PCD Foundation Blog

Johnny L. Carson, Director

Department of Pediatrics Ultrastructure Laboratory

The University of North Carolina at Chapel Hill

While many in the PCD community know the significance of ultrastructural analysis of cilia and flagella in the diagnosis of PCD, most have never even seen one much less have an awareness of how they work and why they are useful for this purpose. Electron microscopes come in two varieties, transmission and scanning. While both varieties use an electron beam to generate an image, they function in fundamentally different ways. Scanning electron microscopes allow us to see surface topography at high resolution and while they are useful scientific instruments for a variety of purposes, they are not used to make the diagnosis of PCD. The transmission electron microscope (TEM) is the type used for analysis of cilia in the diagnosis of PCD. The word “transmission” refers to the transmission of electrons through the specimen to generate an image on a photographic plate. You may ask, “Why can’t we just use a light microscope to analyze cilia for PCD?” The reason is that the structures we need to examine in cilia are small molecules beyond the limits of resolution of light microscopes. What does this mean? In order to produce an image, whether it is on the retina of the human eye, a photographic plate, or a chip in a digital camera, light has to interact with the object being imaged. Visible light moves in the form of waves that interact with an object to be imaged in ways that cause the regularity of those waves to be interrupted. This is called diffraction. It is important that the object being imaged is able to interact with light in a way that causes diffraction. Think of a series of waves washing up along a beach. If the waves crash against a boulder, the interaction creates a large splash. In contrast, if these large waves wash over a small seashell, hardly a ripple is evident. While the cilium might be considered analogous to the boulder, the small structures within the cilium (the seashell) are too small to interact with light waves in order to image them effectively. For that purpose, we choose electrons. Electrons exhibit some of the same properties as light waves but they have much smaller wave forms capable being diffracted by much smaller structures. Thus, electrons can interact with the “fine structure” of the cilium in a way that makes it possible to obtain images of exceedingly high detail down to the molecular and sometimes atomic levels. The benefit of the electron microscope is not that it can magnify thousands or millions of times, but rather at those magnifications, one can appreciate the fine details of the specimen.

Electrons are not easy to produce, at least for electron microscopic purposes. The electron beam itself is generated from a tungsten filament, not much different in appearance from an ordinary light bulb filament. The hard part is focusing this beam of high energy electrons coherently. The path of an electron in air is less than two inches. However to be effective for electron microscopy, the beam must be focused through a series of large electromagnetic lenses. In order to generate the electron beam, electrons are literally boiled off of the filament tip under high voltage, anywhere from fifty thousand to one million volts. To accommodate the large lenses in doing their job, the beam must be directed through a long tube, the column, constantly maintained under a high vacuum. The specimen is positioned in the column among the lenses and the image focused onto a phosphorescent screen for examination. Historically, a conventional plate film camera was positioned directly below the viewing screen but in newer microscopes digital cameras are used. Depending on the application, transmission electron microscopes range in price from approximately $100,000 to millions of dollars. The purchase price is just for starters. The operation and maintenance of these sophisticated instruments generally requires at least one full time on-site technician for management as well as the purchase of a hefty service contract to troubleshoot complex problems that inevitably occur. For this reason, most electron microscopes are found in large multi-user facilities that allow the operational costs to be shared.

Unlike a light microscope in which a specimen can simply be positioned on a glass slide and examined, the processing of a specimen for TEM introduces a whole new level of complexity to this technology. In order for electrons to generate an image, the specimen must be treated with chemicals containing osmium, lead, and uranium in a way that deflects their “transmission” through the specimen making them “electron dense”. Water is also removed from the specimen by passing it through alcohol and the specimen is infiltrated with liquid plastic that is then allowed to polymerize in a small plastic capsule about the size of a pencil eraser. Processing a single specimen to the point of being ready to examine in the electron microscope usually requires about one week. A specialized instrument, an ultramicrotome, is used to shave extremely thin sections from the block that are then placed on a copper mesh for positioning in the microscope.

Proficiency in electron microscopy is a blend of science, art, eye-hand coordination (and sometimes a little luck). Professional electron microscopists have a good educational background in biology, chemistry, and physics. There are educational programs for aspiring students who should expect to spend 18-24 months to master the basic skills of electron microscopy theory and practice. This may sound like a daunting commitment but for the electron microscopist every specimen is a new and unexplored trail.

