of these arachidonic acid metabolite (prostaglandin and thromboxane), they have very short half-life also. So if the inciting agent is not there any

longer, well the arachidonic acid pathway will be turned off…NFk-B will not be turned on…and these pro-inflammatory cytokines will be turned off and you’ll get the resolution of the inflammatory response. Life was good til about 10 years ago when a class of molecules…which I think are going to be important to our profession…was discovered by Charles Sirhind who was very interested in arachidonic acid metabolite and he started to notice later on in an inflammatory response, some of prostaglandin/arachidonic acid metabolites that were being synthesized started to change to a group of molecules…3 different types of molecules…they’re all derived from lipids and synthesized late in an inflammatory response. He named them lipoxins, resolvins, and protectins. Lipoxins are synthesized by PMNs, endothelial cells and made from arachidonic acid metabolites (leukotriene and heat molecules). As it turns out, you can increase the synthesis of lipoxins through aspirin. He (Charles Sirhind) thinks the pathway is induced by aspirin. But if you’re knocking out cyclooxygenase, you’re going to have more of these types of molecules being made because the arachidonic acid metabolite pool won’t be going off into prostaglandins so there’s more to go in other directions…but that remains to be resolved. Then we have resolvins, they’re synthesized by many cells from omega-3-fatty acids. It was known for a long time that fish oil from fish that lived in northern cold ocean are rich in omega-3-fatty acids and they’re anti-inflammatory. As it turns out, omega-3fatty acids are used in the synthesis of resolvins. Protectins are a family of 20C lipids. Three of these guys together appear to actively promote resolution of an inflammatory response.

Slide 60:What do these guys do? Here is a cartoon that’s been lifted from the journal Nature. These dual acting mediators do two things. They decrease the recruitment of neutrophils into the area and they increase monocyte recruitment. These monocytes come in and become macrophages…they clean up apoptotic neutrophils and tend to restore homeostasis into an area with inflammation. There was a paper that came out in the journal of Immunology, they used a rabbit model of periodonititis and if you looked at the bacteria present… Gram- anaerobic flora that’s associated with periodontitis. These rabbits start getting really bad gum inflammation and inflammatory response…they start to lose their teeth. What they did in this experiment was kind of neat, you go in and you mechanically or chemically change the bacterial flora from a Gram- anaerobic flora that’s associated with periodontitis to a Gram+ aerobic flora associated with health. Inflammation goes away and bunnies keep their teeth. What they did in this experiment was, they used lipoxins which is a lipid and topically apply it twice a day to the bunny’s gingiva and not doing anything as far as oral hygiene and the disease went away. They were actively resolving the inflammatory response and we’ll talk about this in the next course about how this flora uses the host as a mechanism for its life-cycle…it’s dependent on inflammation. Maybe in your clinical lifetime, you won’t be needing to pick up scalers or curettes and use lipoxins or resolvins. Questions on the resolution of acute inflammation?

Slide 61: Chronic inflammationLet’s talk a little bit about chronic inflammation and we’re not going to talk in depth. So chronic inflammation is characterized by an inflammatory cell infiltration of monocytes,

small lymphocytes (B & T cells)…those kinds of things…you may have eosinophils if it happens to be a parasitic challenge. In some disease, an acute inflammatory response and a chronic inflammatory response can exist together. IN some situation like a parasitic or malignant situation, inflammation present itself as a chronic inflammatory response depending on the type of challenge.

