I cannot die

I cannot die

For most ME/CFS patients (about two thirds), the disease has an oscillating course, with some periods of improvements followed by worsening of symptoms. Some of them can even experience recoveries, only to find themselves trapped again, weeks or months later (Stoothoff J et al. 2017), (Chu L. et al. 2019). Some anecdotes suggest that there might be a correlation with seasons, with improvements in summer, but there are no systematic surveys on that, to my knowledge.

As for me, in the last 20 years of pitiful combat with this monster, I experienced some substantial short-lived improvements, mainly during the core of summer. At the very beginning of the disease, I also recovered for a year. It was the year 2001, I was 21 and that year has been the only period of normality in my whole adult life. I spent these 12 months studying desperately and what I am as a person is mainly due to what I learned back then. I had already been very sick for about two years and when I recovered, it was as if I were born again. It was a second chance and I was determined to do all right from day one. I decided what was really important to me and I devoted myself to my goal: learning quantitative methods to use in engineering and – one day – in biology.

When darkness caught me again, I was, among other things, reviewing all the main theorems of calculus (particularly those about differential equations) with my new skills and I remember thinking that I was becoming good at developing my own proofs. I had become good at thinking and so, I reasoned, I could finally start my life! But in a few weeks, my mind faded away, and there was nothing I could do to keep a grip to all my beloved notes and books. They became mute and closed as monolithic gravestones. I remember clearly that along with this severe and abrupt cognitive decline, I developed also orthostatic intolerance, even though I hadn’t a name for it back then. But I couldn’t keep sitting, and I didn’t know why. I was forced to lay as if the gravitational acceleration had suddenly increased. My brain had changed to a lifeless stone, and so did my body.

From that very moment, my only thought has been how could I go back to my books and my calculations. And this still is my first thought, when I wake up in the morning. After almost 20 years.

I have experienced some short improvements in these years, during which I had to learn again how to study, how to do calculations, how to code. I never went back to what I was, though. And my brain is ageing, of course, as anyone else’s brain does. But in these short periods of miraculous come back I experience a rare sense of joy (along with anger and fear). Something that you can experience only if you have been facing death.

I was born and I died dozens of times in the last 20 years, and this gives me the perception that, in fact, I cannot die: I feel as if I were immortal and I had lived for a thousand years while at the same time still being in my twenties, since I have no experience of life.

In fact, I lived only when I crossed these short bridges from one abyss to the following one.

 

Immunosignature analysis of a ME/CFS patient. Part 1: viruses

Immunosignature analysis of a ME/CFS patient. Part 1: viruses

The purpose of the following analysis is to search for the viral epitopes that elicited – in a ME/CFS patient – IgGs against a set of 6 peptides, determined thanks to an array of 150.000 random peptides of 16 amino acids each. These peptides were used as query sequences in a BLAST search against viral proteins. No human virus was found. Six phages of bacterial human pathogens were identified, all but one belonging to the classes Actinobacteria and γ-Proteobacteria. One of these bacteria, Serratia marcescens, was identified in a similar study on 21 ME/CFS cases.  

1. The quest for a pathogen

Scientists have been speculating about an infectious aetiology of ME/CFS for decades, without never being able to link the disease to a specific pathogen. The idea that the disease might be triggered and/or maintained by an infection is due to the observation that for most of the patients the onset occurs after an infectious illness (Chu, L. et al. 2019). It has also been observed that after a major infection (whether parasitic, viral or bacterial) about 11% of the population develops ME/CFS (Mørch K et al. 2013), (Hickie I. et al. 2006).

In recent years, the advent of new technologies for pathogen hunting has given renewed impulse to the search for ongoing infection in this patient population. A 2018 study, investigating the genetic profile of peripheral blood for procaryotic and eucaryotic organisms reported that most of the ME/CFS patients have DNA belonging to the eukaryotic genera Perkinsus and Spumella and to the procaryotic class β-proteobacteria (alone or in combination) and that these organisms are statistically more present in patients than in controls (Ellis J.E. et al. 2018). Nevertheless, a previous metagenomic analysis of plasma by another group revealed no difference in the content of genetic material from bacteria and viruses between ME/CFS patients and healthy controls (Miller R.R. et al. 2016). Moreover, metagenomic analysis pursued in various samples from ME/CFS patients by both Stanford University and Columbia University has come empty (data not published, R, R).

2. Immunological methods

Another way of investigating the presence of current and/or past infections that might be specific of this patient population is to extract the information contained in the adaptive immune response. This can be made in several ways, each of them having their own limits. One way would be to collect the repertoire of T cell receptors (TCRs) of each patient and see if they have been elicited by some particular microorganism. This is a very complex and time-consuming method that has been developed in recent years and that I have described in details going through all the recent meaningful publications (R). The main limitation of this method is that, surprisingly, TCRs are not specific for a single epitope (Mason DA 1998), (Birnbaum ME et al. 2014), so their analysis is unlikely to reveal what agent selected them. On the other hand, the advantage of this method is that T cell epitopes are linear ones, so they are extremely suited for BLAST searches against protein databases. An attempt at applying this method to ME/CFS is currently underway: it initially gave encouraging results (R), then rejected by further analysis.