The electron microscope is one of the key instruments in the scientist’s tool box. In terms of PCD, this disease languished in obscurity and misunderstanding for three quarters of a century awaiting the advent of routine biological electron microscopy. Since that time, many new ultratructural discoveries have contributed to our knowledge of the pathophysiologic basis of PCD. Moreover and of equal importance to knowing what PCD is, electron microscopic studies have been central to achieving an understanding of what PCD isn’t. Also in recent years, studies of cilia characterizing fundamental mechanisms of motility and transport also laid an important scientific foundation that has led to an emerging knowledge of their role as sensory organelles. This has opened up the study of entire new cellular landscapes with the discovery than cilia play a significant role in a number of other human health syndromes collectively known as ciliopathies. While cilia exhibit a certain uniformity of structure and exist widely across both the plant and animal kingdoms, it will be interesting to learn whether there are unifying characteristics that link these motility and sensory functions in both health and disease. The emergence of the electron microscope as a routine instrument of research allowed us to see structures in cilia that are the misshapen product of gene mutations in PCD. Inevitably, the ongoing revolution of molecular biology will reveal to us the genetic basis of PCD and technological improvements in electron microscopes and ultrastructural technologies will continue to be an important tool in this quest.

Copyright Johnny L. Carson 2007

E-mail: jcarson@med.unc.edu

It is getting to be more and more important to understand the distinction between primary and secondary ciliary defects.* Here is a brief overview:

When you Hear PCD, Think Proteins

Primary or inherited defects are the result of genetic mutations that impact the production or expression of specific proteins. In the case of PCD (which includes KS, SI, ciliary aplasia, etc), those proteins are important in building the structure of cilia and flagella. They are also turning out to be very important in regulating organ placement and development. We have yet to discover what other things may be impacted by these proteins. If, because of a genetic mutation, your body can not make or use a protein adequately–EVERY SYSTEM THAT RELIES ON THAT PROTEIN WILL BE AFFECTED. PCD is not “selective.” You won’t have some cilia that work and others that don’t. You either have the functioning protein or you don’t, so it is a global or systemic problem for which there is no fix until we come up with treatments that work at the genetic level–thus the importance of genetic research.

Also, a faulty protein is with you forever so the problems associated with PCD, unlike SCD, are almost always apparent from birth or shortly thereafter. You won’t develop PCD over time–it is with you from day one. Granted, it may take a few months to recognize that the symptoms are not typical, but if your PCD symptoms started when you were an adolescent or only as an adult, you most likely do not have PCD.

Different Ultrastructural Changes

Secondary ciliary dyskinesia mimics PCD in symptoms. There may also be ultrastructural changes in ciliary biopsies in SCD, but these changes are very different and distinct from primary changes and a pathologist familiar with the two conditions will be able to distinguish between them. Every time anyone has a cold, they experience secondary ciliary dyskinesia. Any viral or bacterial insult will damage and/or destroy cilia. Repeated infections resulting in SCD can have the appearance of genetic ciliary dysfunction, but they are not the same. People with SCD can eventually recover fully functional cilia, assuming the underlying infection or condition can be treated adequately–people with PCD can not. Treatments for SCD and PCD may be very similar or even identical, but that does not mean they are the same disorder.

PCD vs. SCD What Difference Does it Make?

So why does it matter? There are several reasons:

1.) The long-term outlook is different. PCD is progressive and, for many, debilitating. If my child had SCD (or I did) and there was the possibility that whatever is causing the condition could be “cured,” I’d want to know that.

2.) PCD is genetic. It runs in families. Telling someone with SCD that they have PCD will give them the mistaken impression that they are at risk of passing on the genetic mutation that causes PCD when they are not.

3.) If someone is sick enough to have symptoms of SCD, it is important to find out what the actual cause is so that it can be treated appropriately. Clearly, something is wrong. If it is mislabeled PCD, then the true problem may never be identified.

4.) New information about the PCD phenotype (physical expression of the protein defect) is coming to light all the time. For instance, recent papers indicate that some of the proteins associated with PCD are also implicated in congenital heart disease. The new recommendation is that all people with PCD have a thorough cardiac evaluation. Because these proteins are not an issue in SCD, this recommendation does not apply to individuals with SCD. People with SCD who have been misdiagnosed with PCD are then put through an expensive, frightening and unnecessary cardiac evaluation.

