Sharktank Blog : Good News for CF Patientshttp://www.sharktank.org/index.htmlCutting edge CF ResearchenMelanie ChildersCopyright 2011 Sharktank2011-07-25T20:36:28+01:00 hourly 1 2000-01-01T12:00+00:00 Mon, 25 Jul 2011 20:38:22 +0100What’s Going On?Melanie ChildersNone2011-07-25T20:36:28+01:00http://www.sharktank.org/melanie_childers/files/whats_going_on_CF_sharktank.php#unique-entry-id-71http://www.sharktank.org/melanie_childers/files/whats_going_on_CF_sharktank.php#unique-entry-id-71
And, yes, of course, I’ve been working at my “regular” business, because that’s how I support myself. But, never, never do I stop working on CF, in my spare time. Sometimes, though, you just have to let things “cook” in your head. All the facts combine together for total confusion at first, and then things start to solidify and you begin to see patterns in the stew. I don’t know why it’s necessary for this to happen, but it’s a period of checking and re-checking facts, and learning nothing new, but instead almost just WAITING for the coalescence of those patterns. You can’t help but think about all of the facts, and try to understand how they go together, and this is what I call “work.” I hate the waiting, I hate not learning new facts, but I know that this is necessary. I just get impatient; I want to DO something, and instead I have to just sit there and think and think and think. When the patterns begin to emerge, though, that’s the payoff. It’s a time when I get excited.

Hopefully, this time, I can finish a paper before I go way beyond the subject of the paper and it becomes outmoded. Sometimes, you get to a point where you actually feel a little bit finished with an area, instead of having to put guesses in the paper. I don’t like guesses, even educated ones. They are unprofessional. And, you can’t base one hypothesis upon another. Not good at all. So far, our new model of the pathology of this disease, is the only new model out there, and so far, it is actually correct. But it definitely had some guesses in it. It just turned out that they were correct. But I don’t flatter myself that all of it will always be shown to be correct. And I like to get more specific, if it’s possible.

Seems that this disease, at least in terms of the lungs, is a disease of the periciliary or sol layer of the lungs. The submucosal serous cells secrete into this layer, and the CFTR is highly expressed in these cells, so when it’s not there, you’ve got a problem with that layer. The CFTR is responsible for that layer, to a large degree. The cilia are spread through this layer, and if it’s thick, and not hydrated enough, they can’t get their beat going. The mucus layer sits on top of the sol and touches the very tops of the cilia.

We’ve got all these diverse theories as to what causes this disease, and actually, they are all right. But where do they converge? In the sol. Make no mistake about it: the sol layer contributes to the thickness and viscosity of the mucus (via HCO3-). And the sol layer in CF is missing all of it’s anions.

I know the fate of most of these anions, and their function. But how, if we are to supplement these anions, do we get them through the mucus layer? You can’t depend the compound coming in from the underside of the epithelial lining fluid, because the cell is missing the protein that normally transports these compounds to the sol. You have to either get another cell, like the blood cell, to deliver them (erythrocytes have functionally redundant proteins in them, but would they deliver the anions to the sol?) Or, you have to get the cells, themselves, to deliver them, (submucosal serous cells express certain forms of multidrug resistance proteins, but they have to be INDUCED, without toxicity) or go through the mucus layer.

Okay, enough. That’s what I’ve been working on. Part of it will be in my next paper, along with some other details that I won’t go into here, or I might be looking at another’s name on my next paper. You know how that goes.

Once I get that paper written and published, I’m going to start on here talking about how to remedy this situation. As much as I would like to have ONE answer, ONE treatment for this disease, I don’t think that that is going to be possible. But I’d much rather have two pills and an inhaled compound that one that is not efficacious at all.

In the meantime, think about this: ALL those anions that are missing in the sol. What are their fates and how would you fix this situation?

]]>Let’s talk about the isothiocyanates.Melanie ChildersNone2011-05-31T07:35:11+01:00http://www.sharktank.org/melanie_childers/files/lets_tak_about_isothicoyanates.php#unique-entry-id-70http://www.sharktank.org/melanie_childers/files/lets_tak_about_isothicoyanates.php#unique-entry-id-70Do they work? Can they help?

The honest answer is that we don’t know. Given the current information on all of the factors involved, they SHOULD work. BUT, we have a problem, and it’s a big one—

The isothiocyanates are like azithromycin. Anyone who has CF and who has used AZM to lower inflammation knows that it only works for so long. There’s a reason for that. AZM induces a protein that is redundant to the CFTR protein, and that functions much like the CFTR protein, but the major problem with it’s use is that it also induces this type of protein in the liver. And, once induced in the liver, this protein filters out the AZM before it can get to the lungs and do it’s job of inducing a CFTR-like protein there.

Same with isothiocyanates. So, what to do? Where do we go from here? Why not just use AZM, and not worry about developing any of the isothiocyanates for treatment for CF?

We don’t do that because there is one major difference between AZM and any isothiocyanates: SIZE.

AZM is 749 in molecular weight and the isothiocyanates vary, but they are all under 200. Anything under 500 is considered a small molecule and thus able to be delivered throughm the cellular membrane. This is good.

The problem is that I can’t figure out how to get past that damn cMOAT (also calledm MRP2) in the liver. Can’t get enough through the dermal layer (which is always difficult, at best, anyway) because most of these isothiocyanates are caustic and that makes it uncomfortable to go that route. Oral is out because of the cMOAT. I wouldn’t even dream of attempting mucosal (via rectum) delivery for the same reason. That leaves inhaled and intravenous.

You’ve got the same problem with the former that you would have with dermal and mucosal, except that you could probably dilute the crap out of it, because you wouldn’t need as much if it’s delivered directly to the airways. It’s feasible, but it’s too dangerous for me to ever even think of. I simply don’t have the expertise in that field. And, intravenous wouldn’t have the same problems, but how long of an effect would it have? Years, days, months? No one knows.

The only other choice is to get a new compound. When I look at those kids that took chemotherapy, and it caused them to induce the expression of redundant proteins, the one thing that rings out, that they had in common, was anthracycline, or one of it’s analogs (ie., epirubicin, doxorubicin, etc.). And anthracyclines are cardiotoxic. Cumulatively cardiotoxic. No one wants healthier lungs at the price of destroying it’s partner, the heart.
]]>Banfi's Latest work on SCN- Transport -- How Does it Further Our Understanding of CF?Melanie ChildersNone2011-05-08T19:10:41+01:00http://www.sharktank.org/melanie_childers/files/Banfis_latest_work_on_SCN-Transport.php#unique-entry-id-69http://www.sharktank.org/melanie_childers/files/Banfis_latest_work_on_SCN-Transport.php#unique-entry-id-69Please review Banfi's latest work on SCN- transport. In that article, Banfi said that the CFTR transported SCN-, but he also said that that channel was not the only pathway for SCN- transport to the airway lining fluid--that Pendrin and the calcium activated chloride channels also transported SCN- to the airway lining. That idea bothered me for several reasons.

First of all, I could not understand why, if Pendrin and Ca2+ activated chloride channels also transported SCN-, there would be pathology associated with an absent CFTR protein in the membrane. And, especially, if the lack of antimicrobial function in the airways in CF was caused by a lack of hypothiocyanite. (Although, it is also caused by the lack of another compound used in the lactoperoxidase system - H2O2 - but Conner thinks that SCN-, itself, should do some good, and I have to give that idea some credence.)

Another thing that bothered me is Denufosol. If chloride channels also transport SCN-, then activation of the calcium activated chloride channels by a UTP-like compound should also cause the movement of SCN- to the airway lining fluid to be used in the lactoperoxidase system, to generate at least some hypothiocyanite. But, since then, I've looked over Banfi and Galietta's work on this topic again and I think I understand better what is going on here (Galietta's work is also attached).

There are three routes for SCN-. It is transported by Pendrin, by calcium-activated chloride channels, and by the CFTR. The first two routes, though, require IL-4. And, if you will look at the work on IL-4 and CF, you can see that there is something different, in CF airways, when it comes to this cytokine.

First of all, IL-4 is responsible for the "switch" to a Th2 type immune response, which is what you see in CF, in response to Pseudomonas A infection (abstract attached). This is NOT good. It sets into motion all kinds of pro-inflammatory responses. (Pendrin, itself, is associated with increased airway reactivity and inflammation (attached).

What if Pendrin and IL-4 are compensatory mechanisms for the ion transport that is lacking because there is no CFTR in the membrane of the submucosal serous cells? I'm attaching an article that addresses ion transport mechanisms affected by IL-4. Not only does it increase Ca2+ regulated ion transport, but in normal cells, it increases cAMP dependent ion transport. You can imagine what this means for CF cells -- you've got all the negatives associated with Th2 immune response (ie., inflammation, airway reactivity), but none of the cAMP dependent ion transport usually associated with an upregulation of IL-4 (or an upregulation of sensitivity to IL-4).

All of this is speculation, of course. But I have been looking for that "switch" for years now. Why is the immune response in these kids, to PA, predominantly of the humoral type? I could understand the lack of antimicrobial function, because of the virtual lack of a lactoperoxidase system, but why the inflammation that never goes away, even after antibiotic therapy?
]]>CFTR: Pump or Channel (or Why didn’t Denufosol Work?) IIMelanie ChildersNone2011-03-19T08:47:13+00:00http://www.sharktank.org/melanie_childers/files/why_denufosol_didnt_work_2.php#unique-entry-id-68http://www.sharktank.org/melanie_childers/files/why_denufosol_didnt_work_2.php#unique-entry-id-68
Denufosol is a compound that is supposed to increase intracellular calcium. This increase in intracellular calcium is supposed to affect the calcium regulated chloride channels. This effect on these channels is supposed to increase the movement of chloride out of the cell. So, if the CFTR is a chloride channel, then replacing that channel with the increased transport of another chloride channel should solve the problem, right?

Absolutely. The problem, though, is the assumption that the CFTR is ONLY a chloride channel and that the lack of transport of chloride from the intracellular compartment to the extracellular compartment is what causes the pathology in the disease. That assumption is wrong.

Let me tell you a little more about the CFTR that you probably already know, but has to be covered for those of you who don’t know:

The CFTR has an R domain. The “R” stands for regulatory and this domain has a regulatory function. What first happens is that cyclic adenosine monophosphate (cAMP) is increased (as a function of inflammation, infection, etc.). Next, protein kinase A (it is cAMP dependent) phosphorylates the R domain. Once that happens, the nucleotide binding domains bind to ATP (adenosine triphosphate). And the channel opens.

This is where I have a problem: ATP is associated with pumps. It is energy; power. Power to drive the pump. In fact, Hanrahan and Linsdell did some experiments on this [See Linsdell, P. and Hanrahan J (1998) J. Gen. Physiol. 111, 601-614, and Am J. Physiol. 275, C323-326, linked here]. What they showed was that the CFTR controlled energy-dependent movement of other ions, such as glutathione.

Now, don’t get me wrong: I am NOT disputing that the CFTR acts as a chloride channel. But chloride channels have distinctive properties. And they transport CHLORIDE; they transport it at a certain rate, that can be measured in the lab. How much and at what rate that chloride is transported has been pretty much nailed down as gospel by scientists who study the CFTR.

But if the assumption that the CFTR is ONLY a chloride channel is wrong, and I think it is, or Denufosol would have worked, at least to a degree, then don’t you think it’s pretty important to figure out what that pump is pumping? And, if the measurement of the success of VX-809 is that it gets the variant F-508 to the membrane of the cell, wouldn’t you say it was important to measure the movement of something OTHER than chloride, as proof of this trafficking to the cell surface? After all, although the mutation associated with F-508 is in the first nucleotide binding domain and it is NOT the same mutation as the G551D, which affects nucleotide binding to ATP.

]]>BellMelanie ChildersNone2011-03-11T12:06:00+00:00http://www.sharktank.org/melanie_childers/files/bell.php#unique-entry-id-67http://www.sharktank.org/melanie_childers/files/bell.php#unique-entry-id-67
She’s got hair the color of a new penny and that distinctive baby curve to her head. You know the curve I’m taking about—the one that cries out for your cupped hand to stroke it. Of course, you don’t get to do this much except when she’s asleep because when she’s awake she’s tearing across the room on her low mileage feet, pushing her baby shopping cart into immoveable objects, baptizing her stuffed animals in the toilet bowl, or bouncing in her bed, her smile so wide and genuine, she looks almost Oriental. Bells a charmer, alright.

She makes me feel like a celebrity. That hard logical place in my brain searches for solid, tangible explanations for my popularity with Bell. I was there when she came into the world and I like to think that maybe she remembers me from the very first moment she breathed air into her lungs. But her parents were both there, too, and they don’t seem to hold the same fascination for Bell that I rate. There’s no doubt about it, I’m the star of Bell’s show and she lets me know it.

Bell is a gift from the universe. She pushes my reset button, making me forget all that I have lost, even if just for a few moments. She reminds me that life does not only consist of the darkness of my grief and anger, or of the labor borne of my struggle to escape that abyss. The light in Bell’s eyes promises that, although there will always be the shadow of loss, it doesn’t always have to be dark. I can see the light once more.

I found it, again, in Bell’s eyes.
]]>Channel or Pump? Or why did Denufosol fail?Melanie ChildersNone2011-03-11T11:19:13+00:00http://www.sharktank.org/melanie_childers/files/denufosol_fail.php#unique-entry-id-66http://www.sharktank.org/melanie_childers/files/denufosol_fail.php#unique-entry-id-66
Okay, okay, boys, it’s a chloride channel! It can transport chloride. So can a lot of other channels, that are not even chloride channels. Cation channels—channels that normally transport positively charged ions (chloride is a negatively charged ion) transport chloride, if one is nearby the channel. Chloride transport is going to get done, one way or another, because we are creatures of the sea—we’ve got saline all through our cells. The cell is not going to leave chloride transport to just one protein.

The point is, there is NO pathology (or almost none—the little they’ve found is not enough to make the differences that we see in CF inflammatory or antibacterial processes, that are the central pathological “tip(s) of the iceberg” in this disease—when it comes to chloride transport, or the lack of it, as is the case with CF. Not enough to bother with this substrate.

As Catherine Fuller and Dale Benos state in their review “CFTR!” [Am J Physiol 1992 Aug; 263 (2 Pt 1): C267-86] “Whether to classify the cystic fibrosis transmembrane conductance regulator (CFTR) protein as a pump or a channel is one of the most perplexing and urgent questions facing cell physiologists working in the field of cystic fibrosis CF.”

WHY? Why is this an urgent question? Fuller answers the “why” question near the end of her review when she talks about substrates. SUBSTRATES.

Can you tell I’m angry? I’m always angry when it comes to this subject. It’s like yelling at a brick wall. HEY! HEY! These kids have a problem with a TRANSPORT protein, that affects the makeup of the fluid that lines the lungs! That fluid is something that meets the world! We can FIX it! It’s so easy to fix something that is OUTSIDE, that we can GET to. Don’t you think it’s a little important to look at the substrates of the PUMP?

What if it’s only a channel? Well, it sure isn’t acting like JUST a channel. And it’s affecting, very apparently, things that have nothing to do with chloride. And those things, those SUBSTRATES, are usually moved by pumps. Pumps that are, by the way, 30 and 50% homologous to the CFTR protein. It’s built like ‘em. Looks like ‘em. Acts on the same things these pumps normally act on. Gosh, could that duck be a duck?

I’m sorry, guys. You don’t know where all of this anger is coming from. It’s frustration, actually. At the slow pace of science. At the closed minds. The plodding ponderous inch by inch progress. How many is it going to be too late for, simply because people can’t make a small leap only in terms of INQUIRY and at least ASK the questions that should be asked? There is more and more evidence piling up that this protein is a PUMP, and yet so few are looking at the any substrate other than chloride. Unbelieveable.

Can’t they just say, okay, IF it is a pump AND a channel, what would it’s pump substrates look like, and moreover, what pathology would stem from the lack of their transport? And if the outside (the airway lining fluid) is behind OUR enemy lines and accessible to us, through aerosolization, then don’t ya think this might be a good area to pursue in terms of therapies?

Download the Papers referenced in this post here

]]>The WaltzMelanie ChildersNone2011-02-15T08:37:57+00:00http://www.sharktank.org/melanie_childers/files/The_Waltz_CF.php#unique-entry-id-65http://www.sharktank.org/melanie_childers/files/The_Waltz_CF.php#unique-entry-id-65
I’m on vacation right now. It’s the first vacation I’ve taken in years. Of course, when you are self-employed, you don’t take many vacations. And this one is not really a vacation in the normal sense of the word, because I’m still here at the beck and call of everyone who works for me. Still here, to do invoices, deposits, payroll, contracts, and everything else that I normally do. The only difference is that I am home for ten days. I don’t have an office to go to here. I simply stay in my little RV on my own little plot of land, down near the Gulf of Mexico.

And, of course, I’m sick. Caught something on the plane coming back from Denver, I’m sure. Isn’t that the way it always goes? Go on vacation, get sick immediately, and return to wellness right when it’s time to return to work?

This dream, though—it was about the peroxidases, and their substrates. Again. The waltz, I call it. The waltz of the peroxidases. I’m dreaming about them almost every night now. I think it’s because I set out to finish my paper during this “vacation.” And the paper, of course, is about the peroxidases.

There was something in this dream; I can’t recall it now, but it was of the utmost importance when I was trying to wake myself up so that I could remember it. I thought for sure that I would remember it in the morning when I woke up, but it’s gone now.

I’m sure that studying will bring it back to me. It’s been bothering me for a while now. (I can’t let go of things that bother me, in case you haven’t noticed.) And that’s why I dream of it, over and over again.

It’s something to do with the neutrophils. Chemotaxis. The process by which neutrophils are drawn to the site of inflammation. IL-8 is implicated in this process in CF.

In CF, there are a LOT of neutrophils in the lungs. It’s called airway neutrophilia and it contributes in a major way to the inflammation seen in this disease. I think that the jury is already in, in terms of what causes this increase in neutrophils in the airways of these patients—IL-8. But what causes the increase in IL-8? You don’t just go “Pow!” and the IL-8 is there. There’s got to be something that causes an increase in the IL-8 (and maybe something that causes a decrease in IL-10, as well).

I think it’s that dance. The waltz. The levels of the substrates of the peroxidases.
]]>ChristmasMelanie ChildersNone2010-12-19T10:56:04+00:00http://www.sharktank.org/melanie_childers/files/Christmas.php#unique-entry-id-64http://www.sharktank.org/melanie_childers/files/Christmas.php#unique-entry-id-64
Don’t get me wrong—I love my family. But I don’t like the end of the year, and I especially don’t like Christmas.

I think that time spent feeling sorry for oneself is nothing but wasted time. I think that we are each here for a purpose, and although we might not know our purpose, we can get a glimmer of it in the events that have happened in our lives, and in the gifts that we have been bestowed upon our birth. So, I take those two things and I use them for what I hope is the best work that I can do. This is especially true at Christmas.

I started last night, on my newest paper. I didn’t get a lot done on it, but I made a start. I want to finish it within the next couple of weeks; although I don’t know if that will happen or not. Once I get far enough into it, I CAN’T stop, and it drives me, itself, to completion. But, to get far enough into it to have that happen…..well, I’m not there yet.

I’m going to talk about a system, of course. The system that the compounds that are transported by the CFTR encompass. They work together, these compounds. To a degree. They involve two peroxidase systems that work together, actually. The systems balance each other out.

I used to think that, in CF, you had an increased, overblown, humoral immunity. But, that is not correct at all. In fact, you have an increased, overblown cell-mediated type of immunity in this disease. I bet you didn’t know that, did you?

There has been a paradigm jump in my knowledge of CF recently. As a non-scientist, I studied this disease kind of from the top down, or rather, from the pathology down. Now, I am studying it from the cells, or tissues, up to the pathology. It is a different view, but as with all added views, it adds to the bank. It feels almost exponential, in terms of completing the picture of this disease. It is an exciting time for me, and I feel a little less stupid than I used to feel. Not much, but a little.

So, that’s what I’ll be doing for Christmas this year. And I’m happy about it, so don’t feel sorry for me. I think that you make a choice in life, for every moment that you are here—to serve a purpose, or not serve it. I choose to serve.

For those of you who still have intact families, I wish you a Merry Christmas and a Happy New Year. Cherish the moments. Store them up in your memories.

And say a prayer for those of us who can’t do that anymore.
]]>My MotherMelanie ChildersNone2010-12-11T07:26:17+00:00http://www.sharktank.org/melanie_childers/files/my_mother.php#unique-entry-id-63http://www.sharktank.org/melanie_childers/files/my_mother.php#unique-entry-id-63
She was the epitome of the Gulf Coast Texan. Independent, strong, extremely intelligent, and a drunk. Of course, I’m not saying here that all Gulf Coast Texas women are drunks. Lots of them are, I’m sure, but not all of them. But, my mother was a drunk. It kind of defined her life, unfortunately, or at least, all of the troubles that she had in her life.

My mom was born into a culture where women were not considered real persons without a man beside them. I guess that’s why she married seven times. She was always looking for love, my poor mom. The problem was that she was not a damsel. She was stronger than every one of the men that she married, and much more intelligent than most people around her. And extremely outspoken. That, combined with her ability to binge drink, made her very bad at marriages.

Her intelligence was phenomenal, though. That intelligence was both a boon and a bust for her, in the culture she grew up in. Women were not supposed to be that smart, and it certainly was not good for a woman to be smarter than her man. And, of course, my mother was held back by this culture, professionally, as well.

When times began to change for women, my mother went to college. She was forty years old when she went back to school, and she majored in math. She loved chemistry and math, but she loved math, most especially. I remember that, unlike most moms, who went to bed reading a book or a magazine, my mother used to prop herself on the pillows of her bed, at night, and work trigonometry problems, to relax. She did this even after she had left school, and was already working for NASA, as a mathematician.

My mother was a very unhappy woman. Her drinking drove her children out of her life as soon as they were able to leave. She defined herself as an alcoholic, but was never able to conquer the hold that alcohol had in her life. She either did not realize how mean and abusive she got when she was drunk, or she could not admit it because of the shame it brought to her, even if she did realize it. She could not say that she was sorry because she could not admit, especially to herself, the hurt that she had caused her children. She could not forgive herself. There were some of us (seven children) who forgave her anyway, and some of us who still have not forgiven her to this day.

I am one of the former. And, fortunately, I forgave her when she was still alive. I formed a relationship with my mother by setting out my limits, after I was an adult, on my contact with her when she was drinking. When she got a little too much in her, the cruelty would come seeping out of her, and I would tell her that she was getting drunk and abusive and I would leave. After a while, she started pacing her drinking when she was around me, and after that, she stopped drinking around me at all. I wish that more of my brothers and sisters had done this with her, because they lost a lot of our mother, by just staying away. I am the better for my relationship with my mother.

Like many alcoholics, my mother had an incomparable ability to empathize with others. Part of the reason for her drinking was her inability to handle the input that was coming in from the world around her; it was a way to anesthetize herself to it. Instead of being able to translate it into drive and work, and frustrated by a culture that did not value her strength or her intelligence, she learned early on that the way to live with herself was to drink herself into oblivion. Still, she was so gifted that I know of no one else I respect more than my mother.

She could (and did) build a house with her bare hands. Her voice at town meetings was one of reason and courage. She could (and did) steer a 42 foot cabin cruiser out into the Gulf of Mexico on a regular basis, and never get caught on a reef. She was neither a round, nor a square peg, but many-sided. And she had her problems, but she was independent, strong, intelligent, and most of all, a survivor.

She was my mother, and I hope that I am just a little bit like her. (Fortunately, I left the whiskey behind me after a wild swing with it, years ago.) She was wonderful. And drunk or not, she made the world a better place for having been here.

]]>On the Road AgainMelanie ChildersNone2010-12-01T22:51:45+00:00http://www.sharktank.org/melanie_childers/files/on_the_road_again.php#unique-entry-id-62http://www.sharktank.org/melanie_childers/files/on_the_road_again.php#unique-entry-id-62
During this trip, which will last three days, I will be staying in roadside rest areas at night, from about 4pm until daylight, and I will have nothing to do but read and think. I read at night, think on the road. These trips are usually extremely productive because I am away from the day to day work in my business, and all I have to do, during my driving time, is think.

I have a ream of articles printed out. Hopefully, they will last me the entire trip, but I doubt it. I’ll probably have to charge the battery of my laptop while I’m driving, and pick up some extra articles along the way. That’s okay, because I’ve got the printer sitting in the front seat of my truck, plugged up to a thingamajig that goes into the cigarette lighter doomahickey, to give it power. (As you can tell, I’m not real mechanically inclined.)

This is my question:

You remember the phosphodiesterase inhibitors? Let me refresh your memory: these compounds are so-called “correctors” of the CFTR. I don’t think they’re correctors at all, of course. In fact, I know that they aren’t correctors. What they do is inhibit the enzymes that normally degrade cyclic adenosine monophosphate (cAMP) and thereby increase cAMP. When you increase cAMP, you are going to increase the translation rate of mRNA CFTR into the protein, even if it IS the variant F-508. When you increase translation of that variant, you are initially going to increase it’s escape from the endoplasmic reticulum and it’s subsequent trafficking to the membrane, and of course, it’s got SOME function once it gets there, so you will increase chloride transport for a bit, as well. The problem, however, is that the molecular chaperones, HSP-70 and calnexin, which hold onto the variant in the ER and won’t let it traffic to the membrane because it’s not folded correctly, eventually “catch up” in terms of THEIR translation into proteins, and you’re left with a mess. You’re worse off than if you’d never increased the amount of protein in the first place.

Why did I suspect this was happening? Because, when you look at those phosphodiesterase inhibitors, you see that they are all across classes. For instance, one compound is known to inhibit a type IV phosphodiesterase, another is known to inhibit a type II phosphodiesterase, another type III, etc. What are the chances all of these compounds, across the classes, are going to have the same chemical structure and “attach” to the variant F-508 CFTR? Not much.

Why does this interest me? Because there is a list of correctors, of different classes of compounds, that supposedly “attach” to the variant F-508: aminoarylthiazoles, aminobenzothiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithazoles. And, I know that they are not increasing translation of the protein. WHAT are they doing to it, then? Are they interfering with the molecular chaperones, or the processes, in the ER that normally “mark” it for degradation? Are they binding to it in a way that increases it’s thermodynamic stability? I don’t know.

But I want to know. And, there’s a compilation of other work, on the nucleotide binding domains, and particularly the first NBD, that is floating around in my head. These two things belong together, somehow. Correctors, in my mind, should also be potentiators. Correct the first nucleotide binding domain, and you should correct the protein’s ability to be activated, as well.

And, no, I have not given up on inducing the functionally redundant multi-drug resistant proteins. I am just waiting on the purchase of the compound necessary to do that. I am the soldier who, having fought one battle, cannot rest until the next presents itself.

I have a toxic blend of curiosity and ignorance, you see. And, I will beat on the door of the cave until the monster comes out.

]]>Forced Marches and other Wastes of TimeMelanie ChildersNone2010-11-20T19:08:52+00:00http://www.sharktank.org/melanie_childers/files/forced_marches-other-wastes-of-time.php#unique-entry-id-61http://www.sharktank.org/melanie_childers/files/forced_marches-other-wastes-of-time.php#unique-entry-id-61
If I had believed, fourteen years ago, that the difficulty of a topic should stop me from learning about this disease, I wouldn’t be where I am, now, in my understanding of the disease. So, I am here, today, to explain to you a little more about the phosphodiesterase inhibitors’ effects on CFTR processing, and why this is not a good avenue to take. Mainly, I think that if you come out with an understanding of what I mean by “forced march,” you will understand this topic. So let’s start a little bit prior to that….