Another possible avenue for having access to the information registered by adaptive immunity is to investigate the repertoire of antibodies. The use of a collection of thousands of short random peptides coated to a plate has been recently proposed as an efficient way to study the response of B cells to cancer (Stafford P. et al. 2014), infections (Navalkar K.A. et al. 2014), and immunization (Legutki JB et al. 2010). This same method has been applied to ME/CFS patients and it has shown the potential of identifying an immunosignature that can differentiate patients from controls (Singh S. et al. 2016), (Günther O.P. et al. 2019). But what about the antigens eliciting that antibody profile? Given a set of peptides one’s antibodies react to, a possible solution for interpreting the data is to use these peptides as query sequences in a BLAST search against proteins from all the microorganisms known to infect humans. This has been done for ME/CFS, and the analysis led to several matches among proteins from bacteria, viruses, endogenous retroviruses and even human proteins (in fact, both this method and the one previously described can detect autoimmunity as well) (Singh S. et al. 2016).  There are several problems with this approach, though. First of all, the number of random peptides usually used in these arrays is not representative of the variety of possible epitopes of the same length present in nature. If we consider the paper by Günther O.P. and colleagues, for instance, they used an array of about 10^5 random peptides with a length of 12 amino acids each, with the number of all the possible peptides of the same length being  20^12 ∼ 4·10^15. This means that many potential epitopes one has antibodies to are not represented in the array. Another important limitation is that B cell epitopes are mainly conformational ones, which means that they are assembled by the folding of the proteins they belong to (Morris, 2007), the consequence of this being that the subset of random peptides one’s serum react to are in fact linear epitopes that mimic conformational ones (they are often called mimotopes) (Legutki JB et al. 2010). This means that a BLAST search of these peptides against a library of proteins from pathogens can lead to completely misleading results.

3. My own analysis

I have recently got access to the results of a study I was enrolled in two years ago. My serum was diluted and applied to an array of 150.000 peptides with a length of 16 random amino acids (plus four amino acids used to link the peptides to the plate). Residues Threonine (T), Isoleucine (I) and Cysteine (C) were not included in the synthesis of peptides. Anti-human-IgG Ab was employed as a secondary antibody. The set of peptides my IgGs reacted to has been filtered with several criteria, one of them being subtracting the immune response common to healthy controls, to have an immune signature that differentiates me from healthy controls. The end result of this process is the set of the following six peptides, for each of which I report the inverted sequence (the reason for that will be clear in a moment).

Patient 102 Forward Reverse
1 ALHHRHVGLRVQYDSG GSDYQVRLGVHRHHLA
2 ALHRHRVGPQLQSSGS SGSSQLQPGVRHRHLA
3 ALHRRQRVLSPVLGAS SAGLVPSLVRQRRHLA
4 ALHRVLSEQDPQLVLS SLVLQPDQESLVRHLA
5 ALHVRVLSQKRPLQLG GLQLPRKQSLVRVHLA
6 ALHLHRHVLESQVNSL LSNVQSELVHRHLHLA

Table 1. My immunosignature, as deteced by an array of 150.000 random peptides 20-amino-acid long, four of which are used for fixing them to the plate and are not included here. For each peptide I have considered the inverted sequence too (column 3).

The purpose of the following analysis is to search for the viral epitopes that elicited this immune response. To overcome the limitations enumerated at the end of the previous paragraph I have decided to search within the database of viral proteins for exact matches of the length of 7 amino acids. Why this choice? A survey of a set of validated B cell epitopes found that the average B cell epitope has a linear stretch of 5 amino acids (Kringelum, et al., 2013); according to another similar work, the average linear epitope within a conformational one has a length of 4-7 amino acids (Andersen, et al., 2006). To filter the matches and to reduce the number of matches due to chance, I opted for the upper limit of this length. I excluded longer matches to limit the number of mimotopes for conformational epitopes. Moreover, I decided to look only for perfect matches (excluding the possibility of gaps and substitutions) so to simplify the analysis. It is worth mentioning that a study of cross-reactive peptides performed for previous work (Maccallini P. et al. 2018) led me to the conclusion that cross-reactive 7-amino-acid long peptides might often have 100% identity.

The idea of using peptides as query sequences in both their directions is due to the obvious observation that if a peptide is a linear epitope for an antibody, then also the peptide resulting from its inversion reacts to the same antibody. This simple argument seems to be often overlooked in studies of this kind.

Sample
Figure 1. For each match, the matching protein and the organism it belongs to are reported. The protein ID has a link to the NCBI protein database, while the name of the organism has a link to the NCBI taxonomy browser. The host of the microorganism is also indicated, as well as its habitat, with links to further information.

4. Results

Table 2 is a collection of the matches I found with the method described above. You can look at figure 1 to see how to read the table.

ALHHRHVGLRVQYDSG (102_1_F_viruses)
9-LRVQYDS-15

QDP64279.1(29-35)

Prokaryotic dsDNA virus sp.
Archea, Ocean
8-GLRVQYD-14

AYV76690.1(358-364)

Terrestrivirus sp
Amoeba, forest soil
GSDYQVRLGVHRHHLA (102_1_R_viruses)
10-VHRHHLA-16

YP_009624599.1(65-71)

Gordonia phage Wait
Gordonia terrae 3612 (HP)
ALHRHRVGPQLQSSGS (102_2_F_viruses)
2-LHRHRVG-8

YP_009619965.1(63-69)

Stenotrophomonas phage vB_SmaS_DLP_5
Stenotrophomonas maltophilia (HP)
SGSSQLQPGVRHRHLA (102_2_R_viruses)
7-QPGVRHR-13

