Transcribed by Jazmin Lui May 6, 2014

CRANIOFACIAL BIOLOGY PERIODONTAL TISSUES: GINGIVAL EPITHELIUM

AND CONNECTIVE TISSUE I DR. CRAIG

1. Craniofacial Biology: Periodontal tissues: Gingival epithelium and connective
tissue

[Loud feedback] Oh wow. Maybe I cant move. Which is a problem. This is
weird. Because if I cant move around this is difficult for me. Maybe I can get
some help. Certain people have certain styles you know when they present
stuff, if I have to stand behind a podium its like death to me. So well do the
best we can. So lets recap. We had lectures on development of PDL tissues,
thats not only important for your basic understanding of teeth, but more
importantly for us as clinicians is the last 20 years or so people have been
really focused in, surgical specialties especially, and dentistry is basically a
surgical speciality, really focused in onyeah Im getting some weird stuff
happening herewhat do I have to do here, change my voice? People really,
in surgical specialities have been really focused in onthank you very much, is
that alright? More importantly I can move around. Now Im a happy guy. And
more importantly folks in surgical specialties have really been interested in
how their tissue of interest develops because the idea is: if I really understand
cellular molecular biology of how that tissue develops, maybe if I go into
surgery to restore lost parts for a patient I can actually recreate some of those
events that occur during development and radically alter the outcome of
wound healing for therapeutic gains. I think, my perception is different than
yours, I think we had an unbelievable example yesterday where the
understanding of how periodontal tissues develop actually allows folks, under
certain conditions, it doesnt happen all the time, allows clinicians to actually
regenerate cementum, PDL, and alveolar bone that has been lost to an
infectious product/infectious immunological process, periodontitis. And thats
been like a holy grail of surgical specialties. Its one thing to remove
Transcribed by Jazmin Lui May 6, 2014

pathology and and try to create a new homeostasis in a tissue, it usually
results in scarring. You dont have the anatomy back, you may not have the
function. But if you can get regeneration, not only can you get anatomy back,
you can get function back. It really makes me proud, perio, is one of the first
clinical disciplines first to do that. Were going to have a whole session this
week on periodontal regeneration, were going to revisit that perhaps from a
different perspective so I can hammer home those ideas cause those are
really powerful. So you go into your practice and you tell Mrs. Jones, gee
theres an infrabony defect and that tooth is loose, but Ive got a shot at
actually regenerating lost periodontal tissue connection apparatus, thats a
powerful thing for a patient. We talked a little bit about development of
periodontal tissue connection apparatus, and now were going to switch gears
a little bit and talk about gingiva, which is the fourth component of
periodontal tissues and remember gingiva doesnt participate in those
epithelial mesenchymal interactions that characterize odontogenesis. However
if you understand the biology thats going at the cellular molecular level you
can do seemingly miraculous things for the tissues. Lets talk about
periodontal tissues. So what are the components of the gingiva?

2. Components of the gingiva

You can characterize the gingival into these four categories. Oral or
gingival epithelium, thats covering the alveolus, when you look in clinically
thats what you see. Sulcular epithelium, as you go around into the sulcus
thats the epithelium thats lining the sulcus. The other side of the sulcus will
be the tooth surface. Junctional epithelium, gingival connective tissue. Oral
gingival epithelium is keratinized or parakeratinized and its a stratified
squamous epithelium. The keratin is really important, because the keratin is
like really thick, its a thick protein, rather, very good for abrasion resistance.
But perhaps most importantly, keratinization as we know today, really
impedes the penetration of water and any products dissolved in water, in
Transcribed by Jazmin Lui May 6, 2014