There are certain things that cause a protein to be activated. The CFTR protein is regulated by a second messenger compound called cyclic adenosine monophosphate (cAMP). In short, this compound causes the protein to become activated, so the more cAMP, the more that protein’s pore is open. EXCEPT in the case of cystic fibrosis, because you don’t have the CFTR in the membrane to respond to cAMP, in most cases, and in other cases (depending on how the mutation of the protein affects the protein’s function, obviously), you can’t get the protein to work because it doesn’t respond to cAMP or ATP (G551D).

Now, the F-508 variant of the CFTR protein cannot make it to the membrane of the cell, to do it’s work. BUT THAT DOESN’T MEAN THAT IT’S NOT SYNTHESIZED IN THE ENDOPLASMIC RETICULUM. This variant doesn’t make it to the membrane because it’s folded incorrectly, but plenty of it is still made by the cell. If you do nothing to the cell at all, you’ve still got CFTR being made, but it is folded wrong and can’t get to where it is supposed to be. If you do nothing to the cell, you’ve got enough quality control mechanisms in the endoplasmic reticulum, to hold on to that mutated protein, and keep it from trafficking to the membrane, where it’s supposed to be, to do it’s job.

Now, something you don’t know: there is a lot of CFTR messenger RNA hanging around in that cell. (This is true, in terms of messenger RNA for all kinds of proteins, not just the CFTR) If you don’t do anything, that messenger RNA (mRNA) is going to be there, waiting to be translated into actual protein. Whether it gets translated or not depends on other factors. What happens if you do something to make more of that messenger RNA (mRNA) translate into protein? What are some of the things that you can do, first; and second, what effect does this have on the quality control mechanisms in the endoplasmic reticulum?

Now, you and I wouldn’t respond to someone over in China being caught in a landslide from a Chinese mountain, would we? Of course not. Chinese mountains are not in our vicinity. They have no effect on our lives at all, those Chinese mountains. But if all the grocery stores closed down within a hundred mile radius of you, because of a landslide from a mountain you live next to, you’d either starve or buy more fuel to get to the stores outside of that radius, or grow your own food and starve while you wait for it to be ready for consumption. That would affect you directly. Conversely, if all the stores, for one reason or another, within a 100 mile radius of you started selling all of their food really cheaply, you’d probably have a little boom in the population of your town, right? More food, more people.

When you increase cAMP, normally, you have activation of that protein. And, it is likely that TRANSLATION of that protein, from the mRNA floating around, is also influenced by the amount of cAMP floating around. The more cAMP, the more protein translated, the more F-508 CFTR. And, the more F-508 CFTR expressed, the less likely it is that the quality control mechanisms in the endoplasmic reticulum can keep up. At least initially, anyway.

So, what you are doing, by using a phosphodiesterase inhibitor (which causes more cAMP to be floating around) is increasing the translation of the mRNA for the protein, into protein, itself. If it is the F-508 variant, more of it is going to escape the quality control mechanisms in the ER and make it to the membrane of the cell. Is this a good thing or a bad thing? After all, we’ve been told for decades now that if only we could get enough of the F-508 to the membrane, we are likely to be able to have a fix for CF, so it must be a good thing, right?

Wrong. First of all, when you increase something like cAMP, it’s going to act on a LOT of other proteins, and other cellular processes that you probably don’t want it to act on. Second, those quality control mechanisms in the ER are going to “catch up” to translation levels. And then you are right back to where you were to begin with, and if you stop the drug, you are in even worse shape, because now you’ve got a load of quality control factors, and less of the protein translated, so you’re getting LESS of the protein to the membrane than you started out with. Third, that variant, the F-508, does NOT work properly, even when a load of it gets to the membrane.

Now, there’s some people who might want you to think that these phosphodiesterase inhibitors are actually attaching to the protein and making it somehow fold better, and THAT’S why more of it gets to the membrane. But let’s think about that:

There’s a bunch of different kinds of phosphodiesterase inhibitors. That’s because there’s a bunch of different kinds of phosphodiesterases. But, in the studies on the effects of at least THREE different kinds of phosphodiesterase inhibitors (for example, one tends to inhibit Phosphodiesterase I, and another will inhibit Phosphodiesterase V, etc.), they ALL caused an increase of the amount of F-508 protein in the membrane of that cell. Do you REALLY think that this effect was caused by every single one of those DIFFERENT compounds attaching to the mutated protein and “correcting” it enough so that it trafficked to the membrane?

Fat chance. One thing that all of these compounds have in common is that they all increase cAMP (or cGMP). Besides the effect that these second messengers have on other things in the cell (not good), you’ve got all of the concomitant problems associated with increased translation of a mutated protein. And, none of the benefits of a protein that actually does the job, because the F-508 cannot be activated enough to perform it’s duties properly.

]]>Phosphodiesterase Inhibitors as Correctors and/or Potentiators of mutant CFTR proteinMelanie ChildersNone2010-11-17T08:02:21+00:00http://www.sharktank.org/melanie_childers/files/Phosphodiesterase_Inhibitors_as_Correctors.php#unique-entry-id-60http://www.sharktank.org/melanie_childers/files/Phosphodiesterase_Inhibitors_as_Correctors.php#unique-entry-id-60
Lately, I’ve been doing more research on potentiators and correctors. Just reading around, here and there. And, finally, something dawned on me—CPX, milrinone, pentoxifylline and sildenafil (Viagra)—they are all phosphodiesterase inhibitors.

What is a phosphodiesterase inhibitor? Well, first, we have to define “phosphodiesterase.” Phosphodiesterase is an enzyme (see the “ase” on the end?) that inactivates INTRACELLULAR “second messengers” cycle adenosine monophosphate (cAMP) and cyclic guanosine monosphophate (cGMP). It does this by degrading these compounds. Now, let’s talk about what “second messengers” are. Why are they called second messengers?

Well, first, “second” messenger is meant to differentiate these compounds from “first” messengers, the hormones, which act EXTRACELLULARLY. (The first messengers, the hormones’, signals are “caught up” by these second messengers INSIDE of the cell.) So, these compounds, cAMP and cGMP act inside of the cell; in the cytoplasm, in fact. They are small molecules.

Thus, when you inhibit a phosphodiesterase, you increase the amount of active second messengers, because a phosphodiesterase degrades cAMP into AMP (adenosine monophosphate). So, let’s look at some of the possible outcomes of using a phosphodiesterase inhibitor:

1. An increase in the amount of cAMP (and cGMP)
2. An increase in the ability to transmit first messengers’ signals (hormones)
3. A decrease in the amount of adenosine monophosphate
4. An increase in the activity of cAMP’s “buddy” enzyme, protein kinase A (PKA)

The latter enzyme is where the fun starts (those of you who’ve studied CFTR activation, I realize, are probably bored to tears, but bear with us). Because guess what cAMP does to PKA? It activates it. And when PKA is activated, it catalyzes the transfer of ATP to certain proteins, which control ION CHANNELS. Are any bells ringing out there now? (By the way, the phosphodiesterase inhibitor, sildenafil (Viagra) inhibits the phosphodiesterase that normally degrades cGMP.)

For those of you who’s bells are NOT ringing, let me spell it out for you a little bit: The CFTR transports chloride ions after cAMP activates protein kinase A, which phosphorylates the protein. Got it?

Okay, now, let’s think about that. Xanthines, quinazolines, they are phosphodiesterase inhibitors; they increase cAMP. And they lead to more of the mature F508 in the membrane. Why? Why does an increase in cAMP lead to an increase in mature protein? Why does more of the protein escape the endoplasmic reticulum and get glycosylated and traffic to the membrane when you have an increase in cAMP? Can you really assume that these compounds are somehow attaching to the protein and helping it to not only escape the ER, but become “mature” and traffic to the membrane? I don’t think so.

I think that there is something else at work here. Something along the lines of butyrate, which causes an increase in the expression of the protein so much so that it overwhelms the “quality control” molecular chaperones (HSP-70 and calnexin) that would normally hold on to the CFTR in the ER. We’re talking about a forced march of that protein, when it comes to the phosphodiesterase inhibitors, and that’s never a good thing.

Now, that is not to say that there are not good potentiators and correctors out there, or that they can’t be found. But not the phosphodiesterase inhibitors.

]]>I can’t tell you what I’m doing right now...Melanie ChildersNone2010-11-15T07:28:47+00:00http://www.sharktank.org/melanie_childers/files/I_cant_tell_you_what_im_doing.php#unique-entry-id-59http://www.sharktank.org/melanie_childers/files/I_cant_tell_you_what_im_doing.php#unique-entry-id-59
Why does it seem like anyone who does stuff like I do—go out on their own and study some disease—is a personality that goes along the lines of some made-for-TV movie? I mean, there’s no doubt about it: people that do strange things are just generally strange, period, aren’t they? But, I am just now seeing that, compared to most folks, I’m pretty weird.

For instance, it’s now 3:30am, and I am wide awake. And it’s not that I’m an insomniac. I have no trouble getting to sleep or staying asleep. It’s just that I wake up after just a few hours, sometimes, and I’m completely rested and ready for the day. So, why not begin it?

And the art thing—I have this compulsion to make art. And my paintings are HORRIBLE. I mean, they are a joke, really. But I can’t stop doing them. Some of them are so bad I just throw them in the trash, because I’m embarrassed for anyone to see them. There is NOTHING redeeming about those paintings. Why do I have such a compulsion to create them? I don’t know.

And furnishing houses. Don’t put me in an empty room. The next thing you know, you’ll start getting boxes and crates filled with furniture, rugs, paintings, bed coverings, lamps and plants. I try to control myself, but I can’t. I’ll furnish your whole house if you make the mistake of letting me see it when it’s empty. (In fact, today, before I leave Ohio, I’ve got some shelving I picked out for this one wall in the living room of a friend’s house. And I’m going to get it; nothing can stop me.)

I’ve got a compulsion to fix things. If I meet a person who needs something, and I can see that what they need will change their life for the better, it’s pretty much a done deal. Beds were a particular theme last month. Four mattresses in one month. This month, it’s already looking like clothes are going to be the theme. Someone, please stop me before I get to cars.

Well, that’s it for this blog post. Thought you might want to know what a nut you have, working for you. As soon as I can, I’ll let you in on my little secrets. In the meantime, I’m off to study nucleotide binding domains.
]]>WHERE HAVE I BEEN?Melanie ChildersNone2010-11-04T15:31:56+00:00http://www.sharktank.org/melanie_childers/files/where_did_i_go.php#unique-entry-id-58http://www.sharktank.org/melanie_childers/files/where_did_i_go.php#unique-entry-id-58
Yes, part of that work has been for my business. But my joy comes from working on this disease, and that’s a large part of the work that I’ve been doing. I’m scheduled, soon, to take a three day road trip, during which I will haul my RV with my truck, from Ohio back to Texas. Those are always good times for the mind to rove, and my mind roves to CF during these trips. I often get some very good information from my ruminations on the road. I am preparing for that, of course; just filling my brain up with facts from the science, and especially facts that don’t “fit.” After the road trip, though, some of it will have “clicked” into place, hopefully.

When people who know nothing about CF ask me what I study, I tell them that I study structure-function relationships of audio binding cassette proteins. Believe me, they don’t ask anything else after that. But, that is, to me, the most succinct way of putting it. Of course, it is not the only thing about this disease that I study. I also study systems, such as the myeloperoxidase system, and the lactoperoxidase and glutathione systems.

But it always comes back to those substrates. And that involves protein structure and function relationships. Therein lies the puzzle. The gold.

Because we are lucky, with CF, you see. I know, I know, anyone that has CF or has a child with CF doesn’t count themselves as lucky, but believe me, in a very big way, you are lucky. Why?

Because the lungs are open to the world. Because the CFTR is a membrane transport protein. Because it is responsible for putting it’s substrates on the surface of the airways. And, by inhaling those compounds into the airways, we can replicate the function of the CFTR protein, at least in the lungs.

But there are people out there, very powerful people, who can help us to find just the right compound to induce the CFTR in the cells that are part of the submucosal glands, who think that the path that remedies the pathology in the lungs only is “incremental.” They are not satisfied to remedy the pathology that actually, most often, kills these kids.. They want that “magic bullet” that will make their child as if they don’t have CF at all. Or as close as possible to that. So, in order to get what I want, I will give them what they want.

To do that, I must go back to protein structure and function and back to those chemotoxic substrates. I hope that you will go there with me. Because I need something from you—

Let me tell you what I need: There are some case studies that I found, one time, when I was about to go meet with some people who might be interested in the isothiocyanates as a way to induce the redundant multidrug resistance proteins in CF patients. They were case studies on these kids who had both CF and cancer. I have looked until I’m blue in the face for them again. I printed them out for my meeting, but didn’t save a copy of them to my hard drive. I need them back. I need to study them, and I need to contact the scientists who wrote them, to ask them some questions.

Can you get them for me? They are by Lellamand (I might have spelled his name wrong here; I don’t know), Lenoir, and a woman named Sermet-Gaudelet. You are likely, at first, to find one case study, and you will jump on that sucker like a prize fighter and send it to me.

I already have it. I’m looking for some others.

So, let’s play a game, okay? Everything gets done by making small goals, leading to the larger one, and that’s what we’re going to do here. I want you to find TWO articles, and send them to me. Not just the first one, but at least two. I’m bound to get the articles I want and need if I put you to work on two, instead of one.

And, yeah, I’m putting you to work. Be an arrow.
]]>To answer Francesco’s questions . . .Melanie ChildersNone2010-10-04T08:52:13+01:00http://www.sharktank.org/melanie_childers/files/ec2bd1a1c459d66a6b713f8bc7bfa020-57.php#unique-entry-id-57http://www.sharktank.org/melanie_childers/files/ec2bd1a1c459d66a6b713f8bc7bfa020-57.php#unique-entry-id-57
My answer: When you talk about the effects of increased intracellular GSH, you must factor in its interaction with the stress protein, metallothionein. GSH bonds zinc to metallothionein, and thus sequesters zinc, inhibiting its delivery to both activation sites and inhibitory sites of important proteins, DNA factors, and enzymes, both intracellular and extracellular. But, the key word here is “stress.”

When you use a compound like Meveol, which contains hypothiocyanite (and thiocyanate, and lactoferrin, both acting to decrease extracellular oxidant stress), you are killing the bacteria in the airways. If you kill the bacteria, you’ve solved the problem of neutrophilic infiltration, which increases myeloperoxidase, and H2O2, both major contributors to that stress. If you lower the oxidant stress, you automatically lower the amount of intracellular metallothionein, and you’ve solved the problem of the over-sequestration of intracellular zinc.

I doubt, at that point, you have to worry about extracellular or intracellular GSH at all.
]]>Let’s talk about GSH “therapy”Melanie ChildersNone2010-10-01T14:22:36+01:00http://www.sharktank.org/melanie_childers/files/f339fe2f7493dbe22a73422b9a4d7fce-56.php#unique-entry-id-56http://www.sharktank.org/melanie_childers/files/f339fe2f7493dbe22a73422b9a4d7fce-56.php#unique-entry-id-56
CF patients are probably missing their entire lactoperoxidase system. This is the system that is responsible for killing the bacteria that we inhale every day, in every breath.

What is the lactoperoxidase system? It is composed of:

Hydrogen peroxide (H2O2)
Lactoperoxidase (an enzyme; see the “ase” on the end of it?)
Thiocyanate (SCN-)

I’ll start from the beginning: lactoperoxidase is an enzyme. Enzymes catalyze the reaction between at least two other compounds. In this case, lactoperoxidase catalyzes the reaction between H2O2 and SCN-. What is the reaction that it makes happen? It moves one oxygen electron to the thiocyanate, and this process produces water (H2O) and hypothiocyanate (OSCN). It is hypothiocyanate that kills the bacteria in your lungs. Without it, you will culture PA, BC, and SA quite easily. As well as other pathogens, such as Apergillus. Oh, in fact, there are a whole plethora of dirty living things that OSCN kills, and you will culture them all, if you have CF long enough..

In cystic fibrosis, because you are missing the CFTR protein, and because that protein is responsible for the transport of the SCN- anion, you can’t make hypothiocyanate in the airways.

And, in cystic fibrosis, because you are missing the CFTR protein, and because that protein is responsible for the transport of glutathione (GSH), you have a surplus of GSH inside of the cell. The cell is where H2O2 is made, for the most part. And, usually, H2O2, which has no charge on it and doesn’t need a transport protein to move it out of the cell, just washes in and out of the cell. The part where it washes OUT of the cell is where it combines with SCN-, normally.

Once out of the cell, it’s supposed to be part of that lactoperoxidase system. It’s supposed to give that oxygen electron (with the help of the enzyme, lactoperoxidase, to catalyze the reaction) to SCN-, to make OSCN.

But, in CF, something happens to much of the H2O2 before it can “wash” out of the cell. That something is the GSH that can’t be transported out of the cell, because there is no CFTR in the membrane. What happens to it?

H2O2 combines with GSH, via catalysis by the enzyme glutathione peroxidase (GPX – see the “ase”? It is another enzyme.) and, the result of this are the products H2O (water, of course) and glutathione disulfide (GSSG). So, in CF, you not only don’t have SCN- to make OSCN, you also don’t have enough H2O2 for the reaction with SCN- (supposing that there is ANY residual CFTR function that would cause SCN- to be transported.)

What happens, then, to the GSSG? Well, I personally think that it, too, is “trapped” within the cell. I think that because when I look at the functions of the functionally redundant (to the CFTR protein) MRP proteins, I see that they transport GSSG. There is another reason, too, and I will post the research to support it.

First, we must ask ourselves, what happens to the GSSG, if the CFTR is not in the membrane to transport it? It is trapped there, too, inside of the cell, isn’t it? What happens to it there?

Well, there is another enzyme, called glutathione reductase, which turns the GSSG BACK into GSH.

What does this mean to someone with CF? It means that there is a “sink” in their cells, because they cannot transport GSH, or GSSG, and into that “sink” goes H2O2. The H2O2 that is supposed to be combined, enzymatically, with SCN-, to make the very compound that is responsible for killing pathogens in the airways. GSH combines with H2O2 and out comes water and GSSG.

How do I KNOW that this is happening? Look at the abstract below (particularly, look at the sentence that I’ve underlined in it):

Am J Physiol Lung Cell Mol Physiol 281: L31-L38, 2001;
1040-0605/01

Vol. 281, Issue 1, L31-L38, July 2001
SPECIAL TOPIC
Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice
Leonard W. Velsor1, Anna van Heeckeren2, and Brian J. Day1,3
1 Department of Medicine, National Jewish Medical and Research Center, and 3 Department of Medicine and Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80206; and 2 Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106

Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and -glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.
epithelial lining fluid; glutathione; oxidative stress

You see that? You see that sentence? “The activities of glutathione reductase and glutathione peroxidase in the lung tissue in CFTR-KO mice were significantly increased compared with those in WT mice (48% and 28%, respectively).”

What does that mean, in terms of H2O2? It means that, inside of that CF cell, where H2O2 is made, glutathione peroxidase is going ninety to nothing, turning H2O2 and GSH into water and GSSG. THEN, glutathione reductase is turning that GSSG BACK into GSH to, once again, be a “sink” for H2O2. A vicious cycle that depletes the extracellular space of the H2O2 that is needed for the lactoperoxidase system.

How do I know that H2O2 is depleted in the extracellular space of CF patients? Well, look at the abstract below. I wish I could cut and paste the entire article here, but I don’t want to take up that much space. I strongly suggest that you get the full article for yourself; you can get it online (I have, and it’s free). The reason that I suggest that you get the entire article for yourself is because, in the full article, the authors make it clear that the results of their testing of the levels of H2O2 in expired air condensates from CF patients was surprising to them. It was surprising to them because these patients, as most CF patients, could be assumed to have much higher levels of inflammation in their lungs. They SHOULD have had higher levels of H2O2 in the air that they expired. But they didn’t. It was lower than healthy controls (although this difference was not statistically significant). The reason for that is what is happening inside of the CF cell, where GSH is trapped. (I am continuing this below the abstract.)

Eur Respir J. 1999 Jan;13(1):103-6.

Expired hydrogen peroxide in breath condensate of cystic fibrosis patients.
Ho LP, Faccenda J, Innes JA, Greening AP.

Scottish Adult Cystic Fibrosis Service and the Respiratory Unit, Western General Hospital NHS Trust, Edinburgh, UK.

Abstract
Stimulated inflammatory cells release large amounts of hydrogen peroxide (H2O2). Breath condensate H2O2 has been shown to be elevated in stable asthmatic children, chronic obstructive pulmonary disease and intubated adult respiratory distress syndrome. In cystic fibrosis airways, where neutrophilic inflammation dominates, it is postulated that H2O2 in breath condensate would be elevated and may be used as a marker of airways inflammation. Expired breath condensate was collected from 16 clinically stable cystic fibrosis (CF) patients (mean age 25.3 yrs, mean forced expiratory volume in one second (FEV1) 50.2%) and 14 normal subjects (mean age 29.9 yrs). Total plasma leukocyte, neutrophil, monocyte and eosinophil counts and lung function were also measured on the day of collection. A method of breath condensate collection excluding the confounding factors of nasal air and saliva contamination was validated and used and H2O2 measured fluorometrically using an optimized assay. The median level of H2O2 concentration in breath condensate of CF patients was lower than that in normal subjects (0.064 versus 0.089 microM), but this did not reach statistical significance (p = 0.20, Mann-Whitney rank sum test). Within the CF group, there was no correlation between H2O2 concentration and lung function. Expired breath condensate H2O2 is not elevated in patients with cystic fibrosis, and is thus not a suitable marker of airways inflammation in these patients. Possible explanations include physical barriers to its detection caused by viscous airways secretions, reaction with other reactive species or increased antioxidant activity caused by trapping of positively charged antioxidants in negatively charged airways secretions.

Another sentence that you can’t see in the abstract (now, above) is this (from the full article): “In the CFTR-KO lung, the activities of glutathione reductase (GR) and glutathione peroxidase (GPx) were increased, whereas the activity of gamma-glutamyltransferase (gamma-GT) was unchanged.”

(By now, you’re probably saying, oh no, not ANOTHER enzyme.) But, quite simply, gamma-GT is just the enzyme that is responsible for the synthesis of GSH, in the first place. And, here, in this experiment, we see that it’s activity is unchanged. The cell is not making more GSH. Probably because it can’t transport it.

When you use GSH (especially inhaled) and you have CF, you are making a bet, and an unwise one, at that. You are betting that you have no residual CFTR function, and that you have no SCN- at all in your airways. You are also betting that you have no lactoperoxidase in your airways.

You have at least three peroxidases in the lungs. (Lactoperoxidase is just one of them.) Each of these peroxidases is a part of a SYSTEM. Each of these systems are made for killing bacteria, and fungus and viruses.

Do you really want to guess at this? There is another system which uses H2O2, for killing pathogens in the lungs (myeloperoxidase (MPO)). You’ve already been born without a functioning lactoperoxidase system in your airways. Do you really want to take away ANOTHER peroxidase system? (And, YES, the MPO system is responsible for MUCH of the inflammation that is seen in CF airways, but hey, do you really want to lose it’s antimicrobial activity, without replacing it with something else?)

In my next post, I will talk about GSH, H2O2 and MPO, another peroxidase that is both central to the inflammation that we see in CF lung disease AND very important in terms of killing pathogens.

]]>The POOFMelanie ChildersNone2010-10-01T13:00:36+01:00http://www.sharktank.org/melanie_childers/files/564997d4aa4a6f843c041b7f0a70b5d8-55.php#unique-entry-id-55http://www.sharktank.org/melanie_childers/files/564997d4aa4a6f843c041b7f0a70b5d8-55.php#unique-entry-id-55Is all lost when it comes to BITC?

No, all is not lost when it comes to BITC. As a matter of fact, the problem with BITC’s efficacy might just be dosage. We are administering it at a VERY low dose. The problem might also lie in absorption. Or, in fact, in storage, and preparation. And, there are other things that BITC seems to be doing, such as normalizing blood glucose levels in CF patients with diabetes, that are very worthwhile goals to pursue.

But we want the POOF. And it is not that BITC at higher doses might not give us that. But why pursue it when we know that we have a better chance of getting that POOF with a different ITC? We are getting dismal, if any, results with BITC at the current levels, there are problems with administration of it, and there is good evidence that it might not be the right ITC for the job.

Why do I say that there is good evidence that it might not be the right isothiocyanate for the job? Because, in going over my work on the lactoperoxidase system, I realized that we should be looking at the exact tissue that IS the problem in CF: submucosal.

THESE are the glands that line the airways. They contain 60% serous cells, and 40% mucus cells. AND, CF is a disease of the airways; specifically, it is a disease of the SURFACE of the airways. These cells, that make up the submucosal glands, SECRETE products onto the surface of the lungs, but in CF, the serous cells, which HIGHLY express the CFTR, do not secrete much of anything. It is THIS area we must address. It is in THIS tissue that we must induce the functionally redundant proteins that can take the place of the CFTR protein. (Sorry for all the caps, but I AM being emphatic here.)

And, in looking at the multi-drug resistant proteins; the proteins that can functionally complement the CFTR protein, the ONLY protein that is expressed in the submucosa is NOT the MRP2, MRP1, or the MRP5. It is the BCRP. The breast cancer resistance protein. And that protein is induced by phenethyl isothiocyanate.

And, yes, BCRP is also an MRP protein. It is the same class as the CFTR, and the other MRP proteins. And it is highly expressed in bronchial epithelium. And, specifically, in the submucosal cells.

It will do us no good at all (well, in terms of the pancreas, it does do us SOME good, and that’s why we see the normalization of blood glucose levels, and the effects on digestion, of BITC) to induce an MRP protein in cells other than the ones that line the airways. This is a disease of the cells that line the airways. This is where most of the pathology is seen, and it is why these children die. The airways.

Recently, as I told you before, I had occasion to go back and look over the research to find the examples of CF patients who had taken chemo for their cancer and were “cured” for long periods of time, of their CF. Since 1998, when Lellamand first described the first instance of this in a CF patient who had a sarcoma, it has happened again and again. WHY? What protein or proteins is the chemo inducing that “cures” CF?

Well, of course, these scientists were looking at multi-drug resistance proteins, because chemo is known to induce these types of proteins. But when you look for proteins, you have to use antibodies to identify them. If you are not using the specific antibody for the specific protein, you don’t find that particular protein. It’s like looking for gold with an instrument that detects only water. You’re not going to find anything but water, if that’s the instrument you’re using. If you want to know if the BCRP protein is there, you have to use the antibody for it. So, yes, these cells were expressing multi-drug resistance proteins, and the scientists found them because they were using antibodies specific for them. But that does not mean that it was THOSE proteins that were effecting the “cure.”

And, yes, it might be that these proteins are expressed in the submucosa, but when I look at the research, the only one that I find that is expressed in the submucosa is BCRP.

We chose, initially, BITC, because there was a lot of research on it, and it was relatively non-toxic. Since that time, there has been quite a bit of research produced on phenethyl isothiocyanate, and it, too, is relatively non-toxic. Since that time, there has been much more research on the multi-drug resistance proteins, their function, and their expression.

We have an opportunity to get specific here. I think we should take it.

]]>Location, location, location (What is wrong with BITC?)Melanie ChildersNone2010-09-30T15:47:05+01:00http://www.sharktank.org/melanie_childers/files/a7ec9d8ed6de1e6ba5a74cc62849dd10-54.php#unique-entry-id-54http://www.sharktank.org/melanie_childers/files/a7ec9d8ed6de1e6ba5a74cc62849dd10-54.php#unique-entry-id-54
When I went on a recent trip to work on Meveol, I had the task in front of me, of putting together some of the work on the effect of chemotoxins (to induce the multidrug resistant – functionally redundant proteins to the CFTR protein) in CF cancer patients. What did I find? MORE cancer patients “cured” for spaces of six months to two years, by the cancer drugs that they were taking.