YP_009047145.1(318-324)

McMurdo Ice Shelf pond-associated circular DNA virus-8
?, Antarctica
ALHRRQRVLSPVLGAS (102_3_F_viruses)
8-VLSPVLG-14

QDB71078.1 (24-30)

Serratia phage Moabite
Serratia marcescens (HP)
SAGLVPSLVRQRRHLA (102_3_R_viruses)
3-GLVPSLV-9

QCW19699.1 (88-94)

Vibrio phage Va_90-11-286_p16
Vibrio anguillarum
3-GLVPSLV-9

AOQ27539.1 (36-42)

Leuconostoc phage LDG
L. pseudomesenteroides, L. mesenteroides (HP, rare)
3-GLVPSLV-9

AGZ86450.1 (190-196)

Hepacivirus C
Homo sapiens
ALHRVLSEQDPQLVLS (102_4_F_viruses)
7-SEQDPQL-13

BAR30981.1 (151-157)

uncultured Mediterranean phage uvMED
Archea and Bacteria, Med. sea
SLVLQPDQESLVRHLA (102_4_R_viruses)
3-HRVLSEQ-9

AXS67723.1 (494-500)

Cryptophlebia peltastica nucleopolyhedrovirus
invertebrates
2-LHRVLSE-8

YP_009362111.1 (74-80)

Marco virus
Ameiva ameiva
6-PDQESLV-12

YP_009342253.1 (385-391)

Wenzhou yanvirus-like virus 2
invertebrates
ALHLHRHVLESQVNSL (102_6_F_viruses)
2-LHLHRHV-8

YP_009119106.1 (510-516)

Pandoravirus inopinatum
Acanthamoeba
4-LHRHVLE-10

ASZ74651.1 (61-67)

Mycobacterium phage Phabba
Mycobacterium smegmatis mc²155 (HP)

Table 2. Collection of the matchs for the BLAST search of my unique set of peptides against viral proteins (taxid 10239). HP: human pathogen. See figure 1 for how to read the table.

5. Discussion

The only human virus among the matches collected in Table 2 is the Hepatitis C virus. It is a false positive, in this case; the same peptide is found in two bacteriophages and so this might be a case of cross-reactivity to antibodies raised to some other virus. Then there are some bacteriophages and six of them have as hosts bacteria that are known to be human pathogens. Bacteriophages (also known as phages) are viruses that use the metabolic machinery of procaryotic organisms to replicate (figure 2). It is well known that bacteriophages can elicit specific antibodies in humans: circulating IgGs to naturally occurring bacteriophages have been detected (Dąbrowska K. et al. 2014) as well as specific antibodies to phages injected for medical or experimental reasons (Shearer WT et al. 2001), as reviewed here: (Jonas D. Van Belleghem et al. 2019). According to these observations, one might expect that when a person is infected by a bacterium, this subject will develop antibodies not only to the bacterium itself but also to its phages.

phage
Figure 2. Half of all viruses have an almost regular icosahedral shape, but several phages present an irregular icosahedral shape, with a prolate capsid (Luque and Reguera 2013). On the left a wrong representation of a phage. It is wrong because the capsid has 24 faces, instead of 20. On the right, the representation of a regular icosahedron made by Leonardo Da Vinci for De Divina Proportione, a mathematical book by Luca Pacioli.

If that is the case, we can use our data in table 2 to infer a possible exposure of our patient to the following bacterial pathogens: Gordonia terrae 3612 (HP), Stenotrophomonas maltophilia (HP), Serratia marcescens (HP), Leuconostoc pseudomesenteroides, L. mesenteroides (HP, rare), Mycobacterium smegmatis mc²155 (HP). In brackets, there are links to research about the pathogenicity for humans of each species. G. terrae and M. smegmatis belong to the class Actinobacteria, while S. maltophila and S. marcescens are included in the class γ-Proteobacteria.

Interesting enough, Serratia marcescens was identified as one of the possible bacterial triggers for the immunosignature of a group of 21 ME/CFS patients, in a study that employed an array of 125.000 random peptides (Singh S. et al. 2016). This bacterium accounts for rare nosocomial infections of the respiratory tract, the urinary tract, surgical wounds and soft tissues. Meningitis caused by Serratia marcescens has been reported in the pediatric population (Ashish Khanna et al. 2013).

6. Next step

The next step will be to perform a similar BLAST search against bacterial proteins to see, among other things,  if I can find matches with the six bacteria identified by the present analysis. A further step will be to pursue an analogous study for eucaryotic microorganisms and for human proteins (in search for autoantibodies).

A leap of faith

A leap of faith

During last summer, I’ve pursued a lot of things. I delivered a speech in Turin, after the screening of the documentary Unrest, about the OMF-funded research on the use of the measure of blood impedance as a possible biomarker for ME/CFS (video, blog post, fig. 1, fig. 2).

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Figure 1. Group photo after the screening of Unrest in Turin, Italy, with the organizer of the event (Caterina Zingale, second from the right) and representatives of two Italian ME/CFS associations.
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Figure 2. Question time after the screening of Unrest. On the screen, a drawing of mine.

Then I flew to London to attend the Invest in ME conference, the annual scientific meeting that gathers researchers from all over the world who shared their latest work about ME/CFS. There I met Linda Tannenbaum, the CEO of the Open Medicine Foundation, whom I had the pleasure to encounter for the first time about a year before in Italy, and I introduced myself to Ronald Davis (fig. 3), the world-famous geneticists turned ME-researcher because of his son’s illness. I presented to him some possible conclusions that can be driven from the experimental results of his study on the electrical impedance of the blood of ME/CFS patients, with the use of an electrical model for the blood sample (R, paragraph 6).

