Autoimmune Liver Disease

>> i think we'll begin. so today we're going to have a discussion and presentation on a very broad problem. everybody at one time or another has had an itch. so i've always been interested in the origin of words, so i looked up this and what i found,

yes. do you know what bridge that is? you don't? do you know? that's the most famous bridge in the world. that's the brooklyn bridge. if you ever go to new york, you should walk across the brooklyn

bridge, preferably with a date because it's truly one of the more romantic experiences. and it doesn't cost a penny. and ther are two very nice restaurants. now why we would put up a bridge for something like this? it's because this whole course

is sort of a logo, and the idea that what we're trying to do is to connect science and medicine, sort of basic reduction in molecular cellular, etcetera, etcetera. the sort of stuff that goes on all around us here. and there's a big gap.

most people know a lot about one thing know little about the diseases, and often the reverse is true. so what we try to do here is to sort of take major health problems and take advantage of the extraordinary faculty on both sides of the bridge.

now these gentlemen whose picture were there, those days you could walk across the catwalk while they were building it and ladies used to go across long gowns and have their picture taken. symbolically we're doing the same thing with long gowns but

we're out on a catwalk. so the idea is to learn as much as you can. this isn't a medical school rector where you're going to be quizzed. you're encouraged to interrupt and at the end ask questions and don't hesitate.

best professor i ever had told me that the person who doesn't ask the foolish question is the fool. so i looked u the origin of the word pruritus and itching. pruritus is latin and i couldn't find out what it means in latin. but in old english, they

introduced the world itching. what it meant was an urge. so it's an urge to scratch but we use it far more often in terms of i'm itching to go see the brooklyn dodgers play again or whatever you're itching to do. so it's an urge to do something

or want something but it's interesting that itching has its origin in urge and for patients itching is linked to the urge to scratch and to relieve the sensation. so we whenever possible begin by presenting a live patient who kindly comes and briefly

explains what the nature of their illness is as related to this topic. and several years ago, one of the post docs said that's one of the great things about the chorus because it puts a human face on the disease. it doesn't matter if you're a

molecular biologist or surgeon, it's still the same. so we're fortunate that mark is a physician and part of the group here at the nih. and he's been kind enough to bring one of his patients who he is going to briefly interview and you can ask questions.

and this is a gentleman who has a disease which is characterized by rather severe itching. >> if i could ask mr. weaver to come up. while he's coming up, the disease that mr. weaver has is called primary daily cirrhosis. itching is a predominant feature

of it. in fact, 20 years ago, 20-30 years ago, itching was probably one of the predominant presenting symptoms occurring in about two thirds of individuals. fatigue is the other common symptom present in this condition.

so i'll let him tell you a little about his symptoms. so, could you tell us when you first started to experience symptoms related to your condition. >> [indiscernible]. at the beginning my [indiscernible].

>> okay. was your itching worse in the morning or the evening or it was not different. >> the same all day long. and when you itch, did you itch enough to break the skin? >> no. >> but you also complained of

something else as well on your skin. you notice your skin changing. >> yes. >> so the skin was also changing color, was getting darker and also getting a little thicker. >> right. >> can you tell us a little bit

about how severe the itching was. >> well he's saying that between 10-1, it was about 8. that would be the goal for itching. >> as you can well imagine we don't have very good scales to measure itching, so it's mostly

subjective what patients report. so what he's saying is that on a scale of zero to 10, even his own internal scale he reports it being an 8. so fairly bad for him. did you want to say something. >> i think that the [indiscernible] helped him big

time. when i brought him in 2007 [indiscernible] right here with me on a stable condition [indiscernible] the treatment he's been offered and given. i think that's making a big difference. i guess that's why we are here

to show that him and people in the clinic are doing a job that is really helping. and he's the proof. he's the living proof. >> so maybe we could talk a little bit, does anyone have any questions? do you mind showing.

so you can see here where his skin's a little darker. if you're up close you could see what we call [indiscernible] where the skin becomes thickened, more like elephant skin especially if you rub your hand on it. so it's a reactive change.

anyone have questions about the come digoxin you would like to know? >> [indiscernible] >> so they would like to know how debilitating it was, how much did it affect your life, the itching. >> he was.

>> can he tell us in what way? >> i have come to my arms all the time scratching. >> did it affect his sleep, his work. >> at nighttime sleeping, yes, it was a problem back then in 2007. >> okay.

>> that's also very characteristic the warm weather would make it worse. were there any do -- dietary components. did food make it worse. >> it's also characteristic. it's not affected by food. no, he's never been jaundiced.

any other questions about character or exacerbating factors of his pruritus? was there anything you could do to make it better? >> the cold. we'll talk about some thing that we tried to make it better. i don't want to, you know, take,

steal his thunder but we'll tell you what he did to try to make it better. what he has is associated associated with -- this is due to elevated -- because of the condition that he has due to t-cell mediated destruction of the intralobular.

this is the condition that he has. so the nurse approach that we took was to use a -- called cole -- and the you are dose is to start at four grams and try to work up to 16 grams. so we tried him with cole sty mean.

can you ask him to tell us whether the cole sty mean helped. these were the packets that were like sand that we gave him. this is what it's like. >> yes, it did. >> did it help his symptoms. >> but there was a problem that

he experienced, right? kidney. >> i have [indiscernible] several times. >> that's characteristic of the cole sty mean, it makes one constipated and limits the amount of dose you can give to patients.

also it's really like drinking sand. it's not very palatable. the most he could tolerate was 8 grams so two packets. so we had to try different strategies because that wasn't working. so the next thing we tried was a

drug called -- and that was the second drug we tried. now did that help you? >> i don't remember. this was several years ago. >> i'll at the you, we tried -- 150 milligrams twice a day and that had no effect whatsoever. so the next drug in the

algorithm that we go through was, well with regards to the pathogenesis of this condition i've alluded that perhaps elevated -- in the skins particularly an irritation of the nerve endings could be a particular explanation to why patients get itching.