The theory is a good one. But is BITC the best compound to induce those proteins?

Strangely enough, this is where Meveol fits in. I had to study, again, the lactoperoxidase system in CF, for my trip. And, of course, I had to do some work on it after the trip. Everytime you work on something, you understand it better. At least I do; and it probably works the same for other people, too.

The lactoperoxidase system is responsible for killing bacteria. Lactoperoxidase, obviously, is an enzyme (see the “ase” on the end of it? “Ases” are enzymes, always.) Enzymes always catalyze a reaction between at least two other compounds. In the case of lactoperoxidase, those two other compounds are hydrogen peroxide (H2O2) and thiocyanate (SCN-). The product of this catalysis is hypothiocyanite (OSCN). It goes like this: SCN- + H2O2 = OSCN + H2O. Spoken in English, lactoperoxidase catalysis the oxidation of thiocyanate by hydrogen peroxide, such that the oxygen electron moves from the hydrogen peroxide molecule and oxidizes (moves to) the thiocyanate molecule to form hypothiocyanite and water. It is the hypothiocyanate that kills the pathogens that most often invade CF lungs: PA, BC and SA.

For the first time in the study of this disease, the pathology AND the pathogens can be linked to protein function. Why? Because one function of the CFTR protein is to transport thiocyanate. Without thiocyanate (SCN-), you have no lactoperoxidase system. You only have lactoperoxidase, the enzyme, and hydrogen peroxide. And you cannot kill those pathogens.

How, you may ask, does the study of the lactoperoxidase system fit into the induction of those multi-drug resistant proteins? Well, unfortunately, as I’ve told you before, I am not a real scientist. REAL scientists study this disease, all diseases, from the top down. I study this disease from the bottom up. I use a different kind of reasoning, in doing this. It goes from observation to theory, not vice versa. (I believe it’s called inductive, but I really don’t care about the name.) I also study from the top down, in a sense that has nothing to do with inductive or deductive reasoning. I have studied what the CFTR protein does in ALL of the cells that they have tested it in, and now it is time to narrow this down.

That’s where lactoperoxidase fits in. I am narrowing down to the tissues; the exact area where these proteins are most highly expressed. The submucosal serous cells, quite specifically.

These are the cells that line the airways. What the CFTR transports to the airway surface, via its function in these cells, is of the utmost importance. But, in terms of the isothiocyanates, WHICH one induces the redundant protein in THESE tissues, by the cells in this gland, is of paramount importance. And BITC does not induce any of the MRP proteins in these cells.

I will post again, in a day or so, and tell you which isothiocyanate works in these particular cells. It is a multi-drug resistant protein of the ABC class that it acts on. And this protein is induced in the submucosal gland mucus cells, which also line the airways. Not the serous cells, specifically (or at least it’s induction has not been tested in these cells yet), but in it’s “sister” cells, that make up 40% of this gland.

It is the location that is the key here.

]]>Something is wrongMelanie ChildersNone2010-09-30T15:38:57+01:00http://www.sharktank.org/melanie_childers/files/575760c55ee09e063cf3d7d805555d77-53.php#unique-entry-id-53http://www.sharktank.org/melanie_childers/files/575760c55ee09e063cf3d7d805555d77-53.php#unique-entry-id-53
One night, I dreamed that I was coming back from a cruise and I had THE drug, but what stopped me was the cargo handlers on the ship that I was on. I had to wait until all of the cargo was off of the ship, and the machines that move the cargo off of the ship kept dropping the cargo back onto the deck.

Last night, it was traffic tickets that were in my way. I don’t know why I had to pay them before I could see him, but that was the deal in this particular dream. I was on the phone, and I was punching in my debit card number to pay the traffic tickets, and always, before I got to the end of the number, I got mixed up, and punched in the wrong numbers and had to start all over again.

When I have these dreams, it is always the rage that wakes me. Blam! Straight up in the bed, angry and frustrated and ready to fight. But, of course, as always, then comes the pain, because it is too late. Too late to save him, no matter if I did have THE drug to do it. My beloved boy; he lies in his grave.

I have a choice; it is the one that awaits me every day and I make it, anew, every single day: to cry or to fight. I guess you know which alternative I chose today, because here I am again.

I cannot tell you where I’ve been, but rest assured, I have been fighting, not crying. And, I am pretty sure that have made some progress. I have been fighting for a drug, based upon my work, to get to the pharmacy shelves. This drug, Meveol, will make cystic fibrosis a non-lethal disease. It is not a systemic fix, but it will stop the pulmonary disease. It addresses the root cause for the pulmonary disease, and it will end the death (caused by pulmonary disease) that we see in these children. I will tell you more about it, and the theory behind it, in a post to follow.

What about BITC, though? I cannot see, quite frankly, that it works, at least when applied transdermally. We’ve got one person that experienced a great effect of BITC, but one person does not cut it, when it comes to efficacy of a compound.

Something is wrong.

]]>CFTR Potentiators & CorrectorsMelanie ChildersNone2010-09-04T11:35:24+01:00http://www.sharktank.org/melanie_childers/files/d2ee482cc95d080f386fab7a67472bf4-52.php#unique-entry-id-52http://www.sharktank.org/melanie_childers/files/d2ee482cc95d080f386fab7a67472bf4-52.php#unique-entry-id-52
First of all, one (VX-809) is a “corrector” molecule for the F508 variant. That particular mutant protein, as you might recall, has a problem with trafficking to the membrane of the cell. That’s it MAIN problem: it’s “misfolded” and the quality control members of the endoplasmic reticulum don’t let it out of that protein making organelle, and it’s cut up into it’s amino acid pieces. VX-809 “corrects” the protein enough so that some of it gets to the membrane, to do it’s job.

But, F508 has another problem—even after it gets to the membrane of the cell, it can’t transport anions very well. Something is wrong with it’s ability to be activated. (It’s got a major problem with it’s nucleotide binding domains, but that’s the topic for another discussion). This is where VX-770 comes into play.

VX-770 is a “potentiator” molecule. Simply put, the problem with G551D’s activation, and it’s ability to move anions out, is corrected by this small molecule. This variant of the CFTR protein has a mutation in one of it’s ATP binding sites (the CFTR requires ATP to open, and to transport anions).

So? What does all of this mean to you? Well, let me tell you:

They have tested G551D patients with VX-770, and it improves lung function. They have tested VX-809 in F508 patients and it doesn’t improve lung function, but it increases chloride conductance. That means that, although they can use VX-809 to get the misfolded CFTR F508 protein to the membrane, once there, it isn’t activated like it should be.

So, they are going to try using both of them at the same time. They think that the VX-809 will get the mutant F508 to the membrane, where it belongs, and once it’s there, they’ll use VX-770 to activate it.

What is the assumption here, then? The assumption is that the gating defects of both proteins are caused by the same thing.

That brings us to our next subject: nucleotide binding domains. The CFTR has two of them: NBD1 and NBD2. Hydrolysis (water splits it and it turns into another molecule; in this case ADP) of ATP at NBD1 opens the channel. NBD2 is responsible for the regulating the closing of the channel. BUT, these binding domains “talk” to each other. There may be some kind of feedback loop here (probably has to do with the fact that this protein moves out negatively charged ions, but I won’t get into that here). It might also be that NBD1 is responsible for opening and closing the channel (think of it like a person whose only job is to hold on to the doorknob) and NBD2 regulates how long the channel stays open (think of it like a person whose only job is to tell the guy who is holding the door knob to push or pull on it).

Where is the mutation affecting gating in the F508 variant? Where is the mutation affecting gating in the G551D variant? If they are both in NBD1, then what are they? How do they affect how NBD1 works?
]]>Effects of BITCMelanie ChildersNone2010-08-24T16:48:52+01:00http://www.sharktank.org/melanie_childers/files/ee02e80c29ce07e4a626fcac1a175b1d-51.php#unique-entry-id-51http://www.sharktank.org/melanie_childers/files/ee02e80c29ce07e4a626fcac1a175b1d-51.php#unique-entry-id-51
I’m going to be as vague as possible here, to protect the privacy of the CF people involved, but I’ll give you a quick rundown of a few of the people using transdermal BITC:

There’s one very sick young man who has both one of the major pathogens that colonize in these patients lungs, and liver problems associated with the disease. At 20mg, he started seeing improvements in the color and amount of his sputum, and improvements in cough frequency. The pathogen that he cultivates is resistant to antibiotics, but he was put on yet another antibiotic at about the same time he started BITC, so we don’t know which was responsible for this improvement. He’s on 27mg right now, and his sputum has recently increased as well as his cough frequency. He was put on another antibiotic that his pathogen was previously resistant to, but he is again showing improvement.

This is not quite the “poof” that we had hoped for, but because this young man was what we call “end stage,” it is difficult to determine what a “poof” would look like in him, at this point. And, since we cannot see any test results and are left to judge only by gross measurements such as sputum color and production, and cough frequency, we really don’t know what’s going on. SOMETHING’S causing an improvement, but we don’t know what, because of the mixed variables.

Another young man also cultured a bacterial pathogen known to favor the airways of CF patients. The only changes he saw were an increase in energy (might be a placebo effect) and a “sinking” of his stools. Anyone familiar with the stools of CF patients knows that they usually float because they have so much fat in them. This young man was used to his stools floating over the years, so it was a bit of surprise to him when they started sinking in the toilet bowl. We’ve seen this time and time again in CF patients who are taking this supplement orally, so it is not a surprise to us that it would happen when taken transdermally. It is caused by an increase in the pancreatic enzyme responsible for fat absorption.

However, this young man has also experienced, recently, a bout of aspergillus. I suspect that there is a relationship between this fungus and bacteria, where if you kill the bacteria, the aspergillus can flourish. This is because there is competition for nutrients in the airways, between these invaders. Whether this is true or not, I don’t know; I’m just guessing. At any rate, this young man is treating the aspergillus that he just now found out that he’s been culturing for a while, and he’s better.

Another young man has CF related diabetes. After an initial normalizing of his blood glucose levels, the last I heard his levels spiked again one morning. I don’t know yet about subsequent readings, but I will soon find out.

There are others to report on, but in the service of brevity, I report on only three. There are no real “poofs” here; there is a reason to suspect that BITC is acting in a systemic manner because the changes are controlled by different systems.. Our problem is that we don’t know much about absorption; there is no real ability to control that variable. We don’t even know for sure how these people are mixing BITC, before it is applied to the skin. Or how they are applying it. These people are scattered over several continents. And, every single one of these outcomes could be due to a placebo effect, or to other variables.

No one has yet gone for PFT testing. We have no info on that yet, and even if we did, we can’t say that it would be definitive, in terms of the effects of BITC, unless it jumped very radically (because these kids’ PFTs don’t jump very radically with current mainstream therapies). So, once again, we are waiting.

If we could have a test as to whether BITC induces the expression of MRP in CF cells (and extrapolate from that, the dosage required), then we would know something. We are, right now, though, simply flying along in the dark.

We’re pretty pitiful, aren’t we? But, at least, we’re flying.
]]>Goiters AGAIN?Melanie ChildersNone2010-08-09T20:02:46+01:00http://www.sharktank.org/melanie_childers/files/787130aadfdb98c003a6e2b9c4943f3c-50.php#unique-entry-id-50http://www.sharktank.org/melanie_childers/files/787130aadfdb98c003a6e2b9c4943f3c-50.php#unique-entry-id-50
I’ll recap a little here and boil it down to just a couple of things:

1. Higher levels of TSH make your thyroid swell and this is a goiter.

2. Levels of TSH go down when there are high enough levels of T4 (thyroxine).

Now, when we see that abstract on those CF kids who took iodine therapy for goiters, we see that those who took the iodine therapy developed goiters and those that didn’t take the therapy, didn’t develop goiters, right? Can we say that iodine causes goiters in CF patients? Or is it more likely that there is a relationship between zinc levels and iodine levels that affects the levels of thyroxine?

The latter is probably the case here. If it were simply a problem with a lack of iodine, you would not see goiters in those CF patients that got the iodine therapy [in the abstract, below] and if there were not a relationship between zinc and iodine, in terms of goiters, you would not see the kids NOT having the iodine therapy NOT developing the goiters.

But there is something else here, too—there is a relationship between zinc, selenium, iodine, and manganese—a balance that, if perturbed, will affect thyroid function in a very fundamental way. And, if you have an imbalance in ALL of them, and you add one of those components, such as iodine, you’ve not solved the imbalance, and you might have exacerbated it to the point where you actually have a pathology (goiter). It is like the B vitamins, where if you have a low amount of two and you supplement one of them, the one that you didn’t supplement is more out of whack than ever.

Now, I’m not saying that BITC or any other isothiocyanate won’t interfere with thyroid hormone levels. I don’t study the thyroid. I don’t intend to. And although I’ve got one person who used this compound who had high TSH levels, and they went down to normal, that one person isn’t enough to make a case for BITC’s ability (or rather, it’s induction of the expression of a functionally redundant protein to the CFTR) to restore that balance.

And if you know that you have a perturbation of the homeostasis in ALL of these compounds, what do you do? Do you try to guess at the amount you should supplement each one? And, what if supplementation doesn’t help? What if homeostasis is determined by the DELIVERY or the CHELATION (opposite of delivery—it means the pick up, or taking away) of one of these elements to an enzyme, or an expression factor, and not simply by levels in the serum? Wouldn’t you think you would fare better, overall, by restoring that SYSTEM of homeostasis, instead of a single element in it?

Volume 79, Issue 4, Pages 684-687 (October 1971)

Complications of iodide therapy in patients with cystic fibrosis**

William L. Nyhan (Editor), Harry C. Shirkey (Consultant), M.D. Thomas F. Dolan Jr., M.D. Lewis E. Gibson

Forty-seven of 55 patients receiving long-term daily iodile therapy as part of the therapeutic regimen for cystic fibrosis of the pancreas developed goiters. Goiters usually appeared 2 to 3 years after onset of therapy, with a range of 3 months to 12 years. Fourteen patients receiving iodides had laboratory or clinical evidence of hypothyroidism; 2 of these 14 did not have a goiter. No goiters were observed among 55 patients with cystic fibrosis not receiving iodides. Fourteen of 55 children receiving iodide therapy developed nasal polyposis whereas no polyps were seen in 40 patients not receiving iodide. This may be due to the age distribution of patients studied, since most children in the 5 to 15 year age range received iodide therapy.

]]>Science and SentimentalityMelanie ChildersNone2010-08-01T16:06:03+01:00http://www.sharktank.org/melanie_childers/files/a652f5f102f5918cc2185532bad3bc61-49.php#unique-entry-id-49http://www.sharktank.org/melanie_childers/files/a652f5f102f5918cc2185532bad3bc61-49.php#unique-entry-id-49
I don’t know. I send posts in, and if they are too personal, I find myself editing them over and over again, until the guy who is responsible for posting them says, “Enough! I’m posting it, Melanie!”

And, some people do like the personal parts, I guess. But, if I were still a CF parent, I’d have to say that I wouldn’t like those parts. I would come here, to this blog, and say, “Okay, what can I understand better today?” What has Melanie said, recently, about CF science, that I can put to use to make my CF child’s life healthier? Those people can quickly skim the personal posts and get to the real meat of things.

What bothers me, though, is this: Suppose people reading this blog read the science AND the personal posts and think that my sentiment colors my science? What if they think that, in my quest to stop this disease, in my pain over losing my child, I am jumping to conclusions?

I am not fool enough to think that that cannot happen. It is something that I must constantly guard against. It is part of the reason that I continually state that I am stupid, and ignorant (the other “part” of the reason that I state this is because it’s true!), and it is why these posts are often peppered with more questions than answers. And, finally, it is why I post abstracts of articles to underpin my postulates.

No matter how much I guard against sentiment coloring my science, there is one sure truth: Dead kid or no dead kid, no science is completely objective. People want to make money. People want fame. People want to publish. And people fall in love with their theories. As we say in Texas, they get “stuck on stupid.”

To digress a bit here, I’ve been “stuck on stupid,” myself, previously. When I was studying metallothionein, I was stuck on stupid. I just KNEW that the CFTR protein MUST be transporting metallothionein. I don’t know how many CF scientists I wrote to and asked about this, before I finally thought to write to Linsdell, who told me, unequivocally NO, the damned thing is too big to be transported through the CFTR pore opening. Until I accepted that fact, and threw away the old idea, I couldn’t understand quite a few things, collateral to the transport properties of the CFTR. They didn’t make any sense at all, until I was able to put metallothionein into its proper place (and leave it there).

I think that what makes the difference between whether someone colors their science with sentimentality, or the quest for fame, fortune, or simply keeping their job in academia, is the degree to which they have one trait: curiosity.

If you are curious, you can’t help but keep on digging. And one thing that is wonderful about science is that you can turn your postulates around and try to prove them wrong. You have to read it all—articles that would disprove your postulate, and articles that give it credence. And, even if you can’t find an article that exactly states that you are incorrect (or correct), if you ARE wrong in your idea, you eventually will come to a place where you don’t understand anymore. Nothing fits. The science that HAS been proven experimentally begins to look incorrect, at that point, and you know that you’ve lost your way, that something in that postulate is wrong.

But OF COURSE I mourn the loss of my son. Of course I do.

And do I think that my science is colored by sentiment? Sure it is. But not in the way that you would commonly think.

You see, I want to know what my son’s killer looks like. I want to know it’s face. I want to know the shape of it’s teeth and the texture of it’s scales.

So, the sentiments are both there, no doubt. But, there is a difference, in the quality of them. The pain that you see in my posts is only the engine.

The curiosity is the tool.
]]>Stripping GSH?Melanie ChildersNone2010-07-26T10:24:32+01:00http://www.sharktank.org/melanie_childers/files/d2ef85dfb55fd1dbfc734e613ab82919-48.php#unique-entry-id-48http://www.sharktank.org/melanie_childers/files/d2ef85dfb55fd1dbfc734e613ab82919-48.php#unique-entry-id-48
Years ago, when I first began studying this disease, I came upon the information, published by Hanrahan and Linsdell, that the CFTR transported glutathione. Well, actually, the article stated that the CFTR protein was “permeable” to glutathione, and I extrapolated from that that the CFTR transported the tripeptide. Many, many experiments have since shown this to be absolutely correct. The CFTR transports glutathione.

At this time, some went on their merry way of championing the use of glutathione in the treatment of cystic fibrosis. And Sharktank went on its way of actually studying this disease in its entirety, because we knew that glutathione was only part of the answer. I recently read a few things about CF and BITC on the internet. I came across some sites where people were questioning if BITC “stripped” glutathione from the cells.

I hope that I have taught you enough about the science of these proteins, and CF, to realize how funny that idea is. Don’t get me wrong—I know it’s a scare tactic—but I want you to be able to see it, logically, alongside all of the other things that we know about the CFTR protein, and if you can do that, you will see how funny it is.

Let’s begin to line up the facts here, and you will see the joke:

1. The CFTR transports glutathione

2. The MRP transports glutathione

3. The “stripping” of glutathione from the cells is caused by the transport of glutathione from these cells, by both of these proteins.

So, it logically follows that if the CFTR transports glutathione, and MRP transports glutathione, and this transport is characterized, in other terms, as “stripping,” then BOTH proteins do this. If you can say that the MRP’s transport of glutathione is “stripping,” then you must also characterize the CFTR’s transport of glutathione as “stripping.”

If one is good, then how can the other be bad, if they are the same?

The key here is the word “strip.” It is a word laden with emotion. I won’t even begin to guess why someone would use such a word in this context, but I would suggest to you that you consider their motives. And then you determine whether you want to believe anything else that this person says about BITC.

I am not in love with my theory. That is not the way to finding the truth, and to crushing this disease. It is the way of the zealot. And no one wins if we stop seeking the truth, and, instead, begin to try to convince people by politicking.

I know, however, that there are people out there who know just little enough to be afraid when they hear words like “strip.” This post is for you. There are the facts; they are well known and well documented, in the scientific literature.

Put them together and come to your own conclusion.
]]>Supplements and Butt RupturesMelanie ChildersNone2010-07-19T14:49:24+01:00http://www.sharktank.org/melanie_childers/files/cc40fbe2c771e82659602b11889608a6-46.php#unique-entry-id-46http://www.sharktank.org/melanie_childers/files/cc40fbe2c771e82659602b11889608a6-46.php#unique-entry-id-46
I’ll start out by telling you a story—

I used to be a weightlifter. Not in any professional capacity, but I lifted weights to stay in shape. I believe in hard work, but I also believe in efficiency, so of course, before I began to lift weights, I studied the biochemistry of the cells comprising muscle tissue. And, obviously, I had to make a study of the processes by which ATP was produced and used by the cells. And, then, I studied, of course, the fuel itself: protein, fat, and carbohydrates.

I formed an opinion, as most weightlifters do, that carbs make you fat. You eat carbs, they turn into glucose, insulin is produced to pick up that glucose, and then it is stored in your cells as fat. I wanted muscle, not fat.

But, after cutting carbs to the bare minimum, I began to crave them. Believing in the adage, “better living through chemistry,” I began to look for a method by which I could eat carbs and not process them as fat.

When you read articles on this topic, you come across loads of crap products on the internet; with cute little names, like “Carb Busters, Carb Blockers,” and “Carb Blasters,” etc. It’s always amazing to me the load of bullshit that people will buy.

These articles always start out the same way. First, they tell you that they’ve obtained some breakthrough scientific product that is “all natural.” That’s supposed to make you feel safe about taking the product. Then, they start quoting little snippets from real scientific articles about the main ingredient in their product. That ingredient usually has a long, scientific name, but it always comes from a “natural” source in which dedicated scientists with large foreheads have just recently managed to discern miraculous properties.

And the articles they cite are real. The studies have been done. The results are in. That’s supposed to make you feel like you’re making an informed decision.

If you stop at this point and buy the product, you’re a fool.

Because, first, the product that they are selling has very little of the ingredient mentioned in the article. In fact, often, these products only have trace amounts of the ingredient in them. And, often, they have none of the ingredient at all.

Now, this one particular product that I found, that was supposed to block the absorption of carbohydrates, supposedly had one of these miraculous ingredients in it. I don’t remember the name of the product, or even the name of the miraculous ingredient anymore. But it did come from a natural source; I believe it was a type of lima beans. I do not like lima beans, but even if I did, I wanted the name of that chemical.

But first, I read the articles that were cited in the sales pitch. This “carb blocker” ingredient had been tested in mice. It did, indeed, block carbs.

Was it a good thing to block carbs? No. Because if you used enough of that miraculous ingredient on those poor mice, it blocked the carbs so well that they ruptured their anuses. That miraculous ingredient gave them mice butt ruptures.

Now, fortunately, this “carb blocker” didn’t have enough of that natural ingredient, probably, to rupture any weightlifters’ butts. It was, most probably, ground up lima beans that they were selling for $40 a pop, so to speak. Heh.

The moral of this story is to watch out for supplements. This industry is almost completely unregulated. Know what you’re buying. Always look for the amount of the main ingredient, as tested by some accepted measure, such as HPLC (high performance liquid chromatography). Read the articles. Do your homework before you go putting just any old crap into your body.

Butt ruptures are not pretty, you know.

]]>Of course I took an entire bottle of the cyanide! After all, it’s NATURAL!Melanie ChildersNone2010-07-13T07:45:53+01:00http://www.sharktank.org/melanie_childers/files/ed6c3e3b6a765065afbf70824eb9ccc5-45.php#unique-entry-id-45http://www.sharktank.org/melanie_childers/files/ed6c3e3b6a765065afbf70824eb9ccc5-45.php#unique-entry-id-45
Some of you out there might (I sure hope not, though), at this point, be saying, “What? The water was poisoned?”

No, the water wasn’t poisoned. But the contest winner was poisoned by it because she drank too much of it, too quickly. Yes, even water can poison you, if you have too much of it at any one time. The poison, as they say, is in the dose.

Remember that: The. Poison. Is. In. The. Dose. the next time you think about using a “natural” compound indiscriminately.

There are times when you should take more of any natural compound, in order to restore a balance, of course. In fact, those of you out there who take vitamins are assuming that you HAVE an imbalance in whatever vitamin you are taking, and so you are supplementing those particular vitamins, with a One-A-Day, a Flintstone, a Centrum, etc.-- or an ADEK (if you have CF).

More is not better. And simply because it comes from a natural source doesn’t mean it can’t hurt you. Even water can kill you if you take in too much of it at one time.

]]>The Carrier TestMelanie ChildersNone2010-07-06T18:56:21+01:00http://www.sharktank.org/melanie_childers/files/b71438ae8531dff3e3fbb585620a20c5-44.php#unique-entry-id-44http://www.sharktank.org/melanie_childers/files/b71438ae8531dff3e3fbb585620a20c5-44.php#unique-entry-id-44BITC moved the levels. It moved arachidonic acid slightly higher, which was unexpected (and might be statistically insignificant, as well), but it moved DHA quite a bit. Wonderful news! That means that it's being absorbed ENOUGH to do what it's supposed to do. I'm ecstatic.

We don't yet have the page up where people can post information about their use of BITC, but it's moving towards completion very quickly. And we have more people using this compound as a supplement, transdermally now. With some preliminary reports. Good ones.

So, things are moving right along. Our "poof" is becoming a reality for more than just one person.

I am going to try to get a friend of a friend to teach me some chemistry. I don't have that under my belt, believe it or not. I studied molecular biology enough to know that plasmids were not a viable method for the transfection of the CFTR protein. And I gleaned quite a bit of collateral information from that study, but I really need to know more basic chemistry.

You guys see what a dufus you have teaching you? I really am so very ignorant, and I hate that. But, that's the reason that you need to get your doctors involved if you decide to use BITC as a supplement. Your doctor protects you from this mad scientst, you see? Luckily, there are some very compassionate and informed doctors practicing CF medicine, and the smart ones have learned to listen to their patients, because they know that their patients have learned to listen to their own bodies.

Anyway, I will be posting more frequently in the next few days. Much, much hope to you, my CF family.
]]>Let’s Talk About Goiters (cont’d)Melanie ChildersNone2010-07-06T18:52:03+01:00http://www.sharktank.org/melanie_childers/files/ce1017111b2aebe3ac84592ffbf2e6a0-43.php#unique-entry-id-43http://www.sharktank.org/melanie_childers/files/ce1017111b2aebe3ac84592ffbf2e6a0-43.php#unique-entry-id-43
We know that T-4, or thyroxine, is the “signal” that “tells” the pituitary gland to lower the amount of TSH it is releasing. So, you lower T-4, you increase TSH, and you increase T4 and this results in a decrease of TSH. It’s an inverse relationship between T-4 and TSH. And, it’s the elevated TSH that causes goiter. And, in much simpler terms, if you have enough T-4, you don’t get goiters.

But whether you have enough thyroxine (T-4) or not depends not just on how much iodine you have in the system. More specifically, it depends on how much iodine the thyroid can absorb from the system. That’s where isothiocyanates (and thiocyanates) poke their noses into this system. Both the isothiocyanates and the thiocyanates inhibit iodine uptake by the thyroid, which leads to decreased production of thyroxine.

Now, I’m going to be a little redundant here, but I want you to remember something: The level of T-4, or thyroxine, has an effect on whether or not you get a goiter (it’s actually a persistently high level of TSH that causes goiter, but this is caused by a persistently low level of thyroxine).

So, we can look at thyroxine and say what effects the levels of thyroxine? You’ve already got:

1. Iodine deficiency

2. Interference with the thyroid’s ability to absorb iodine

But just thinking about this logically, without referring to the research, there’s something else that would effect levels of thyroxine, isn’t there? Remember that thyroxine is changed into T-3 (tri-iodothyronine), the active form of the protein. What IF you were changing too much thyroxine too quickly, into tri-iodothyronine? You’d have less thyroxine, wouldn’t you?