aqueous solution. It impedes them from penetrating deep into connective
tissue. As it turns out the inflammatory state of the connective tissue is real
important. Sulcular epithelium, so now were going to go around the corner
into the sulcus, were going to climb into the sulcus. This epithelium can be
non-keratinized or parakeratinized depending upon the inflammatory state of
the underlying connective tissue. You as therapists can modify the state of
keratinization if you get your patients to practice really great oral hygiene. As
we go more apical in the periodontal sulcus we go against this magical
epithelium that adheres onto tooth surface, junctional epithelium, thats
always non-keratinized, its a stratified squamous epithelium. One side
attaches to the tooth or tooth analog, and the other side attaches to the
underlying gingival connective tissue. Yes? [Student: is it more keratin with
inflammation?] oh, just the opposite. What happens to epithelium when its in
an inflammatory area, especially in the mouth, it goes through mitosis much
faster. As its going through mitosis much faster it doesnt have time to
express the genes associated with keratin. So youll see this over and over
again. You want to try to get the inflammatory state as low as possible for
your surgical patients. If you dont youll start having this hypertrophy of
epithelium in the area thats not keratinized which allows the penetration of
bacterial products deeper into the connective tissue, further fuelling the
inflammation and things get kind of out of hand. Well talk about that more,
well talk about inflammation, general pathology, and I think I gave you some
lectures on pathogenesis of periodontal tissues. Where was I? You destroyed
my chain of thought. Underlying gingival connective tissue. So underlying
connective tissue, in Tony Nancis book, Tan Cattes oral histology, he divides
it into deep and superficial connective tissue. were not going to do that for
our purposes. Were just going to talk about gingival connective tissue as
gingival connective tissue.

3. Components of the gingiva (oral (gingival) epithelium)
Transcribed by Jazmin Lui May 6, 2014

Heres some histology from one of my teachers. He probably did the best
oral histology I know if. His name is Max Litzgarden. He was the chair of perio
at University of Pennsylvania and a few other things. And Max did these
beautiful histological sections, and well talk about them as we go along. This
is human and Ill tell you how, living human, and Ill tell you how you got
these. You probably cant do these any longer so thats why I showed them.
Heres the oral surface up here, right? [points to dark strip on right] so heres
the oral or gingival epithelium, and this deeply purple layer is the squamous
cell layer and this is all keratin out here. A really good barrier for penetration
of bacteria and bacterial products in the underlying connective tissue. Then as
we go along the sulcus here all of a sudden this purple stuff, this is sulcular
epithelium [points to left side of tissue, white triangle] this is pretty healthy
gingiva because I dont see any inflammatory infiltrate down here in the
connective tissue. Notice you have vessels down here but no vessels up here.
Epithelium doesnt have any vasculature. So it depends on diffusion across
basement membrane for its nutrient and waste exchange. You dont see it on
this slide but further down is sulcular epithelium. This is a really nice
histological section of gingival connective tissue.

4. Components of the gingiva (oral (gingival) epithelium)

So this I lifted from the Tan Catte book and it bothered me. And I couldnt
put my finger on it for the longest time but now I think I know whats going
on. In my opinion theres some problems with the Tan Catte illustration
although artistically its beautiful. So heres the cartoon and heres actual
histology. So heres the marginal epithelium, so this is, when you look in this
is the most coronal extent of the gingival tissue, they call it the gingival crest,
I have no idea why they call it the gingival crest, marginal epithelium is what
most people talk about. And this portion, heres the sulcular epithelium and
heres kind of like sulcular epithelium here. This is not attached to tooth
surface in this case, enamel, and since this is not attached sometimes this is
Transcribed by Jazmin Lui May 6, 2014

called free gingiva. And the gingiva thats further down thats firmly attached
to tooth or bone this is called attached gingiva. And when you get your
lectures on periodontics, depending on if youre talking about implants or
teeth, the amount of attached gingiva can be important to some folks. What
else is here. So we have those wonderful gingival fibres that are attaching to
the cementum of the root surface. These are going from the tooth out to the
gingiva the dentogingival fibres that we talked about yesterday. And here
are some, going from the tooth to the periosteum, dentoperiosteal fibres. And
heres some fibres of the principal fibres of the connective tissue proper, so
these are the [points to very bottom] crest fibres, horizontal fibres. And now
Im looking at this cartoon stuff here, and I dont know what this stuff is
[points to purple fibres joining the junctional epithlium to tooth]. Maybe this
is cementum here but its the same colour as these fibres, that sort of bothers
me but thats alright. You can have cementum overlaying the enamel surface.
But when I go over here, the other thing Im supposed to tell you, heres the
crest [points to alveolar crest] mind you, in primates, you need to have about
2 mm cementum exposed so you can have attachment of the gingival fibres
or at least 3 of the gingival fibres, 3 of the 5 gingival fibres. Now I go over
here and I have a problem. Heres the osseus crest and heres the CEJ, and I
can believe this is epithelium, but theres a lot of epithelium here [points to
junctional epithelium region on histological section] and what characterizes
the junctional epithelium is that one of its basement membranes, the so
called external basement membrane is somewhat linear, and then when you
are getting up at sulcular epithelium it becomes more, you start to see rete
pegs. But you dont really see this much here until you get way up here. And
the bone is coronal to the CEJ, so this is a violation of biologic width. Im
looking at this and saying, this is weird! I look up here and look at these rete
pegs. They dont look like the human tissue that Ive been showing you. I
think this is dog. Because canines, carnivores, will have osseus crest thats
coronal to the CEJ. I think Nanci stuck in dog histology here. So when you
look at this make believe osseus crest is down here, unless youre going into
Transcribed by Jazmin Lui May 6, 2014