Slide 62: Chronic inflammation – CausesSo what are the causes of chronic inflammation? A type of infective agents that the host can’t deal with with an innate or adaptive immune response and the classic one would be an infection with Mycobacteria. Immune-mediated inflammatory disease or prolong exposure to toxic agents that you cant rid yourself of through the innate immune response

Slide 63: Chronic inflammation role of macrophagesThe prime cellular regulator of chronic inflammation is macrophages. They’re derived from monocytes recruited into the area and depending upon the signaling molecules that this tissue macrophage encounters in the site, these macrophages can go in 1 of 2 directions. They can become so called M1 macrophages that are associated with chronic inflammatory response that’s associated with tissue destruction and generation of reactive oxygen/nitrogen species. This is the kind of destructive chronic inflammatory response that you see in periodontitis. The signaling molecules that tend to do that are IFN-g and TNF-alpha to make the macrophages go into M1 line. The M2 line is usually associated with other cytokines principally IL-4 and these macrophages tend to be more associated with repair process. They will be responding to growth factor and synthesizing things like PDGF, FGF…this is the cytokine usually associated with repair…TGF-beta (was first found in neoplastic cell cultures…it’s a very large protein family of which bone morphogenic protein is a member). These M2 macrophages tend to come in and debride the area and stimulate repair and resolution whereas the M1 macrophages come in to the area and perpetuate the inflammatory response usually with destructive sequalla (not sure about sequella???). M1s are associated with periodontitis and other kinds of chronic inflammatory response.

Slide 64: Granulomatous inflammationThere’s a type of chronic inflammation that you need to know about. It’s called granulomatous inflammation because from a histopathology viewpoint, this type of inflammatory response is associated with the formation of granulomas. So what if the host encounters something that it can’t get rid of like Telk?? from a surgeon gloves, or asbestos or Mycobacterium tuberculosis (oldest known infectious agent known to man) and some fungi and foregin bodies. Granulomatous inflammation is characterized histologically by something called epitheloid cells…you’d think it’s derived from epithelium but it’s not. So it’s characterized by the presence of macrophages, lymphocytes especially T-lymphocytes, epitheloid cells (which are derived from monocytes) and also a proliferation of fibroblastic cell around this group of cell that tries to wall off these lesions and those cell types together are called a granuloma. You can kind of see them histologically if you look at a tissue that has granulomatous inflammation

Slide 65: Granulomatous inflammation (flow chart)This is a little flow diagram kind of showing what’s happening here. Granulomatous inflammation… you have an injury you can’t resolve…so if you can’t resolve it, the body says I’ll wall it off…I’ll try to sequester it. It’s a failure of the acute response to really deal with this agent and there’s an adaptive immune response that is generated. But still the effectors of the adaptive immune response are basically the effectors of the innate immune response. So what happens is you get sequestration within macrophages, they eat up this infectious agent to try to keep it at bay…with the formulation of these granuloma as soon as T-cells start to come in and organize the tissue.

Slide 66: Granulomatous inflammation (microscopic slide)This is a low power of Granulomatous inflammation. Here’s one granuloma, here’s another. The macrophages are kind of sitting in the center here. There are fibroblastic cells that are on the outside sort of making a capsule. And this whole tissue is just filled with small lymphocytic inflammatory cells infiltration.

Slide 67: Granulomatous inflammation (higher power microscopic slide)Higher power. So here’s the fibroblastic capsule or at least a part of it. Here are macrophages inside this granuloma and here are some macrophages (monocytically derived cells that have fuse together in a giant cell and that’s what these epitheloid cells are called). Why don’t we call them giant cells but no the early histologist thought they looked like epithelium so that’s why they’re called epitheloid cells. And sometimes these epitheloid cells can look a little weird.

Slide 68: Picture of a womanSo here’s man’s oldest infectious disease, TB.

Slide 69: Picture of lower lip infectionHere’s a lesion on the tongue. When I was a dental student, they thought they had cured TB in this country and in our pathology course, we all had to go down to Philadelphia General Hospital and go into the infectious disease unit and one by one, we peered through the window and there was this poor solitary soul in one bed and they said see that’s TB and you’ll never see TB in your clinical lifetime. How wrong they were, it has come back with a vengeance.