the second explanation is that perhaps there's overstimulation of opioid receptors. so one strategy was to use opioid antagonists. so we tried an oral one called -- and initially started him on 25 milligrams and then increased it to 50 milligrams.

now did the mal trexone help you? so that was the one drug that actually gave him the most improvement. because of that we actually pushed the dose up to 75 milligrams. and he is taking that for about

five or six years. and it's only within the last year that we discontinued it. and the reason for discontinuing it was because his symptoms began to improve. he complained of less itching. and also he's taking many other medications and he's beginning

to have drug interactions. so we actually stopped that. and then he was put on prednisone. now prednisone is not the standard therapy for the condition that he has. the standard therapy is -- but he wasn't responding to it.

his liver biopsy was already consistent with the diagnosis of -- billiary cirrhosis but some patients may have an overlapped condition where they have autoimmune billiary obstruction. we put him on that thinking this may help because he was

improving but he was still symptomatic. we put him on prednisone 10 milligrams for a period of two years. can you tell us if the prednisone helped? >> it [indiscernible] my appetite to begin with.

it was much better. >> that's also typical of prednisone, it makes people increase their appetite, gain weight but there are other side effects. and we tried them for a period of two years. and his liver bio chemistry did

not improve much on it. so we actually decided to stop it because of he wasn't getting much benefit and there were a lot of side effects associated with it so we discontinued it. so right now he's only on -- colic acid and -- his itching he says is much better.

does anybody have any questions they would like to ask about the therapeutic approach or anything else about his condition? before we turn it over to dr. arias. >> so if we use objective measures, those minimal improvements from the

medications. typically with these drugs what you see is normalization or significant improvement in the al kind phosphatase -- the two blurry enzymes. he had maybe about a 20-30% improvement which is not much. also his seample -- minor

transferase or -- did not improve significantly. they're still elevated. he also had two liver biopsies which continued to show bile duct loss and bile duct destruction. so all the objective data suggests that there's not much

improvement with these. subjectively though he does report improvement in his symptoms. all right, thank you very very much. >> so i'm going to talk for a brief while about this problem of itching and liver disease and

some of the things i'll mention i'll refer back to the patient who presented many of the features. and them mark hoon is going to speak. mark is a tenure track investigator at nidcr. he got a ph.d. in bio chemistry

at the university of leeds. came to the nih in 1992 and has done extraordinarily interesting work on the problem of tapes. there's a remarkable article in science which we put up on the website that you can refer to on the circuitry for itch response in mice that was published in

2013. by the way, all the power points, background and everything else are available to you hopefully before each session on our website. okay. so i've got a disclaimer because much of the data that i'm going

to show you is from the group of -- who is at the academic medical center in amsterdam. and the two key references on the bottom of this slide and they are also on the website papers. he was a fellow with us many moons ago at albert einstein and

one of the thing we were thinking about is what causes this itching which can become extremely severe in people with liver disease. and we made a few observations and that has sort of peaked my interest through the years and followed through but mostly the

data i'm going to show you. i'm going to talk a little bit about the signs, the symptoms, who gets itching with liver disease. some clinical observations will point toward possible mechanisms, that's the birds business.

the current treatment has been discussed a little bit. we'll comment about it. mechanisms, old and new. and some new therapeutic considerations. and then mark hoon is going to discuss in more detail the neurobiology of itching.

now, pruritus and liver disease itching is quite varied. and as is pointed out, it's kind of subjective. but in many diseases, including this one, primary billiary cirrhosis, the itching can be really severe. now the patient has those

significant pruritus, and as you could see the scratching and a lot of the secondary changes mean it's been going on for a long time. but even patients where it's infinitely worse. there's one patient reported in the literature with liver

disease who would not have had a liver transplant for the disease at least at that time. it was so disruptive that a liver transplant was actually performed through the process. so the pruritus can be severe. it's usually at night. people often describe it like an

animal under their skin and they kind of dig so they excoriate, meaning they have local bleeding. and they get relief with rubbing and medications help a little bit not much. most itching doesn't involve the palms and the soles of your feet

but itching in severe liver disease can and that's a real disability. it's unaffected by sleep. people who are exhausted fall asleep and still continue to itch. so who gets it? well, not everybody with liver

that's why i asked this patient if he was ever jaundiced because this is precisely the circumstance under which the itching is severe. this is a very important clinical observation. i'll explain why. so when people have conventional

hepatitis, acute hepatitis, abc, etcetera, etcetera, pruritus is not usually a part of that picture. at least not a significant one. and people are deeply jaundiced due to a complete obstruction of the bile duct, say cancer of the pancreas or something.

it's a complete obstruction. itching is usually not present and if it is, it's very mild. so when does it occur. it occurs in diseases where there's incomplete billiary now, the billiary tree starts way up at the hepatocytes with the small branches which then

come together and ultimately inform the common bile duct that their liver is bile into the intestine. that tree can be affected by different diseases that partially obstruct and reduce the flow, there's still some flow of bile.

whether diseases that affect the liver cells themselves and the secretory processes that contribute to bile. and so what often happens is people such as this patient may have pruritus for years. and before dermatologists for example knew about primary

billiary cirrhosis, they never thought of liver disease. now any patient who itches a great deal particularly a premenopausal woman because it's more common in woman than men they are immediately referred to a gastro enterologist or a liver doctor.