veterinary dentistry youre probably going to dealing with mostly humans and
not dogs. So I think we covered that. So you kind of have an idea of where
these structures are in space.

5. Formation of the dentinogingival junction
Development of the dentogingival junction. When crown formation ends,
the enamel organ that formed the tooth regresses into something called the
reduced enamel epithelium. It has basically has two layers. It has the inner
layer which hosts the ameloblasts and they attach to enamel surface by
hemidesmosomes. Epithelium attaches to substrates with hemidesmosomes.
And then theres another layer, the OEE, the collapsed stellate reticulum, the
striate intermedium, thats all been collapsed into the outer layer of the
reduced enamel epithelium. And were going to have a lecture on tooth
eruption. So as this tooth begins to erupt into the oral cavity, slowly it comes
into contact with the overlying oral epithelium, theres a number of cytokines
released especially from the outer reduced enamel epithelium, also there is
ingress of bacterial products and antigens, from the oral cavity into the sight,
so inflammatory mediators like prostaglandins, and those kinds of things start
to be elaborated, and youll learn more about inflammation in general
pathology. But just like the question over here, when you start to have
inflammation start to occur, what happens? Epithelium starts to proliferate. So
thats kind of neat. So this outer reduced enamel epithelium starts to
proliferate can you see how its getting thicker here, if we had a higher power
image of this area here wed see its inflamed with inflammatory cells. And we
have this thick layer of overlying outer oral epithelium and the two meet. And
the idea is if you want, I think, thinking histologically, you want to have an
epithelial lined eruption cuff through the tooth can erupt into oral cavity. So
you dont have open connective tissue that allows for infection and ingress of
bacterial antigens. How many of you have kids here? Well youll realize this
idea doesnt work very well. Theres a phenomenon called teething. Children
can be nasty individuals, or at least some of them, when they start to have
Transcribed by Jazmin Lui May 6, 2014

teeth erupt into the oral cavity, because these areas may become infected or
may become inflamed, theyre not very comfortable little campers at that
point in time. The idea is to have an epithelial lined eruption cuff through
which the tooth can erupt into the oral cavity. So lets take this a little further.

6. Formation of the dentinogingival junction (after amelogenesis)
Again this is all human. Notice how this doesnt look like the Tan Catte
picture at all? It looks really like canine material. So anyways, now this tooth is
erupting further into the oral cavity. So heres the overlying oral epithelium.
Theres some epithelium here that lines this developing sulcus. If you had
maybe a periodontal probe youd be able to put the probably all the way
somewhere down here, I guess, very easily. So this epithelium is not attached
to enamel surface. It lines the sulcus, so we call it sulcular epithelium. Notice
how we really developed over here on the oral epithelium. And you still have
developed rete pegs all the way down to about here [points to junctional
epithelium] and then right about here all of a sudden the rete pegs tend to
go away and you have a more linear interface between the junctional
epithelium and the underlying connective tissue. So on histological sections
you can always tell where junctional epithelium begins. And just like the book
says, tissue that Max got, junctional epithelium goes all the way down to CEJ.
All the way down to the CEJ. So this junctional epithelium initially derived
from reduced enamel epithelium. And there were two very famous
periodontal scientist types. One was called Bearhog. I love that name. Id love
a name like Bearhog. He stated that this initial adherance of the junctional
epithelium to the crown of the tooth, the enamel of the tooth, is called the
primary epithelial attachment. Primary epithelial attachment. And the
sulcular epithelium derived from oral epithelium but the junctional epithelium
is derived from reduced enamel epithelium, initially thats called the primary
epithelial attachment. As time goes by these epithelial cells tend to migrate
into the sulcus and theyre lost, theyre desquamated. And stem cells that are
present in this area give rise throughout life to another succeeding generation
Transcribed by Jazmin Lui May 6, 2014