Slide 70: 2 microscopic slides side by sideThe granulomas associated with…actually there’s a long colorful history with TB….goes back to SLIDE 68

Depending upon the site of where the granulomas formed, they have different names. So if you have disseminated little tiny granulomas throughout your appendages and body…it looks like little particles of millet seed…miliary tuberculosis. This is called scrofula, it happens to be in the lymph nodes of the neck. Anyways there’s a long history of TB…goes back to SLIDE 70

So if you look histopathologically, the epitheloid cells…the foreign body giant cells…all the nuclei assume this peripheral horseshoe kind of shape. Langerhan’s cells are characteristic of TB.

Slide 71: Summary: The inflammatory responseIn these 3 lectures, what have we talked about. We’ve talked a little bit about characteristics of inflammation. Inflammation is the reaction of a tissue and its microvasculature to an insult. Three events occur. Generation of inflammatory mediators. Those inflammatory mediators go to the endothelial cells and the microvasculature. Endothelial cells and microvasculature are the ones that control tissue perfusion, tissue edema, and control which innate immune cells they want to recruit from the extra-vasculature space. We talked a lot about mechanisms to increase vascular permeability…means to generate edema. Why? Because in our profession, the pharmaceutical industry has given us a lot of drugs to deal with inflammation. We talked about cell derived ones either de-novo or synthesized like the arachidonic acid metabolites or pre-made blood derived mostly from the liver. We talked a little bit about how inflammatory cells are recruited to the site of infection. We talked about how they kill…oxidative and non-oxidative mechanisms. Especially the oxidative comes with heavy collateral damage. We talked about the potential for host damage. We also talked about mechanisms for recognition. We talked a little bit about chronic inflammation. One thing I didn’t mention when I was talking about resolving inflammation was if you look at this class of drugs (protectins, resolvins, lipoxins)…what they’re doing is allowing inflammation to run its course because maybe inflammation in most instances is a good thing, but they’re terminating inflammation at the tail end not allowing inflammation to go on. Most of the drug we have at our disposal if you think about it…aspirin, Cox-2 inhibitors, NSAIDs, and corticosteroids. They all inhibit the inflammatory response whereas lipoxins as a group stimulate resolution of inflammation. Let’s take a 10 minute break and we’ll talk about systemic effects of inflammation and whether it has relevance to our profession

Systemic Effects of Oral Infections and The acute phase response

Slide 1: Systemic effects of oral infections and the acute phase responseSo your Kumar text has a really nice discussion of the inflammatory response. So I would highly suggest you take a look at it and as you’re reading it have my lecture handout there cause they go into a lot of detail on stuff that perhaps I don’t think is that important to us and he goes into NO details about MMPs or regulation of bone resorption. He does talk about resolvins which is pretty amazing. This next part, there’s a litte bit in your textbook but not very much. This topic has been evolving the last 15-20 years. But there’s a couple of nice original articles that I put on the handout that you might want to look at. So I usually start this by saying…if you have a peripheral limited inflammatory response like periodontitis, does it have systemic effects. You already know that’s true because we talked about those pro-inflammatory cytokines (TNF-alpha, IL-1, IL-1 beta). They can go to liver, bone marrow, and hypothalamus. What I want to do in this next discussion is talk a little bit about systemic effects of local inflammatory response. The

first 4 topics, we’re going to run through really quick. We’re going to spend majority of time on something called the acute phase response. In fact, Kumar talks a little bit about the acute phase response in his text also but he doesn’t talk about the dental stuff. So that’s kinda like the outline we’re going to follow

Slide 3: Systemic effects of inflammation: FeverAs we already mentioned, especially with resident tissue macrophages or monocytes that have been recruited into the area through PAMPs, DAMPs, and other receptors for PAMPs. There’s a stimulation of NFk-B and in this situation it will increase the synthesis in local concentration of TNF-alpha, IL-1, IL-6. If any one of you gets this question wrong on the midterm, shame on you. As it turns out, IL-1 goes to the central thermoregulatory center in the hypothalamus and turns on arachidonic acid metabolite synthesis. Cox-1 is turned on which stimulates the synthesis of prostaglandins E2 and in this situation prostaglandin E2 is not used for increasing edema or for recruitment of specific cells. This acts centrally to increase the body’s basal metabolic temperature. This is a primitive attempt to suppress infections especially viral and bacterial infections. It turns out that if you fool around with Cox-1 or try to inhibit Cox-1 with aspirin…irreversible acetylation of Cox-1 and 2, you can inhibit fever. Can you inhibit it totally? NO, if a mechanism is important in the body, there’s usually several pathways to affect it. IL-6 and TNF-alpha directly stimulate the hypothalamus. It doesn’t need to go through arachidonic acid metabolism to affect it. You can partially inhibit fever. So that’s another wonderful function of aspirin. It’s an analgesic, anti-inflammatory, anti-coagulant and might even stimulate lipoxins and now it’s anti-pyogenic