it also occurs in pregnancy. in pregnancy there's a curious syndrome. like in the beginning women have often vomited, an upset stomach -- vomiting when you're pregnant. in the second or third trimester of pregnancy, they often begin

to itch. not jaundice. very small proportion of them become jaundice due to obstruction of bile -- process. and the amazing thing is that within 48 hours after the birth of the baby, if not sooner, it all disappears.

a very important clinical observation. so these are the entities. and if you look at what's at the top, what that cartoon is, those are the days -- ways two liver cells come together. those dark lines or tight junctions.

the domain of two liver cells come together to make it two. and the two is like a capillary and that's the smallest branch of the billiary system. there were millions of them. and then they connect down into the secondary and tertiary branches and then finally the

big duct, that takes it all into the duct. there are diseases that affect the canaliculi include membrane. so this stands for benign recurrent -- hepatic homeostasis, an incredible disease where people get episodic severe itching, some

usually jaundice and then for reasons we really don't understand, it goes away and then it recurs and the liver remains morphologically normal. this is progressive intracellular -- that happens in children who have a defect in one of the two atp binding

cassette transporters, one that pumps bioacids out of the cell into the canaliculi her and the other pumps are responsible for phosphocholine in the bile and that's the form and protects the billiary system. there are many hormones. during hormone pregnant sea, the

secretory capacity of the liver is reduced by 40%. you never know it because there's a huge functional reserve unless something else is going on. some women when they take estrogen obtaining oral contraceptionsives is all of

thinking and even jaundice. there are many drugs that have this effect, some have morphologic features and some don't. it sometimes occurs in alcoholic patients and it's not uncommon and chronic hepatitis b and c to have this itching syndrome.

now the first in that red circle that's where primary billiary is, it's an immune mechanism we think t-cell mediated is point out that it affects the smaller branches but not all of them. the pipeline is reduced substantially. that's very important, i'll

explain why in a moment. then there was some other diseases including ones involving starring sclerosis of the bile rededuct -- billiary duct. for the most par it's incomplete. the thing you always have in

common in incomplete billiary now we've got to go to a little bit of physiology. you recognize the cartoon has the canaliculi those small boxes of the small bile duct and the intestines. compounds that are secreted by the liver are reabsorbed and

recirculated. the classic one is by i don't -- bio acids. it's the major way of getting rid of cholesterol in the body. bioacids are a detergent that facilitates fat absorption and they do a lot of other things, signaling molecules.

they're going to come back to that. so 85% of the bile acids that are secreted are reabsorbed. when you pete a meal -- eat a meal and when the bio acids gets down to the ilium it's reabsorbed. that cycle goes on every time

you eat. if you eat six times a day, circulation keeps up with it. it's quite an amazing phenomena. bio acids is not the only thing that enters patterns of association. drugs do. years ago we had some studies to

suggest that some drugs like -- which was very commonly used in those days, we showed has a big hepatic circulation and we tried to argue that the reason with a why people had -- because the drug never went away. whatever went through kept coming back and causing

problems. we don't know. now when there is a disorder, cole stasis means bile secretory failure. and it could be at the level of the canaliculi, where the transporters are. it could be at the level of the

small bile duct and the bigger ones. the net effect is it's reduced billiary sceation into the when that happens it's like a partial obstruction in certain areas of the liver. and components of the bile gain access to the blood.

we don't really know how it happens. there's some evidence that goes through the liver and become depending on what the substance is. in others it goes between the cells where tight junctions become leaky.

one thing is clear that the plasma level of these substances that would be secreted goes up. not the bile pigment. now why aren't these people jaundiced? they are jaundiced because the bile pigment is not it's a waste product.

you probably know it comes from the degradation of heme. so once it's in the intestine the bugs converted the water soluble -- and it's gone. so people can have big time and there's a ten fold capacity to excrete the bile pigment than is used in the ten fold excess.

it's a safety valve. so you really have to have substantial liver disease in something like pbc in order for people to become jaundice. that's a bad prognostic sign. but all these other conditions, pregnancy and whatnot. you get the picture like a

gradation that occurs. and in all of those circumstances of incomplete billiary obstruction, most of them are not jaundiced but they are itching. and that is what i said leads to sometimes clinical detours before you get to someone who

recognizes this. let's talk about the -- of pregnancy. the it occurs in 1% of all pregnant women. and the major symptom is itching and it can be very severe. it occurs in the second to third trimester.

there are mild elevations of the plasma bile acid. they are elevated, they can go up to four or five times mueller. -- four or five times more. it's not a structural thing if it can turn on and turn off like what are the clinical clues

about the mechanism. one is the rapid disappearance of pruritus and -- after the delivery of a baby. the other is something that we did 25 years ago but now it's become very popular, it's become refined. that is if you pass it through

into the stomach and into the duodenum and you can get that tube to go into the bile duct and it's coming out of your nose. so it's called naso billiary drainage. basically any bile from getting into the intestine.

as i'll show you the pruritus and almost always rapidly disappears. and has been mentioned as partial relief with these non-absorbable residents i call story mean is basically like trapping bioacids and other things into the intestine that

prevents the interhepatic circulation or the bile acid -- which promote billiary secretion and it's thought maybe that may accelerate the relief. and with -- we don't have a clue how that works. then there's partial relief and sometimes pretty dramatic with

now those are the clinical observations. now this chap has a tube coming out of the nose into the bile you won't volunteer for it but if you're itching like crazy it's a wonderful thing. because look what happens. here itching is measured as

intensity, qualitatively the patient's word. but it's obvious when you see it someone who is itching, it's like my gosh within 24 hires i don't feel it. and that's what happens and as long as the drain you can continues it's fine.