Well, guess what happens in the case of zinc deficiency? Yep. Exactly. Here an abstract to peruse, on this topic:

Drug Nutr Interact. 1987;5(2):113-24. Effects of zinc deficiency on thyroid function.
Oliver JW, Sachan DS, Su P, Applehans FM.

Abstract
Interactive combinations of altered zinc and thyroid states were studied in rats to assess pathophysiologic effects. Clinical signs of zinc deficiency or thyroid alteration were limited to effects on growth rate. Changes in organ and glandular weights and serum thyrotropin levels reflected changes in serum thyroid hormone concentrations. Significantly (probability less than .001), zinc-deficient rats had enhanced hepatic thyroxine-5'-monodeiodinase activity. In addition, the zinc-deficient state was found to be protective against thiouracil-induced suppression of the microsomal-monooxygenase and thyroxine-5'-monodeiodinase enzyme complex. This protective effect was evident by greater thyroxine-5'-monodeiodinase and reduced nicotinamide-adenine dinucleotide phosphate cytochrome c reductase activities, as well as cytochrome P-450 content, in zinc-deficient/thiouracil-treated animals. Thus, the enzyme complex had increased triiodothyronine-generating capacity in conditions of zinc deficiency, which may be important because of the greater biological reactivity of triiodothyronine. Primary zinc deficiency conditions of the magnitude seen in this study and in this-age rat did not appear to alter serum thyroid hormone levels or organ/glandular function. However, concurrent zinc deficiency and altered thyroid status did change thyroid hormone response and disposition, which may be important to populations at risk because of thyroid dysfunctional states.

Here are a couple more you might want to look up, on your own:

1. Fujimoto S, Indo Y, Higashi A, et al. Conversion of thyroxine into tri-iodothyronine in zinc deficient rat liver. J Pediatr Gastroenterol Nutr 1986;5:799-805.
2. Hartoma TR, Sotaniemi EA, Maattanen J. Effect of zinc on some biochemical indices of metabolism. Nutr Metab 1979;23:294-300.

Now, keep in mind that I’m not saying that this is exactly what is happening. I don’t know for sure. But I do know that zinc deficiency, in case after case, in research article after research article, lowers thyroxine levels. So, it is not only iodine, or the lack of it, that is linked to the formation of goiters. Zinc plays a large role in that system.

And, in the case of CF, you have MAJOR problems with zinc homeostasis. And, please, PLEASE do NOT go out and buy some zinc supplements and start eating them like candy. The problem is with zinc HOMEOSTASIS. Zinc delivery and the uptake of zinc by various enzymes and proteins is what is screwed up in CF, and if you take too much zinc, you’re only going to exacerbate the problem, not help anything.

Zinc is REQUIRED for maintenance of the proper level of thyroxine. Look it up.

I’m going to end this blog post with yet another apology. I’ve been working on this post and left it on my computer, open, for the last several days, because I could not get back to it. I’ve been traveling, which is a large part of my job.

But I am here, now, for a few days, and I intend to finish up some of my other blog posts, and get them on here. I’ve also answered a couple of comments in the blog, from Francesco and Paul. Please see my answers there.
]]>Let’s Talk About GoitersMelanie ChildersNone2010-06-28T07:56:41+01:00http://www.sharktank.org/melanie_childers/files/9538a04aaee47055eaaef6841c869608-40.php#unique-entry-id-40http://www.sharktank.org/melanie_childers/files/9538a04aaee47055eaaef6841c869608-40.php#unique-entry-id-40
What are goiters and why do they come into existence? A goiter is a swelling of the thyroid gland, in the neck. Sometimes, a goiter is also caused by nodules on the thyroid gland. (These are usually not cancerous.) Either way, you got a lump in your neck and that ain’t good.

Let’s talk about the thyroid hormone and the pathway by which it is regulated: It is a self-regulating process, involving a feedback loop.

Not enough iodine, which causes less T-4, which results in an increase in TSH.
IN RESPONSE to more TRH, the pituitary gland releases a hormone called thyroid stimulating hormone (TSH).
TSH causes the thyroid gland to trap iodine from the blood and this iodine is an essential component of thyroxine (T-4)
Thyroxine (T-4) can be converted into the biologically active form of hormone, called tri-iodothyronine (T-3).
This is the feedback loop: If you have enough T-4, the pituitary gland becomes less sensitive to TRH, and secretes less TSH, slowing down the cycle.

Now, suppose you can’t get enough iodine? What happens? The pathway is obstructed, of course. The system breaks down because the thyroid gland can’t trap iodine from the blood. You got the TSH,to TELL it to trap more iodine, but if the iodine ain’t there, it just ain’t there.

What is the result of this? The result is that you don’t have enough T-4, and of course, that you don’t have enough T-3, either.

What happens when you don’t have enough T-4? Well, the pituitary gland is secreting, probably, more TSH. And, in fact, this has been borne out by the research, specifically in terms of CF patients. [Eur. J. Pediatr. (1982) 138: 327-330] The same workers also showed that T-4 is lower in CF patients, which is exactly, again, what we would expect.

Now, hopefully, by now, you’re asking: Melanie, why are you centered on the lack of iodine causing these changes in the thyroid-hormone balance in CF patients? Well, because, as Philippe pointed out, the CFTR TRANSORTS IODIDE. And that’s exactly where the system breaks down in CF patients.

But, suppose you replace the CFTR with another protein that can transport the same things that the CFTR is supposed to transport? Such as iodine?

Now, remember why we are on this topic in the first place: Thiocyanates inhibit iodine uptake by the thyroid, leading to reduced iodination of amino acids, which results in the decreased production of T4.

So, with isothiocyanate, you’d end up with more iodine output into the extracellular space, where the blood cells can pick it up, but you also don’t have as much uptake by the thyroid, of the iodine. Do they balance each other out? I don’t know.

You’ve got two different mechanisms here, causing the same thing:

1. Not enough iodine causes not enough T4, which causes less T-4, which results in an increase in TSH.

2. Downregulation of uptake of iodine by the thyroid, caused by isothiocyanates, which causes less T-4, which results in an increase of TSH.

You end up with more iodine circulating, but less taken up. Are they a wash? I doubt it (most things aren’t).

Which one wins? Zinc wins, boys and girls. Zinc wins. You didn’t expect that answer, did you?

And, that will be the subject of one of my later blog posts. Here's a hint: BOTH of the two above RESULTS in less thyroxine (T-4). But what if the amount of thyroxine that you have is dependent on something else, too? Something else that is replaced (normal homeostasis is restored, in the case of CF) by having a functional ABC transporter protein, such as the MRP, in the place of the missing CFTR? Hmmm?

I want to apologize to you guys for having a dry spell here, and having to wait so long for another post. Sometimes I get real busy in my day job and it takes me away from CF for a little while.

In the meantime, peruse this VERY interesting article (WOW! That's a hell of a lot of goiters in CF patients treated with iodide, do you SUPPOSE that it's not the problem here? Duh :)

Volume 79, Issue 4, Pages 684-687 (October 1971)

Complications of iodide therapy in patients with cystic fibrosis*

*

William L. Nyhan (Editor), Harry C. Shirkey (Consultant), M.D. Thomas F. Dolan Jr., M.D. Lewis E. Gibson

Forty-seven of 55 patients receiving long-term daily iodile therapy as part of the therapeutic regimen for cystic fibrosis of the pancreas developed goiters. Goiters usually appeared 2 to 3 years after onset of therapy, with a range of 3 months to 12 years. Fourteen patients receiving iodides had laboratory or clinical evidence of hypothyroidism; 2 of these 14 did not have a goiter. No goiters were observed among 55 patients with cystic fibrosis not receiving iodides. Fourteen of 55 children receiving iodide therapy developed nasal polyposis whereas no polyps were seen in 40 patients not receiving iodide. This may be due to the age distribution of patients studied, since most children in the 5 to 15 year age range received iodide therapy.

]]>Trash cansMelanie ChildersNone2010-06-14T19:10:54+01:00http://www.sharktank.org/melanie_childers/files/73d20787ad413aa1d10e79553a1490f6-39.php#unique-entry-id-39http://www.sharktank.org/melanie_childers/files/73d20787ad413aa1d10e79553a1490f6-39.php#unique-entry-id-39
I told my daughter, a while back, that I loved trash cans. She thought that was really funny. I guess it is a bit funny, but let me tell you about trash cans, before you think I’m nuts—

When you can crumple something up in your hands, and throw it in the trash can, you are done with it. I like that. I truly enjoy throwing things into the trash can because that means I am done with it. It’s processed, stored, or just dealt with, and I don’t have to look at it again. It’s ACCOMPLISHED. That’s the main thing that I like. Throwing something into the trash can means that something has been accomplished. And I’m hooked on that feeling of accomplishment equally as much as I’m hooked on science.

You guys read my sad musings about my lost son, and I wonder if you might think that I am just walking around, hoping for rain. to hide my perpetual and everlasting tears. I’m not. And it’s not that I’m NOT sad about losing my son, or the suffering that he had to go through before he left this earthly plane of existence. I AM. But, like I told you before, pain comes in waves. They wash over you, and then, for a while, they are gone.

When they are washing over me, this blog is an outlet to get them out. Those posts are really hard for me to write, for some reason. The guy who posts my blog posts for me is forever getting requests from me not to post those posts yet, so that I can edit them, once again. I’d probably be editing forever if he didn’t stop me and just post the damn things.

I think that it’s important to write them, though. And, most of all, I want them OUT. I will never be able to crumple them up and put them in the proverbial trash can, but I can’t let them “cook” inside of me. But the reason that I want to edit them over and over again, is because I view this blog as something that serves hope, not despair. And, my CF family, I do not want you to take any sadness from my posts. I want you to have only hope. You have every reason in the world to justify much hope.

There are quite a few people taking BITC now. (I will say, here and now, that I hope that every one of them are using it under their doctor’s supervision.) Those taking it orally are experiencing the typical changes in fat content of their stools and they are, of course, able to lower their enzymes, in response to the effects of BITC on their pancreas. This is to be expected; it’s not something that we haven’t seen before. We are currently working on a website that these people can log on to anonymously, and that will chart certain indices, such as color and amount of stools, mucus production, etc. And there are some who are just beginning to use BITC transdermally. So far, so good.

It is difficult to report on any numbers, at this point. We don’t have the ability to do the lab tests, to get the numbers, and since so many have just started their use of BITC, not enough time has passed to get any lab reports from their doctors yet. And, the results of the carrier testing will not be out until mid-July. So, we are waiting.

As soon as I know, you will know. That’s a promise. So far, we’ve only seen the effects of transdermal BITC in one person. And one person does not prove anything at all, of course, but it gives us much to hope for. The one thing that I really like about it is that a CF patient over twenty years old has blown a lung function score of over 130%. It’s a regression back to over five years ago for this guy, in terms of lung function. I want that. I wanted it for my son, and I want it for the older CF patients now. And it’s what should happen, if you are able to replace the non-functioning CFTR protein with a functionally redundant protein, like the MRP2.

In the meantime, let me finish up by telling you that I am not some sad and lonely woman with no life. I have a beautiful and loving daughter, Penny, and a world-shaking miracle of a granddaughter, Aera Bella. When I am not doing this work, I have a growing business and career. I have a big sprawling family that I am honored to love and to have in my life. And, I have you, my CF family, and this work. In the little bit of spare time that I have left, I study physics and I paint oil paintings.

My life is full. It is only my heart that has a hole in it.
]]>How are the CFTR and the MRP different? (Or what are their substrates?)Melanie ChildersNone2010-06-07T07:20:34+01:00http://www.sharktank.org/melanie_childers/files/f9f7aa00cf3488e86ed549a695bb1413-38.php#unique-entry-id-38http://www.sharktank.org/melanie_childers/files/f9f7aa00cf3488e86ed549a695bb1413-38.php#unique-entry-id-38
Someone asked me today, in an email, if BITC would make their child taste less salty. I had to tell him that I did not know. But that got me to thinking about substrates again, and about the CFTR and the MRP proteins, themselves.

This is the thing, with the “salty” question—See the article below (and there’s another one using azithromycin, with which I’m not going to fill up this blog post.) These compounds, that are substrates of the MRP proteins, cause also chloride efflux. I don’t know, at what point, they do this, because they have other substrates, which are going to compete with chloride ions, dependent on the structure of the protein. But the CFTR, itself, also transports chloride anions ALONG with other substrates, such as glutathione. But how much? I don’t know. Are the amounts of chloride anions transported by each protein comparable? I don’t know. So, will the kid taste salty, if he takes BITC? I don’t know.

Biochem Pharmacol. 2004 Jul 15;68(2):253-61.

Increased chloride efflux in colchicine-resistant airway epithelial cell lines.
Dragomir A, Roomans GM.

Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden. anca.dragomir@medcellbiol.uu.se

Abstract
Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing. Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP. Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug. While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells. Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells. Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes. The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels. Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients.

Let’s think about glutathione adducts….Remember? I told you we were going to cover them.

Now, glutathione (GSH) adducts are compounds combined, bound, to GSH. And it is this form, an adduct, that is transported by the MRP proteins. What compounds bind to GSH and are transported by the MRP? Colchicine, yes. Maybe azithromycin, too. Certainly chemotoxins, [Lancet. 1997 Sep 6;350(9079):711-2.] but also, probably, the isothiocyanates. And, the oxidized form of GSH, glutathione disulfide, is also transported. What do all of these things have in common?

THEY ARE NOT ANIONIC COMPOUNDS. Adducts of GSH are not anionic. Neither is GSSG. Now, look again, at the Colchicine article above. See this: “…while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels.” Since we know that colchicine is transported by the MRP protein [Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8822-6.], and we know that the CFTR is responsible for cAMP-stimulated chloride efflux (transport out of the cell), then one of two things must be true:

1. MRP transports chloride ions along with other non-anionic compounds, and/or

2. MRP controls chloride transport via alternative chloride channels—(non CFTR, of course, but not directly transported by the MRP, either.)

I have no idea which it is, or whether it is both. How MUCH chloride it is responsible for moving out, I have no idea. I do know that chloride transport is important to neutrophil function, ie., the killing of bacteria (See the abstract below):

J Leukoc Biol. 2008 Jun;83(6):1345-53.

The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils.
Painter RG, Bonvillain RW, Valentine VG, Lombard GA, LaPlace SG, Nauseef WM, Wang G.

Gene Therapy Program, Department of Medicine and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.

Abstract
Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.

… but this is not the entire arsenal of the airways. It is highly engaged in CF, because of increased neutrophilic migration, but that is not something that should be, if you can pack some “big guns” in, elsewhere in the system.

I’m sorry to give you so many “I don’t know’s” in this blog post. That’s just the way it is, sometimes. In studying this disease, it is of first importance, the questions that you can ask. The more the merrier, as they say. It is begging for trouble if you decide on a course of inquiry and can’t get off of it because you think that you already have the answer. It is the pure idiot who thinks he/she knows everything.
]]>Stupid PeopleMelanie ChildersNone2010-06-07T07:23:32+01:00http://www.sharktank.org/melanie_childers/files/08fcd7b4d3f9930c6a631b3210f085c5-37.php#unique-entry-id-37http://www.sharktank.org/melanie_childers/files/08fcd7b4d3f9930c6a631b3210f085c5-37.php#unique-entry-id-37
And then there are the people who try to tell you that you “must let go.” Let go of what, exactly? The love? The memories? What the hell are they talking about? And you know something? If pain were the only thing that I had left, from my life with my son, I’ll take it. I’ll take the pain. I’m not letting go of a damn thing. I’ve “let go” of enough.

My ex-boyfriend, Bubba (yes, that’s really his name; he was a Texan), told me, three days after my son’s funeral, “Melanie, it’s been three days; you should be over this by now.” Needless to say, the relationship didn’t last long after that.

He wasn’t much different from the well-meaning idiots who ask you, “How long has it been now?” and then tell you that that’s long enough. Oh! Okay. Let me just turn this thing here to the “off” setting and we’re done. It’s like a pot of beans. When it’s done, it’s done. Cooked long enough. Time to “let go.”

These people have one thing in common, besides stupidity: cowardice. They can’t stand to see the pain so they try to discount it. “Oh, but they mean well,” my sister tells me. NO, they DON’T “mean well.” They are cowards. And thoughtless assholes. Your pain is something that they don’t want to come face to face with, because if they do, they might have to imagine that they’ll have to face it, too, one day. They might have to recognize that there are some things that can’t be fixed; that have no solution; that the only thing that they can do that will make any difference is to share your pain.

The shortest verse in the Bible is “Jesus wept.” [John 11:35] He was weeping with the people; sharing their pain. Cowards can’t do that; they won’t do it. And STUPID people show their cowardice by saying things like “Oh, he’s in a better place now.” Or, they tell you that it’s been long enough, and it’s time to “let go.”

No, it’s not time to “let go.” And it never will be. So, the next time you think about “comforting” someone who has lost a child, or another family member, and these words of “comfort” come to mind? Just shut the hell up, okay?
]]>The BoxMelanie ChildersNone2010-06-03T14:58:28+01:00http://www.sharktank.org/melanie_childers/files/21195ac1781a3e3ab6f4e0fa642057d1-36.php#unique-entry-id-36http://www.sharktank.org/melanie_childers/files/21195ac1781a3e3ab6f4e0fa642057d1-36.php#unique-entry-id-36
I have this truck; it’s a big truck. I don’t really care for it, because I can’t park it very well. I always end up in two parking spaces, kind of swayed over into someone else’s space, at the back. Actually, I’m not a good driver, either. Everyone remarks on it. And lots of people give me the “one finger salute” on the freeway. . I’ve run over the tops of two cars backing that stupid thing up. There’s a lot of “Oh My God!” from my passengers, too. In fact, now that I come to think of it, I don’t get that many passengers volunteering to ride with me.

Anyway, I got this truck. And my big brother likes that truck. I leave it at his house, near Houston, when I have to fly across the country and stay for a few months, on business. And, while it’s there, he cleans it up. I don’t like cleaning it, either. To me, it’s just a truck. I keep all kinds of things in it, and it often needs to be cleaned out. But my brother cleans it for me, when I leave it at his house. He’s a Texan and he likes trucks.

There’s this box in the truck. I travel with that box. It’s just a cardboard box, sealed up with tape. On the top and on the sides, I wrote on it: “Keith’s Stuff.” It’s not a big box. About the size of a couple of shoe boxes, put together. But it’s got some of Keith’s stuff in it. A couple of his shirts, a pair of blue jeans he always wore. Even a used Kleenex I can’t seem to part with. That box is always in my car, whatever car I have at the moment, or truck, as the case has been for the last few years.

Anyway, the box broke. Split at the seams. I guess it had something heavy on it, and the seams gave way. My brother knew enough not to touch it. He left it on the backseat, seams split, contents about to tumble out.

So, I got another box, a new one. And I’ve got it in the living room of my RV right now.
Tomorrow, I will take those precious things out of the crumpled and broken box, and I will pack them again, in a new box. And I will write “Keith’s Stuff” on the new box and put it back in my truck.

I don’t know how the contents of that box are faring, though. One of the pieces of paper that I saved looks like it’s about to fall apart; IS falling apart. But there is a shirt in there; I saw it when I brought it into the house tonight. Keith’s shirt, one he always liked to wear; one he looked particularly good in. I recognized the pattern of the material and I could almost see him again, wearing it.

And there’s the first alcoholic drink he ever bought. We agreed that we would save it, to commemorate the occasion. A Zima beer. I saw it, caressed by old yellowing newspapers, it’s old and yellow now, too. I remember Keith had it on his coffee table, in his apartment. I had bought him a small Christmas tree, a Santa Claus attached to it, with a tape recorded message that would play anytime someone moved near it. Keith said that he was going to change the message to: “Move away from the beer. Step away from the beer.” I wish he had. I would have saved that, too. How I would love to hear his voice again.

All of it put together probably wouldn’t fetch ten cents at a garage sale, but it’s more precious to me than oceans of gold. It’s as close as I will ever get to my son again, in this lifetime. And, somehow, tomorrow, before I set off on the road, in my truck again, I have to go through that box, and put everything back together, without leaving a mark on it. It has to be as close as it can be to the way that it was when he was here.

It just has to.
]]>It’s Time for a BreakMelanie ChildersNone2010-06-03T14:57:54+01:00http://www.sharktank.org/melanie_childers/files/06e4b0fdc972d574cd921f72b89392c6-35.php#unique-entry-id-35http://www.sharktank.org/melanie_childers/files/06e4b0fdc972d574cd921f72b89392c6-35.php#unique-entry-id-35
Let me give you some background—I was the next to the youngest out of seven children. A child of divorce. Well, actually, the child of quite a few divorces. My mother, God bless her soul, was born before her time. She was brought up during a time when women were not supposed to be smart. She didn’t go to college until she was in her forties, and then she majored in math. She actually worked for NASA in the control room, doing something having to do with math for a few years. She loved math. When most people were watching television, right before bed, my mother was sitting, propped up on pillows, in her bed, working math problems. She was quite strange in that way.

But, mostly, she married. And married, and married. And married. I think, at last count, she had married seven times. I think that the state of Texas only allows you to marry seven times. At any rate, her last marriage was performed in Louisiana.

She was always looking for love, my mother was. I don’t know if she ever found it, except maybe with my dad, who died when I was barely six years old. So, for the most part, I was a child of a single parent, with a parade of sometimes abusive stepfathers, coming and going.

I fell in love with reading at a young age. My mother had a set of encyclopedias that she kept on the bottom shelf of the bookcase, and as soon as I learned to read, I read those encyclopedias. My mother always had five or six books that she was reading, at one time. She’d go from one to another, until she finished them all. I cannot imagine a house without books.

I finished high school after almost dropping out when I decided to skip the whole first semester of my senior year, hanging out with a boyfriend who was already out of school. My mother badgered the school officials to let me finish my senior year, although I got all “F’s” for the first semester (you could only have so many unexcused absences in those days, and they failed you for the semester, no matter how good your grades were), and all “A’s” the second semester, finishing out with a “C” average, and graduating with my class.

I got a scholarship to go to college. I don’t know whether it was based on need or on aptitude, but I was corralled by my counselor to take some tests, along with an auditorium full of other students, and I’ll be darned if I didn’t win the damned thing.

I did not want to go to college. And, of course, the first semester of college, I just didn’t go. Instead, I met a guy, a few years older than me, and got married. My mother had taught me by her actions, if not by her words, that I needed a man, in order to be a person. Three years later, I had two children, less than a year apart, and five years later, I was a single parent.

When my kids were growing up, the laws were not so stringent on fathers who did not pay child support, and I was never paid a penny. Instead, I worked constantly, sometimes three jobs at a time, in order to take care of my children. I am sure that many of you, while reading my posts here, will see regret, guilt, and remorse, written all over them. And, yes, I feel all of those things, for my children’s, and especially Keith’s, childhood. It was very rough; we were constantly in survival mode.

When I was in my thirties, I decided to go back to school. I finished a four year degree in four years, at the very top of my class, and won another scholarship—to law school. By this time, I was sick, again, of school, and dropped out after my first year.

I got a job as a bill collector and made some pretty good money. For the first time in my life, I was able to support myself and my children by working just one job. But, by this time, Keith was getting sicker. And, so, of course, I began to study cystic fibrosis. It wasn’t something that I wanted to study. It was something that I had to study. You all know how that turned out. Keith died, and I’m still here, still studying cystic fibrosis.

After a few years in the collections industry, I got a call from a man for whom I had worked part time in sales. He had a new product, and he wanted me to help him sell it. It was a good product, in an industry that was desperate for it, and I learned everything that I could about selling it. After a few years, I became so successful, I forced the owner of the company to make me his partner. And, like many commercial enterprises, when you have a good product, and you do a good, honest job, you grow. And my company is growing very fast now. It’s good to see it. It’s good to look behind me and know that I did this.

I’m not sure when I crossed over the line and became a workaholic. If Keith were still alive, I’d seek out therapy. But, because he’s not, I need work too much, now, to try to do anything about my addiction to it.

Whether it’s work for money, or work for CF—it’s like my and my mother’s five and six books at a time. You get to a place in one, you start on another, and then you move on to another and another and another, and then back to the first one, or the second, or the fourth one, until you’ve read them all, and you start on a new batch of books. I don’t read many books anymore; I read about systems having to do with CF.

I’m not a genius. I’m not a scientist. I’m probably only moderately intelligent. And the only reason I’ve been able to reach the place I’ve reached before anyone else could get there, is because I never give up and I never stop, and every spare minute has to be filled with work.

Now, excuse me, I have to get back to work. A very nice gentleman sent me a book that is likely to teach me some things that I don’t know about the lactoperoxidase system (Remember that I did not understand the relationship between iodine and thiocyanate?), and I’m making notes on it. When I am done with it, I will explain it to you.

]]>To Philippe and FrancescoMelanie ChildersNone2010-05-26T19:14:52+01:00http://www.sharktank.org/melanie_childers/files/042280c1cf0459386a4ceaa45f05762b-34.php#unique-entry-id-34http://www.sharktank.org/melanie_childers/files/042280c1cf0459386a4ceaa45f05762b-34.php#unique-entry-id-34
Your comments and your questions are so interesting, though, they beg for a response. So, I will answer them here. You comments are listed as such, and my answers are below them:

Philippe
Melanie, don't forget I- and Br- (2 halogens). I- is not in big amount but, imporatnt to manage SCN.
2 days ago, 1:55:01 AM

Francesco
Hallo Melanie,
On this argument, i found that prof. Galietta from Italy have already understood some of the mechanisms. In the paper "Thiocyanate Transport in Resting and IL-4-Stimulated Human Bronchial Epithelial Cells: Role of Pendrin and Anion Channels" they found out that Cl- and SCN- are probably transported by the same protein, that they call Pendrin. This protein is stimulated by Interleukine4 (IL4) (a cytokine). this study underline again that SCN is important with lactoperoxydase as antibacterial system.
3 days ago, 2:53:39 PM

Francesco
This might be usefull:
http://www.jimmunol.org/cgi/content/full/181/7/4883 talks about what you illustrated us about the system LPO - SCN -H2O2 (the latter stimulated by IL-13 IL-4). And talks about pyocyanine produced by Pseudomonas.A. and its effect on the h2o2 depletion.

http://www.jimmunol.org/cgi/content/full/181/3/2203
this instead talks about the role of Pendrin in ASthma and the Role of stimulation of pendrin by Il-4 and by Il-13 on the ASL.

To Philippe, first: I was looking at an abstract, the other day, where the authors showed that iodide efflux via CFTR was increased in response to the LPS (a component of the bacterial wall) from the pathogen, PA. What is interesting is that the iodide efflux, under these conditions, is time and dose-dependent. See:

http://www.ncbi.nlm.nih.gov/pubmed/20346919

I had not paid much attention to iodide efflux previously, at all. Do you suppose that this is the purpose of it? To “manage” thiocyanate (SCN-)? If so, please explain this mechanism. The authors of the abstract linked above think it has something to do with phagocytosis.

To Francesco: The article that you refer to:
“Thiocyanate Transport in Resting and IL-4-Stimulated Human Bronchial Epithelial Cells: Role of Pendrin and Anion Channels” [J Immunol. 2007 Apr 15;178(8):5144-53 ] is not about the CFTR transport of thiocyanate. It refers to a different anion channel that the CFTR probably controls indirectly, from a family of transporters in the SLC26 class. See: EMBO J. 2002 Nov 1;21(21):5662-72. This is the same mechanism which controls HCO3- transport.