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Figure 3. Talking to Ron Davis about a possible explanation for the increase in electrical impedance in the blood of ME/CFS patients in London, during the last Invest in ME conference (blog post).

In London, I was able to visit the National Gallery and while I was passing by all these artistic treasures without being able to really absorb them, to get an enduring impression that I could bring with me forever, I decided to sit down and to copy one of these masterpieces (I can’t draw for most of the time, and when I improve for a few weeks in summer, I usually have to carefully choose where to put my energies). I sat probably beside one of the least important portraits collected in the museum (Portrait of a young man, Andrea del Sarto, figure below) and I started copying it with a pen. When I finished, the museum was closing, so that I missed all the works by Van Gogh, among many other things.

We were at the beginning of June, I was experiencing my summer improvement, a sort of substantial mitigation of my illness that happens every other summer, on average. But because of these travels, I elicited a two-month worsening of symptoms, during which I had to stop again any mental and physical activity: I just lay down and waited. At the beginning of August, I started thinking and functioning again and I almost immediately decided to quit what was my current project (a 600-page handbook of statistics that I commenced in 2017) and I started studying mathematical modelling of enzymatic reactions (figures 4 and 5).

Cattura.JPG
Figure 4. The plot of the reversible Michaelis-Menten equation for ribulose-phosphate 3-epimerase. The intersection of the surface with the plane is the state of equilibrium of the reaction (the rate of production of X5P is the same as the rate of its transformation into Ru5P) (R).
sa.png
Figure 5. The plot of the concentrations of S, P, ES and E for the transformation of L-tryptophan into N-formylkynurenine by IDO-2. This is a semianalytical solution that I found using the approximation of the Lambert function. I also pursued numerical solutions, of course (R).

I knew that these reactions were described by ordinary differential equations and that I could solve them numerically with the methods that I studied just before I got sick, about 18 years ago. I was interested in the metabolic trap theory by Robert Phair, an OMF-funded researcher. So I downloaded a chapter of one of the most known books of biochemistry and a thesis by a Turkish mathematician on metabolic pathways simulation and I started my journey, working on the floor (I have orthostatic intolerance even when I get better in Summer, so I can’t use a desk, figure 6). I ended up learning the rudiments of this kind of analysis, also thanks to a book by Herbert Sauro and to some suggestions by dr. Phair himself! Some of the notes I wrote in August are collected here.

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Figure 6. In August I was studying mathematical modelling of metabolic pathways, sitting on the floor: because of my orthostatic intolerance, I can’t sit for long, especially when I need to think. You can see the book by Sauro opened in the foreground, the thesis by a Turkish mathematician (red cover), and the handbook of Octave (blue/red cover).

At the beginning of September, I was absorbed by the problem of how to study the behaviour of the steady states of tryptophan metabolism in serotoninergic neurons of midbrain as the parameters of the system change. This kind of analysis is called bifurcation theory and I literally fell in love with it… In figure 6 you can also see a drawing: I was drawing a picture I have been thinking about for the last 20 years. It is a long story, suffice it to say that in 1999, just before my mind faded away for 18 months, I started studying the anatomy of a man who carries a heavy weight on his back (see below). That was my first attempt of communicating what was happening to me, of describing my disease.

Only recently I considered to not represent the weight, which is a more appropriate solution since this is a mysterious disease with no known cause, and I made a draft (the one in figure 2) that I then used as a starting point for the drawing below. I finished this new drawing at the beginning of September, in a motel room of San José, in California, just in time for donating it to Ronald Davis (figures below) when I moved to the US to attend the third Community Symposium at Stanford (see here). In California, many surprising things happened: I met again Linda Tannenbaum and Ronald Davis, and yes, I encountered also Robert Phair! But this is another story…

resilience 2

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In the following pictures, you can see how the drawing evolved. Notably, the figure in the centre changed his face and some part of his anatomy. The three figures are meant to be a representation of the same figure from three different points of view. It is more like a project for a sculpture, a monument that is much deserved by these patients.

At Stanford, I had the chance to be face to face with one of my preferred sculptures ever: The Thinker, by Rodin, in both its version: the model moulded first, on the top of The Gates of Hell, and the big one (crafted later), now considered the iconic symbol of Philosophy, but likely originally meant to be a metaphor for creative thinking (I say that because the original sculpture included in The Gates of Hell is a representation of the Italian poet Dante Alighieri, depicted in the act of imagining his poem).

At the end of September, my mind started fading away again. I knew that would have happened, even though I had an irrational hope that this year would have been different. At that point, I was in Italy and I asked some friends to help me organize a trip to the southern hemisphere, in order to live another summer. It required more time than I would have hoped. I am going to leave from Italy only tomorrow. My goal: Argentina. I have been able to do something, at a highly reduced speed, in October, though. I have developed a model for solar radiation at sea level, in function of the day of the year, of the latitude, and of the distance from the Sun (I have considered the actual elliptic orbit of our planet). The main problem has been the modelling of absorption and of diffusion of radiant energy from our star by the atmosphere, but I solved it. Part of these notes are here, but I want to self-publish the end product, so I keep the rest to myself. In that period, I was also able to find the exact solution of the improper integral known as the Stefan-Boltzmann law, something I tried to do in the summer of 2008, in vain, in one of my recovery-like periods. In figure 6 you can see one of the results of my model for solar radiation: the monochromatic emissive power at sea level in function of the day of the year, for the city of Buenos Aires.

emissive power.png
Figure 7. The monochromatic emissive power of the sun at noon, at sea level, at a latitude of -32° N, in function of the wavelength and of the day of the year. Note that the vertical axis is expressed in W on microns multiplied by square meters.