you take it out and it takes actually a couple weeks before it goes back to where it was. i'm going to leave this but the general concept is that something is getting into the circulation that is stimulating the itch neurons which you're going to hear a good bit about

in a moment. so we're going to leave that. now, the -- was very much interested and trying to pursue this and find out what it is in the serum of people with severe itching and liver disease that can cause problems. so what they did was they took,

because i figure itching is neurons. so they took a bunch of neuronal cell lines and added serum from people with itching, without itching, severity and whatnot. and what are they going to look for? well they look for calcium.

why was the calcium. he goes calcium is a major signaling event that regulates all kinds of pathways within a cell. it's a pretty good thing to look for. what they discovered was quite surprising.

you can see it over on your left there. that's a dose response of adding serum to these neuroblast them awe cells and you're measuring calcium within the cells. so you add serum from somebody who has the cole static itching depending on the amount you add,

the calcium goes up. the signaling calcium release. and it increases serum calcium that's shown here and it happens primarily with serum in people who have been intrapathic co-low stasis pregnancy or on the other side primary billiary cirrhosis etcetera.

this was the first clue. so the question is well can we find out what it is. so in a series of very interesting papers, they identified what it is in the serum of these individuals that causes itching. in brief here's what they did.

first you put it there a molecular sieve, you treat it with a pep said, it doesn't go away so it's not a peptide. you treat it with -- why would you do that. well because -- blocks g protein mediated coupled receptors. a whole bunch of them.

probably about 40 or more within the cell. and this thing blocked. well that was interesting so that gave you a clue that there was something that was interacting with g protein coupled receptors. and so -- off, he knew the

literature and one of the thing that was actually discovered in holland in 2005. no, much earlier. that lie so phospho-- acid was it. so they started looking for a lipid. and they used various techniques

to eventually do it which i won't go through. if you're interested i'll give you the reference. what they found is that the factor that caused the calcium increase was lie so phosphocytic acid. and there's a formula.

so what's lie so phosphoitic in the 80's or 90's of the last century to show that -- acid has a lot to do with the cytoskeletal arrangement of cells with activating platelets and actually with migration of cells. it interacts with pip 2 to help

with the centralling process. and it acts on six different known g protein link receptors. an important role in neuropathic pain and are present in the literature at that time. more are cently there were descriptions of reprogramming gene expression in nerve

endings. that all sounded very interesting. so how does lipo phosphocytic a said made. where does it come from? it comes from -- choline which is over on the far side which is then acted upon by two

enzymes -- which then slits the molecule into -- and then there's an enzyme or taxing that splits off choline. that's how you get lie so phospholytic acid. so they decided they would look in the serum of patients who had pruritus.

icp with the interpathic co-low stasis of pregnancy. these are 13 women who really were itching very seriously. and they measured lie so phosphocytic acid which was increased in their serum. but they persisted. so what's r toxin.

r toxin is a -- and it was first described as being over expressed in several cancer cell lines where it promotes motility metastasis. if you knock it out, mice don't develop properly as in an genesis and neuronal development.

where is it. there's some disagreement about it but it seems to be expressed in hepatocytes. it certainly is in the -- endothelial cells. those are the cells that line the blood supply within the liver that feeds into the

hepatocytes. and in fat cells. and there are a couple reports in the literature that people with chronic hepatitis have increased amounts of r toxin actually circulating in their blood. so they measured r toxin by a

biochemical means essentially looking at the cleavage of choline. and they used serum from patients who are controlled. that's the one on your far left. those are, i forgot what pc is. pregnant control, yes. and women who are pregnant in

their third try semester who aren't itching. and then those with intrapathic itching of pregnancy. there's a pig significant differ. there's more r talk -- toxin circulating in the blood. they looked at other patients,

non-pregnant ladies. here are a bunch of co-low static patients. this is severe itching these people had not just a little scratch as compared to cole owe static patients who didn't itch and healthy control. again toxin levels were

substantially higher. and if they look at itching in other states, it looks as if the cole static patients, those with liver itching have much higher levels of r toxin than do the co-low static patients that aren't itching or patients with chronic renal failure.

renal failure also gives rise to this itching syndrome or people who were not. so this was sort of guilt by there are a lot of diseases that cause itching. hodgkins disease, lymphoma is a very common one. and the itching can be very

severe. it can be almost the initial symptom. but here toxin levels in the blood and people with chronic the people with wound healing or you cut yourself everybody knows you itch where the new skin is being formed.

that didn't make any ditches. and also with itching, people with atopic dermatitis. they thought it was specific for this. now what happens if you put this tube down and you drain the billiary system? and here those patients would

they symptomatically improved associated with it. there was a 50% decline with the serum r toxin levels. can you correlate itching with these blood levels? well, they tried. itching is subjective. that's the best that we have.

so you see that by itching intensity, it seems to correlate with the serum r toxin levels but certainly not with the bio acid level which are the ones on your right. now other people have described varying degrees of correlation of bile acid levels and

different bile acids, a whole bunch of it with itching. so this isn't necessarily the end of the line for it. so then they tried to reproduce this in animals. so they can have something to work with. they developed a very, i thought

ingenious. they put a magnet under the siliconnized magnet. the mice are in a cage and the cage is surrounded by a magnet. so every time the mouse does something, you get a tracing. and that happens mainly at night.

so most investigators don't want to sit there in the dark trying to figure out if a mouse is scratching or not. let the machine do it. and it works. i'll show you a tracing of it. they then took some lie so phosphocytic acid in various

amounts and injected it subcutaneously and looked up what happened a number of actual scratches that take place every 15 minutes. this is not molecular science. and as you see, there's a dose response curve. so why don't we make co-low

stasis in a mouse. well you can tie off a bile you can take amounts that's effective in one of the transporters and you can also give them some bile acids. that may accelerate it. or you can make the mouse pregnant and give them bile acid

or not. and you measure the serum r toxin levels and you measure their scratching. and so in pregnancy in a mouse, nothing happens with any of those models. toxin levels stayed the same. they don't itch.