Slide 4: Systemic effects of inflammation: ShockSo that’s fever. Next, shock. So what if you have a massive infection or massive trauma so that in many places in the body, pro-inflammatory cytokines are being turned on in mass especially TNF-alpha. If you have TNF-alpha being synthesized throughout the body, it’s going to affect the vasculature in that area. And so much edema can be generated perhaps you fall below a certain level of blood pressure now that most fluids are outside in the periphery and not in circulation. BP falls until you cross a threshold where the circulatory system is not able to perfuse to tissue and the most important tissue is the brain and you go into shock. A failure of the circulatory system to maintain adequate tissue perfusion of vital organ.

Slide 5: Systemic effects of inflammation: LeukocytosisSo depending upon the type of challenge, you can get an inference by looking at a patient’s differential blood cell count. From looking at what kinds of WBCs are being stimulated. IL-6 and TNF-alpha go to the hemopoietic stem cell niche in the bone marrow and depending upon the inciting agent, you’ll have different kinds of WBCs increasing in the blood. Neutrophilia is an increase in neutrophils in peripheral blood is usually due to a bacterial infection. Lymphocytosis, the small WBCs as oppose to NK cells, increased peripheral lymphocytes usually associated with viral infections. Increase in eosinophils probably…the eosinophils and IgE access evolve to take care of parasitic infections but in our society we don’t have a lot of parasite so it becomes subverted to reacting against harmless allergens.

Slide 6: Systemic effect of inflammation: LeukopeniaLeukopenia in folks who are poorly nourished or in disseminated malignancy seems as if the bone marrow starts to give up and there’s a decrease in WBCs

Slide 7: Systemic effect of inflammation: The acute phase responseOne of the things that happen when there’s an increase in serum levels of pro-inflammatory cytokines, there’s something called the acute phase response. So the acute phase response is really primitive. Probably predates the innate immune response in many respects. It’s a primitive response to lots of different insults: infections, trauma, inflammation or malignancy. The effectors of the acute phase response are really diverse and very non-specific as oppose to the innate or adaptive immune response. And the components include altered lipid metabolism. If you start watching a patient under acute phase response over a fair amount of time, you’ll start to see total cholesterol starts to go up and LDL (bad cholesterol) goes up and HDL (good cholesterol) goes down, trigylcerides go up. If you watch for the total Fe content and it starts to go down, bacteria need Fe to replicate. Skeletal muscle and adipose tissue start going through gluconeogenesis and blood sugar levels start to go higher. The 4th component is the induction of acute phase proteins. They’re not all proteins. Sometimes when you read the literature, they’re called acute phase reactants. So this lipidemia and gluconeogenesis start to look like metabolic syndrome that’s kind of plaguing us in this country. Is there a component of inflammation that underlies this? I guess we’re going to start talking about that.