5.) Millions of dollars are going into defining the “phenotype” of PCD–what the disease looks like and how it responds over time. This effort will be compromised if people who don’t actually have the genetic mutation are included in the data collection.

If You Think You May be Misdiagnosed

It is frustrating for all of us that the diagnostic picture in PCD is so mangled right now. However, the clinical picture of PCD, and the fact that it is a distinct clinical entity that can be distinguished from other conditions like SCD, is getting clearer all the time. Hopefully, this information will start to make its way into clinical practice so patients don’t have to face the frustration of misdiagnosis as often as they do now–and it is very common. The best way to be sure of your diagnosis is to participate in the research program or to contact one of the six PCD clinical centers who have expertise in this area. These centers are also valuable resources for information about collecting, preparing and analyzing ciliary biopsies for centers not affiliated with the PCD studies.

*PCD—Primary Ciliary Dyskinesia (inherited)

SCD—Secondary Ciliary Dyskinesia (acquired)

Michele

One of our biggest challenges, as people who care about PCD, is raising awareness about the disorder–not just in the medical community, but also with the general public. We all have had the experience of saying PCD or Kartagener’s and seeing eyebrows raise in confusion. For me, I generally try to describe PCD by saying “it’s like CF” or “it’s a little like really bad asthma,” trying to find a common frame of reference, but I’m always aware that those comparisons don’t do justice to accurately describing PCD. I’m convinced that one day PCD will have the same name recognition as cystic fibrosis–we just have to be persistent and creative in finding opportunities to raise awareness. Here is a really neat story from Kansas City that illustrates how patients can find/make these opportunities.
High school student Ashli from Olathe, Kansas was diagnosed with PCD last October after a lifetime of illness. Like many PCD families, Ashli and her mom, Vicki Bicknell, were very frustrated about the delay in diagnosis and the pervasive lack of awareness about PCD, even among the specialists Ashli had seen throughout her life. This frustration translated into motivation to get involved in PCD awareness. Ashli’s Ethics Class at Olathe South High School was looking for ideas for a community service project for the students. This was a comprehensive project, getting the students involved in choosing a cause, fundraising for their selected cause, and providing all the support services (marketing, public outreach, etc) that go into creating a successful event.
Bravely sharing her personal experience, Ashli spoke to her class about PCD and about the importance of patient organizations to support disease education and research efforts. Her presentation moved her class to choose the PCD Foundation for their Ethics Class project. Ashli, her mother, her classmates and her teachers worked very hard to organize a fundraising effort, soliciting donations from local companies and selling donated items on behalf of the PCD Foundation. They also arranged for an educational seminar to be held in the Olathe South High School auditorium and invited speakers from the PCD Foundation to talk about PCD, the importance of rare diseases research and why patient organizations are important. Local media outlets were invited and the session was taped for possible use in future news or print media stories. About 300 students, faculty and interested individuals attended the seminar. Johnny Carson, PhD (UNC) voluteered his time to come to Kansas City and explain what happens in PCD and how he got involved in PCD research. He highlighted that rare disease research makes a positive impact on everyone–not just the people with the rare diseases, and encouraged the students to get involved in fundraising and support for rare diseases research and also to consider pursuing education in the research sciences. Meghan Manion and Ashli Barker spoke about their personal experiences and what it’s like growing up with a rare chronic illness They were very open about the challenges and encouraged students to ask questions. I shared my experiences from the parent perspective and talked about starting a patient foundation (and took the opporunity to share darling baby pictures of Meghan!).

Ashli, with the full support of her mother and teachers, put a tremendous amount of time and effort into this event and did a phenomenal job of organizing a very comprehensive PCD awareness program. As a result, $4,000 was raised for the PCD Foundation and now the young people of Olathe, Kansas are more informed than many medical experts about PCD!
It was a real honor for us to be invited to speak about PCD (my favorite subject) and to hopefully encourage the next generation to consider choosing education/career paths that may help people with rare diseases. Special thanks to Johnny C., Meghan M., Mrs. Ruth Ann Falls, Mrs. Birch and the rest of the Olathe South High School administration and especially to Ashli Barker and Vicki Bicknell!