My intention was to use that model to choose the perfect place where to move in order to have environmental conditions that closely resemble the ones that we have in Rome from June to September (the period in which my improvements happen). I also wanted to quantitatively study the effect of both infrared radiation and ultraviolet radiation on my biology. There are several interesting observations that can be made, but we will discuss these subjects another time, also because I had to quit this analysis given my cognitive deterioration. The video below is a byproduct of the geometric analysis that I had to pursue in order to build my model for solar radiation at sea level.

Dawn and dusk at a latitude of 42 degrees north, during three years of the silent rolling of the Earth on its silken ellipse. Three years of adventures, suffering, joy and death.

So, by November my mind was completely gone and my physical condition (namely orthostatic intolerance and fatigue) had worsened a lot. This year I have been able to try amphetamines: I had to go from Rome to Switzerland to buy them (they are restricted drugs that can’t be sold in Italy and can’t be shipped to Italy either). One night I felt good enough to take a train to Milan and then to take another transport to the drug store. And back. I managed to do the travel but I pushed my body too far and I had to spend the following month in bed, 22 hours a day, with an even worse mental deterioration. It is like having a brain injury. Amphetamines have been useless in my case, despite two studies on their potential beneficial effect in ME/CFS.

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Right now, I am collecting all the books and the papers that I need with me in Argentina (figure above), in case I will improve enough to study again. But what am I going to work on?

  1. I want to finish my model of solar radiation, with some notes on the effect of infrared radiation, ultraviolet radiation and length of the day on the immune system. There is a mathematical model published recently that links the length of the day to the power of the innate immune system, and I want to write a code that calculates the relative activity of the innate immunity in function of latitude and day of the year. I would like to self- publish it as a booklet.
  2. I want to finish my handbook of statistics.
  3. I need to correct a paper submitted for publication (it has been accepted, but some corrections have been required).
  4. I want to deepen my understanding of the bifurcation theory for metabolic pathways and to continue studying tryptophan metabolism with this new knowledge.
  5. I want to complete my work on autoantibodies in ME/CFS (see this blog post) and to submit it to a journal. I have been working on that for a while, inventing new methods for the quantitive study of autoimmunity by molecular mimicry.

Should I improve again in Argentina, several avenues can be followed in order to understand the reason why summer causes this amelioration in my own case. I have many ideas and I’ll hopefully write about that in the future. Of course, I also want to read all the new research papers I have missed in the last months. I will bring with me my handbook of anatomy for artists because I hope to be able to draw again, and I won’t miss this opportunity to leave some other handcrafted images behind me for posterity, that can’t care less, obviously! I would really like to finish the drawing below because I feel that in this draft I have found a truly elegant (and mechanically correct) solution for the hip joint of a female robot.QR-A_000026

Now I am useless, my mind doesn’t work and I am housebound. I can’t read, I can’t draw, I can’t do calculations, I can’t do coding, I can’t cook… This has been the quality of my life for most of the last 20 years. This is a huge waste: I would have used these years to perform beautiful and useful calculations and to pursue art. I would really make people understand how tragic this disease is in its cognitive symptoms, what we lose because of it. This is, in fact, the reason behind this blog post: I wanted to give an idea of what I can do when I feel better, and of what I would have done if there had been a cure.

I have lost most of my adult life, but I will never accept to waste a day without fighting back.

Ronald Davis at Columbia University

In evidenzaRonald Davis at Columbia University

All the following studies have been made mainly thanks to private funding. Please, consider a donation to the Open Medicine Foundation, in order to speed up the research. See this page for how to donate to OMF.


OMF Scientific Advisory Board Director Ronald W. Davis, PhD, has just delivered a speech about ME/CFS at the Albert Einstein College of Medicine at Columbia University in New York. In what follows you find several screenshots that I have collected during the lecture, accompanied by a very short description. I imagine that a video will be soon made available but in the meantime let’s take a look at these slides.

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Indoleproprionate is reduced in ME/CFS patients. This molecule is not produced by our own metabolism, it comes from a bacterium of the gut (Clostridium sporogenes) which is low in patients. It has a neuroprotectant effect. Indoleproprionate is currently used in some clinical trials for other diseases and it might be available in the next future as a drug.

Hydroxyproline is high and this is believed to indicate collagen degradation. Ron Davis talked about the case of a ME/CFS patient who turned out to have a problem in the craniocervical junction which was fixed by surgery. Is there a link between high hydroxyproline and abnormalities of the joints (the neck among them) that some patients seem to have?

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The increase in electrical impedance in blood samples (as measured by the so-called Nanoneedle device) only happens when cells from ME/CFS patients are incubated with plasma from patients. When these same cells (white blood cells) are incubated with plasma from healthy donors, the impedance is normal. For an introduction to this experiment, click here. The published work is here.

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The Nanoneedle study has been extended with 20 more patients and 20 more controls.  This device can be used for drug screening, other than for diagnosis.