As to the second article you mentioned:
The Pseudomonas Toxin Pyocyanin Inhibits the Dual Oxidase-Based Antimicrobial System as It Imposes Oxidative Stress on Airway Epithelial Cells1 [The Journal of Immunology, 2008, 181, 4883 -4893],
the jury is still out on whether CF patients have not enough H2O2, or too much of it, and where this excess is located, ie., intracellular or extracellular. Look at this: PLoS One. 2008;3(10):e3367. “Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production.” And this: Eur Respir J. 2000 Jul;16(1):95-100.

“Hydrogen peroxide and nitric oxide in exhaled air of children with cystic fibrosis during antibiotic treatment.”
I don’t agree that you can assume that pyocyanin, from PA, inhibits Duox enough to deprive lactoperoxidase of enough of it’s substrate (H2O2), to say that H2O2 and it’s precursor, superoxide anion, are not major contributors to the inflammatory component of this disease.

I can’t find it right this moment, but someone on Sharktank recently posted an article on the issue of whether infection or inflammation comes first in CF. And inflammation was the winner. I think that it is very important to remember that thiocyanate not only has antibacterial effects in it’s oxidized form (OSCN), but that unoxidized, it acts as a buffer for these oxidants.

Hey, on a side note, are you guys from Alaxia Biotech? Your names seem familiar….

]]>ExperimentsMelanie ChildersNone2010-05-28T14:32:57+01:00http://www.sharktank.org/melanie_childers/files/c00b9959cad984ec4f1b340b13d30f25-33.php#unique-entry-id-33http://www.sharktank.org/melanie_childers/files/c00b9959cad984ec4f1b340b13d30f25-33.php#unique-entry-id-33
Don’t get me wrong—we were sure of our theories. I check, recheck, check again, recheck again, check another time, recheck my recheck. I’ve got whole entire abstracts memorized, I’ve checked so many times. I’m continually checking, because I am so scared of hurting someone. But, no matter how much checking you do, theories are often just the starting point, and you have to go back to the proverbial drawing board, over and over again, usually, no matter how good your theory is. It is, in fact, very unusual, to hit the jackpot and be right the first time around. That old adage, “A little knowledge is a dangerous thing,” rings very true, when it comes to theories.

Sooner or later, though, you come to a point where you have to test. No one in the mainstream scientific world was paying attention to our work. Oh, of course, there were some scientist writing to us their congratulations on our first paper. I guess they didn’t know who we were and that we had no credentials. But aside from that, no one was really interested. And, I think, too, that no one wants to test someone else’s work, because it costs money to test, and they couldn’t make money off of a patent that they didn’t have. So, we had to do the testing ourselves.

And we didn’t want to test on CF patients, so we tested on the next best thing: a carrier. But, what to test for? Carriers are not sick. Aside from the problems with the vas deferens, in some heterozygotes, carriers don’t have the pathology that’s seen in CF patients. And, of course, we don’t have the ability to test for potential difference, or chloride ion transport, or any of the other anions that are moved through the MRP or the CFTR. What to test?

We searched around the databases and saw that, sometimes, carriers had different fatty acid profiles than normal people. I don’t want to go too far into the reasons why, but it has to do with zinc transport to certain proteins that are responsible for the processing of fatty acids out of the membranes of the cells. Because the zinc homeostasis is screwed up in CF, the proteins that are responsible for the processing of arachidonic acid (AA) and docosahexaenoic acid (DHA) don’t work properly. Zinc is supposed to fit into one inhibitory spot on the protein that moves AA out of the membrane and it’s supposed to be picked up by the protein responsible for moving DHA out of the membrane. These proteins don’t work right because zinc homeostasis is messed up in CF. The end result is that you have too much AA, which is pro-inflammatory, and you don’t have enough DHA, which competes with AA for the cyclo-oxygenase cycle which, in turn, results in more pro-inflammatory compounds, prostaglandin E2 and leukotriennes. Long story, short, CF patients have more inflammation because of this. Now, don’t go and rush out and buy DHA. It helps, but because it is way down in the pathway for inflammation, and there are other factors that increase inflammation, it doesn’t work THAT well. And it doesn’t address the core problem. But, suffice it to say that, if you can effect a change in the fatty acid profile of a carrier, and especially in terms of these two fatty acids, you know that your compound is doing what it’s supposed to do.

So, we decided to test the fatty acid levels of our carrier. To start, he went in to his doctor and got some blood drawn, and sent it out to the lab, to get a fatty acid profile done on it. That gave us a baseline.

Then, he took BITC for one week. And he had his blood drawn again, and a fatty acid profile done on it. No change. So, he increased the dose – doubled it—for one week. Then, he had his blood drawn again, and the fatty acid profile completed on it. Voila!

Guess what happened? Can anybody out there guess? Which one went up, and which one went down?
]]>Substrates and ConjugatesMelanie ChildersNone2010-05-17T14:53:34+01:00http://www.sharktank.org/melanie_childers/files/fe5923f56111cdf500281424137007a0-31.php#unique-entry-id-31http://www.sharktank.org/melanie_childers/files/fe5923f56111cdf500281424137007a0-31.php#unique-entry-id-31
Substrates are the substances that a protein, or an enzyme, acts upon. If you have a protein, like the CFTR and the MRP, that is supposed to transport a substance across a membrane, into the extracellular space, then it’s substrates are the substances that it transports. For instance, the CFTR transports glutathione, while the MRP transports glutathione attached to another compound, such as a chemotoxin. Both glutathione and glutathione attached to another compound are the substrates of the proteins CFTR and MRP, but the latter—glutathione attached to a chemotoxin—is both a substrate and a conjugate.

In the case of the CFTR protein, it’s substrates are all anions—negatively charged ions. In the case of MRP, it’s substrates are usually the same anions that are transported by the CFTR protein, but they are attached to another compound. Together, the anion and the other compound, are conjugates. Another term for these conjugates is “adducts.” For instance, when you have a glutathione adduct of a chemotoxin, you call it the glutathione adduct of whatever chemotoxin you are talking about. You can have the glutathione adduct of any compound that will accept the extra electron on glutathione. Once they form a bond, they are a conjugate, or an adduct.

The boy who had both CF and cancer; the one who started us all off on this venture, was taking chemotoxins. His cells began to express MRP proteins, because of these chemotoxins, and the glutathione in his cells formed conjugates with the chemotoxins and these conjugates were transported out of the cell by the MRP proteins.

The cool thing about this is that the MRP proteins also have, as substrates, the oxidized form of glutathione, called glutathione disulfide, or GSSG. And, in the place that the GSSG is transported, the extracellular space (the airways), there are chemicals that turn GSSG right back into it’s reduced form: GSH. So, when the boy who was taking chemotoxins to fight his cancer started making MRP proteins, and those proteins started transporting glutathione adducts of the chemotoxins, they also transported GSSG. And, once that GSSG got to the extracellular space, it was turned right back into GSH.

That’s the idea behind aerosolizing GSH into the airways. But, it’s not a good idea. Why? Because the amount of GSH in the airways is up and down, depending on other conditions. When you aerosolize it, you are giving GSH not just to the CF patient, but you are giving it to all of the pathogens that use it to conjugate antibiotics that are supposed to kill the pathogen. The pathogen uses it, too, especially if there is a lot of it out there. Normally, the amount of GSH in the airways would be regulated by pump proteins, like the CFTR and the MRP. But, that regulation is gone, if you aerosolize it directly into the airways.

Once you have those proteins in the membrane of the cell, you can pump out not just GSH and GSH conjugates, but all of the substances that are substrates of that protein; and they compete with each other. You won’t be just pumping out GSH conjugates, but also sulfate conjugates, thiocyanate, bicarbonate, glucuronic ion and chloride. The trick is to get those proteins into the membrane of the cell.

In CF, the CFTR is mutated, and in most cases, never makes it out of the organelle that it’s made in—the endoplasmic reticulum—because it’s folded improperly and the chemical messengers that take it through the endoplasmic reticulum, to get it matured, sense that it is not folded properly, and hold it back there, to be chopped up and destroyed there. It never “traffics” to the membrane, where it’s supposed to sit and do it’s job. But this is not the case with the MRP proteins. They make it to the membrane.

And, once there, they use what would normally be CFTR substrates to combine with other substances, to form conjugates, and these conjugates are pumped out via the MRP. The great thing about this is that those CFTR substrates are still pumped out to the airways, in a different (conjugate) form, but they are still getting to where they belong.
]]>SubstratesMelanie ChildersNone2010-05-17T09:17:28+01:00http://www.sharktank.org/melanie_childers/files/a71424d3d48830dcfcb8a45e09821439-30.php#unique-entry-id-30http://www.sharktank.org/melanie_childers/files/a71424d3d48830dcfcb8a45e09821439-30.php#unique-entry-id-30
The current literature tells us that it transports:

1. chloride ions (Cl-)
2. thiocyanate (SCN-)
3. bicarbonate (HCO3-)
4. glutathione (GSH)

But. Let’s look at the literature on MRP—

First of all, it is known for its role in detoxification processes. What are these processes?

Well, there’s Phase I, and Phase II, and then there’s Phase III antiporter processes. It is the latter two that we are interested in, when it comes to the function of MRP proteins. The Phase II detoxification process is, first and foremost, a conjugation process. (Conjugation is where two compounds are joined together, to form another compound.) That compound is then transported out of the cell by the Phase III antiporter process—it’s pumped out, in other words.

So, what this means is that something is conjugated with, most likely, an anion, and then the MRP pumps it out of the cell. Let’s address the current literature, to see if the above listed anions are used in this process:

Chloride – There’s not much on this topic—MRP (or MDR, which is sometimes used interchangeably, with MRP, to refer to multi-drug resistant proteins) and chloride transport. There is this:

Am J Physiol. 1996 May;270(5 Pt 1):C1370-8.

P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody.
Han ES, Vanoye CG, Altenberg GA, Reuss L.

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.

Abstract
The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.

And this:

Phys Med Biol. 2008 Jan 21;53(2):N1-7. Epub 2007 Dec 28.

Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells.
Duncan L, Shelmerdine H, Hughes MP, Coley HM, Hübner Y, Labeed FH.

Centre for Biomedical Engineering, School of Engineering (H5), University of Surrey, Guildford GU27XH, UK.

Abstract
Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.

Which tells us that the multi-drug transport proteins are involved in ion transport, and probably chloride ion transport, but that’s not really the question that we want answered. We want to know if chloride is conjugated to any other compound, and then transported out. Chloride transport mediated by multidrug resistance proteins was of interest, first, in cancer therapy, when it was noticed that cancer patients who were given chemotoxins had changes in their volume activated chloride channel activity, but this tells us nothing about chloride as a conjugate. Perhaps it is simply transported by the MRP proteins, while the protein is transporting conjugates. In fact, that would seem to be the case—

Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L24-30.

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.
Gao L, Broughman JR, Iwamoto T, Tomich JM, Venglarik CJ, Forman HJ.

Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

Abstract
Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value),
whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.

Now, this doesn’t mean that chloride is moved out along with a conjugate, specifically. And it certainly does not mean that chloride is conjugated with anything else, and is then transported by the MRP protein. But what it does mean is that where glutathione goes, so go chloride ions. They share a transport mechanism; and probably are both substrates of the same transport protein.

I will leave you here, for now, with those thoughts. The next time, I will cover glutathione and it’s conjugates.
]]> “Hey, everybody! Look at the fat guy with the groceries!”Melanie ChildersNone2010-05-09T17:34:46+01:00http://www.sharktank.org/melanie_childers/files/e80a35b0594afb672e092b90fdb5ac4d-29.php#unique-entry-id-29http://www.sharktank.org/melanie_childers/files/e80a35b0594afb672e092b90fdb5ac4d-29.php#unique-entry-id-29
Anyway, I thought I’d write a little about Keith. Just to give you a hint of his personality. So, here goes:

At school, Keith had to have bodyguards. Seriously. Walking from class to class, in the lunchroom, everywhere. I let the teachers and the administrators at the school know that, if my son was hurt, they would pay big time.

Why, you ask, would someone hurt him? Well, because Keith was tiny, for his age, but his mouth was really big. He didn’t mince words, and he didn’t consider the size of those he was talking to, when he decided he needed to say something. He was just like that. The acidity of his wit could curl your toenails off.

When he went camping with my brother and his boys, one summer, my brother stopped at the store and was buying groceries for the trip, and he left the boys in the truck, to wait for him. My brother is a big guy; both tall and overweight. He looms.

Keith, waiting in the truck, saw my brother exiting the grocery store and coming into the parking lot, and yelled out, “Hey, everybody! Look at the fat guy with the groceries!” My brother’s kids were astounded that Keith would have the nerve to do this to their father, but Keith got a big kick out of it. And so did my brother.

I don’t know why he didn’t have a healthy fear of those who were larger than him, but he didn’t. Or maybe he did, but he just had to speak his mind. He wasn’t fearful, either, of anyone in authority. He was in a special class at school, for kids who acted up (of course) and when the teacher’s aide (a total idiot) told the boys that they stunk and that they needed to go home that night and take a bath, Keith told her to go “f___ herself.” She sent him to the principal’s office, where Keith told the principal that if he, the principal, had told that him he stunk, then he (Keith) would tell him to go “f___ himself,” too. Strangely enough, the principal agreed and sent him back to class.

Anyway, that’s just a little flavor of my son, Keith. I wish you could have known him.
]]>Another 27 MilligramsMelanie ChildersNone2010-05-08T18:38:05+01:00http://www.sharktank.org/melanie_childers/files/2fefcf6312eaf6ca08d936e0844aab71-28.php#unique-entry-id-28http://www.sharktank.org/melanie_childers/files/2fefcf6312eaf6ca08d936e0844aab71-28.php#unique-entry-id-28
First of all, let me digress a little bit and tell you about our experiences with oral benzyl isothiocyanate—

To start out, let me tell you that, as far as I know, everyone who took this dietary supplement took it under the care and supervision of their personal physicians. It is not the purpose of Sharktank, or myself, to undertake the medical care of CF patients. If a patient wants to change their treatment regimen, that’s their and their physician’s business. But, the fact is that they do it, and they often fill us in on the details.

When Keith was in the hospital, I often found myself out on a back step of some door of the hospital that no one ever went into, and that was always locked, smoking cigarettes, and thinking about the science of CF. I knew that my son was so damaged by the disease that his only hope of surviving was going to be as if some angel came down from the sky and passed a wand over him (mixing my metaphors a bit here, I admit) and said, “Poof! You no longer have cystic fibrosis.” That’s what I was searching for, you see; something to stop the ravages of the disease and let him heal.

Well, of course, we all know, now, that that didn’t happen, but we must ask ourselves what would the outcome be, if it had happened? Would he, all of a sudden, have been a normal kid, rising up out of his sickbed, and running through the nights, partying with his friends?

Of course not. Even if the “Poof!” magical wand had passed over him, he would have still been damaged. His lungs, especially, and of course, his pancreas, to a degree.

Let us talk about the pancreas, first, and then we’ll get to the lungs. The pancreas has two types of functions: endocrine and exocrine. Both are affected in CF, to varying degrees, but it is the exocrine function that we will discuss first. Anyone who takes pancreatic enzymes knows that the purpose of these is to replace the fat metabolizing enzymes that the CF pancreas cannot excrete. The longer you have the disease, the more enzymes you have to take to digest the fat in your diet, because the pancreatic cells that perform the exocrine function are slowly being destroyed. So, if you have cystic fibrosis and you get the “Poof!” wand passed over you, you’re still going to need to take pancreatic enzymes, depending on how many of those cells have been destroyed. It largely depends on how old you are, as to how much of the exocrine function has been destroyed. You will get some back, because the body has a way of healing itself. But never all of it. And, certainly, no matter how much you get back, it is dependent on the time you spend healing. And, of course, healing can only begin to take effect once the conditions causing the damage are stopped. You gotta have the “Poof!” first.

Now, the endocrine function of the pancreas—if you have CF and CF-related diabetes, you’re familiar with this function. You can’t damage one kind of cell in the pancreas, without also damaging others. When enough of the cells responsible for the endocrine function of the pancreas are damaged, they stop producing insulin (and glucagon, too, but that’s a subject for another day). I suspect that the lack of insulin production by the pancreatic B cells has more to do with the nerve cells than it does with the B cells, themselves, because certain drug treatments have been known to restore insulin expression in these tissues, and bariatric surgery has been shown to be curative, in many cases of non-insulin dependent diabetes, but again, that’s the subject for another day. The reason that I mention this, though, is because I suspect, as well, that at least some insulin expression might be restored, if the “Poof!” can take place in CF patients. I have concrete reasons to suspect this, but they are the subject of another paper, that I will write in the future, so I won’t talk about them here.

And, now, to the lungs. Obviously, as we’ve all been told, time and time again, once you do damage to the lungs, it’s done. There’s no going back. And cystic fibrosis is a disease of the lower airways—the alveoli, which you can picture as little machines for oxygen exchange—are damaged beyond repair.

But, if you were to take the alveoli out and stretch them on a flat surface, you’ve have enough to cover at least one tennis court. Lots to work with there. And where there’s life, there’s hope. The human body has enormous restorative and compensatory abilities. If you’re lucky enough to experience the “Poof!” and you’re still alive and functioning, you’re going to undergo a healing process and get at least some of the function back. What you don’t get back, you’ll compensate for. Depending on how long you’ve been sick and the damage done, though, you’ll never get it all back. I didn’t see my son running marathon races, if that angel with the wand had come down, but what I did envision was him coughing up all that gunk that was in his lungs.

And that’s exactly what happened when CF patients took oral benzyl isothiocyanate. Their enzyme intake had to be radically lowered. The ones that had CF-related diabetes began experiencing normal blood glucose levels. And they coughed up some really old crap from their lungs (they described it, variously, as a “flushing out” and as a “flushing of the toilet.” Heh.). They got their “Poof!” It lasted a couple of weeks, and then it stopped. The reason that it stopped is the same reason that azithromycin and colchicine, administered orally, don’t do the complete job of inducing a functionally redundant protein in CF patients—the liver proteins, and particularly, MRP2, filters these compounds out of the bloodstream, before they can induce these same proteins in the lungs.

So, what does “27 milligrams” have to do with anything here? Twenty seven milligrams is the amount of benzyl isothiocyanate that caused the “Poof!” in an 80kg CF patient, when administered transdermally. And it continues to do so, even after more than three months. Why doesn’t it stop, like the oral benzyl isothiocyanate stopped? Because it bypasses first pass hepatic metabolism and goes directly into the bloodstream.

Poof!
]]>27 MilligramsMelanie ChildersNone2010-05-08T18:34:48+01:00http://www.sharktank.org/melanie_childers/files/c1c68f8f822229e13b4d2d533e4b3a31-27.php#unique-entry-id-27http://www.sharktank.org/melanie_childers/files/c1c68f8f822229e13b4d2d533e4b3a31-27.php#unique-entry-id-27
Antimicrob Agents Chemother. 2007 Mar;51(3):975-81. Epub 2007 Jan 8.

Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.
Cigana C, Assael BM, Melotti P.

Cystic Fibrosis Center-Azienda Ospedaliera di Verona, piazzale Stefani 1, 37126 Verona, Italy.

Abstract
Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-kappaB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-alpha mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-alpha mRNA levels (P < 0.05) and a 45% decrease in TNF-alpha secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-kappaB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-alpha and propose inhibitions of NF-kappaB and Sp1 DNA binding as possible mechanisms of this effect.

Levels of tumor necrosis factor – alpha (TNF-a) and the activity of SP-1 (SP-1 is affected by zinc homeostasis, which is, in turn, affected by glutathione transport –or non-transport in the case of CF) are just two of the players that affect inflammation in CF lung disease. You can’t look at this disease in terms of inflammation and not see the effects of those components on increasing inflammation. And AZM tamps them down. What could AZM be doing that would cause that?

Well, I’ll give you a little hint: AZM also increases chloride ion transport. That should give you an idea right there. If you are increasing chloride transport in these cells, and you’re tamping down inflammatory factors, chances are pretty good that you’re inducing a protein that it functionally redundant to the CFTR protein, such as the MRP proteins. And here you go, boys and girls:

J Chemother. 2005 Aug;17(4):393-400.

Long-term azithromycin in cystic fibrosis: another possible mechanism of action?
Pradal U, Delmarco A, Morganti M, Cipolli M, Mini E, Cazzola G.

Cystic Fibrosis Center, Azienda Ospedaliera di Verona, Verona, Italy. ugo.pradal@azosp.vr.it

Abstract
Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are not completely understood. Besides its antiinflammatory and antimicrobial activities, one possibility could be the overexpression induction of the multidrug resistance-associated protein (MRP), which could affect chloride transport, thus overcoming the ion transport defect of cystic fibrosis. Seven patients were evaluated before and after 4 weeks of azithromycin treatment (500 mg once daily). Ion transport was studied in vivo by measuring nasal potential difference (NPD). MRP mRNA expression was studied in nasal cells by an internal standard-based semiquantitative RT-PCR assay. NPD was consistent with cystic fibrosis before treatment. After azithromycin treatment, sodium transport was still impaired, whereas a significant increase in chloride conductance was observed (p = 0.03). A significant direct correlation was found between MRP mRNA expression levels and NPD chloride response after azithromycin treatment (p = 0.04, r = 0.78). In conclusion, azithromycin may induce MRP overexpression and restore chloride conductance in the airways of cystic fibrosis patients. These findings suggest a new potential role of azithromycin in the treatment of cystic fibrosis pulmonary disease, i.e. the possibility to upregulate proteins whose function may, at least in part, compensate for the basic defect of cystic fibrosis.
Now, no doubt, AZM plays a role in the treatment of this disease. If it induces the expression of a functionally redundant protein, why are people who are on this drug still suffering exacerbations? Why are they still colonized with those same old nasty pathogens?

The answer to this question lies in the method of delivery of this drug. It’s given orally. And, anything given orally has to go through the liver—it’s called first pass hepatic metabolism. What this means is that if AZM can induce the expression of MRP in the lungs of CF patients (thereby replacing some of the missing CFTR protein with another protein that can do the same job), it can also induce it’s expression in the liver of these patients. Once that happens, less AZM can get into the bloodstream, and get to the lung cells, to induce the MRP. To overcome this, you have to get around first pass hepatic metabolism—in other words, get around the liver. The only way to do that is to administer the drug in a different manner—either IV or transdermally.

Obviously, you don’t want to go the IV route. Cystics have to do enough of that already, and although AZM does induce MRP expression, it is not necessarily the best inducer of it. The chemotoxins that were used in the young man to kill his cancer, that Lallemand wrote about, in 1997, are better at inducing MRP, and the induction lasts a lot longer, too. But, of course, you can’t take chemo drugs on a regular basis. They’re toxic, obviously.

Same story with oral colchicine as a therapy for CF patients. It induces functionally redundant proteins, and restores some of the chloride transport. See here:

Biochem Pharmacol. 2004 Jul 15;68(2):253-61.

Increased chloride efflux in colchicine-resistant airway epithelial cell lines.
Dragomir A, Roomans GM.

Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden. anca.dragomir@medcellbiol.uu.se

Abstract
Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing. Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP. Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug. While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells. Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells. Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes. The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels. Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients.

And here:

Mediators Inflamm. 1999;8(1):13-5.

Interest of colchicine for the treatment of cystic fibrosis patients. Preliminary report.
Sermet-Gaudelus I, Stoven V, Annereau JP, Witko-Sarsat V, Reinert P, Guyot M, Descamps-Latscha B, Lallemand JY, Lenoir G.

Service de Pédiatrie II, Hôpital Necker-Enfants Malades, Paris, France.

Abstract
Cystic fibrosis (CF) lung disease is characterized by persistent inflammation. Antiinflammatory drugs, such as corticosteroids and ibuprofen, have proved to slow the decline of pulmonary function although their use is limited because of frequent adverse events. We hypothesized that colchicine could be an alternative treatment because of its antiinflammatory properties and upregulatory effect on cystic fibrosis transmembrane regulator (CFTR) closely related proteins. We herein present results obtained in an open study of eight CF children treated with colchicine for at least 6 months. Clinical status was better in all patients and respiratory function tests significantly improved in five. Median duration of antibiotherapy decreased significantly. These preliminary results support our hypothesis of a beneficial effect of colchicine in CF patients and stress the need for a controlled therapeutic trial.

But, it’s got the same problems as any other ORAL drug that is used to induce MRP proteins—it also eventually induces these same proteins in the liver, which will then filter out the drug before it can get to the lungs.

So, hopefully, you’re curious enough by now, to ask yourself, “What the heck does ’27 milligrams’ have to do with anything I’ve read here so far”? Why that title?

Stay tuned, cowboys and Indians, and you’ll find out in the next blog post.

]]> I Am Not a ScientistMelanie ChildersNone2010-04-26T07:50:33+01:00http://www.sharktank.org/melanie_childers/files/8fb44ffaceef4e5c9a3de6bed57c8ed1-26.php#unique-entry-id-26http://www.sharktank.org/melanie_childers/files/8fb44ffaceef4e5c9a3de6bed57c8ed1-26.php#unique-entry-id-26
But I’m a voracious reader, and I’m a damned good guesser. And, once I’ve made a good guess, I can either find it borne out already, in the literature (which means that I’m catching up to mainstream science in one place), or others test it in the lab, after I write about it, and my guess was on the money (which means that I’m ahead of the curve). Or, there’s a third possibility, where I’m totally wrong. I learn from those situations. I make the mistake count, instead of ignoring it.

It’s pitiful, really, sometimes I find myself having “eureka moments” about things that scientists back in the 60’s have already figured out; stuff that is common knowledge, you know? In other words, I’m learning things the hard way. But it’s the only way I know of doing things. I can’t go back to school, at this point in my life, and learn all of these things (although, sometimes, I’m sorely tempted to do so), so I just read everything, old and new.

Have you ever had a “eureka moment’? That’s one thing that studying science does for you. It gives you thrills. It really does. When something clicks into place, and you can suddenly see things that you haven’t seen before, that someone before you saw, or that no one yet sees, you get that eureka moment. It’s like drinking twenty cups of coffee in ten minutes. It must be what runners get, when they feel euphoric, after running for a while. I’m totally hooked on it.

But I’m not a scientist. Remember that.

In this world that I am in, I am an outsider. I am not apprised of the consensual order of things. That is both a plus and a negative. I remember how long it took me to get it into my thick head that metallothionein could not be transported by the CFTR protein. And, to understand cyclo-adenosinemonophosphate’s role in the whole scheme of things (which, incidentally, CF scientists have known about for years, and I’ve only figured out sometime in the last three years). Pitiful.

But, it cuts both ways. Because I am not apprised of the consensual order of things, I am not boxed in to studying areas of this disease that are not directly related to stopping it. I don’t have to study PA. That’s not my life’s work. I don’t have to study zinc homeostasis, past the point that I’ve already studied it. I don’t need that job, because I’ve already got one that supplies for my basic needs. If I’m interested in any of the anions that are transported by the CFTR and the MRP protein, or if I’m interested in the proteins, themselves, I can pursue that interest. I am free, you see? And scientists are constrained by politics, by the pursuit of funds to carry on their research, and any number of other reins on their natural curiosity. I am not.

And neither are you. What is important here, given that particular situation, is the internet, and how the internet makes available and accessible the information that a patient might use to change their own lives; to buffer the ravages of a disease like cystic fibrosis, or even to ameliorate the ravages of the disease.

When people have that power over their own lives, and the way that they live them, they grab that power. They do so because they know, intuitively, that they are the only ones who will suffer if they do not, and that the people that they heretofore had handed these reins of power, will not, cannot, suffer as they do.

So, I am not a scientist. Neither are you, my CF family. But the information that you need to control your lives, and the course of your disease, is there, at your fingertips.

Use it. Shrug off the old paradigms of dependency; the authoritarian view that only the select few can understand what your body is doing to you. You are living this disease. Because of the internet, the science, no matter what you might label those who seek to consume it, now belongs not only to the select few, but to all of us. I am not the first to embark on such a journey.