Slide 8: Systemic effect of inflammation: Acute phase proteinsTo be an acute phase protein, you have to be synthesized by liver and your concentration during an acute phase reaction has to go up or down by 25%. So a lot of these proteins are really common like albumins. You have high concentrations of albumin in your blood. So for albumin to go up or down by 25%, you’re talking about a relatively significant change. Most acute phase proteins are synthesized in the liver and regulated by our pro-inflammatory cytokines. In the humans, acute phase proteins are divided into those that increase during an acute phase response and those that decrease in an acute phase response. The major acute phase proteins that go up are C-reactive protein, serum amyloid A, fibrinogen, haptoglobin and the ones that go down are albumin and transferrin. Most of this is a primitive response to a infectious challenge

Slide 9: Systemic effect of inflammation: C-reactive protein (CRP)Let’s concentrate in on CRP. CRP was initially named because it was a protein that was present in blood that reacted with C polysaccharide of the pneumococcus bacteria. But since then, it’s been found that it binds other things with higher avidity. So it binds very avidly with phosphotidyl choline, phosphotidylethanolamine, componenets of cell membranes…yours and bacteria. And also chromatin, laminin, and fibronectin. When it’s bound, it has two interesting functions. One is it activate complement by the classical pathway. So CRP is a family of proteins called pentraxin. There’s 5 subunits to the CRP molecule. Looks a lot like IgM. IgM is really good at stimulating complement activation and there are folks who believe that CRP is probably the fore-runner of IgM class of

immunoglobins. So it will activate the complement cascade and all phagocytic cells (macrophages, monocytes, PMNs)…they have receptors for CRP on their cell surface. So CRP is a true opsonin. It makes a particle suitable for phagocytosis

Slide 10: CRP is associated with atherosclerotic complicationsThis protein was discovered before WW II. It’s said that post-surgically you’d watch CRP in patient’s blood to see whether they came down with a secondary infection. So this was routinely monitored in hospital as a monitor for systemic inflammatory status as a marker of systemic inflammation and classically, up until 2000 or so, values less than 10mg/L were considered to be “normal”. If you have happen to have a surgical patient who CRP levels spiked over 100mg/L, they probably have a bacterial infection and you brought that patient back into OR. But recently, within the last 15-20 years or so, people have in the high normal range (below 10mg), they seem to be associated with increase risk of heart attack (MI) and stroke up to about 4.4 times. So we’re going to take some time and we’re going to look at this paper that actually started all this.

Slide 11: Inflammation, aspirin and the risk of cardiovascular disease in apparently healthy menSo there was a study called the Framingham study, they followed this community in Framingham, MA and they followed people for heart attack and stroke and consequences of atherosclerosis. They really focused in on lipids. So from this study…that’s why physicians are so keyed in on lipid profile. But as it turns out, about ½ the people who have heart attack have normal lipid profiles. So there was a study that was started a long time ago called Physician Health Study. They followed physicians prospectively, and physicians could get their own blood sample and they were good at being health historians. So what they did was fill out a questionnaire and they sent some blood to Harvard. You had to be 40-86 years of age during entry and you followed yourself every year. If you didn’t send in your blood, there was a letter sent to you. So Paul Ridker…if you ever wanna be really depressed someday, just look up Paul Ridker CV. It’s like this guys has these publications. I don’t even think he can sleep or sit down or anything and they’re all fantastic…New England Journal of Medicine. Paul Ridker was wondering around the halls of Harvard Medical School and he heard about the Physician Health Study and he kind of got involved when they were analyzing some of the data. He was really interested in physician who had no history of atherosclerotic complications and he followed them. They have several arms to this study. We’ll only talk about two arms. One arm received a tablet of aspirin a day. And the other arm received the placebo. Their outcomes that they’re looking for are MI, heart attack and stroke. These are due to atherosclerosis. Venuous thrombosis…that’s not due to atherosclerotic pathology. It was only a 4 year study duration. He wanted to go on further but the results were so striking that they stop it. During this time period, 543 physicians/dentists/health care professional experienced one of these outcomes: vascular disease. And then they went back into the study and for each case, they pulled out a control…so it was a prospective case-control study.