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The peptide called Copaxone, now used in Multiple Sclerosis, seems to work in reducing the impedance in the nanoneedle device (click on the images to enlarge). Suramin also has some effect (on the right). It doesn’t seem as good as Copaxone though, to me…

SS-31 is an experimental drug for the mitochondrial membrane. It does work when used in the nanoneedle device! (click on the images to enlarge).

Nailbed capillaroscopy could be a new instrument for ME/CFS diagnosis. Inexpensive and already in use in hospitals.

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No new or known pathogen has been found in patients, so far. This project is still in progress. It is updated as new technologies for pathogen hunting become available.

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All the severe patients have at least one defective copy of the IDO2 gene. The same applies to 46 additional ME/CFS patients that have been recently tested for this gene. This is a common genetic problem in the general population, but it is ubiquitous in these patients. And a statistically significant difference is thus present between ME/CFS patients and healthy controls. This discovery has lead to the development of the metabolic trap hypothesis, which has been recently published (here). For an introduction, read this blog post of mine. They are planning to test the metabolic trap hypothesis in vivo using cellular cultures!

72687448_10219023729760631_936263077656002560_n.jpg

Patients have high mercury (maybe from fish in their diet) and low selenium in hair. Low selenium can reduce the conversion of T4 to T3 in the liver. Low T3 might be a cause of fatigue. High uranium was also detected!

78209825_10219023656558801_2413390815666634752_n-e1574360171978.jpg


All these studies have been made mainly thanks to private funding. Please, consider a donation to the Open Medicine Foundation, in order to speed up the research. See this page for how to donate to OMF.


 

 

C’è del marcio a Bologna?

C’è del marcio a Bologna?

I disabili possono costituire una fonte di reddito cospicua per chi lavora nell’ambito della assistenza a queste persone. Per le disabilità riconosciute, infatti, il sistema sanitario offre (giustamente) dispositivi costosi e personale con le più varie mansioni. Ma in molti casi appalta questo genere di servizi.

Come funziona un appalto? Si redige un capitolato di gara, che elenca i requisiti che i candidati devono possedere per poter partecipare alla gara. Questo capitolato viene reso pubblico solo al momento del bando. Il vincitore sarà l’ente che, tra i candidati, meglio soddisfa i requisiti del capitolato di gara.

Un anno fa, dopo una indagine di vari mesi, la Guardia di Finanza di Bologna ha iscritto nel registro degli indagati quattro dipendenti Ausl e due rappresentanti della Associazione Italiana Assistenza Spastici (AIAS Bologna) con l’accusa di aver truccato un appalto. Infatti il file del capitolato di gara sarebbe stato corretto più volte – prima ancora del bando – dai rappresentanti di AIAS Bologna, in modo da cucirsi addosso il concorso e assicurarsi la vittoria [1], [2].

Si parla di due milioni e 130 mila euro circa, in tre anni, con possibile rinnovo per altri tre anni. Si tratta della gara per il Centro Regionale Ausili (che include anche il Centro Ausili Tecnologici) e per il Centro Adattamento Ambiente Domestico. La gara è stata sospesa [3], [4].

Dopo ulteriori accertamenti, il procedimento contro uno dei dipendenti Ausl è stato archiviato, ma per il resto l’indagine è ancora in piedi, che io sappia [5].

Io stesso sono il beneficiario dei servizi di una di queste aziende appaltatrici, che assistono le persone con disabilità. E non potrei fare a meno di questi servizi, non sopravviverei.

Proprio per questo mi infastidisce non poco venire a sapere di tali illeciti (presunti, fino a che il procedimento giudiziario non termina il suo corso). E perché sono spiacevoli e pericolosi questi illeciti? Perché in fondo suggeriscono che le disabilità più remunerative sono quelle a cui si presta più attenzione e a cui si offrono più servizi.

Lettera aperta all’Osservatorio Malattie Rare

Non sarebbe necessario scrivere una nota all’articolo pubblicato dall’Osservatorio Malattie Rare (O.ma.r.) sulla malattia di Lyme alcuni giorni fa [1], in cui si liquidano i possibili esiti cronici della patologia in parola come non esistenti o riconducibili ad altre patologie, “non ultime le patologie psichiatriche”. Non sarebbe necessario, ho scritto, perché coloro che si occupano professionalmente della malattia di Lyme, nonché i pazienti, sono consapevoli che in realtà le possibili sequele della infezione acuta da Borrelia burgdorferi sono ben documentate in letteratura e costituiscono un problema di enorme portata su cui si concentra oggi molta ricerca, spesso di alto profilo [2].

E allora perché questa preterizione? Perché un articolo divulgativo pubblicato da O.ma.r. ha notevole risonanza e – se incompleto o impreciso – rischia di alimentare false credenze tra i neofiti. L’articolo in questione è encomiabile nel divulgare nozioni preziose sulla fase acuta della malattia e nel mettere in guardia contro test e trattamenti non provati, ma è del tutto fuorviante nella parte dedicata alle sequele croniche (penultimo paragrafo).

La malattia di Lyme risponde alle attuali cure antibiotiche nel 90% dei casi. Questo significa che un decimo di coloro che ogni anno, in estate, contrae la malattia per il morso di un vettore (principalmente l’Ixodes ricinus in Europa) andrà incontro a una condizione cronica (cioè con durata superiore ai sei mesi) e debilitante, nota in seno alla comunità scientifica con il nome di post-treatment Lyme disease syndrome, PTLDS [3], [4]. Il nome scelto (che si potrebbe tradurre come Sindrome della malattia di Lyme dopo trattamento) sta a indicare che i pazienti sperimenteranno sintomi, nonostante i trattamenti della fase acuta.