And I will not be the last.

]]>Unfinished BusinessMelanie ChildersNone2010-04-26T07:47:44+01:00http://www.sharktank.org/melanie_childers/files/3fc321305c8f28e980b917855285e69f-25.php#unique-entry-id-25http://www.sharktank.org/melanie_childers/files/3fc321305c8f28e980b917855285e69f-25.php#unique-entry-id-25
My family hates this about me. They never see me. I Criss cross the country, not seeing what was my home except for a few minutes once or twice a year. Now, the road is my home. The money I make I spend on othersÆ needs. My family, first, of course. And then the occasional stranger that I meet along the way, who a small chunk of two or five hundred might help to dig out of a hole. I spread it around like the manure that it is, trying to make things grow; to make the world a little better place for my having been here.

It is not so much that I am altruistic. I'm just a pragmatist. Why do bad when you can do good? And why keep something that means so little, when it means so much to others, and really does them more good than it could ever do me now.

I've actually been called an angel a few times. Usually by people who don’t know me very well. My family and my friends, who have known me through the years, would very much disagree with that assessment. My enemies would, too. I consider anyone an enemy who has done something crappy to me. The crappier the deed, the longer I hunt them down. Ha!

If they screw me in business, I'll haunt them for life, sticking the proverbial knife in them at various moments of vulnerability, until I run them out of business. If they mess with my family, may God, Himself, be watching out for them.

There was a big brouhaha a few years ago, when the doctors at St. LukeÆs Episcopal Hospital tried to use the Texas Futile Care Law to disconnect my sister, Andrea Clark, from a respirator that she chose to be on. Having been around hospitals quite a bit, with Keith, I knew that the meeting that they were calling us in on, to Consult about AndreaÆs care, would be a tag team of hospital administrators, social workers, and doctors trying to talk my family into pulling the plug on my sister.

So, at the beginning of the meeting, I sweetly asked for all of their business cards, ostensibly so that I could remember who I was talking to. After I’d pocketed all of their business cards, I turned into the [insert expletive here] that I truly am, and told them that if they had an Ethics Committee meeting and decided to unplug my sister, my family would picket their hospital.

And we did just that. And I posted every single persons name in that meeting on the internet. Along with their phone numbers. And what they were trying to do to my sister.

Needless to say, Andreas doctor left town, the hospital did a one-eighty, and my sister lived for a few more weeks, without the Sword of Damocles hanging over her head. And with her family being able to say goodbye to her properly. I was her arrow. I shot straight and true, and I hit my mark.

I am your arrow, too. Shooting straight and clear, for my prize kill, for my bounty, CF. You can say that itÆs romantic, and all that crap, woman on a mission bunk. That's all bull. Whether you succeed or you fail comes down to one thing: how much and how hard you work at something. Focus and persistence wins the day, every time. Do the work? The answers will be there, if you do the work to find them.

Sharktank, and all who are members of it, have done that for you. They worked, right alongside me, for over a decade. With much hope, of course, but with the humility, too, to admit when we were wrong, and to seek the truth, only, and not some ego edification, or money at the end of a rainbow. We did it for our kids. We did it in love. The only question that remains now that we are on the brink of success, is what are YOU going to do?

Will you be an arrow, too? No matter how straight and true one's aim is, there's only so many arrows in the quiver, you know. We need your help. And, no, we are not asking for your money. We are asking you to talk to your doctor. Educate him or her about BITC. Ask for their help; their supervision, if you want to use transdermal BITC. And, first and foremost, educate yourselves.

I am about to post another entry on thiocyanate, itself, and it's role in this disease. It's going to put together quite a few things that we've already talked about. I've been working on it for a few weeks now, and it's a bit long, and an arduous read for the scientifically uninitiated. But, if you have to, read the former blog entries, because if you can "get" them, you can get the next one.

Be an arrow.
]]>The Rest of My LifeMelanie ChildersNone2010-04-26T07:47:22+01:00http://www.sharktank.org/melanie_childers/files/1f3b8fbe67259bfbf506497daeabf979-24.php#unique-entry-id-24http://www.sharktank.org/melanie_childers/files/1f3b8fbe67259bfbf506497daeabf979-24.php#unique-entry-id-24
The next day, I drove by her house until I saw that her car wasn’t in the driveway and I walked up on the porch and took out the little pane of glass in the front door, that was always loose. I reached in and opened the door from the inside. And I got the gun, replaced the pane of glass and left.

It takes courage to blow your brains out. And I’m just like most people when it comes to pain—I don’t like it. So, of course, I resorted to the easy kind of courage one finds in a bottle of whiskey. Kentucky bourbon, to be specific.

I got drunk. And, like the dying calls of most animals for help, I called out, too. I went on the internet and found a room of people who had lost their loved ones, and began posting there that I was going to kill myself. Most people thought I was crazy, many berated me, but one guy wrote to me, in a private message, that he had felt that way before, and asked me if he could talk to me on the phone. I gave him my number. What, after all, did I have to lose? If he was some nut who was going to come to kill me, he’d just be saving me the trouble of doing it myself.

He called me in the next few minutes and we talked. He told me that his mother had died when he was in his teens. He believed in reincarnation and he told me that Keith had accomplished his purpose on this earth, but that if I took my own life, Keith and I would have to come back and go through all of the pain again, to accomplish mine. I could not bear the thought of it. So, for that moment, I lived. And, for another, and another, in a long unending line of moments.

This is not to say that I gave up the gun. I put my mother through hell. She knew I had it, and I would not give it back and she knew why. I needed that gun for insurance. If the pain got too great, I was going to use it. And, a few times, it did. I remember one time, sitting on the edge of the bed, with that gun in my hand, completely sober, ready to give it a go. But I felt some other presence in that room that day. I don’t know what it was, but it wasn’t good. It was dark and it wanted me to eat the barrel of that gun. I’m not a religious person, or a person prone to seeing or feeling things that can’t be measured by the physical. But there was something there that day. I could not see it or hear it, but I could feel it’s presence and it was black and I knew that it would be there, to welcome me, once the bullet hit my brain.

And, so, I live, until another kind of darkness comes to visit me. I will not stop for death, but I’m sure that it will stop for me, sooner or later. In the meantime, I am left to consider my purpose. I am sure, at this point, there is not one among you who cannot say what that purpose is. I have made myself, as they say, abundantly clear.

]]>The Balancing ActMelanie ChildersNone2010-04-26T07:36:00+01:00http://www.sharktank.org/melanie_childers/files/1630376667fd5e039ab17b9d6866e4fb-23.php#unique-entry-id-23http://www.sharktank.org/melanie_childers/files/1630376667fd5e039ab17b9d6866e4fb-23.php#unique-entry-id-23From here on out, I want you to think of cystic fibrosis as a disease of circles. Waves, actually, descending on out and originating in the anions exiting (or not, as in the case of cystic fibrosis, where you are lacking the protein (the CFTR) that causes the exit of these anions from the cell) the cell. Have you ever noticed how, when you throw a rock into water, it’s not just one wave, but smaller, and then ever smaller, waves, coming out of the middle? For the purposes of studying cystic fibrosis, the first of those waves is the lactoperoxidase system. Let me tell you what happens with it, and what doesn’t, in the disease of cystic fibrosis:

There are enzymes (you can tell an enzyme from another protein by the “ase” on the end of it), called lactoperoxidase and myeloperoxidase. In case you didn’t know it, enzymes make something happen between two other components. Take, for instance, lactoperoxidase—it uses hydrogen peroxide and thiocyanate and makes hypothiocyanate. And myeloperoxidase can do this, too, but usually it does some other pretty nasty chemical reactions, as well.

For instance, myeloperoxidase converts hydrogen peroxide and chloride ions into hypochlorous acid (HOCl-). Ever read anything about hypochlorous acid and CF lung disease? Let me show you a couple of things:

Am J Respir Crit Care Med. 1995 Dec;152(6 Pt 1):1910-6.
Neutrophil-derived long-lived oxidants in cystic fibrosis sputum.
Witko-Sarsat V, Delacourt C, Rabier D, Bardet J, Nguyen AT, Descamps-Latscha B.
INSERM U25, Hôpital Necker, Paris, France.
Abstract
We evaluated long-lived oxidant potential in the sputum of patients with cystic fibrosis (CF) by quantitating the methionine-inhibitable, long-lived oxidant fraction of sputum, referred to as the chloramines. Taurine, the preferred amino acid substrate for chloramine formation, and myeloperoxidase (MPO), the chlorinated oxidant-generating enzyme, were also quantitated. As compared with the sputum of asthmatic subjects, the sputum of CF patients contained high concentrations of chloramines along with high levels of taurine and active MPO. A negative correlation between chloramine and taurine was found in the sputum of CF patients. No correlation was found between the density of Pseudomonas aeruginosa and the level of chloramines, taurine, or MPO. In contrast, respiratory parameters (%FEV or %FVC) and a nutritional index correlated positively with chloramine levels, whereas negative correlations were observed with taurine and MPO. In addition, the effect of antibiotic therapy, which significantly increased chloramine and decreased taurine levels, supported a beneficial effect of chloramines on overall clinical status. Our findings support a dual role of long-lived oxidants at the site of airway inflammation in CF, one component of which is their ability to mediate oxidative stress and the other a beneficial effect that may be partly explained by their inhibitory effect on antiprotease defense systems.

See that article there? That shows that chloramines, which are the product of myeloperoxidase (and enzyme, remember? It's got the "ase" on the end of it, like all enzymes do), chloride ions and hydrogen peroxide (HOCl-) and an amine, such as taurine, are long lived oxidants which are correlated (this means that where you see one, you see the other, so they have some relationship to each other) with higher lung function in CF patients. In other words, they serve a positive purpose. And that's great.

But, chloramines cut two ways. (See the article below this paragraph.) For one, they are long-lived oxidants. For two, they are not very specific when it comes to killing the major pathogens that infect CF patients' airways. So, while they are doing some good, they are also doing some damage. All oxidants do damage, because they cause inflammation, which leads to damage. There are different kinds of chloramines, depending on what kind of amine they use, and some are more damaging than others, but we won't get into that here.

Cantin AM.
Unité de Recherche Pulmonaire, Centre Hospitalier Universitaire de Sherbrooke, Quebec, Canada.
Abstract
Airway secretions of cystic fibrosis patients were found to contain high concentrations of taurine, which decreased with antibiotic therapy during acute respiratory exacerbations. Taurine, in a 1:1 molar ratio with HOCl/OCl-, caused a 10-fold increase in the amount of HOCl/OCl- needed to induce cytotoxicity to the cat lung epithelial cell line, AKD. Although DMSO protected cells against HOCl/OCl(-)-mediated injury, the presence of an equimolar concentration of taurine with HOCl/OCl- prevented DMSO from protecting cells and sulfhydryl groups against oxidation, suggesting the formation of taurine chloramines. Spectral properties confirmed the formation of monochloramines and dichloramines. Chloride-free buffer, DIDS, and low temperature (4 degrees C) each protected the cells against taurine/HOCl/OCl-, indicating that taurine chloramine uptake through anion transport pathways was required to induce cytotoxicity. A molar excess of taurine inhibited cytotoxicity, to induce cytotoxicity. A molar excess of taurine inhibited cytotoxicity, by decreasing taurine dichloramines and increasing the formation of less toxic taurine monochloramines. We conclude that taurine can protect lung epithelial cells by converting HOCl/OCl- to anionic monochloramines, but that taurine dichloramines can be toxic to respiratory epithelial cells through mechanisms that depend upon epithelial cell anion transport.

I present these articles to you so that you can see the imbalance here. In CF, you've got the chloramines doing all the work. That's not how nature intended it. They are long-lived, they cause damage to normal tissue, and they are not very "on target" when it comes to the pathogens that are invading these kids' airways.

You see, you can look at the relationship between these factors in another way, and ask another question, that puts you closer to pinpointing the problem--you can say, "What is it about these particular pathogens, that would normally be solved by normal airways, and isn't, in CF, because an anion is not being transported to the surface of the airways?" Once again, coming from the other side of the equation, you find thiocyanate. How do you know that thiocyanate is the competitor, and the missing component here? Because thiocyanate is a competitor for myeloperoxidase. It competes with the enzyme here--myeloperoxidase. It uses both the product of the enzyme, which makes HOCl- (hypochlorous acid, in case you forgot) from H2O2 and chloride ions, plus an amine (in the two articles above, they present the taurine amine, but there are others) , to make hypothiocyanate, instead of the chloramines. And hypothiocyanate IS specific to killing these pathogens, and is NOT damaging to lung tissue.

I hope I haven't totally confused you yet. If you look at it as a math problem, think of the enzyme, myeloperoxidase, as the equal sign. And each of the things that it does with each of the other components as parts of the equation, but on different sides of the equal sign. You have pathology and these pathogens that are normally killed in the normal lung are not killed in CF airways, because the equation is lop-sided when you are missing one component on the other side of the equation. It's like a fat person sitting on one end of a seesaw, and a skinny person on the other side.

And, one really cool thing about hypothiocyanate? These pathogens cannot form resistance to it, like they do antiobiotics, because it kills them in a different manner.

I'm going to end this post now, and I will tell you why, next time, pathogens such as PA, BC, and SA can't form resistance to hypothiocyanate. But, in the meantime, can you see the wave? Because one anion, thiocyanate, is not transported to the epithelial fluid, lining the lungs of these patients, there is no competitor for myeloperoxidase, the enzyme that is the equal sign, in the equation. That causes yet another problem--an increase in inflammation caused by long-lived oxidants, which are also not good at killing the pathogens. The equation, one way or another, is going to be balanced, because nature abhors a vacuum. What do you suppose is the balancing factor here, if you don't replace thiocyanate in the airways? Of course, it's the pathogens!

]]>LAST MOMENTSMelanie ChildersNone2010-04-17T19:59:17+01:00http://www.sharktank.org/melanie_childers/files/556ef3546c6ec874cd23ecb018a1b86e-22.php#unique-entry-id-22http://www.sharktank.org/melanie_childers/files/556ef3546c6ec874cd23ecb018a1b86e-22.php#unique-entry-id-22
So, now, I am the tortured one. And it is nothing more than what I deserve. I was his mother and I could not save him.

I wish I could tell you all of the memories that curl like smoke inside of me; his last months, weeks and days on this earth. When we watched his life, his hopes, and his dreams die, as his lungs were dying inside of him. He asked the nurse, the day before he died, “Am I going to die?” He could not ask me, you see, because he knew I could not face it, and I would not be able to tell him the truth. The nurse started to cry and ran from the room. I wanted to do the same, but of course I could not. I would not.

We slept very little those last days. He fought to breath. On the last day, he woke from a few minutes sleep and starting coughing. I knew, as I rose from my cot beside his bed, that that day was the day. I don’t know how I knew, but I knew. He rested his forehead on his thumbs, as he often did. He couldn’t hold it up anymore. His oxygen sats tumbled down to the 70’s.

I begged the doctor to put him on a respirator, but he refused. Keith and I had talked about this previously, with his doctor—he wanted to be put on a respirator--but this was the doctor on call on the weekend, and he refused. I asked him to give my son some morphine, instead he put a Do Not Resuscitate order in front of my dying child and asked him to sign it. Keith put his “X” on the form. It was his last act, giving permission to the doctor to let him die. He did it because he wanted the morphine and he thought that the doctor would give it to him, to ease his suffering, if he signed the DNR.

That was August 1, 1998. The day that my son died. George W. Bush, governor of Texas, signed the Futile Care Law, in 1999, a year later—this law gives doctors the ability to withdraw or refuse treatment to patients if they see that it is futile to continue treatment. That law was not the law of the land the day that my son died, but the doctor treating him was.

I’ve often thought about writing to the doctor that let my son die that day, and telling him what a piece of dirt he is. But, of course, I can’t bear to look at the death certificate, and find out his name. What a guy, huh? What kind of doctor makes you sign a DNR in order to give you pain medication? And you can say, Ah, well, he was going to die anyway. It was just a matter of time.

Yeah, and so are we all going to die anyway. None of us escape from this life alive. Every moment on this earth is precious. Our lives are nothing more than moments strung together and no one has the right to deny us even one.
]]> I’m really pissed. [angry if you are from the UK;)Melanie ChildersNone2010-04-16T07:29:59+01:00http://www.sharktank.org/melanie_childers/files/9a50a87bfaa062e6f7755bd81b435e68-21.php#unique-entry-id-21http://www.sharktank.org/melanie_childers/files/9a50a87bfaa062e6f7755bd81b435e68-21.php#unique-entry-id-21
When I first saw that chart that the CF patient who had been taking 27mg per day transdermally of BITC sent to me, all I could focus on was the sky high PFT scores. Last night, I took the time to read over the chart in detail. A gradual, albeit rapid, disappearance of mucus viscosity. A gradual, albeit rapid, change in the color of the mucus, from green, to yellow, and then finally to clear and clear and clear again. And, yes, it makes me happy. It makes me ecstatic, actually.

But look at the patents—everybody is lining up for a place at the trough. Banfi, the French. All of them wanting a piece of the pie. That’s how our system works. I know that.

But look at “Example 5” below. They filed, Lellamand, and his collegues, for the patent on using chemo drugs to cause the expression of the MRP proteins, in 1999! They filed in 1999! Then, they tried colchicine, to induce the protein. It was the safest anti-tumoral chemotoxin they could think of, of course. But, of course, it didn’t work very well in many patients. And, of course, they tried it ORALLY.

You see why I’m pissed off? WHO followed things out, among them? Nobody. WHO said, “Hey, this works, maybe we can figure out a better way.” Nobody.

These guys are pros. They’ve got the education. They have the funds. They have the ability to confer with other very highly educated people in the field. And they just didn’t GET it. And they didn’t bother to follow through. Look at the drugs that they patented. Every one of them already on the market for another disease—cancer. Why didn’t they go back and try to figure out something else, that wasn’t as toxic? And why didn’t they think about what would happen if they gave this orally? (In case I haven’t told you often enough, oral administration of compounds that induce the MRP also induce it in the LIVER, which very quickly begins to express MRP, which filters out the drug, which then causes you to move right back to square one.)

Little tiny Sharktank, and me, the ignorant in-the-dark pseudo scientist, has to take the helm and guide this ship into port. Jesus, what a sad commentary that is on the system that is supposed to find answers for the suffering.

So, yeah, I’m pissed. And you should be pissed, too. They were handed the answer when they saw that CF patient with a sarcoma, respond to the chemo drugs, by getting rid of his CF symptoms. They were handed the answer. And they rushed to the patent office to ensure that they could make money off of that happy accident, and then stood by and did nothing else. The sorry, greedy, non-curious bastards.

I’m not here to make money off of this. I have the patents, but I care not one whit for making any money off of them. I have those patents because I know that if I didn’t, they would be rushing to the patent office to get them, and then they would, once again, sit by and do nothing. And, then, we would not have control over this compound. We couldn’t use it as a supplement. But, because we have the patents, and the DSHEA laws on our side, we can.

It’s going to be a bumpy ride. Big pharmas do not like it when you knock the need for their products off the market. And when you have a compound that changes the biochemistry of the patient in a systemic manner, you’re doing just that.

Strap on your boots and spurs, boys and girls. We’re riding this bronco to the end of the race.

EXAMPLE 5

Treating Cystic Fibrosis with Chemotherapy Which is Prescribed for a Lymphoma.

The patient, who was born in 1969, suffers from cystic fibrosis, which was diagnosed at the age of two months by means of a positive sweat test. His clinical picture is typical for cystic fibrosis (exocrine pancreatic insufficiency, polyposis of the sinuses, chronic pyocyanic colonization). He is following a standard basic treatment for his cystic fibrosis: respiratory kinesitherapy, several courses of treatment with antibiotics and vitamins. In August 1992, he presents with a stage IV lymphoma. He undergoes four courses of treatment combining adriamycin, cyclophosphamide (Endoxan Asta®), cytorabine (Aracytine®), vincristine (Oncovin®), methylpred-nisolone (Depa-medrol®), Bleomycin and Methotrexate in October 1992 and November 1992. In January 1993, he undergoes a new treatment combining methotrexate, cytarabine and methylprednisolone. All chemotherapeutic treatment is stopped in April 1993. The patient is judged to have been transformed at the pulmonary level from March 1993 to December 1993, both by himself and by the doctors who are monitoring him; no more coughing, no more respiratory kinesitherapy; he exerts himself physically without difficulty and he has several normal pulmonary examinations. In the absence of maintenance chemotherapeutic treatment, some symptoms resumed from the end of 1994, such as an increase in the coughing. However, a marked improvement in the condition of the patient was noted over a period of one year following the aggressive treatment for the lymphoma.
The treated patient was homozygous for a mutation in exon 20 in the NBF2 domain.

]]>It’s really not a big dealMelanie ChildersNone2010-04-14T09:30:06+01:00http://www.sharktank.org/melanie_childers/files/54d5059a6e5a3aac8ba763fd1d988b59-20.php#unique-entry-id-20http://www.sharktank.org/melanie_childers/files/54d5059a6e5a3aac8ba763fd1d988b59-20.php#unique-entry-id-20
I guess if you’re just entering the world of CF and if you’ve never been a member of Sharktank, it might seem that way to you. But, geez, we’ve been at this job for over fourteen years now. To me, and I’m sure to the rest of the long term members of Sharktank, we’re all just thinking, “Hey, it’s about time we hit some kind of mother lode!”

This group has been through decades of published research. We’ve not just studied the new stuff for fourteen years, but dug back into the old research, from before they found the gene that causes CF, and studied that, too.

When I look back on all that we’ve learned, it’s no surprise to me that we finally found a compound that would induce a functionally redundant protein. For God’s sake, the Lancet letter describing the miraculous “cure” for the CF patient that had cancer, and was given chemo drugs, was published in 1997! What surprises me is all the various pathways that we went on, in order to create a new disease model, and THEN, finally, to this point, where we can do something about this disease.

I know it’s simplistic to say that all we really did was figure out how to manipulate protein expression. Because, of course, we had to learn about all kinds of things before we could finally figure this out. After all, it’s not as if you can just look up the answer to that question on Google.

Still, like all complicated things, when you finally figure them out, they really seem simple, don’t they?

So, here we are. And, really, it’s not that big of a deal. I just wish we had not lost so many during our journey. And, I wish that I could say that my long search was motivated more out of love for my CF family, than out of hate for this disease. It is not true, though.

But I will soldier on, as they say. I will take that hate, and that anger, for the murderer of my son, and I will cut a swathe through the battleground that CF creates in it’s sufferers lives. I will soldier on.

And I hope you will join me by joining Sharktank. We are not asking for your money. (When we need it, we are not shy about asking for it.) We are asking for your support; for you to talk to your doctors. Because, if Sharktank is correct, it will be possible, with your help, to eradicate this murderer called CF, from the lives of our families and of our children, while I’m still alive to gloat over our victory.
]]>It WorksMelanie ChildersNone2010-04-12T09:57:33+01:00http://www.sharktank.org/melanie_childers/files/f2251a8d70724dec8be6170ae9325e37-19.php#unique-entry-id-19http://www.sharktank.org/melanie_childers/files/f2251a8d70724dec8be6170ae9325e37-19.php#unique-entry-id-19
Now is as good a time as any to tell you what I just found out: transdermal BITC works in at least one person, exactly as we predicted it would, in exactly the ways that we predicted, and for the long term, instead of the short term, as it did in the oral form. Apparently, the liver MRP is not induced when you take it transdermally, and it’s getting to the lungs, where it does the job of inducing the functionally redundant MRP protein.

It’s only one person, of course. But it’s wonderful news to hear. Wonderful. I hope you all know how lucky you are, to receive this news in your lifetime.

Our carrier test failed to produce any movement in the fatty acids of the carrier, but it was a very low dose, of course, and I didn’t expect much of anything from it. Now, we will double the dose, to 20mg, and see if it has any effects.

I have, of course, inside information, from my source, who used more than that, transdermally (not much more), so maybe we’ll have to increase it by about ten milligrams before we see an effect on the fatty acids, but we’re sure to see some results soon, on the carrier end of things.

Anyway, put that in your pipe and smoke it. I’m both happy and very sad, at the same time. I so wish I could have had this for my beloved son.
]]>The CFTR transports “stuff” OUT of the cellMelanie ChildersNone2010-04-07T10:44:44+01:00http://www.sharktank.org/melanie_childers/files/52d5f058f225fffaece8e39df5c5e0d0-18.php#unique-entry-id-18http://www.sharktank.org/melanie_childers/files/52d5f058f225fffaece8e39df5c5e0d0-18.php#unique-entry-id-18
I don’t know why, but people who have been studying this disease with me for years have gotten that little fact confused. And it’s important to remember.

Think of it like this: You’ve got the airway and the tissue that lines it, and then you’ve got the “stuff” that the CFTR transports onto the airway lining. The CFTR sits in the membrane of the cells that line the airways, and transports ions (or anions, which are negatively charged ions) onto the surface of the lungs. This “stuff” is what makes up part of what is called the epithelial lining fluid (or ELF, for short).

Now, there is little doubt that the CFTR also controls, in some unknown way, the transport of sodium ions (Na2+) INTO the cell. But it does NOT transport anything, itself, into the cell. It ALWAYS transports OUT of the cell.

What happens, I think, is this: In CF, the CFTR protein is not in the membrane, and not doing it’s job of transporting chloride anions out of the cell. So, the cell, wanting equilibrium, has some way of controlling some other channels, called epithelial sodium channels, which are in charge of reabsorbing sodium ions (cations, which are POSITIVELY charged). So, if you don’t transport chloride ions, which are negatively charged, you get your equilibrium by reabsorbing sodium ions, which are positively charged. This creates NaCl, which is salt. Where salt goes, water follows, so you pull water out of the fluid that lines the lungs (presumably the mucus that is there, too), and you have a mucus without much water in it. This theory has been disputed, but I think it’s correct. It certainly explains why these patients have mucus plugs.
]]>Miscellaneous StuffMelanie ChildersNone2010-03-27T17:59:39+00:00http://www.sharktank.org/melanie_childers/files/243383734ce5e0fd7c240366067e3352-17.php#unique-entry-id-17http://www.sharktank.org/melanie_childers/files/243383734ce5e0fd7c240366067e3352-17.php#unique-entry-id-17“Extraordinary Measures.” I didn’t like this movie. And I’ll tell you why. This is a movie that relegates ownership of the power over our lives to professional scientists. It makes us completely dependent on them. It has the message that if you throw enough money (and maybe a lot of power) at something, it can get done. God knows, that’s not true when it comes to cystic fibrosis.

Money, contrary to popular belief, doesn’t make the world go round. It helps it a little bit, but it’s not the end all, be all, when it comes to getting things done. It takes people to get things done. And people don’t always require money; although they do require time and effort, to get things done. And, I think that, if a person cares enough, they can always steal a little time, here and there. And, if they’re focused enough, they can do something extraordinary with it. And the more people, the more time and effort, the more they can get done. Get enough people together, all focused on one thing, and you’ll get the job done.

I used to say, when Sharktank first started, that we wanted a bunch of people to get together to study the science of CF because each one wore a different tint of glasses. Each person sees the world in a slightly different way, and it was important to get all of those visions together, to see this disease. I still believe that—it took all of us to get to where we are today. All of us having a vision of ending this disease. And each of us took a little time here and a little time there, and put the effort into it, to reach our goal.

The only purpose I, personally, played in this was that I was the person who put stuff together. And, believe me, I’m stupid and hard headed, so I was not the best person to do the job, but I was the one who was angry enough to be persistent enough to do it. And, believe me, scientific articles or no scientific articles, I’m still an idiot. I can see everything wrong with my work. I know what’s right about it, but I can still see where I made mistakes, and they still embarrass me.

