Those of you who don’t know me probably think that you haven’t heard from me on this blog for a long time because I’ve been farting around, or lost interest, or something else just as silly. Those of you who DO know me will not be surprised to hear that, of course, I’ve been working.

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?

Do 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.

Please 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?

Okay, last time I was on a rant. Forgive me. I sometimes get ahead of myself. Let me explain:

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.

Bell is my granddaughter. Her name is actually Aera Bella Odeye, but I call her Bell (pronounced Bay-ull in Texan). Bell is a wonderful young woman. Well, as much of a woman as a girl can be at 17 months old. But wonderful, nevertheless.

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.

Chloride transport. It bothers me. It sucks, quite frankly. For over fifty years, we’ve been battered with chloride transport. The CFTR is a chloride channel, we’re told, ad infinitum, ad nauseum.

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

I had a dream last night. I have a lot of them, actually, and remember them more than most people, probably. I don’t give them much credence, except in regards to what they say about my own state of mind. Except, when they are about biochemistry.

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.