La causa della PTLDS è al momento non nota. Alcuni studi supportano l’ipotesi che ci sia una disfunzione immunitaria in questi pazienti. Due studi hanno dimostrato la presenza di anticorpi contro il sistema nervoso centrale nella metà dei pazienti PTLDS [5], [6], solo per citare i più recenti. Per approfondimenti su questo argomento si legga qui e qui. La ricerca in questo campo continua [7].

Altri gruppi hanno dimostrato la persistenza del patogeno – dopo trattamento – sia nel modello animale della malattia di Lyme [8], [9], [10], che negli esseri umani [11], [12]. Presso la Columbia University è stata avviata una raccolta di tessuti provenienti da individui deceduti, che abbiano avuto una ben documentata infezione da Borrelia burgdorferi (link) proprio per indagare ulteriormente questo aspetto.

Numerosi sforzi e investimenti sono stati profusi recentemente nella ricerca di nuovi agenti antimicrobici per questa infezione, nella speranza di scongiurare le sequele croniche, da parte della Università di Stanford [13], [14], della Università Johns Hopkins [15], [16], [17], della Università Northeastern [18].

Nell’articolo di O.ma.r si legge che la sindrome in questione – chiamata impropriamente post-Lyme dall’autore – è caratterizzata da “sintomi soggettivi – e dunque non quantificabili – quali affaticabilità e difficoltà a concentrarsi”. A questo proposito è quasi superfluo ricordare che molti sintomi sono soggettivi, finché l’ingegneria non ci offre uno specifico strumento di misura: si pensi alla risonanza magnetica nella sclerosi multipla o all’elettroencefalogramma nella epilessia. In secondo luogo, questi sintomi sono solo in parte soggettivi, infatti i pazienti PTLDS presentano alterazioni quantificabili nel sistema immunitario [5], [6], nella espressione genica [19], nel metabolismo [20], etc.

Per quanto riguarda il riferimento all’ambito psichiatrico, vale la pena fare delle considerazioni. E’ senz’altro vero che le principali patologie psichiatriche (i disturbi dell’umore da un lato e le psicosi dall’altro) contemplano gli episodi infettivi come fattore di rischio [21], [22], tuttavia la PTLDS semplicemente non si sovrappone alle patologie psichiatriche, né nella presentazione clinica, né nella epidemiologia, per questo è una categoria nosografica a sé stante (vedi qui). Per fare un esempio concreto: negare l’evidenza, come sembra fare questo articolo, è una delle possibili manifestazioni della schizofrenia paranoide [23], ma né questo tipo di sintomi né altri sintomi patognomonici per malattie psichiatriche sono menzionati nella definizione di caso della PTLDS [4].

Paolo Maccallini

Riferimenti

  1. Orzes, E. (2019, Nov. 7). Malattia di Lyme, attenzione alle false credenze. O.ma.r. (link)
  2. Instutute of Medicine (2011, Apr. 20). Critical Needs and Gaps in Understanding Prevention, Amelioration, and Resolution of Lyme and Other Tick-Borne Diseases: The Short-Term and Long-Term Outcomes – Workshop Report. Cap. 7 (link).
  3. Centers for Disease Control and Prevention. Post-Treatment Lyme Disease Syndrome. (link).
  4. Aucott, J., Crowder, L., & Kortte, K. (2013). Development of a foundation for a case definition of post-treatment Lyme disease syndrome. Int J Infect Dis, 17(6), p. e443-e449. (link).
  5. Chandra A, Wormser GP, Klempner MS, et al. Anti-neural antibody reactivity in patients with a history of Lyme borreliosis and persistent symptoms. Brain Behav Immun 2010;24:1018–24 (link).

  6. Jacek E, Fallon BA, Chandra A, Crow MK, Wormser GP, Alaedini A. Increased IFNalpha activity and differential antibody response in patients with a history of Lyme disease and persistent cognitive deficits. J Neuroimmunol 2013;255:85–91 (link).

  7. Maccallini, P; Bonin, S; Trevisan, G. Autoimmunity against a glycolytic enzyme as a possible cause for persistent symptoms in Lyme disease. Med Hypotheses. 2018 Jan. (link).
  8. Hodzic E, Feng S, Holden K, Freet KJ, Barthold SW. Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Antimicrob Agents Chemother. 2008 May; 52(5) (link).

  9. Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment. APMIS. 2010 Sep 1;118(9) (link).
  10. Embers, M et al. Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection. PLoS One. 2012; 7(1). (link).