For instance, in that first paper, that was in the Medical Hypotheses journal, I thought that the CFTR transported the very same things as the MRP proteins. That is not the case. The CFTR transports the anions, and the MRP transports the adducts of the anions. For example, take thiocyanate—I believe that it is correct that the MRP transports adducts of thiocyanate (actually glutathione adducts of thiocyanate), while the CFTR just transports the thiocyanate anion. You see? Perhaps it’s a small thing for others to see as a mistake, but to me, it’s glaring.

And, I remember the time when I was stuck on metallothionein for two years. I just knew it had to be transported by the CFTR. Of course, that was totally wrong. It was entirely too big to be moved through that pore, but it took a while to convince me of it. Finally, once I accepted the premise that it couldn’t be moved through that pore, I was able to move on and figure out it’s part in the disease. But my own hard-headedness held me back from that for a couple of YEARS! That truly pisses me off. Of course, in the meantime, I learned a lot of other things about the disease, and about biochemistry in general, so I suppose I shouldn’t get too pissed off at myself.

I think that it’s really important to get a kind of “big picture” about things. I think that, as human beings, we always want to break things down into their smaller components, but when you have a disease like cystic fibrosis, and you see all of the pathology associated with it, you have to be able to put everything into the big picture, to be able to figure it out. Yes, of course, it comes down to a small thing, like one single protein, the CFTR, but when that protein moves out multiple anions, you have to learn the system, in order to understand the disease. And multiple systems, even. Not just the respiratory system, although that’s a hell of a good place to start. To put it simply, once you learn a whole bunch of things, those things start to coalesce, into a picture, and it is that bigger picture that gives you cues about where to look, so that you can point out the smaller things, like which anions are transported.

Not that I’m congratulating myself, or Sharktank, at this point. I will always hold off judgement until I see concrete results. That’s part of the humility thing, you know. Keeping your mind open to the fact that you very well might be wrong. Of course, I don’t think that we’re wrong because of what we saw BITC do in CF patients when they took it orally, even though the effect was short-lived. I know we have the right compound, and that even other compounds in the same class should work, to varying degrees, in the same way, in the CF system. And I know why the effect was short-lived, and how to overcome it, by changing the delivery method. I just don’t know the dose. . .

God and the universe willing, we will know that soon. We did a smart thing, using the same carrier as we used last time. (This guy is a hero, by the way.) We KNOW that his fatty acids move in the right direction, because of BITC, because they already moved, when he was taking it orally. We just don’t know how much it will take, transdermally, to get them to move. I am on pins and needles, waiting for the results.

]]>Old and new thingsMelanie ChildersNone2010-03-26T17:17:11+00:00http://www.sharktank.org/melanie_childers/files/2b4ce092d3db89a05cf36f19bb4b1a78-15.php#unique-entry-id-15http://www.sharktank.org/melanie_childers/files/2b4ce092d3db89a05cf36f19bb4b1a78-15.php#unique-entry-id-15
After my work on glutathione was stolen, and written up without giving me credit, I got very stingy with sharing my work on this disease. I kept everything “close to my chest” and didn’t write to anyone, or tell anyone but a few trusted friends on Sharktank, what I was working on, or thinking, until after I had found thiocyanate. Then, I published.

Now, though, I feel as if I have accomplished enough so that it can stand for me and my work. And at 55, I have to ask, what will happen to this work, if I leave it and I’ve kept things to myself? All that I know will be lost to you. It will be in the hands of mainstream scientists, who take a lot longer to accomplish things, because of the system that they work in, and the burdens that it puts upon scientists, to justify their paychecks.

I’m at the point in my life now where I don’t mind too much if something is taken from me and written up as someone else’s, quite frankly. In fact, I think it might be necessary, and a good thing, just in case I’ve missed anything.

So I want to talk about the CFTR a little bit more—

Remember: it’s a transport protein. It’s job is to transport “stuff” out of the cell, to the lining of the lungs—the airways, and that “stuff” is part of the epithelial lining fluid. What you don’t know yet is that it is what is called a “multi-anion” pore protein.

What does that mean? It means, simply, that the CFTR transports more than one kind of anion. We have talked about, thus far, four of them:

1. Chloride anions (Cl-)

2. Glutathione anions (GSH)

3. Thiocyanate anions (SCN-)

4. Bicarbonate anions (HCO30)

But, there are two more:

5. Glucuronic anions

6. Sulfate anions

You’ve heard that saying, “Out of the mouths of babes”? That’s me—I’m the babe. Ha! Just kidding there. But, I am a “babe,” in the sense that I am very simple. I think in a very simplistic way about cystic fibrosis. But, sometimes, by thinking about something in a very simple way, you can chance upon some pretty important facts.

Those last two anions—glucuronic and sulfate—they’re important. They’re important to the extracellular milieu. And, I think, particularly, they’re important to the mucus properties seen in this disease.

How do I know that? Because of the serous cells. What are serous cells?

Well, they’re two kinds of cells in the submucosa of the tissue in the lungs. Or, at least, two kinds of cells that produce mucus. And, we all know that we have a problem with mucus in these patients, so it’s pretty important that we look at those cells.

And there’s something awry when it comes to the serous cells in CF patients’ lungs—they don’t work. They don’t put out anything. We know that the CFTR is localized in these cells. There is work out there that proves it. And, there’s work out there that shows that serous cells from CF patients don’t put out any mucus. (I’ve posted citations to it, below this paragraph) All of that mucus production is left to the mucus cells. And they go absolutely nuts, trying to make up for the lack of mucus production by the serous cells. They overcompensate, in CF (probably in response to an increased calcium level in these cells, but we’ll get to that another time). And, something is wrong with the mucus; it’s not structured right. It’s got bonds in the wrong places, and the wrong kinds of bonds.

Am J Physiol Lung Cell Mol Physiol. 2007 Jan;292(1):L304-11. Epub 2006 Sep 22.
Acinar origin of CFTR-dependent airway submucosal gland fluid secretion.
Wu JV, Krouse ME, Wine JJ.

Proc Am Thorac Soc. 2004;1(1):47-53. Submucosal glands and airway defense.
Wine JJ, Joo NS. [This is a review, which just goes over what we know about the topic, but it’s good to read it, if you want to know more about what I’m talking about.]

Mucus is structured like a matrix. Do you know what a matrix is? Think of it as a piece of screen, like on a screen door. All those little squares, joined together. Now, I doubt that mucus is structured like a bunch of squares; it might be triangles, or rectangles, or hexagons, for all I know. But the important thing is the bonds. They are chemicals, and they have properties that cause them to join other chemicals together, into a structure, and that structure is mucus. Let me interject here and tell you that HCO3- has a lot to do with the composition of the epithelial lining fluid, of which mucus is a part. But I have no idea whether it has to do with mucus, itself. I’m sure it does, but I cannot fathom, at this point, how that works.

I do know, though, that there is a problem with glucuronic anion and sulfate anions. And the problem with their lack of transport shows up in the mucus that’s secreted from the glands (not the acini serous cells, which don’t secrete at all in this disease).

When you look at CF mucus, you find some very interesting things. For instance, there’s a bunch of hyaluronic acid. (And, by the way, that’s an anion, too.) Glucuronic ion is used to make hyaluronic acid. And hyaluronic acid is used to make some really big, long mucus chains called glycosaminoglycans. These patients’ mucus is different from normal mucus, no doubt about it. They’ve got more of the stuff used to make the glycosaminoglycans and they have more glycosaminoglycans in their mucus.

And, all of this is very interesting to me, of course, but THE most interesting thing is the sulfates. They’ve always scared the holy crap out of me, quite frankly. They were mentioned in the first years I began studying this disease as “a problem with sulfation,” and I never could figure out what that meant, for years. Until now.

The thing with sulfates is that there are not just one kind of sulfate. You’ve got different kinds. With GSH, you can just say “glutathione” and that defines it. Same with glucuronic anion. But there are different kinds of sulfates, and the research on a couple of them interests me—chondroitin sulfate and heparin sulfate. (“Heparin,” in this case, I believe is supposed to be spelled with an “a” after the “r,” but this spell check thing is on, I guess, and I am not going to waste my time trying to figure it out. You get the picture, though, and know enough to search it on google if you want to.)

I have to leave you now, but I want to ask you something. I want you to ask yourself something: Why is it that if the CFTR protein is supposed to transport chloride ions, when you take a sweat test, it’s considered positive, for CF, the more chloride ions you have in it? Haven’t you ever been curious about that?

]]>I'm a stalkerMelanie ChildersNone2010-03-26T17:05:41+00:00http://www.sharktank.org/melanie_childers/files/b7d8ba1202408b3d786401fbe311296b-14.php#unique-entry-id-14http://www.sharktank.org/melanie_childers/files/b7d8ba1202408b3d786401fbe311296b-14.php#unique-entry-id-14
Yes, I used to be a bill collector. I think that that’s where I developed the ruthless part of my personality. Don’t get me wrong—I’m a nice person. Really, I am. But when I want something; when I set a goal, I’m GOING to get there. I have my eyes on the goal, and I have problems seeing anything but my target. I WILL run over a few people along the way, but I will honk before I hit ‘em, and if I have time, I’ll go back and help ‘em up off of the ground.

I used to work in a tall building, called the Arena Towers, in Houston. I don’t remember how many floors it had, but it was set up so that each floor was laid out the same, and my company was on the twelfth floor. Each morning, I had to park my car in this parking garage and walk to the building, ride the elevator up to the twelfth floor, get out, go down a couple of halls and to my desk.

Before I finally went to bed at night, during those years, I would pick out the articles that I wanted to read during that walk to my office in the morning, and during every break during the day. I’d print them out and have them with me, whenever I was doing anything else, but working. So, I’d have this big sheaf of them when I got out of my car in the morning, and I would begin reading them as soon as I began the trek from the parking garage to my desk.

It’s funny how the brain and the body work together. Sometimes, on the way to my desk, I would find myself stopped, and I would come up out of my reading, to see that I was on the curb of the driveway where cars went into the parking garage. There would be no cars coming, but I would look up from my papers and find myself just stopped there automatically.

Sometimes, when the “system” wasn’t working too well, at the end of my walk to my desk from the parking garage, I would find myself on a completely different floor in the building, but where my desk should have been, and all these strange people I didn’t know, looking at me curiously, wondering, I’m sure, what the hell I was doing there.

After a while, people got so used to me reading and walking, they would stop me when I tried to get off on the wrong floor. Like a child, they would say to me, “No, Mrs. Childers, you can’t get off yet,” and pull me back from leaving the elevator precipitously.

I tell you this story about me because I want you to know who I am. And, at 55, I am just now realizing that I am very different from other people in some very distinct ways. I'm not sure if it would matter what the topic is--CF or something else--I can't let go of things. I have never been able to give up on anything that was of the least bit important to me. I'll worry it until I drag it to pieces. My brother says I'm like a bulldog at someone's pants' legs. I drive people nuts.

I always thought everybody was like this. But they're not, I'm finding out. I'm the weird one.

Despite what you may want to think, what many people think, it's not passion that drives me; it's not decency, or love for my fellow man. And maybe it's not even grief. And, certainly, it's not intelligence. It's some innate thing that some psychologist will come up with, some day, a name for--that inability to stop; to let go; to give up. I am lacking that ability. I'm the quintessential stalker personality. And the puzzle that can't be left alone, for me, is cystic fibrosis.
]]>A Brief Summary of where we’ve come from and where we’re at….Melanie ChildersNone2010-03-23T10:24:30+00:00http://www.sharktank.org/melanie_childers/files/c09ba18668102c25e75388947b1b9cf4-13.php#unique-entry-id-13http://www.sharktank.org/melanie_childers/files/c09ba18668102c25e75388947b1b9cf4-13.php#unique-entry-id-13
This whole thing started because my son, Keith, had cystic fibrosis and I finally realized that the promises of a “cure just around the corner,” weren’t going to pan out and my son was going to die if someone didn’t do something. As usual, if you want something done and done right, sometimes you have to do it yourself, so I began my journey.

Lots of people joined me in the journey, and Sharktank was created. There are some very talented people on Sharktank—doctors, lawyers, research scientists, etc. They got together and created a non-profit entity called Share International Research, so that people could donate to the project and get their donations as a tax deduction. I think they did this mostly for credibility reasons, but either way, it works. We’ve raised enough money to pay for the things that we need, although maybe not enough for what might be needed in the future.

We saw a kid “cured” from cystic fibrosis. This happened by accident, because he was taking chemotoxic drugs. And, we’ve traveled this long road, trying to figure out how to replicate that “cure” for the rest of CF sufferers. Obviously you can’t take chemo drugs all of your life, or they’d kill you, but we thought that there might be something more specific and less toxic, and we were right.

We had to figure out why the kid with cancer experienced such a turnaround in his disease, and we found the MRP proteins. They can substitute for the missing CFTR protein. We found an entire CLASS of compounds that can cause the MRP protein to be made by the cell.

We tested one of those compounds and it worked, when taken orally, on a carrier. Then, when people with CF started taking it orally, it worked for them, too. But it only worked a short while.

We figured out why it only worked for a short while, and now we’re testing it in the same carrier (whose fatty acid profiles will show the same change, if we can just get the right dosage) transdermally. We are currently waiting on the blood test results of our first experiment with transdermal BITC. If there is no change in the fatty acid profile, we will double the dose.

Next, I will talk about the class of compounds, both generally, and specifically, that cause an induction of the expression of the functionally redundant (to the CFTR) proteins, MRP
]]>Keith Lanier Johnson ChildersMelanie ChildersNone2010-03-21T09:13:36+00:00http://www.sharktank.org/melanie_childers/files/13bf1a4e5bb7e5a6a58d8d0e3c64f1b9-12.php#unique-entry-id-12http://www.sharktank.org/melanie_childers/files/13bf1a4e5bb7e5a6a58d8d0e3c64f1b9-12.php#unique-entry-id-12
I’ve tried to give you a sense of Keith, because he’s a big part of this story, by writing about things that happened in his life. The thing is, you just can’t distill a person down to a few stories.

Moreover, Keith didn’t want to be defined by his disease. He wanted to be as normal as possible. And I really don’t think that he would like it if I made him a gigantic part of this story. He is, of course, and I would love to honor him by telling you more about him, but I think that I honor him more by respecting his wishes.

It is the worst possible scientist that allows their personal feelings to influence their work. And, as much as I want a cure for this disease, I have to guard against letting my emotions get involved in any decisions. I think that, if Keith were alive today, I would have a much more difficult time doing that.

I promised Keith I would stop this disease. And I’ve always been very angry at God because Keith is not here and yet I am still doing this work. Couldn’t He have given me a little break, and let me keep my son, after all?

The fact is, though, that I do this work because I have no other choice. Not because I am a great person that cares so much for other people. (Although I do care for other people; it remains to be seen whether I am a great person or no—I’m a work in progress, in that regard.) The fact is that I am engulfed in an unending anguish that is unfathomable, except to a parent who has lost a child. The fact is that the only surcease I get from that pain IS during the time that I am studying this disease.

It is the only thing that I can do now, for my son. I am trying to keep my promise. After decades of taking care of my son, this is the only thing left to me, besides putting flowers on his grave—this work.

I will tell you a funny story—and, by way of that, introduce you to a friend of mine, Ed Taboada.

When Keith was still alive, and in his teens, I was interested in genetic manipulation to cure this disease. At first, being an idiot, and knowing almost nothing about proteins, etc., I asked around about inserting the missing amino acid into the protein. Of course, the scientists that I talked to told me that that wouldn’t work. You cut into a protein at an amino acid and it’s like a necklace of pearls that breaks. All of it falls apart.

So, of course, when the work on genetics came out, I was really interested in replacing the faulty gene. I got really interested in the work on plasmids. These are little pieces of DNA that you can “infect” a cell with, that cause the cell to make the protein that you want. I thought that if I could put the corrected CFTR DNA in them, and aerosolize them into Keith’s lungs, I could cure his CF.

But, of course, I figured I would have to learn about plasmids, first, and second, find out how to get the correct DNA to put into the plasmids. My ultimate plan was to commit burglary on a research institution that had some CFTR DNA and put the DNA into the plasmids, and put the plasmids into Keith. It wasn’t something I put into concrete terms, but it was something that was possible.

First, I had to learn the territory, though. So, I’m faced with the first obstacle of learning quickly. The best way to do that is to get a teacher. But I have to hide what I’m planning from that teacher.

So, I get a friend of mine, Joan Maricle, to get on the internet (remember, at this point, I know only how to access PubMed and email on the internet), and post an ad on a science newsgroup. I wanted a grad student who had his degree in molecular biology to teach me.

I made up a story to hide my intentions. I “interviewed” the grad student via email, and told him that I was the parent of a home schooled 14-year-old girl, Monique, who was interested in molecular biology and I would pay him $10 an hour to teach her, every day, via instant message, the subject. He bought it, hook, line and sinker. And “Monique” became a student.

I didn’t have much money, or much time, so I thought that I could get a jump on things by going to the medical library in Galveston, and checking out books on molecular biology, and reading them in my spare time. I read through about five books a week on the topic, and although I didn’t understand everything that I read, I understood enough to finish my teacher’s sentences in our “classroom” every day. He told me, later, when I told him the truth about everything, that it kind of frightened him that I was able to do that. He thought he’d run across a fourteen year old genius, or something.

Of course, I found that transfecting with plasmids was not a feasible route to go, but I made a friend along the way, who has helped me though the years of studying this disease.

He taught me something that has been served me well thoughout this journey, without which I could not have traveled far at all -- humility. Without humility, you cannot be a good scientist. When you believe that you know everything, you lose the ability to learn anything new. When you think that you are right, and you cannot perceive that you can be wrong, you have lost the ability to find the truth.

Thank you, Ed Taboada, for teaching me that humility is the first skill of a good scientist.

So, all of you guys and gals out there, reading this blog, who think that this science thing is a bit overwhelming, you’ve got the first and perhaps the most important part down pat: you’ve got humility. Now, the only thing that stands between you and your goal is the work that you are willing to put into reaching it.

Stay tuned…..next, I WILL get into the brass tacks, and bring you up to date on what we are doing right now.
]]>Sharktank ExperimentsMelanie ChildersNone2010-03-15T08:00:42+00:00http://www.sharktank.org/melanie_childers/files/75f66b0914c44964fe253c49c6c72e18-10.php#unique-entry-id-10http://www.sharktank.org/melanie_childers/files/75f66b0914c44964fe253c49c6c72e18-10.php#unique-entry-id-10
It’s all under a doctor’s care and supervision, of course. Let me tell you what we did and why we did it:

As you know, if you are a parent of a child with CF (or a CF patient), cystic fibrosis is an autosomal recessive disease. You don’t really need to know the definition for autosomal recessive, because I’ll tell you about it right now—all it means is that both parents have to give a copy of the faulty gene to a child in order for that child to have CF. The gene is recessive, and only one copy of it cannot cause the disease. You have to have two copies of it—one from your mom and one from your dad—in order to have the disease.

So, people who have only one copy (parents of children with CF always have at least one copy of the gene) are what we call “carriers.” They carry the gene, but they don’t necessarily have the disease. They are called “CF heterozygotes” because they carry the gene. (CF sufferers are called “CF homozygotes” because they carry two copies of the gene). For our purposes, we will call these people carriers.

Carriers don’t always come out unscathed, when it comes to carrying a CF gene. There are some carriers who don’t develop a normal vas deferens (this is the item that men get a vasectomy on, when they don’t want to have anymore children; it carries sperm) and who are sterile because of it. And, they also have other differences in their biochemistry that aren’t so obvious.

One of the things that differs, often, in these carriers, is their fatty acid profiles. Kids with CF have some really screwy fatty acid profiles, and sometimes their parents do, too.

So, if you are wanting to know if the compound that you are taking is having an effect on the biochemistry of the cells, one of the things that you can test is the fatty acid profile of a carrier. And that’s what we did.

One of our carriers (who is a hero, by the way) took benzyl isothiocyanate. First, he had his doctor take a sample of his blood and get a fatty acid profile done on it. Then, he took a certain amount of benzyl isothiocyanate for a week, and had his blood tested again. No change in the fatty acid profile. So, he took a doubled dose and had his blood tested again, a week later.

The fatty acids that were skewed in CF patients went in the opposite direction, one of them quite significantly, after the second dose. Because of that, we knew that benzyl isothiocyanate was having an effect on a basic level in, at least, people who were carriers of the CF gene.

What happened next was up to individuals. Many parents of CF children went to their doctors and explained this science to them and asked for permission to try benzyl isothiocyanate for their child. One, in particular, is the person who manages this blog. His daughter is still taking benzyl isothiocyanate. Many others are, as well.

Let me digress a bit here and caution you—I am not telling you to use benzyl isothiocyanate, or to give it to your child with cystic fibrosis. I am not advocating it as a therapy. I am simply telling you what happened. And, hopefully, you will notice that the parents who did give their child this compound did so under the care and supervision of their physicians.

Did benzyl isothiocyanate help these patients? Yes. There were quite a few astounding stories from these people, at the outset. One parent called me to ask me to talk to her daughter, who thought she was cured, in order to caution her that she needs to follow the doctor’s orders and stay on her antiobiotics.

But, then, something happened. Or rather, something did not happen. The good effects stopped (except for the digestive effects). The people who were taking BITC began to lose all of the good effects on their lungs. Previously, their lungs seemed to be clearing out, but then BITC stopped having any effect at all, except on digestion.

We almost gave up on it, at that point. We couldn’t figure out what was going on. Why did it stop working?

Hopefully, you are interested enough to tune in to the next blog post. I hate to leave you hanging, but I don’t want to make this post too long, and what happened next is a longer story. I hope you tune in…..
]]>A short history of SharktankMelanie ChildersNone2010-03-15T07:45:06+00:00http://www.sharktank.org/melanie_childers/files/85650f9a26ec217cbd221e9bb99495b8-9.php#unique-entry-id-9http://www.sharktank.org/melanie_childers/files/85650f9a26ec217cbd221e9bb99495b8-9.php#unique-entry-id-9
About three years before my son died, a few people from Cystic-L got together and decided to start studying cystic fibrosis. (I’ve gone over this before, but for those of you who are just now tuning in, I’ll do it again.) We started reading PubMed, and I started going to the medical library every weekend, to get articles about cystic fibrosis. We began learning what the scientists knew. That initial group consisted of me, Richard Young, George Eckel, and Valerie Hudson.

Richard died of cystic fibrosis. And, then, Keith, my son died, of it. On the day that Keith died, an article was published in Current Contents (this is a resource that you have to pay $600 a year for, and it gives you the articles before they go on PubMed; it takes another month for them to get on PubMed, but I paid for this resource because I was in a hurry). Here is the abstract of that article:

Am J Physiol. 1998 Jul;275(1 Pt 1):C323-6.
Glutathione permeability of CFTR.
Linsdell P, Hanrahan JW.
Department of Physiology, McGill University, Montréal, Québec, Canada H3G 1Y6.
The cystic fibrosis transmembrane conductance regulator (CFTR) forms an ion channel that is permeable both to Cl- and to larger organic anions. Here we show, using macroscopic current recording from excised membrane patches, that the anionic antioxidant tripeptide glutathione is permeant in the CFTR channel. This permeability may account for the high concentrations of glutathione that have been measured in the surface fluid that coats airway epithelial cells. Furthermore, loss of this pathway for glutathione transport may contribute to the reduced levels of glutathione observed in airway surface fluid of cystic fibrosis patients, which has been suggested to contribute to the oxidative stress observed in the lung in cystic fibrosis. We suggest that release of glutathione into airway surface fluid may be a novel function of CFTR.
PMID: 9688865 [PubMed - indexed for MEDLINE]

That article caused an epiphany for me. I knew just enough about CF to know that some anti-inflammatory component was missing, and when this article was published, I knew I had my component.
Remember: this happened the day that my son died. I was LIVID. I was so angry that this was just coming out and that scientists, REAL scientists, who had the benefit of a formal education in science, knew this and were just now publishing. I mean, they had to know about glutathione, or else why would they bother to test it’s permeability in the first place? And, now, my son was dead and couldn’t benefit from it.

I began writing really mean emails to Cystic-L. I’m from Texas, and I cuss like a sailor. We’re brought up that way in Texas. Curse words are a part of our vocabulary at a young age. And I didn’t spare any curse words on Cystic-L. I railed against the scientific establishment. I railed against the parents of children with CF. I urged them to look at this work and to get off their butts and do something, because the scientists were doing nothing. I soon was asked to leave Cystic-L.

But, there were some people on Cystic-L who were listening. Jim Caldwell, who is a computer expert, was one of them. He formed a group called WLGroup. It was a mailing list for people who were interested in studying CF.
We were all enthralled with the information on glutathione, and we all worked our butts off, trying to find more information, and where glutathione fit into the picture of CF pathology. One of our group, [I am deleting her name here], went off on her own, and published our work on glutathione. We tried to tell her that glutathione was not the entire picture in CF, but she would not listen. When problems began to surface with the use of glutathione, we divorced ourselves from her and her insistence on “treating” CF patients with an untested therapy, and became Sharktank.

Sharktank and it’s members have continued to study cystic fibrosis. We followed out the pathology stemming from the lack of transport of glutathione, and other ionic compounds transported by the CFTR protein. We were the first to publish on the transport of thiocyanate by the CFTR. And thiocyanate is THE compound that explains the lack of antibacterial function that is seen in this disease.

The fact that we were able to get this far is astounding. We are truly a revolutionary force. We are the only group of our kind, and we exist because of the internet. For the first time in the history of mankind, people who suffer from a disease can get together and actually study their disease and drive the science of it forward.

There are people, every day, who thank me for the work that I am doing on Sharktank. But, I am not responsible for this work. Sharktank is. Any idiot can come along and, if they have enough perseverance, study for fourteen years and have a good chance of hitting the proverbial jackpot. (Even a blind hog, as they say in Texas, can find an acorn now and then.) But they can’t study without the tools. They can’t study without access to the material--full articles. And they cannot persevere without a team of people willing to ask questions and to participate in that endeavor. That team, now comprised of over 300 parents of children with CF, and CF patients, themselves, is Sharktank.

We don’t have to spend our time filling out paperwork, to apply for grants, to fund our work. We don’t have to hang our heads, and worry about tenure, if we hit a brick wall in the science, and find that we are wrong. We simply turn around, and go in another direction. There is a freedom in that, that doesn’t exist in the mainstream scientific world.

It is my hope that other Sharktanks form out there in the cyberworld. We need a Sharktank for Duchenne’s Muscular Dystrophy, which kills boys in their early twenties. We need a Sharktank for cancer, which kills everybody, constantly.
We are far too dependent on mainstream science and scientists. We look at our doctors and researchers as if they are gods. And, yet, we are the only ones who will suffer the consequences of their failures. Sharktank exists because a group of people chose to stop abdicating responsibility for the lives of their children who suffer from cystic fibrosis.

This does not mean that we give no credit to the scientists who have gone before us, and who go on, daily, doing the work of science. We stand on the shoulders of these scientists, in fact. Without their work, we could nothing. But we will no longer be totally dependent on them.

The next time I post, I will tell you what some of the parents on Sharktank did. We did everything legally, of course, but we performed experiments on ourselves. I want to tell you about the results of those experiments. Stay tuned for some interesting stuff.

]]>More personal stuffMelanie ChildersNone2010-03-08T08:58:46+00:00http://www.sharktank.org/melanie_childers/files/8c1462c3b2faa09b1936abcff9a5ecfb-6.php#unique-entry-id-6http://www.sharktank.org/melanie_childers/files/8c1462c3b2faa09b1936abcff9a5ecfb-6.php#unique-entry-id-6
I was once described, by my old college biology professor (at the time I had no interest in biology) as “relentless.” I’m not sure what she saw in me that would cause her to describe me in this way, but as I’ve grown older, I’ve noticed that other people see me this way, too. In fact, when I get into fights with my family, this is the trait that they bash me with. It’s not a particularly socially redeeming trait, I’m sure. It’s not easy to be around someone so single-minded and focused.