Slide 12: Incidence of cardiovascular disease

Here’s the incident of cardiovascular disease as a function of what their CRP levels were at study entry. As we go through some of these studies, you’ll notice that the values change but that’s because the sensitivity of the assay has changed. Of the folks who didn’t have a vascular event in those 4 years, the CRP values were here. If you had any vascular event (1.37)…highly significant. 264 of them had a heart attack. We had a 1.5 CRP level at study end. Again highly significant. Not many had stroke. Still significant. Venous thrombosis elevated but not significant. That’s not what stopped the study

Slide 13: Relative risk of myocardial infarction subset by CRP quartileThis is what stopped the study. As it turns out, CRP is not normally distributed. So you can’t use parametric statistics to describe it so you have do these fun and games things. So one of the thing biostatisticians did is they looked at outcomes based on quartile. So along this axis, they put the physician here…we had the lowest CRP. Way up here was the physician with the highest CRP values. And then I just made cuts here…here’s the 25% that had the lowest, here’s the 25% that had the highest. What they found was really striking, that within 4 years, this is for heart attack, your risk of getting a heart attack normalized back to the 1st quartile…increased remarkably. So if you were in the highest quartile of CRP values, 4 years later you had a 4x greater risk of getting a heart attack than those that had the lowest. This is what stopped the study. If you were given one tablet of aspirin a day normally, you can knock that risk by ½. So in Dr. Katz course, they tell you about data safety monitoring boards. Now when you do a clinical trial like this with really bad outcomes like death, you have to with your own money appoint series of experts one of which is a biostatistician. They have access to your data but you don’t. They were following this study and at 4 years, they saw that the risk of having a heart attack was cut in ½ with aspirin. They stopped the study after 4 years. So this is a heart study…it was probably funded to go on a long time. But because they found such an incredible effect, I’m sure they had to give back the funds they didn’t use. You know how hard it is to get money for studies and to have to give the money back…it’s terrible. So this study, not only lipids but now systemic inflammation is highly associated with adverse outcomes as far as heart attacks. You can use anti-inflammatory, again aspirin… pleiotropic effects…you can knock that in ½.

Slide 14: “LUMEN”So what’s going on here. This is Dr. Craig quick explanation of atherosclerosis. You’ll get much more later on in this course. What happens especially where the arteriole tree branches…there’s these problems with fluid flow in these areas. If there is an inciting event, usually a ROS that triggers this, an area of inflammation occurs within the intima…cells began to die…in come PMNs, then comes monocytes…muscle cells are dying in the area releasing their cells…and these monocytes come in and becomes macrophages…and start engulfing these dead necrotic cells and their cholesterol. Under a microscope, they look like “foam cells”. So the earliest histopathological lesion of an atheroma is the presence of “foam cells”. Half of these foam cells in here will stay within this lesion and this necrotic lipid laden core that’s in here…and if this has access to the vasculature, it will precipitate massive blood clotting…and patient has heart attack or stroke. What we have evolved in our population…because all of us have these even at your age…I guarantee it that you have atheromas with our lifestyle. Smooth muscle cells

proliferate over this area and they synthesize Type I collagen. So this becomes a fibrous caps impregnated with smooth muscle cells and when these things rupture they usually rupture near the shoulder area and there’s an inflammatory response that’s here and monocytic cells come in and they start to release MMPs which destabilize this cap and if you happen to be unfortunate enough, this cap will rupture and this necrotic core has exposure to the blood clotting cascade and you end up with a heart attack or stroke downstream. So this is the question. Are the folks with elevated CRP levels…is the CRP levels going up because of the inflammation that’s present…just reflecting the inflammation that’s present in these unstable atheromas OR does this patient have high levels of CRP from perhaps other sources and if there’s a bit of inflammation that starts here because you have more CRP in the blood and more CRP gets deposited in this area…more complement becomes activated…more phagocytosis occurs so it focuses and stimulates the inflammatory response.