co-low static mice don't itch. you can make them cole low static but they're not itching. this was trust tating but they also don't have very high serum lie so phosphocytic acid or r toxin. so how and this potentiate itch. it is interesting that an itch

receptor, a family of them and some of the factors that activate those receptors experimentally and directly as you'll see very much participate in the neuronal response that is manifested as itch. now, one of the things is protein tgr5.

now tgr5 is essentially a bile acid receptor. and it's present in macro phages, endothelial cells. it's not there for -- and we could have a whole session on it much here's from a recent paper jci in 2013 on the potential role of tgr5 and itch.

and so what you see these are neuronal cells and those positive ones are positive toward tgr5. what they've done is to measure scratching by a similar technique i guess. i don't recall. and those have the wild type

which is the gray bars. those where they over express tgr5. that's the blue bars. and those where they knock out that's thread bars over a period of time. so you see on the far left, when no bile acids are given, you

over express tgr5. and the scratch index in the mouse goes up. if you add bile acids in addition, this is the oxy collate similarly. in the over expressed ones it goes up and the others take place.

at any rate, they argue at the localizing tgr45 to the neuron, it interacts with a whole cluster of parts of the itch receptor signaling mechanism. and that's what those circles are which mark is going to talk about. so there's a few more clinical

observations that come on. this is a very interesting thing because you know like with much of medicine, information comes from both sides and it's not only from one. one is nasal billiary drainage and -- co-low static patients rapidly develop a decline in the

serum phospholpa to mueller. that one's reported last year. the oral regimens had a minimum effect. in pbc patients the bile acid can reduce pruritus and there's one report suggesting that the levels -- it's interesting that the -- levels in pregnant women

in co-low stasis rapidly declined to normal after delivery of the baby. what does this mean. what's been suggested is this kind of mechanism. we have on one side the so-called co-low static bile duct, that's the very small bile

that's where the are this needs to be. something goes from there ultimately into the circulation. factor x. which they interact perhaps in the hepatic site or certainly could be in the -- endothelial to activate r toxin

transcription and expression and secretion. and the blood level goes up. when that happens, lie so phosphocytic acid is formed. and lice owe phosphocytic acid down stream signals into nerve receptors and stimulates the response that we identify as

but there's got to be more because there are situations where the lyso -- there's got to be some cofactor that augments this process. no you that cofactor could very well be bile acid and if in fact tgr5 are present and the bile acids are elevated, they don't

have to be too high or maybe it's a specific bile acid we just don't know. this is the state of the art a at the moment. so what do they conclude. the hypotheses is that in cole owe stasis, the toxins locally, we're not sure where locally is

general lyso phosphocytic -- and other factors perhaps bile acids, i think through tgr5 may also play a role in initiating or potentiating this -- toxin antagonists have now reached the stage where they're beginning clinical trials in cancer it took a long time to get

this was four or five years ago. wouldn't it be amazing fan lpa inhibitor or an r toxin inhibitor actually -- this process. we'll find out eventually. so that's the story. i introduced mark to you who will continue and appears all

the questions which you have and i have, right. >> can you hear me? i'm not sure -- okay. so this is actually the title -- but i really like it because i probably use it to go through the different parts of the story and to introduce some background

elements as well. so first of all i'm going to talk about diseases. so chronic itch. there's actually a very large number of people, really diverse group of people with chronic these people are a lot like most [indiscernible] think of itch as

something really annoying that happens -- if you scratch for half an hour and you forget about it. for these people it's like having a mosquito bite but continuous all the time. i went to a meeting on scratch this summer and the patient

groups there's basically people who all their lives they go to bed, wake up in the morning and the bed sheets are covered in blood because they scratch all night long. so mechanically the group meets together. the first group are clearly

people who have various skin conditions. there's clearly a large number skin conditions including ones like this which are genetically controlled. and then there's other conditions -- will have had infections from various -- again

incredibly itchy. then there's the group that includes liver disease patients. but also includes a bunch of other diseases that seem to be systemic. this is in a particular hodgkins lymphoma. a large number of patients now

that actually live that basically have non-functional kidneys, 85% of the people with kidney failure on dialysis have drawnic itch. and 15% of that have severe chronic itch that is life changing. other diseases like --

understand why these people have chronic itch. there's also another group grouped together here of people which have a variety of neurological disorders that also show pruritus. i'll go to in a moment that's perhaps mechanically a little

easier to understand. and i would like to point this out that the chronic itch is somewhat similar to another condition that my group is very interested in and that's chronic pain. we all heard of people with chronic pain.

also many people have heard of people with chronic itch but just like with chronic pain people with chronic itch there seems to be no purpose for all this condition. it just seems to be self driving. there's really no added point

for many of these patients. so i'm essentially a biologist and i work with the neurons. this is now my transition into the world of nerve cells. and i started out by showing the end of another cell. this is the end of the nerve cell which is found in the skin.