I don’t believe in saying, “Oh well, that’s just the way that I AM.” If you are hurting other people and doing harm to them emotionally, you should change your behavior. And, I do try to curb my relentlessness in social situations.

But, in some situations, such as work, or study, it serves me well. So, I can’t flush it down the toilet entirely.

I like to think of myself as a fighter. But, I have lost a battle that was central to my happiness—I lost my son to cystic fibrosis. And my life will never be the same again. Never.

I used to be a bill collector. I was a very good one. I worked for the collection arm of Equifax, and I was their top collector. Being relentless serves you very well in that situation, as you might imagine.

After my son died, though, I just couldn’t do that job anymore. For some reason, I had to change everything about my life. And, so, I changed jobs, of course. I went back into sales for a very small company involved in the newspaper business. Being the relentless person that I am, I worked my way up to becoming a partner in the company that had previously been owned by one person. Now, it’s owned by two. And, now, it’s not so small anymore.

I travel constantly. I have been home less than 24 hours in four years. I rent cars, stay in hotel rooms, and run this business. In the warm months, I drive a Dodge dually diesel truck and pull a Titanium RV all over the United States. I don’t want to go home. There are too many memories there, and too much loss.

I don’t care much about money. It’s a tool. I don’t save it. I give as much of it away as possible, doing the most good as possible, in the world that I inhabit. Some day, someone will find me sitting, dead, in front of my desk in a hotel room, and my family will have to ship my body home for burial. And that’s fine with me. I’ve got enough life insurance so that I won’t be a burden to anyone when I die. But, I will, with what’s left of my life, serve others.

I want, for myself, only one thing, and I can’t have it: Keith. I don’t expect happiness anymore. It is not possible for me. And I know all of the old bromides, and the clichés, and the psychologists’ advice: Life must go on. I agree, in fact. But no one says that it goes on with happiness. And anyone who has ever lost a child knows that.

I don’t mean to paint a dreary picture here. There is some happiness in my life, but all joy for me must be experienced in and through others’ joy. A vicarious sort of pleasure. Maybe it is borne from guilt, that I could not save my son. I don’t know. I don’t care. It just is what it is. That part of me that is relentless says, “Hey, I want what I want, and I won’t be happy without it.”

And that’s okay. I look around the world and I see people being blown up by bombs, hacked to death in front of their families by machetes. All kinds of misery that just “is what it is.” Life isn’t fair. Not everyone’s life is to be lived with pleasure, or with joy. And I am very fortunate to be able to serve, and to, perhaps, make this world a better place for my having been here. And, too, maybe this was why my son was here with me, and graced this earth with his wonderful presence for a short time.

I don’t know. But, for the rest of my life to have meaning for me, I have to operate on that basis. And, so, I study cystic fibrosis.

I hope that you are interested, and relentless enough, to make the journey with me. . .
]]>The Lactoperoxidase System and the Third Anion (Thiocyanate)Melanie ChildersNone2010-03-06T15:14:47+00:00http://www.sharktank.org/melanie_childers/files/9ef35751cdadde037f74b7cc6a9cf761-5.php#unique-entry-id-5http://www.sharktank.org/melanie_childers/files/9ef35751cdadde037f74b7cc6a9cf761-5.php#unique-entry-id-5
There are many natural compounds that are lethal, or at least, very damaging to these pathogens, but those compounds often damage normal tissue to the same extent that they kill these bacteria. OSCN does not. This compound is a killing machine when it comes to these pathogens, but it does nothing to normal tissue.

For many decades, scientists have been trying to figure out why CF patients can’t kill pathogens like the rest of us do. Why do these same ugly bugs invade these kids’ lungs and end up destroying them?

They studied beta defensins, which are small peptide molecules that assist in killing bacteria and fungi. This was all the rage in CF research when my son was still alive in the 1990’s. They thought that the salt composition of the CF ELF had something to do with inactivating these peptides. This would have provided a direct link between the lack of chloride anion transport and the pathology seen in the disease. Didn’t pan out.

They studied lymphocyte function, receptors on the cell surface, mucus properties, all kinds of things that could be directly associated with chloride transport (or the lack of it, as seen in CF patients) and explain the lack of ability of these patients to fight these particular pathogens, and nothing panned out. Why?

Because chloride transport is not the gold standard of CF pathology. It’s the wrong anion. I’m not saying that the CFTR doesn’t transport chloride anions. It does. But the transport of chloride anions, and the lack of it, is not central to the disease process in cystic fibrosis.

The lack of transport of thiocyanate is. Moreover, thiocyanate is lethal to the very pathogens that are the harbinger of death in these patients. I’m not trying to be melodramatic here, because melodrama should not be a characteristic of a good scientist, but I am stating a fact. Here is a great article on this topic:

The lactoperoxidase system links anion transport to host defense in cystic fibrosis.
Conner GE, Wijkstrom-Frei C, Randell SH, Fernandez VE, Salathe M.
FEBS Lett. 2007 Jan 23;581(2):271-8. Epub 2006 Dec 19.

For the first time, anion transport can be linked to the pathology seen in this disease. Put that in your pipe and smoke it, boys and girls. The bell is beginning to toll, and it marks the end of the rule of cystic fibrosis in the lives of it’s victims.
]]>The Third AnionMelanie ChildersNone2010-03-05T20:16:53+00:00http://www.sharktank.org/melanie_childers/files/d36d39c0a8ddd53cc3dd12c46b88a99c-4.php#unique-entry-id-4http://www.sharktank.org/melanie_childers/files/d36d39c0a8ddd53cc3dd12c46b88a99c-4.php#unique-entry-id-4
These are important because, first of all, when you have no CFTR protein to transport them, these compounds don’t get to the surface of the airways. And that results in the damage that we see in the lungs, in this disease.

So, so far, we know that the CFTR transports chloride ions, but we can’t link that up with any part of the pathology seen in the disease. At least not in any way that is understood well yet.

And, then, we know that the CFTR transports glutathione ions (GSH). Let’s talk about how the lack of transport of these kinds of ions causes some of the pathology seen in the disease, and then we’ll go on to some other transported compounds.

Glutathione is the most prevalent antioxidant in most biological systems. But, what’s an antioxidant? Well, for our purposes, (and for GSH, specifically) let’s just say that it is a compound that is negatively charged. A negatively charged compound is one that has an extra electron on it. By “extra” we mean that it needs another electron in order to make it neutral. So, there it is, out there, in the ELF (normally, but not in CF, because the CFTR is not there to transport it to the ELF), looking for some other compound to supply that electron. When it picks up that electron, it has become oxidized. And that’s why it’s called an antioxidant. Because it is able to pick up an extra electron and become oxidized. It’s like a sacrificial lamb, to oxidants. Those oxidants could cause, and do cause, in CF, major damage to the tissues that are the lungs. But, if GSH is there, to pick up their extra electrons, they can’t do that. So, GSH is pretty important to the antioxidant status of the lining of the lungs.

Let’s not forget, though, the rest of the system. We’ve got a cell, and that cell is a part of the tissue that lines the airways. Then we’ve got the CFTR, which is supposed to sit in the membrane of the cell, and transport compounds to the lining of the airways. So, if you don’t have the CFTR, you’ve got

(1) Not enough GSH in the airways, and

(2) Too much GSH inside of the cell

Think of it as I do: Inside and outside. You’ve got problems associated with inflammation in the airways, and that’s because the CFTR is not transporting GSH. GSH is not in the airways, to act as an antioxidant, and oxidants create inflammation. So, in CF, you have airway inflammation. That’s the OUTSIDE of the cell.

But, on the INSIDE of the cell, you have too much glutathione. And, that glutathione acts on other things inside of the cell. Part of our first paper addresses this; particularly in the case of metallothionein.

Metallothionein is a stress protein that is 60 kiloDaltons in size. It’s expressed in the cell in response to stress, and in response to zinc. I bet you didn’t know that CF patients had a problem with zinc, did you? Here are some citations to articles that are about zinc and CF:

Abnormalities in zinc homeostasis in young infants with cystic fibrosis.
Krebs NF, Westcott JE, Arnold TD, Kluger BM, Accurso FJ, Miller LV, Hambidge KM.
Pediatr Res. 2000 Aug;48(2):256-61.PMID: 10926304

Low plasma zinc concentrations in young infants with cystic fibrosis.
Krebs NF, Sontag M, Accurso FJ, Hambidge KM.
J Pediatr. 1998 Dec;133(6):761-4.PMID: 9842040

Now, this does NOT mean that you should go out and chew up a bottle of zinc supplement. In fact, even if you just supplemented zinc a little bit too much, you’d have a real problem if you have CF. Because the problem, in CF, is not that you don’t have enough zinc, but rather, that you can’t deliver it, or pick it up, from the enzymes and proteins that use it. It’s called homeostasis. Zinc delivery and chelation represent the ways that your cells handle zinc and that’s called zinc homeostasis.

Hopefully, you are asking yourself: What in the heck does zinc have to do with glutathione? Well, that’s where metallothionein comes in. Glutathione forms a bond between metallothionein and copper, zinc, and nitric oxide. Metallothionein can hold all of these compounds on it, in the INSIDE of the cell. And, in fact, metallothionein delivers these compounds to where they need to be. And, when you’ve got too much glutathione inside of the cell, it’s like having too much glue for these compounds. They can’t get delivered like they should be delivered. And there’s a problem, in CF, and pathology associated with every single one of these compounds: copper, zinc, and nitric oxide. There are what I call “failsafe mechanisms” built into the cell for copper homeostasis, so there’s not as much of a problem with that metal. But few for zinc. And few for nitric oxide.

I could write another three or four pages explaining everything that screwed up zinc homeostasis causes in CF, but I already did that, in the article cited below, and it is not absolutely central to the topic (the CFTR doesn’t transport zinc), although it contributes substantially to the lack of immune function that is seen in this disease:

Med Hypotheses. 2007;68(1):101-12. Epub 2006 Aug 24.
A new model of cystic fibrosis pathology: lack of transport of glutathione and its thiocyanate conjugates.

So, we know the inside/outside problems with glutathione in CF. Or, at least one of them. We’ll get back to that later.

I want to tell you about a great screw up. Mine, and Sharktank’s. (But mostly mine, I admit it.) Science is filled with these sorts of things. Serendipity, I think it’s called. Where you’re looking for one thing, and you find something else by accident.

We weren’t looking for another negatively charged compound, you see? We knew there must be something else there, but we knew that we didn’t have the instruments to find it. A lack of transport of glutathione and a screw up of zinc homeostasis couldn’t explain, all by themselves, the things that happened in this disease. But, we were so centered on finding out how to cause the increased expression of the MRP proteins (that can replace the mutated CFTR, in the membrane of the cell), we centered on that, and figured that we might come upon some other significant information through the continued study of the MRP proteins.

We accidentally found out about thiocyanates, the third anion. We were studying isothiocyanates, as an inducer of the MRP proteins, and we found out that thiocyanates are transported by the CFTR. Well, we didn’t actually find that out. We guessed it, because we read these articles:

Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Linsdell P.
Can J Physiol Pharmacol. 2001 Jul;79(7):573-9.PMID

Thiocyanate secretion in sweat in cystic fibrosis.
GIBBS GE, HUTCHINGS RH.
Proc Soc Exp Biol Med. 1961 Feb;106:368-9. PMID: 13705074

[Thiocyanate--a pathogenetic factor in cystic fibrosis (mucoviscidosis)?]
Weuffen W, Hein J, Below H, Gülzow HU.
Padiatr Grenzgeb. 1991;30(3):205-10. German. PMID: 2067870

But, it turned out to be absolutely, astoundingly correct! And, CENTRAL to the pathology seen in this disease.

Now, isn’t that special? We will talk about thiocyanates in our next blog post, but in the meantime, think about how nice it would be for a CF patient to be able to be rid of all the nasty pathogens, like pseudomonas, burkholderia, and staph? THAT’S where thiocyanates come in (SCN-, for short). The third anion.
]]>SharingMelanie ChildersNone2010-03-04T09:26:36+00:00http://www.sharktank.org/melanie_childers/files/ce4f456be481bbe75c792eb3e82b78ea-3.php#unique-entry-id-3http://www.sharktank.org/melanie_childers/files/ce4f456be481bbe75c792eb3e82b78ea-3.php#unique-entry-id-3
What kinds of things, though? What are some of the characteristics of these compounds?

Well, we know for a fact that the CFTR normally moves chloride ions to the outside of the cell, right? So, because we know that the chloride ion (or anion, in this case, because it’s negatively charged—as opposed to a cation, which is positively charged) is negatively charged, we can safely assume that, whatever the CFTR transports, it’s probably going to be negatively charged, too.

And, because there is no known link between a lack of chloride ion transport and the pathology (disease state, ie., damaged lungs, pancreas, etc.), then we must also at least suspect, if not assume, that the chloride anion is not the only thing that is transported by this protein. So, we’re probably looking at, at least, one more substrate of the CFTR, if not two more.

To find out the answer to which substrates those might be, we can look at the other protein, the MRP. The reason that we look here is because this protein has been extensively studied by cancer researchers. The reason that it is studied by cancer researchers is because they want their chemotoxins, which kill cancer cells, to remain in the cells, to get at an optimum level, so that the cells will die. And MRP is the enemy of that goal, because it is made inside of the cell, and it’s job is to pump OUT chemotoxins. If you have cancer and your cells respond to therapy by expressing MRP, you are in trouble if you think that many forms of chemotherapy will work for you. Your cells will just use the MRP protein to pump them right back out. So, researchers have studied this protein, and others like it, for much longer than they’ve studied the CFTR protein.

And we are very happy about that, because we can take some things that we know about the MRP and apply them to the CFTR protein and make some good guesses as to what those other substrates are. Because they are highly likely to share substrates. Not share as in I’m going to share my lunch with you, but share as in having some of the same ones. In other words, whatever the MRP proteins pump out is probably close to what the CFTR protein pumps out. And, by looking at these pumped out substances, we can get a good idea about what the CFTR pumps out, besides just chloride.

We know that the MRP proteins pump out something that is called an “adduct” of glutathione (GSH, for short). An adduct is simply a way of saying that something is combined with something else, and joined together. So, an adduct of glutathione is nothing more than a chemical, bound to glutathione. Some examples of adducts of glutathione are ATP-GSH, and GSNO (this is glutathione bound to nitric oxide).

So, when you are guessing about substrates, you can make a good guess that while the MRP proteins pump out ADDUCTS of glutathione, the CFTR probably pumps out glutathione, itself. And, interestingly enough, GSH is an anionic compound. Once something else is bound to it, though, GSH loses it’s negative charge, by accepting an electron from whatever it is binding to.

Remember: the CFTR transports negatively charged compounds. So, we are looking for negatively charged compounds that the MRP is pumping out, combined with GSH adducts. That’s how the two proteins are different. One pumps out negatively charged compounds, and one pumps out adducts of negatively charged compounds.

I’m closing this post for now, because I want you to be able to consume quite a few bits of information in it. But, before I close, I want to tell you that we made a very lucky guess and we weren’t even trying to guess. We were trying, instead, to find compounds that were enough like the chemotoxins that caused the MRP proteins to be expressed in that boy with cancer, to cause these same proteins to be expressed in all CF patients. In trying to find the answer to that question, though, we found another answer, that is central to the reason that CF patients get sick in the first place, and go on, getting sicker and sicker, throughout their lifetimes.
]]>Chemotoxins are bad, but the proteins that transport them are good—at least for CF patients. Why?Melanie ChildersNone2010-02-28T14:40:53+00:00http://www.sharktank.org/melanie_childers/files/8a43658ab6b064fa29ddc173fdf78b8f-2.php#unique-entry-id-2http://www.sharktank.org/melanie_childers/files/8a43658ab6b064fa29ddc173fdf78b8f-2.php#unique-entry-id-2
Now, obviously, we don’t normally take in chemotoxins, such as cyclosporine, epirubicin, etc., so we don’t normally make the proteins, such as the MRP, to transport them. These proteins are only made by our cells when we NEED them, to transport these chemotoxins.

By now, you should be saying, “Hey! Wait a minute, if the job of the CFTR and the MRP proteins is to transport compounds to the surface of the lungs, to comprise part of the ELF, why would the ELF use chemotoxins?” It doesn’t. The ELF doesn’t use chemotoxins. The transport of the chemotoxins is obviously a way that the cell has to get these bad and poisonous compounds out of it. But, while it’s transporting these bad compounds, it’s also transporting things that the ELF needs, to protect the tissue of the lungs.

I’m going to introduce you to another new word here: substrates. Substrates, simply put, are everything that a particular protein transports (they can also be something that a protein ACTS UPON, in any way, but for our purposes, they are what our favorite proteins – the CFTR and the MRP -- transport). So….if the MRP transports chemotoxins, then these chemotoxins are it’s substrate. If the CFTR transports something, then those compounds are it’s substrate, too. Got it?

So, the “happy accident” showed us that the MRP proteins have substrates that are shared with the CFTR protein. Since the CFTR protein is missing in action in cystic fibrosis, and the MRP proteins can take it’s place, they must have at least one common substrate.

What are they? What does the CFTR normally transport that the MRP transports, too?
]]>The Happy AccidentMelanie ChildersMelanies Blog2010-02-21T21:55:12+00:00http://www.sharktank.org/melanie_childers/files/339e0e4b27f1c99431f792ccd83bf91b-1.php#unique-entry-id-1http://www.sharktank.org/melanie_childers/files/339e0e4b27f1c99431f792ccd83bf91b-1.php#unique-entry-id-1
First, though, I want to talk about the OUTSIDE of the cell--the "stuff" that lines the airways, and why it is so very important to this story, and to the wonderful accident that you are soon to know about....

Think about breathing in air. We don't get to choose what goes into our lungs. And the world is a very dirty place, filled with bacteria, dust, all kinds of particles that don't do a thing for our lungs. And we can't live in a bubble. So the lungs have a really good system to get this dirty stuff out, or to kill it, if it's biological matter.

It does this by making mucus and other "stuff" that lines the airways. All of this "stuff" has to be made INSIDE of the cell. And it has to be transported to the OUTSIDE of the cell. I bet you can guess what transports it, by now, can't you? If you can't, then I'll tell you: the CFTR protein. Yes! All (well, not ALL of it, but some pretty important part of it) that nice "stuff" that is there, that coats the insides of the "tubes" that are our airways, is put there by the CFTR protein. The CFTR protein acts like a "pump" to put this "stuff" on the surface of the airways.

By now, if you're a parent of a child with CF, you're saying, "Hey! Wait a minute! MY kid has a lot of mucus in his/her lungs and you're telling me that the CFTR is responsible for transporting mucus, too?" Well, yes, parts of mucus are made inside of the cell, and a certain kind of cell called the serous cell, is responsible for making mucins (components of mucus) and the CFTR is in the membrane of these serous cells and transports these mucins to the OUTSIDE of the cell, to help make up part of that "stuff" that coats the airways. BUT, there are other mucus cells, that are not serous cells, that also produce mucins. And they ARE going ninety to nothing, producing mucins, to make up for what the serous cells don't produce. We will get to them later on and talk about why, even though these mucus producing cells are trying to "help," they are doing a lot of harm. As a CF parent, we know the harm, because most of us do postural drainage to get this mucus out, because we know that bacteria love to live and grow in it. But, that's for much later.

Hopefully, by this point in our story, you are curious enough to ask, "What the hell is in that "STUFF"? Asking that question is the same as asking the question: "What does the CFTR protein transport?"

And THAT'S where we get to our happy accident. Because that accident answered part of that question, and gave us the key to the rest of the picture of what the murderer we call Cystic Fibrosis is.

The accident happened in 1997. We read about it in a letter to a very respected medical journal, in the UK, called The Lancet. Here is the information on it, if you want to look at the letter:

Lancet. 1997 Sep 6;350(9079):711-2. Induction by antitumoral drugs of proteins that functionally complement CFTR: a novel therapy for cystic fibrosis? Lallemand JY, Stoven V, Annereau JP, Boucher J, Blanquet S, Barthe J, Lenoir G. PMID: 9291908

But I will explain it to you here. Lallemand, a doctor in the UK, had a patient that had both CF and cancer. He wanted to get rid of the cancer, so he began the usual cancer treatment of a cocktail of drugs, called "chemotoxins." These drugs, commonly a part of a course of therapy called "chemotherapy," kill cancer cells, and other normal cells, too.

But, something happened to this patient with CF, while he was being treated with these drugs. He got rid of CF. He declared himself "cured." And, indeed, something very wonderful had happened to him. He had cultured Pseudomonas Aeruginosa for many years of his life, and yet they were gone in a few weeks. Totally eradicated. He no longer had to do postural drainage. He became as normal as any normal person. And this patient was in his early twenties. What happened to make him well?

Your first guess would be that the CFTR protein became normal, wouldn't it? Wrong. My temptation here is to leave this for my next post, but I just can't do that to you, my friends. I'll give you the answer right now:

His cells had begun making a protein that could do the same things as the CFTR protein could do. This protein, and in fact, the whole CLASS of proteins, called the multi-drug resistance associated proteins (MRP), also sits in the cell and transports out "stuff." And that "stuff" is enough like the "stuff" that the CFTR protein transports so that if you have the MRP protein in the membrane of the cell, it can do the job of the CFTR protein. The scientific way of putting this is: The MRP proteins are functionally redundant to the CFTR protein, which means, again, that the MRP protein can substitute for the CFTR protein in terms of their function. It can do the very same things that the CFTR protein does.

NOW, you're probably asking, "Well, why don't we all have those proteins? And why, if this CF patient had them, didn't they work before he got sick with CF?" I'll tell you why: Because these proteins are only made by the cell when the cell has something toxic, like chemo drugs, given to it. Obviously, we can’t start feeding our CF kids chemo drugs; it would kill them, but what was "happy" about the accident is that we found out that another protein could take the place of the function of the CFTR protein.

And, this was really, REALLY a cause for happiness because this class of protein, the MRP proteins, have been studied for many more years (in cancer research) and we can find our more about them, from that research, than we know about the CFTR protein. We could find out more about the "stuff" that these proteins transported. And, maybe, we could replace that "stuff," if we knew what it was, by having these patients inhale it into their airways?

But, that's not what we did. There are reasons why we didn't go this route, and I will get to them much later on, but the most important thing, here and now, is that you know that we now have a way to find out what's in that "stuff" that the CFTR protein is transporting to the lining of the airways.

See you next post, where I will tell you what's in that "stuff." For now, I want you to ask yourself, what in the world could be in that "stuff" that is transported by both proteins? Why would a protein that is made to move toxic compounds out of a cell also help to move out things that could be helpful in the lungs? How in the world could these two things have anything in common?

And, by the way, if you feel a little silly talking about that “stuff,” you can also call it “epithelial lining fluid,” or ELF, for short. That’s what I will be calling it from now on, too.

Melanie Childers

]]>My name is Melanie Childers and I study Cystic Fibrosis.Melanie ChildersMelanies Blog2010-02-21T14:36:42+00:00http://www.sharktank.org/melanie_childers/files/c0421e328e4243b45cec5e28bd3ec7ae-0.php#unique-entry-id-0http://www.sharktank.org/melanie_childers/files/c0421e328e4243b45cec5e28bd3ec7ae-0.php#unique-entry-id-0
I don't have a science background and I don't want one (after fourteen years of studying this disease, I guess I now have a science background, though, whether I want one or not). I saw, about three years before my son died, that those studying this disease were not at a place where they could stop it. They were, in essence, in that regard, failures. I thought that if I followed their path, exactly, that I would be in the same place that they were in--failure--at the end of my studies. So, I didn't go to school to learn about Cystic Fibrosis. I started at the beginning and took it from there, wherever my curiousity took me. I began at the cellular level and started comparing the differences between normal cells and CF cells. The purpose of this blog is to tell you that story--the story of the path that I followed to the point where we are now, in our understanding of Cystic Fibrosis.

I want to point out that I did not get to where I am alone, in my understanding of this disease. A group of people that I met on Cystitc-L started Sharktank, a mailing list for parents of CF patients, and CF patients, themselves, whose goal was the same as mine: to understand Cystic Fibrosis. Sharktank grew to over 300 members (really quite small, in terms of mailing lists, I think), and we incorporated a non-profit entity, called Share International Research, to fundraise for our project. Many, many people on Sharktank contributed to this quest, in many small and large ways. Without each one of them, we would not be where we are today. Anyway, this is the story of that quest. I hope that you find some hope and even some pleasure, in the reading of it, but also, I hope that you are edified by it. Knowledge is, indeed, power, and it is empowering, as well. Nowhere is that more true than in the fight for life. So, here we go....

Of course, I followed the mainstream research, but I soon saw that, when it came to the "gold standard" of CF pathology--chloride ion transport--it took me nowhere in terms of pathology. I could not find where the lack of transport of chloride ions caused the disease in these kids. No one else could, either.

But, you can't understand anything about CF unless you understand the protein, itself. And the CF protein (Cystic Fibrosis Transmembrane Conductance Regulator, or "CFTR," for short) is the protein at the center of this disease. The most prevalent mutation of this protein causes the protein to be misshapen. Because it is not shaped normally, the cellular "quality control" mechanisms don't let it get to the place it should arrive at, in the cell: the membrane of the cell.

The CFTR protein is supposed to be made and matured, in the endoplasmic reticulum (ER) of the cell. In CF, the most prevalent mutation (F508) causes the protein to be shaped incorrectly (kind of like if you were born with an arm coming out of your head) and so the protein is stopped before it can "traffic" to the membrane of the cell.

The membrane of the cell is where the CFTR is supposed to come to rest. And that's where it does it's job. It's job is to transport ions out of the cell, onto the lining of the lungs. For a very long time, it was thought that the most important ion that was NOT transported onto the lining of the lungs was the chloride ion. Scientists are now realizing that it is not, and that other more important negatively charged compounds, are more important than chloride ions. It is these ionic compounds that are at the heart of the disease.

So, now you know just enough about the CFTR protein to understand it's job. Oh, one other thing--pretty important--the CFTR sits in a particular KIND of cell's membrane--the epithelial cell. These cells line the insides of organs. Look at it like this: Say you have a lung, and that lung has "tubes," (called the airways) that air comes into. Something needs to be lining those tubes, to protect them from the outside world. And the cells that make up the part of the airway that is the lining are epithelial cells, and the stuff that lines the airways (sits on the surface) secrete compounds that are there to protect the airways. This "stuff" is there for the purpose of keeping the airways clean of bacteria, among other things. So, what it's made of is pretty important. The CFTR is responsible for putting a part of that "stuff" there, and in CF, the CFTR is still stuck inside of the cellular organelle that makes it--the ER.

Without a doubt, the most important cellular processes take place inside of the cell. But, in order to understand CF, you have to understand it in the world it is in--the lungs. So, even though you can look at the cell, and see the CFTR in it's membrane, you have to recognize that, because the CFTR TRANSPORTS "stuff" to the surface of the lining of the lungs, the fact that it moves this "stuff" OUTSIDE the cell, makes that OUTSIDE "stuff" pretty important. In fact, it is this "stuff," which protects the airways.

So, now you know a little bit about the world that CF lives in. And you know that we are going to be looking at the inside of the cell, and the outside of the cell, and at the organ that holds both, in terms of the major system that is targeted for pathology in this disease--the lungs.

Tune in next week, and I will tell you about a wonderful accident that led to a big discovery about CF, and that all important "stuff" that lines the airways. This accident, and particularly what was learned from it, signals the beginning of the end for this disease.
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