of junctional epithelial cells and those cells or that attachment is called the
secondary epithelial attachment. Ok so, the initial one is from reduced
enamel epithelium as the tooth erupts, as life goes on those epithelial cells
are replaced by stem cells in the basement membrane and that becomes the
secondary epithelial attachment. Why do they talk about the primary and
secondary epithelial attachment? I have no idea. One of those things youve
got to know. Now lets see if we can go further on this.

7. Formation of the dentinogingival junction (after amelogenesis)
Kind of focus in on this junctional epithelial attachment. So heres the
sulcular epithelium, notice the convoluted basement membrane or basement
cell layers. And then you start to get around here and it sort of levels out.
And this is a very unusual epithelium because I cant think of another
epithelium that does this in the body. When you think of the gastrointestinal
tract, and I find myself thinking about my gastrointestinal tract quite often
during the day, and I cant think of another place in the gastrointestinal tract
where you have this structure, this hard structure, this non desquamating
structure that starts deep in connective tissue, pierces through the mucosa
and is sitting in one of the filthiest spots of the body, the oral cavity. Teeth
are kind of unusual if you think about it. It gets colonized with this biofilm.
Some of the bacteria that can be in this biofilm is really nasty bacteria. So this
junctional epithelium serves an incredible purpose. Not only by
hemidesmosomes attaches to tooth surface, but because of its length and
continuity it impedes, its not keratinized, it impedes the ingress of bacterial
products into deep underlying connective tissue in health when its doing its
job. If It doesnt do its job youll have gingivitis and if youre susceptible
youll get destruction of the periodontal connective tissue attachment
apparatus, which is way down here somewhere, and you start progressing into
periodontits. So this interesting thing about the junctional epithelium is if you
want to talk histologically theres 2 basal lamina. So the part that attaches
tooth is called the internal basal lamina, the conventional basement
Transcribed by Jazmin Lui May 6, 2014

membrane that unites the epithelium with the underlying connective tissue is
the external basal lamina.

8. Oral epithelium
Lets talk a little bit about oral epithelium. Youve had lectures on
epithelium, Im going to concentrate on things more specific to oral gingival
epithelium. Its a stratified squamous epithelium, keratinized, ortho or para-
keratinized. Consists of a basal layer, a spinal cell layer, a granular cell layer
and a squamous or sometimes called cornified cell layer. And lets go through
each cause theyre kind of important.

9. Cytokeratins expressed in periodontal tissues
If you look at, if you want to characterize epithelial cells, and this came
directly out of cancer therapy, you can characterize how primitive a carcinoma
is depending on what cytoskeletal elements in expresses. Cytokeratins. Oral
gingival epithelium is very differentiated epithelium, this is the constellation of
cytokeratins it expresses, junctional epithelium, reduced enamel epithelium,
our friends the cell rests of Malassez, these are all simple epithelium, relatively
primitive epithelium, and all you really gotta remember is that they express
cytokeratin 19. So relatively primitive epithelium for those three types. Oral
gingival epithelium, highly differentiated. A typical stratified squamous
epithelium.