and the cells which are in the skin clearly within the skin there are immune cells as well. it's an interaction between various factors that drive for instance those patients with rare skin conditions. there's an abnormality that basically drives activation of

the peripheral nerve endings to activation of various receptors. so on this slide i've shown a table of receptors that's been suggested that are present in the peripheral nerve endings. at the very top here, the one that you all heard of, histamine receptor we all know that

histamine, if you go for a skin test, the control they put on your skin is histamine produces a wheel and also incredibly itchy this is released from mass cells found in the skin that are activated. so i remember other receptors i've noted these two here, these

are ones that have been identified and relatively recently by -- group so compounds what's found endogenously but very useful tools for neuroscientists interested in an itch. and then there's another process of these are up here -- but

there's also a number of other receptors. in particular this one -- interleukin 31. i'll come back with that a little later. so i guess this is the periphery. so these are remarkable cells,

very different from any cells in the body that they have the nerve terminals at the skin. they have this enormously long axon that stretches all the way to where the cell bodies are and the cell bodies are found right by your spinal cord. if you think of a giraffe that's

kind of scratchy, it's here. this can be several meters long. so these are remarkable cells. and the other end cell is found where it forms a contact sign up and this is found in the spinal cord. it's at this stage that the information from depolarization

at this end of the cell is transmitted into the release of neurotransmitters which then cross and activate the next partner in the nerves. nerve pathway. so that's what my group is interested in how we detect. these neurons are found in the

spinal cord with the ganglion neuron are responsible for more than itch clearly. there's a mechanical stimulate through your skin, you feel temperature, you feel pain and you feel -- so one of the questions that we, where we have in the beginning is how do you

actually distinguish between all these different types of stimuli. is that there is multiple difference, there's multiple cells in there and they respond a bit to all these different types of stimuli and somehow they send a code on to the

nervous system. or in fact this case where actually there are groups of cells that are molecularly defined by a particular but then are responsible for detecting certain types of response. in a particular, we were

interested because of -- in the class of neurons that express this particular molecule the trip v1. it's an ion chanel found in the plasma membrane and identified as the receptor of the capsaicin found in chile pepper. it's not only responsible for

both heat and also if think about hot chile pepper it's responsible also for pain. this is a -- through the ganglion. you can see the neurons -- this is in black that expresses the receptor is heterogeneous. the cells which are probably

small dialysis -- now there's also evidence that in fact trip v1 and the trip v1 neurons are required to itch. so for instance antagonists for trip v1 block itch. agonists can cause itch. and nerve help for trip v1 has basically lots of responses to

histamine. what's interesting is this knockout didn't have complete loss of responses to other exownltdz. our question was those neurons, these neurons expressing trip v1 encode for all itch or do they in fact have other receptors for

the other types of itch compounds. so we took a different approach and in this approach we generated animals. but now where we can express a transgene. this is a promoter that's driving two transgene gfp which

is to mark the cells and then the r toxin receptor. so in my sense you have a natural r toxin receptor. if you inject normal mice, they could care less. they could drive specifically in humans receptor in specific sector neurons.

you can eliminate just certain cells within the whole body. this is exactly what we did. this is now a panel of -- we're looking at distant populations of neurons. the only neuron that are lost in these animals after r toxin ablation are the ones that

express the trip v1. sure enough those animals are now not as expected responses to histamine or greatly reduces responses to his mean through there are also lots of responses to other sets of compounds that we know cause itch in animals. so we now know the neurons.

as i told you earlier, the trip v1 neurons are not only just involved in itch. we tested these animals. we know very well that they are also required for some types of temperature response and some types of pain responses. so still, how do we distinguish

between pain increase and itch. we favored a hypotheses that there are in fact select populations of neurons within the trip v1 cells that are i'm coded for resconses. we set that out to try to identify molecular markers especially ones that would allow

us to functional define itch. and to make a very long story short, we identified this molecule, a molecule called ntpb -- identified 30 years ago as a peptide release from the -- extract. it's involving controlling as its name implies.

what we found that expressed in a small subset of neurons -- and importantly in this mouse -- this mouse notably has no itch responses. so this is the clue that we had. this maybe was a molecule that was important. and first of all we wanted to

show it to establish whether or not it was expressed in the trip v1 cells. so this is now i'm showing you a label of hybridization. over here you could see that the cell is pression he ising ntv1 and this is pressioned and this is the image.

you see that all the v cells also express trip v1. not all terrain v1 -- about a fifth of the neuron that express train v1 express mtb. these are a very distinct population. they have a certain size with neurons that express trip v1.

so, this is the question that was asked i don't recall. i show that the -- neurons are suspect of the trip v1 cell. importantly when we now perform experience where we look now for the issues i talked about at the very beginning. this is one receptor that's much

related gcbr class and this is a related issue. importantly these receptors are found and co-expressed with ntpb. so it looks like this with molecular marker defines that the white cells that require to itch and in the same cells as

expressed a functional receptor. so we generated enough and we saw this characterized knockout. the first thing we tested the knockout for all the responses to a whole array of different -- temperature, touch, painful touch and propo receptor sensors.

these are completely normal they have no defect at all. remarkably these animals have attenuated responses almost complete responses to all the compounds we could throw at them. so it looks like -- a is a great functional marker of peer

now these of which -- are a member are known to be molecules that release some cells and call activation of cells outside of the cells. the first neurons -- of a variety of different neuro peptides including this compound tgrp and another compound that

we identified. and these molecules are known to not only to be required but this end of the cell but also are responsible for a variety of different responses are found within the skin. we all know that around the cut there's a red swelling.

this is actually called by relief of cglp -- from the end of the peripheral neuron. this is called -- and the swelling there. the first one we did was inject ntvb into the skin. and unlike responses to these neuro peptides we saw no

responses at all. now when we injected mtvb into the spinal cord we can now enlist the responses just like the histamine injected into the by injecting directly into the spinal cord we could get and then until knock outs we could also elicit scratch

so these knockout animals -- because of the developmental defects or the defects in the h pathway because we can regenerate by injecting it directly into this region where this acts as a transmitter. this shows that ntb is not only required but also sufficient for

a scratch response. so how does this fits in with what we've known already in the field. so a number of years ago -- group had shown another neuro peptide was also required in this pathway where it's shown that -- peptide receptor

knockouts and ablation of the -- release peptide receptor neurons basically a late each response. again, to make this story short, i'll show you the pathway that allows us to bring together what we found to what was already published. so the peer receptors at the

periphery have their theirterminals in the skin -- can activate a neuron in the spinal cord and a receptor for ntbp. this neuron releases a releasing peptide and activates a tertiary -- peptide receptor. this model makes all predictions.