Slide 15: Picture of fatty tissue/plaqueSo stated another way…this is the interior of an aorta that has multiple infarcts

Slide 16: Central question to be addressedSo the question becomes is the elevation in CRP literally reflecting the inflammation in these unstable plaques or is increase in CRP from the peripheral sources predisposing this individual for heart attack and stroke? See the difference. Paul Ridker doesn’t know but perhaps dentistry can address one of these questions. That’s what we’re going to do with the rest of our time together this afternoon

Slide 17: Slade GD, Offenbacher S….So at the turn of the century, Steven Offenbacher group in North Carolina got access to the NHANES III and about 13000 of the folks in that survey also had a periodontal exam. He wanted to know if there was any association between CRP levels and periodontal disease from this study. This was a landmark study.

Slide 18: CRP values subset by periodontal pocketing: NHANES III dataWhat he did is he looked at this data and he subset it into 3 groups. One group…none of these subjects in this group had any sites that had any pocketing greater than 4mm and this is their mean CRP levels. And you had folks who maybe had mild periodontitis, folks that had a lot of pocket depth; they all of a sudden had elevated CRP levels.

Slide 20: Risk assessment for periodontitis So this was kind of an interesting study and so as luck would have it, we had just finished the study on risk assessment for periodontal disease progression and I had these bloods sticking around in my freezer and I read this paper and wondered if we could replicate that with our study. So we saw patients at baseline, we gave them a complete periodontal exam, so we knew at the beginning who had periodontitis and who didn’t and their severity. And 2 months later, we repeated the exam and we knew who’s periodontitis actually progressed and we took blood to measure the serum markers

Slide 21: Mean serum IgG antibody titers in periodontally healthy and disease subjects

One of the things we did was we looked at antibody to these 6 bacteria…they’re all associated with periodontal disease. But P. gingivalis was highly significant. So those that had periodontitis had significantly elevated antibody to P.gingivalis compared to those that were healthy

Slide 22: Mean (+/- SEM) serum chemistry values subset by periodontal statusSo if you just sequester them into 2 groups: healthy and diseased. The ones that were disease, they have elevated CRP levels, elevated cholesterol, depressed HDL…this wasn’t significant but that’s statistics for you and…LDL was increased. We kind of made the observation from this that periodontitis is associated with the acute phase response.

Slide 23: Mean (+/- SEM) serum chemistry values in quiescent vs. progressive periodontitisIf you looked at the few people that actually had more than one site progressed, the values got even more interesting. So other people have done this study more eloquently and robustly than we did

Slide 24: Associations between oral and systemic diseasesNot only is periodontitis associated with an elevation of CRP but also associated with components of acute phase response. As it turns out, periodontitis is not only associated with heart attack and stroke but the first observation of pre-term, low birth weight. Babies’ mom that had periodontitis tend to give birth to pre-term, low birth babies. Type II DM and heart attack and stroke. And then there’s been this grab bag of other diseases that are associated with periodontitis…chronic kidney disease, Alzheimer’s, rheumatoid arthritis. The weirdest one was…there’s a radio show that the medical center puts on called Dr. Radio. This spring I got this invitiation from a physician who’s in Neurology and he wanted me to talk about periodontitis and erectile dysfunction. Another reason to brush and floss your teeth.

Slide 25: Proposed mechanisms for the association between oral and systemic diseasesSo what are the mechanisms for all these associations between periodontitis and disease/conditions. The most ancient one is called anachoresis. Seeding thorugh the circulatory system of bugs from one body site to another. In the beginning of the 19th century, a lot of people lost their teeth because they thought oral bacteria would populate the coronary sites and be associated with heart attack/stroke. Innate immune cells go into area of chronic inflammation like periodontitis and get primed and then when they travel to unstable atheromic sites…are more likely to become activated…so this is the group at North Carolina…they like this idea. Presence of confounding factors such as smoking and age are common in both diseases. So my friend Felipe Pujol, he’s a guy who doesn’t’ believe any of this stuff. Finally, a group of us thinks it’s the acute phase response for moderate to severe periodontitis that’s behind it