10. Oral epithelium: basal cell layer
So this is some incredible histology from Max. And lets talk a little bit about
the basal cell layer. Not only is the basal cell layer convoluted, but the
processes of the cells are convoluted [points to white/purple squiggly border].
Not only is there no vasculature in the epithelium, all vasculature comes from
underlying gingival connective tissue. Heres a red blood cell cross section.
Unbelievable histology. You wont see histology like this very often. So lets
talk about basal cell layer. The big old basal cells that are sitting out here.
Transcribed by Jazmin Lui May 6, 2014


11. Basal cell layer (compartments)
Sitting on the basement membrane are 3, actually 3 different types of
keratinocytes. And you can kind of differentiate them by the types of integrins
or cell adhesions molecules they express. In basal cell layer there are stem
cells and they remain firmly tethered to underlying basement membrane
because of the 3 integrins expressed, and these are the ligands, you dont
have to remember this stuff. The stem cell under appropriate conditions will
go through mitosis, and the daughter cells can either remain stem cells, but
more likely they go into this compartment, this transit-amplifying cell. These
cells can still retain the ability to divide, though diminished, but theyre
thought because theres down regulation of these integrins. Whats beginning
to happen is these transit-amplifying cell are beginning to lose contact or
their affinity for attachment to underlying basement membrane. And the third
compartment you can kind of see in the basal cell layer are committed cells.
They have stopped expressing the integrins completely, and like a balloon
thats being lost into the atmosphere, begins to migrate off basement
membrane and go into the spinous cell layer and start turning on genes
appropriate to spinous cell layer.

11. Basal cell layer (electron micrograph)
So heres an electron micrograph. And heres a portion of a keratinocyte
sitting on the basement membrane. Heres the underlying gingival connective
tissue [points to light tissue beneath basement membrane] and if youre a
histologist youll be saying to yourself Boy theres a lot of complexity here, I
know these things, these are mitochondria right? [points to round cells in
upper half of slide] These things in here, these are cytoskeleton right? and
Im going to call these tonofilaments. Why? Because I like the name. I have
no idea chemically what these things are or what they do. I kind of noticed
down here in the basement membrane there seems to be different layers. This
layer seems to look electron dense, well call it the lamina densa [dark grey],
Transcribed by Jazmin Lui May 6, 2014

and this layer looks electron luscent, so Ill call it lamina lucida [light layer],
still have no freaking idea what these things are or what they do, and then I
see these little things come out, sort of like fingers of Velcro [extend down
from lamina densa], and Im sort of imagining in my mind because I know
that all of these things are collagen fibrils, and are cut in crossection, and it
looks like these guys are sort of going around the collagen, like vicromesh. So
Im going to call them anchoring fibrils. And then I noticed that the cell
membrane is not uniform. In certain areas its denser (dark black patches) so
Im going to call this a hemidesmosome. And I notice these tonofilaments
seem to attach to these hemidesmosomes. And so Im going to call this the
internal attachment plaque and the external attachment plaque because it
sounds like I know what Im talking about. But I still have no idea what Im
looking at or what their functions are. How were these dissected out at the
molecular level?

Transcribed by Jazmin Lui May 6, 2014




12. Basal cell layer: basement cell membrane ultrastucture
So were going to go through some of this. This is a little cartoon showing
morphology. So heres the hemidesmosome and the attachment plaque. And
heres the cell membrane of one keratinocyte. And here is this
hemidesmosme with tonofilaments attaching into it. And then theres some
magic here, haha, whoever did this has no idea what theyre talking about
[points to lamina lucida]. This is the lamina lucida, I have no idea what a
lamina lucida is but I can point it out on a micrograph. Heres the lamina
densa. And I have these things called anchoring fibrils and fancily I have these
Type I collagen fibrils are that are lacing through, kind of attaching
Transcribed by Jazmin Lui May 6, 2014

everything. So all is good with the world but if youd like to move ahead and
push the bounds of science perhaps you want to do something else. So now
we know what the proteins are. And I just want to go through one protein
cause it has clinical implications in practice.

14. Bullous membrane pemphigoid
You will see folks who have this. This is a lady, this is a terrible photograph,
should get a new one. This is a lady who presented to the clinic here, the oral
medicine clinic. Her gingival is really red and inflamed. If you took a 2 by 2
vase and put it there and rubbed it really lightly, all the epithelium would
come off underlying the under connective tissue. And this woman at this time
is not a happy camper she is really in pain. This disease is called bullous
membrane pemphigoid. Bulli is nothing more than a blister. But if you said
blister to your patient theyd know exactly what youre talking about so
medicine and dentistry always try to have a different name so we can have
some semblance of authority on this subject. So this is membrane
pemphigoid. Now theres a second autoimmune disease called pemphigus.
Youll learn about this when you get into general pathology. This is bullous
membrane pemphigoid. If you didnt know what you are looking at, youre
taught to take a bard parker blade, take a biopsy, lets see what this looks like
histologically. So if you did that, this is what these lesion would look like
histologically.