Michele

I spoke with Johnny C last night about putting a patient survey together looking for clues to other cilia-related phenomenen in PCD–particularly cystic lesions in the liver, pancreas and kidneys, splenic abnormalities, congenital heart defects and eye problems.  There is increasing evidence of the importance of ciliary function in skeletal development, as well.  The problem is that some of these issues in patients are “sub-clinical” and may not rise to the level of being diagnosed, even though they may be compounding the respiratory problems faced in PCD.  Coming up with a survey that is easy enough for patients to participate in, yet provides enough valid information may be difficult.  Asplenia is of particular concern because it does happen with PCD (very rarely, however) and if it is missed, can lead to sudden overwhelming infection and even death.  I think there is valid reason to do thoracic  and abdominal evaluations on all people who carry a diagnosis of PCD, but doubt that will become standard for many years.
Back to the survey–there are also privacy concerns.  We would want to make the information available to researchers, but won’t have individual details to match a survey to a particular person.  Not sure how we will get around these issues, but it is information that really needs to be collected somewhere and we could at least make a good start.  More on this as we get closer to having a completed survey.

The PCD and Diseases of the Cilium session was interesting.  There were two talks on right-left laterality determination and how ciliary activity (of a specific kind of cilia called nodal cilia) most likely contributes to the proper development of “sidedness.”  In the absence of this activity, left/right asymmetry is random.  This appears to be true for both DNAH5 and DNAI1 defects.  The evidence for how this works is pretty compelling for total situs inversus.  It’s just not so clear for some of the other situs defects seen in PCD (in at least 6% of the population).  These other defects, called situs ambiguous or heterotaxy syndromes are concerning because they are associated with a much greater risk (200 fold) of congenital heart defects and other problems. Most of these congenital problems are picked up right away after birth, so this is not to say you should suspect you or your child has a serious undiagnosed heart defect.  It’s just something to be aware of in case symptoms should develop.  This paper will be published soon and available to share with your personal physicians.

This Gordon conference is a little different in that the clinical session included PCD, cystic diseases of the kidney and diseases that result from genetic mutations in the basal body of the cilium rather than in the dynein arms or ciliary ultrastructure (like PCD).  Basal body ciliary disorders are very serious and in some cases lethal in the neonatal period.  One common factor in understanding all of these diseases of cilia is the concept of intraflagellar transport (IFT).  IFT refers to the actual chemomechanical movement of proteins, lipids and enzymes from the surface of the ciliated tissue, into the basal body, and up the ciliary shaft (axoneme) to the appropriate place on the cilium.  When these proteins don’t “localize” to their proper area, you get functional problems.  In one known PCD genetic mutation, improper protein placement results in cilia that beat super fast at the tip, but have no functional stroke at all closer to the surface.  This is an interesting discovery because these cilia appear to beat just fine under the microscope and biopsies taken from the functioning tip of the cilium will look ultrastructurally normal.  It’s only by using high-speed video beat analysis that the defect can be detected.

There is some evidence of overlap between PCD and non-motile or basal body ciliary disorders.  The development of cysts in the kidneys, liver and pancreas are one potential area of overlap, as is retinal dystrophy or degeneration syndromes.  I’ve been working on a patient survey to get at how common these issues are in the patient group and in blood relatives of patients.  Mike Knowles thought this would be a useful project for us as a patient group and referred to it as an “enabling” survey because it would enable them to follow families who appear to have overlap syndromes.  I’ll be working with Johnny Carson and Joe Sisson on this project and we’ll provide updates on survey progress as they become available.

So far the only consistent clinical indicator of PCD, regardless of defect or gene, is low nasal NO.  I’m looking forward to the discussion about this tomorrow.

The official PCD session is due to start in about an hour.  I just got back from the poster session where I had the opportunity to speak with several of the researchers.  The Gordon Conference has strict rules about sharing unpublished material–they want to protect the work of the scientists, so I will have to limit my posts to things that are published or that I have received permission to share.  Mostly I was stunned, again, by the complexity of the cilium and the processes that go into producing functional cilia,whether motile or non-motile.  There are so many steps along the way and one mistake shoots the whole deal!

Networking is another benefit to attending these conferences.  I spoke with Scott Randell, the UNC person who handles tissue sample collection.  He has communicated with several members of the group regarding tissue donation post-transplant or in the case of death and he very much wants to set up an easy process for PCD patients to be able to make tissue donations.  The key is to have the pieces in place prior to the event, and he suggested getting consents signed and pathologist contact information as soon as a PCD patient is on the “Go” list for transplant.  We are going to try to make the necessary forms, from his end, available on our website.  Other institutions may require additional forms.  I was very glad that he tracked me down about this, though, because it seemed that the PCD community was willing to do whatever was needed–we just needed information from the other end.