Slide 26: Periodontitis and atherosclerotic complicationWe’re just going to finished up with a couple of things because when you get into clinic, your patients are going to ask you about this because this has gotten a lot of press. So what question do you want to address? What’s the strength of the association? Are we

talking about something that’s really robust or something that’s associated? What biologically plausible explanations have been forwarded? And I’m going to push the acute phase response. Does periodontitis precede atherosclerotic complications? And most importantly for our profession, if you have a patient who’s at risk of heart attack/stroke and they have periodontitis and you treat their periodontitis…will their risks go down for having heart attack or stroke. These are the kind of things your patients are going to ask

Slide 27: Incidence of fatal MI in the USCardiovascular disease is about 1/3 of the deaths in the US so it’s important

Slide 28: Relative risk of CHD in periodontitis: Prospective studiesThere’s a lot of paper out. This is a really good one. This is from the American Heart Journal and they did a meta-analyses on all studies done up to that time and if you take patients from 5 studies and put them together. It’s a prospective study so folks who had periodontitis today, what are their risks for getting heart attack in the future. And there’s an increase risk but nothing to ride home about. 95% confidence levels. Don’t go around 1…so it’s correct…it’s highly significant.

Slide 29: Relative risk of CHD in periodontitis: Cross-sectional What if you looked at cross–sectional studies, folks that have a heart attack, do they also have severe periodontitis. Now the risk factor goes up by 1.5 or so and then…

Slide 30: Relative risk of CHD in periodontitis: Control StudiesFinally case control. If you do a case-control type of matching study, then the odds goes up to about 2 or so. So what you can tell your patient is, there’s seems to be this relationship between the two. It’s not 4 fold increase…it’s more 1.5 or around there.

Slide 31: Additional studiesSo there are additional studies. So people have taken this idea and gone a little further. So there’s a number of treatment studies that are out at this point in time. Very few people look at death or heart attack. But most people look at pseudo outcomes. So if you take patients that had periodontitis and elevated CRP, and you give them treatment…can you lower the CRP values and the answer is YES. There’s one study from Australia…they took folks that were going to have full mouth extraction and they have periodontitis…so they followed them as they extracted all of their teeth and sure enough CRP went down. So you can lower CRP values. If you lower CRP values, it can result in lower incidence of MI. This is a Paul Ridker study. He was using drugs to decrease inflammation not treatment. Finally, this is a study you have to remember, prevention of atherosclerotic events…this is a study that the NIH funded…and periodontal therapy for folks who were at risks for MI did not have an effect on them having a subsequent MI. However, there’s a lot of comment about the study design and how the study was conducted

Slide 32: Potential significanceWhy are people so interested in looking at this? Think about what this would mean for our profession? If you could treat a patient who was at risk for heart attack or stroke, treat

their periodontitis…and it seems to be only for patients who have moderate to severe periodontitis…if you successfully treat that inflammation and decrease their systemic inflammation/CRP and it was actually shown that it would decrease the incidence of heart attack and stroke. That would mean periodontal therapy would not be an add-on but a medical necessity. That’s why people are so interested in this. We have a disease associated with Gram- anaerobe bacteria…really generates a lot of systemic inflammation and it would be lovely to see if it could be effective from more than just a dental perspective but also a medical perspective too.

Slide 33: ConclusionsI hope I convinced you that local inflammation if it’s significant enough can have systemic effect. Periodontitis has been associated with several systemic diseases some of which are really crazy like erectile dysfunction. The association is greatest with increasing periodontal disease, severity, and progression. It’s real important that you understand that periodontitis seems to contribute to systemic inflammation through the acute phase response and that effective periodontal therapy seems to decrease some of those components such as CRP. In big cardiovascular study, decrease CRP values resulted in the decrease in atherosclerotic complications. This is the take home for your patient if you see them in clinic, no studies at this point in time to my knowledge has been effective in actually decreasing a hard outcome…the incidence of heart attack/stroke/pre-term low weight babies. There was a paper showing that periodontal therapy did not decrease blood sugar levels or at least glycosylated hemoglobin. So even though the mechanisms seem to be there, no one has been able to show the hard outcomes. Questions on today talk? Have a great evening.