15. Bullous membrane pemphigoid: histology
So heres epithelium (points to dark red tissue) and heres the connective
tissue (points to pink tissue). And these are all lymphocytic cells, B and T cells,
mostly T cells. So its enormous, the inflammatory infiltration thats occuring.
This epithelium is beginning to lift off the connective tissue, making a blister
(points to white space) or bulli. But then if you came in with an antibody with
a fluorescent tag on it, and that antibody was specific for human IgG
Transcribed by Jazmin Lui May 6, 2014

antibody, one of the five classes of human antibody. This is what youd see
under fluorescence microscopy.

16. Bullous membrane pemphigoid: immune fluorescence
Youd see the overlying gingival epithelium (points above green) and
underlying gingival connective tissue (points under green). Theres a whole lot
of this patients IgG antibody right at that interface of the basement
membrane. So this patient is making an antibody against and antigen, some
self antigen. We dont know what it is. If I was in charge of this the story
would end right here. However this a person named Elaine Fuchs whos at the
Rockefeller University just up the road, and Elaine Fuchs had, a long time ago,
about 20 years ago, did the following using antigenic mice. I want to walk
you through this to give a flavour of how these people unravel this stuff,
there are some really clever people in the field. So all Elaine has is antibody
from these patients against some protein she believes. So what does she do?
She doeswhat happened??? I lost all my stuff! Huh, somehow, it didnt
getthats amazing. Ok so Im going to have to walk you through this. So
what she did, she went and she made a cDNA library. She took all the RNA
thats being expressed and she cloned it into an expression vector. If you tell
the plasma if you have the right promoter for your plasma, you can actually
drive synthesis of that piece of cDNA. She has the antibody against this bolus
pemphigoid antigen. So with the antibody she can pull up colonies of
bacteria with recombinant. So now she has a piece of gene to the bolus
pemphigoid antigen but she doesnt know what the gene actually encodes
for. So what you can do is take embryonic stem cells from mice and
microinject this micro DNA into stem cells. And then you can go into a female
adult mouse and induce a state of pseudo pregnancy in that mouse and
implant stem cells. The mouse is going to allow development of that fertilized
egg, hopefully it has your recombinant protein in it, take a little bit of the tail
from the littermates and look for the mutated protein, so youre going to
knock out that bullous pemphigoid antigen so you can get heterozygote, and
Transcribed by Jazmin Lui May 6, 2014

then youre going to do some incest here, you take littermates that are
heterozygotes and mate them together. You can imagine all this stuff that
comes with trying to mate these poor animals. And you can actually come up
with animal that has both the genes knocked out. And if you do that,

[switches to slide 12 basal cell layer cartoon)

If you do that, and you look, well first off, if you look at the mouths of
these little recombinant mice that had the bullous pemphigoid gene knocked
out, what you dont see is you dont see this structure (points to lamina lucida
unit). As it turns out, this structure in here happens to be a collagen, its type
17 collagen. And thats the antigen that people with bullous pemphigoid
antigen make antibody against. Antibodies gets decorated around this
structure, this brings about an immune response, this area is destroyed, if its
destroyed then underlying epithelium lifts away from the connective tissue
giving these blisters. One of antigens which turns out to be the internal plate
which is collagen 17. And in your text, it shouldve been, I dont know what
happened to this in the hand out. I have this in the hand out. Just a second. I
hate making a mistake because I never make mistakes. I thought I made a
mistake once but I was wrong. Ah! I see it. I see the mistake. Watch this is
going to be much better. Im feeling better already.

[Opens new powerpoint]

17. Bullous membrane pemphigoid (bullous pemphigoid antigen knockout)
Life is good. So basically this is what the Fuchs lab did. And those of you
that are kind of into recombinant DNA technology can look through this.