the first is that grp induced does not require npvb. second nppb receptor neurons don't require grp. and the third it requires the grp pathway in cells and finally the grp and the nppb receptor should be in the same cell. we'll go through those

the first is grp induced itch does not require nppb. if we inject into the spinal cord grp, this also causes itch. so does it require -- so we have the knockouts we can inject grp into the spinal cord and we have the knockout. second, the nppb receptor neuron

we can ablate these neurons. this is a panel which shows that we can specifically eliminate the cells that express the receptor. this is untreated animal following ablation. this is a very specific ablation because we don't eliminate the

grp expressing. what's also very interesting is this is a very effective ablation. as we expect we eliminate histamine and nppb because we're basically cooking the pathway 59 this point here. but what really is very

interesting is that when we analyze and we test these animals we find that there's no effect on pain on further sensation or on such. this is a very specific aspect. and as the prediction states the grp induced itch is not affected by ablation of these neurons

because grp is down stream. and thirdly, if we now these are blocks or ablate these neurons we should also block nppb itch. again we can ablate the grp receptor neurons. and as was reported previously this blocks the induce the itch it blocks the grp induced itch

and it also blocks the nppb and them finally the grp and nppb should be co-expressed. first of all, this is now a hybridization in this spinal cord which shows the expression of the receptor and the grp expression pathway. it's very similar indeed when we

performed the ablation with the nppb we get a reduction in the number of neuron that express the grp. and indeed when we do the labeling, we can see co-expression of the receptor for the nppb and the grp. this is the pathway for itch.

and numbers are important pieces of information. first of all, this is a dedicated itch pathway. it doesn't require -- responses that's very useful for treatment because clearly if you wanted to treat itch, you don't want to get rid of responses to pain.

it also tells us another bit of useful information. and that is that the two neuro peptides that are required at the itch pathway it's not only nppb but grp. and finally, our work is showing that in fact at the periphery selective populations of neurons

it's just been in responses to again, a hint to possible ways to treat these conditions. and so this final three slides i wanted to go through possible ways and mechanisms for treatment of chronic itch. and first i want to talk about this group of treatments that go

via the peripheral nerve endings, the peripheral neurons. and there are a number of different treatments that are available and ones which are in the first is of course antihistamine which we all use when we are bitten by a mosquito.

it's fairly effective. you put on antihistamine cream if you get bitten by a mosquito. i get relief but patients are -- completely ineffective. because this is not -- by which those people get that chronic there are other tumor developments so i think the

people that have atopic dermatitis has been found to have interleukin 13-1 and there's a new humanized anti-bodies in clinical trials that's been used to treat those types of patients. there's also some papers out, i read recently an ige monoclone

anti-body that's been used also with people with chronic itch. again these are people with skin conditions and you can actually start the mechanism by which these types of treatments might but clearly, this is a difficult area to work in because first of all you have to identify the

molecules of the periphery. and them find ways to block those. another approach is that of affecting an itch centrally. and we heard from the introduction the clinical side that's inhibiting opioid when you talk about these, they

are found that central neurons which are found in the spinal and in fact, one of the classical symptoms of -- one of the classical symptoms of epidurals is intense itch. opioids can cause really really strong itch responses. and they very often patients

with administered -- because it's so bad. clearly one potential area for development of new anti-itch is antagonists are these two neuro peptides, grp and one that we have ntpb. and clearly in order to be effective, then you really need

to have something that's very specific. for both of these neuropeptides are found in different areas of the body. so that could easily be or might easily be reasons for not using these as targets for developmental itch.

what is really remarkable about this itch pathway is that they both grp and nppb are required. and one thing that may in the future be possible to actually use a mix antagonist of these two compounds at the level of antagonism where they don't have -- because they're both

required an itch pathway. that clearly is something in the future. and the third class of treatments to itch really stems from the correction of how does pain itch. we all know from mosquito bites at least i do if they are

so bad i'll scratch them until it becomes painful. and then the itch goes away. this is well-known if you scratch long enough it becomes painful and you don't have any itch anymore. so how does that happen? so it's believed that there's an

inhibitory pathway that stems from this pain pathway that goes into the spinal cord. and branching off of the pain pathway there's activation of inhibitory interneurons. and these release some factors that inhibits this pathway. and this pathway, there's two

very strong pieces of evidence that this is indeed the pathway. first of all in animals where there's a conditional ablation of the neurotransmitter glutamate, these were two conditional knockouts. those animals exhibit spontaneous unsubstantiated

after about six weeks they have to be euthanized because they scratch themselves where they basically have large lesions all over their body. another piece of evidence in this pathway is basic helix transmitter five, b5. and these animals have lost this

class of inhibitory interneurons. and those animals, just like the conditional knockouts showed spontaneous and substantiated so if you get rid of this inhibition in animals, you not only don't get, the chances of actually having this -- in fact

this pathway actually acts as a inhibitory pathway. it works all the time and prevents itch from happening. this is a type of arrangement that's found in many sensory systems between different sensory modalities. and again in the case of the

patients told us that in fact cold inhibits itch. again there's good actual evidence that this occurs in the spinal cord. in fact, we all know that cold also inhibits pain. and that's why we put a cold compress on something that's

painful because again these pathways have crossed between the different pathway. it's drops another pathway that's cold and another set of inhibitory neurons that would act on this matt way here for main and al -- for pain and also on the itch pathway.