I also had the chance to speak with Mike Knowles about a new bronchiectasis registry project we’ve been invited to participate in.  It is a joint effort with the NIH (specifically the MAC study) and the COPD Foundation.  It sounds like an excellent opportunity for PCD patients because the goal is to identify therapeutic targets for non-CF bronchiectasis–and we certainly fit right in to that category.  It’s tricky right now because the NHLBI is not willing (or able) to set aside research dollars for bronchiectasis, so individual groups and researchers are trying to step in to the breach.  Because pretty much 100% of our patient population develops bronchiectasis at some point in their life, this seems like a very good place for us to focus therapeutic research dollars (if we ever have them!).  The COPD Foundation is closely associated with the Alpha-1 Foundation and they do a terrific job managing resources so I look forward to working with them on this project.
There is a Gordon Conference on nitric oxide (NO) just down the road.  We have barely scratched the surface regarding the “why” for low NO in PCD, but there is compelling evidence that very low NO, in a patient who has been positively ruled out for CF, is diagnostic for PCD.  Under rare circumstances NO can be very low in bad sinusitis, but PCD and CF are the only other conditions that show low NO (and PCD is usually lower than CF), so if you are absolutely sure a patient with low NO doesn’t have CF, it leaves PCD as the likely culprit.  NO analyzers are becoming more common in large clinical facilities, so this may help us with earlier diagnosis.  We will meet with the NO group on Wednesday and I’ll let you know what comes of it.
I have to run, but I just need to say that intraflagellar transport (IFT) is really cool.  I’ll post about it later.

So I’m in the hotel cafe having my pre-breakfast coffee.  There are about 200 people at the conference and we have all meals together, but since I’m pretty useless until my first cup of caffeine, I try to fit one in prior to meeting with everyone.

I estimate that the attendance is almost half and half, American and international.  It is amazing to see the collaborative spirit in the cilia research community.  Many projects are jointly sponsored by labs all over the world.  Last night, the speakers focused on the extreme complexity of the structure of cilia and flagella.  This has been recognized for years, but there is seemingly new information coming out all the time.  Dynein arms themselves are very complex structures and the goal is to use what is being learned in Chlamydamonas (a single-celled algae) flagella because this information translates into the human genome quite nicely.

Again the researchers pointed out that not only do the structures in the cilium look different (outer dynein arms vs. inner dynein arms vs. central pair), but they are biochemically distinct which is why it is so difficult to find all the potential areas of dysfunction.  I’ve seen these models and heard the talks for years now, but when you see the complexity of ciliary ultrastructure, it actually is amazing that cilia EVER work they way they’re supposed to!

The UNC group was pretty much dragging last night–all day in planes or on shuttles and a three hour time difference to boot, so we’ll have more time to talk today.

That’s it for now–need to get back to my IV caffeine infusion–

It is Sunday evening and I’m sitting in the lobby of the Ventura Beach Marriott.  I’ve been told there is an actual beach nearby, but I’ve yet to find it!  The first session of the Gordon conference begins in about an hour, but already I’m very glad I came.  At registration, we received a schedule that includes all poster presentations and there are about 20 scientific posters related to motile cilia function.  In some ways, the posters are even more important than the formal talks because they represent the “cutting edge”–material that has not even been published yet.  It’s a great opportunity to see what’s coming up in the future for research.

Saw Johnny Carson (ultrastructure guy), Maimoona Zariwala (geneticist) and Milan Hazucha (NO guy) from UNC already.  Dr. Hazucha is in Ventura for a Gordon Conference on NO which just happens to be taking place about 2 miles from the Gordon Conference on Cilia.  We will be having a joint meeting with the NO people Wednesday afternoon.

Tonight’s session is called “Generation and Control of Ciliary Movement.”  There will be six speakers covering topics including how the structural anatomy of a cilium contributes to ciliary motility and how the actual cilium is built.  I’ll try to post after the session, if I am still awake.

The Gordon Conference on Cilia, Mucus and Mucociliary Interaction begins today.  Michele Manion is attending as a representative of the PCD Foundation.  Look for her informative updates in this blog.