18. Bullous membrane pemphigoid (DNA blots)
Now this is what I wanted to show you, this is so neat. So this is panel A,
this is panel B. So a southern blot of what happened here. 6.7 So this is the
Transcribed by Jazmin Lui May 6, 2014

bullous pemphigoid antigen (points to 6.7), and what they did was transfected
a gene that had that gene knocked out, so its smaller (points to 2.6). So they
put it into a pseudo pregnant mouse, they looked at the littermates.
Unfortunately I watched them do this in Nick Partridges lab, and they do this
without anaesthesia, they take a bit of the tail and then, just like that. You can
tell how many times they do this because the tail gets shorter and shorter
and shorter. So here is a mouse that didnt get knocked out gene (+/+) and
heres a mouse that did get a knocked out gene (+/-), thats intact and ones
knocked out, and then they get these two guys together, hopefully ones
male and ones female and they have a night of incestuous love, and the
littermates if youre lucky and the live, come out as the homozygous
transgenic animal (-/-). This is a RNA northern blot (D). So here is the intact
animal, has both alleles that are intact. Here is the heterozygote, I love this, so
the RNA is half (+/-), I love this, you never see this. So this is the
homozygous, both alleles knocked out, nothing. And when you look at (E),
heres the protein, the homozygous, the one thats completely knocked out is
missing. So you look to see whats happening. They have the blistering just
like the human phenotype

19. Bullous membrane pemphigoid (panel legeds)
Here histology is not as good as Maxs but shes going to get a Nobel
prize, so whos the greater fool? So up here we can see, heres the lamina
densa, lamina lucida (LD, LL) heres the hemisdesmosomes, heres the
hemidesmosomes. High power here, and here are these cytokeratin or
tonofilament (inset top right box) coming in and attaching. And in the guy
thats knocked out, the transgenic knockout you dont have that internal
plaque (inset bottom box). So it turns out when you biochemically
characterize that gene it turns out to be collagen 17. And YES!

20. Basal cell layer: BPAG1 is COL XVII
Transcribed by Jazmin Lui May 6, 2014

Heres the diagram. Heres the old diagram, heres kind of like the new
diagram. In the so called tonofilaments here is the cytokeratins, and here are
these desmosomes. Turns out to be these anchoring fibres right here, thats
collagen VII, you gotta remember that. It was discovered by Bob Burgenson,
almost worked for Bob Burgenson. And heres the underlying type I collagen.
And right in here is collagen XVII and its forming a sort of an attachment into
the hemidesmosome and into the lamina densa. The lamina densa has a lot
of type IV collagen. And these laminens and stuff they tend to populate the
lamina lucida. Through these kinds of molecular workings people have begun
to decipher out their actual biological chemical component and their
functions for a diagram like this. So when you get into oral medicine and they
start talking about bolus pemphigoid. Raise your hand and say I think the
antigen is type XVII collagen just watch your professors, theyll go nuts.
Theyll say really? And thats enough for bolus membrain pemphigoid.

21. Basal cell layer (non-keratinocytes)
What else is in the basal cell layer? We also have non-keratinocytes in the
basal cell layer. Some of which are melanocytes and in some patients can be
very extreme. These are real important: Langerhan cells are dendritic cells
that sit there they can be infected with virus or pick up bits of antigen.
Once they do that they change their phenotype, go into lymphatic drainage
and go into lymph nodes, where they help with antigen presentation to be in
T cells. Youll learn more about this in general pathology but these are real
important, these dendritic Langerhan cells. We also have Merkel cells which
are a tactile sensory cell. Depending upon your state of periodontal health
youll have various forms of lymphocytes which youll learn about next year,
which participate with the innate and adapt immune response. Some quick
pictures of melanocytes and then well take a quick break.

22. Basal cell layer: melanocytes
Sometimes the melanin can be quite dramatic.
Transcribed by Jazmin Lui May 6, 2014


23. Basal cell layer: melanocytes
Heres a little diagram, so heres a little melanocyte and the basal cells there,
it has these little processes that go up in to spinous layer. What you see are
these little packets of melanin and they give the dark colour in these areas.
Lets take a 5 minute break and then we can go through the next portion of
the next hour.