what we still don't know is what is the transmitter here that this acts to inhibit this. and really a clue from that came fairly recently. and this is a different -- this is a kappa opioid receptor. in fact, this compound is now in the clinical trials as the

treatment for a type of itch, the itch that's found in renal failure patients. so this would be my third compound that could potentially be used for treatment of chronic itch that basically the itch pathway trying to prevent it by inhibiting the at some stage

here in the pathway. so now many questions of course that remain and i'll just name some of them here. the first is that a lot of the work that we've been doing and the neuroscience field that's been doing it is basically the acute itch.

because all the experiments we perform is that the cells and molecules are the same -- that might not be a correct assumption. secondly, these systemic conditions where we can't understand what's really happening, is this through

modulation of intermediate parts of the pathway. are these akin to neuropathic is there a similar neuroneuropathic itch that can ache for those types. and another open question, at least for the patients that have skin conditions, what is

happening in the skin that's actually causing the chronic and there's a lot of evidence that in fact the dysfunction of the interaction with the beta cells and this is what drives the current conditions. i would like to say i think

there's definitely hope by identifying the molecules and i think they are now in a unique position that we're actually using -- all the stages of the pathway and hopefully this will lead to new therapies and treatments. finally i would like to thank

people in my group that are involved -- that did a majority of the hard work i talked about with the nppb story. thank you. [applause] >> if you would repeat the question. >> if i could hear it.

>> [indiscernible]. so we would say that this is why -- nppb like most neuropeptides act as a neuro module later. there's very good evidence that these neurons -- is also so we didn't eliminate glutamate.

there is some low level transmission that this requires the full response that you need to have both both together. >> do people who have neurologic inheritable disorders characterize by a failure to appreciate pain. is that itch?

>> so i don't know about that per se but i know there's a group of people that have no sponses to pain. and the knockouts as the same gene that's affected in those individuals has been made recently and i know both animals have no itch response.

it's indirect. it's very rare individuals that have no pain. they actually tend to die early because pain is useful. >> are these neurons present in late fetal life. these neurons, the dorsal, are they present in late fetal life.

the receptors? >> are they present in vitro you're asking. this is the political question. >> i don't know. >> along the same lines there are people who don't itch. are you aware of whether what the expression of an nppb are in

those indiduals. >> ion't know. i didn't realize that there are actually people -- >> i mean, this is anecdotal. i know there are people who normal stimuli that would induce itching, at least it's much more difficult to induce itching in

>> that would be interesting but clearly that's a long pathway -- >> it's interesting that they also have the juice responses to capsaicin as well. probably because nmpb is one of those genes that is needed for more than just itch. it is actually required.

it's produced by the atrium in the heart and disstresses. i think those people would have many other health problems as well. their blood pressure wouldn't be controlled and they would have problems with their heart. there's probably an explanation

but clear threely it's affected in that pathway. >> is there reciprocal truth. let's say treating with histamine reduce pain. >> so histamine is very complicated because if you inject histamine into the dermal layer, it causes itch.

if you actually inject under skin it's painful. there are receptors in the pain pathway as well as itch. so it's not clear cut. histamine is not the clean agent for the itch. so -- again is found in both. this is one thing that 've

started to actually think when carefully because a lot of people think that the receptors are found in the peripheral neurons but in fact there are in the case of insects biting you, if you think about that, the receptor is actually on the -- because when you're first bitten

by mosquitoes as kids we don't have a reaction. after we generate the ige against the antigen and saliva from the mosquito that we actually. so in that case the receptor's really on the immune cells and the immune cells then release

this and activates the neurons. a lot of these big questions are sort of somewhat at least for me open. i got to where the initial receptor is because it could be some other -- that's then -- or in some of these systemic conditions they could also be

compounds that activate. so for instance the treatment for colostasis is -- there are receptors at least at high lels in he peripheral neurons an found in the spinal cord. so you've shown us today that itch is associated with a specific cellular molecular

compartment. and you also just reminded that pain serves as a function. so what is the function of itch? that's a good question. it will be one of the questions i would have had. i can give an opinion, i can give an idea.

the thing is that clearly we don't know what. one explanation i've heard and i think it's reasonable is this is evolutionary. mechanm to move parasites from the skin. so if you get, because this is a reaction just to the very

surface area. so you can't get itch. if you inject compounds deep within the skin, you don't get an itch response -- activity delivered within that very surface area of the skin to get histamine response. and so the argument is that this

is an evolution of mechanism to basically get those parasites out of your skin. quickly. and the argument is that basically you want to scratch until it's painful because then you know you've got -- passes the skin where you've got pain

and you don't want to remove anymore because you'll get infections and have problems. >> what's the role cannabinoids. >> i don't know -- all kinds of places in the influence and the peripheral nervous system. there is some evidence in the pain pathway.

>> maybe -- >> some of these diseases that are really tractable that's crazy. >> this is why for instance patients at least being put forward the reason why patients before they are going to bed seem to feel that itch is worth

it because they have nothing else to think about. they've now gone down -- they haven't got any other sensory input. itching because they actually notice it. and so there's a whole field in the chronic pain arena which is

basically are -- for people with chronic pain without -- basically a distraction. and a distraction is basically sedation. if you can basically sedate the individual, they won't want to scratch or have any pain. it's gone down, and in fact

people with pain and i were given drugs that are -- against, these are drugs that basically would use -- it reduces the target and reduces the level of sensory impulse and processing. >> are you interested in looking at this tgr5? >> i'm not focused to actually

returning -- you go to the dentist because you have a pain -- >> people are itching not to go to the dentist. >> i want to thank you very very much and thank you all. i hope you enjoyed it as much as i did.

>> thank you. >>