  11. Marques, A. et al. Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clin Infect Dis. 2014 Apr; 58(7):937-45 (link).
  12. Sapi, E. et al.The Long-Term Persistence of Borrelia burgdorferi Antigens and DNA in the Tissues of a Patient with Lyme Disease. Antibiotics 2019, 8(4), 183. (link).
  13. Wagh D, Pothineni VR, Inayathullah M, Liu S, Kim KM, Rajadas J. Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition. Drug Des Devel Ther. 2015 Feb 11;9:805-16. (link).
  14. Venkata Raveendra Pothineni et al. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening. Drug Des Devel Ther. 2016; 10: 1307–1322. (link).
  15. Feng J, Wang T, Shi W, Zhang S, Sullivan D, Auwaerter PG, Zhang Y. Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library. Emerg Microbes Infect. 2014 Jul; 3 (7). (link).
  16. Jie Feng, Megan Weitner, Wanliang Shi, Shuo Zhang, David Sullivan, and Ying Zhang. Identification of Additional Anti-Persister Activity against Borrelia burgdorferi from an FDA Drug Library. Antibiotics (Basel). 2015 Sep; 4(3): 397–410. (link).
  17. Feng J, Auwaerter PG, Zhang Y. Drug combinations against Borrelia burgdorferi persisters in vitro: eradication achieved by using daptomycin, cefoperazone and doxycycline. PLoS One. 2015 Mar 25;10(3). (link).
  18. Sharma B, Brown AV, Matluck NE, Hu LT, Lewis K. Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells. Antimicrob Agents Chemother. 2015 Aug;59(8):4616-24. (link).
  19. Jerome Bouquet et al. Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease. mBio. 2016 Jan-Feb; 7(1). (link).
  20. Fallon BA et al. Regional cerebral blood flow and metabolic rate in persistent Lyme encephalopathy. Arch Gen Psychiatry. 2009 May;66(5):554-63. (link).
  21. Benros ME et al. Autoimmune diseases and severe infections as risk factors for mood disorders: a nationwide study. JAMA Psychiatry. 2013 Aug;70(8): 812-20. (link).
  22. Benros ME et al. Autoimmune diseases and severe infections as risk factors for schizophrenia: a 30-year population-based register study. Am J Psychiatry. 2011 Dec;168(12). (link).
  23. Rajiv Tandon et al. Definition and description of schizophrenia in the DSM-5. Schizophrenia Research, Volume 150, Issue 1, October 2013, Pages 3-10. (link).

     

 

 

Is it all in your neck?

Is it all in your neck?

1. Introduction

Recently there have been some anecdotal reports of patients with a diagnosis of ME/CFS who met the criteria for a diagnosis of craniocervical instability (CCI). After surgical fusion of this joint, they reported improvement in some of their symptoms previously attributed to ME/CFS (R, R). After some reluctance, given the apparently unreasonable idea that there could be a link between a mechanical issue and ME/CFS, I decided to look at this avenue. So here I am, with this new blog post. In paragraph 2, I introduce some basic notions about the anatomy of the neck. In paragraph 3, I describe three points that can be taken from the middle slice of the sagittal sections of the standard MR study of the brain. These points can be used to find four lines (paragraph 4) and these four lines are the basis for quantitative diagnosis of craniocervical instability (paragraph 5-10). In paragraph 11, I describe CCI. In paragraph 12, I discuss the possible link between craniocervical instability and ME/CFS. In paragraph 13, there is a collection of measures from the supine MRIs of some ME/CFS patients. In the last paragraph, I propose an alternative definition of CCI, with the introduction of Euler’s angles.

Sagital section 1 bis.jpg
Figure 1. Left. The midline slice of the set of sagittal sections of an MR study of the brain of the author of this article. In the lower part of the image, we can see the section of the axis, with the odontoid process wedged between the anterior arch of the atlas and the ventral layer of the dura. The posterior arch of the atlas can also be seen just below the posterior edge of the foramen magnum. Above the foramen magnum, the brainstem with its three components: the medulla (or medulla oblongata), pons, and midbrain. Right. Lateral-posterior view of the atlas and the axis. They make up a one degree of freedom kinematic pair with the rotation axis corresponding to the axis of the odontoid. By Paolo Maccallini.

2. Basic anatomy

The craniocervical (or craniovertebral) junction (CCJ) is a complex joint that includes the base of the skull (occipital bone, or occiput), the first cervical vertebra (atlas or C1), the second cervical vertebra (axis or C2), and all the ligaments that connect these bones (Smoker WRK 1994). This joint encloses the lower part of the brainstem (medulla oblongata) and the upper trait of the spinal cord, along with the lower cranial nerves (particularly the tenth cranial nerve, the vagus nerve). Since the CCJ is included in the series of sagittal sections of every MR study of the brain, its morphology can be easily assessed (figure 1, left). It is worth mentioning that the CCJ is the only joint of the body that encloses part of the brain. The atlas and the axis are represented with more detail in figure 1 (right), where their reciprocal interaction has been highlighted. From a mechanical point of view, these two bones make up a revolute joint, with the rotation axis going through the odontoid process. This is only a simplification, though, because while it is true that the atlantoaxial joint provides mainly axial rotation,  there are also 20 degrees of flexion/extension and 5 degrees of lateral bending, which means that spherical joint would be a more appropriate definition. Other degrees of freedom are provided at the level of the occipital atlantal joint, where 25 degrees of motion are provided for flexion/extension, 5 degrees of motion are provided for one side lateral bending and other 10 degrees are provided for axial rotation (White A. & Panjabi M.M. 1978).

3. Points

The measurement of the Grabb’s line and of the clival-canal angle is based on a simple algorithm which starts with the identification of three points on the midline sagittal image of a standard MRI scan of the head (figure 2). In order to find this particular slice, search for the sagittal section where the upper limit of the odontoid process reaches its highest and/or the slice with the widest section of the odontoid process. This algorithm is mainly taken from (Martin J.E. et al. 2017). In looking at T1-weighted images, always keep in mind that cortical bone (and cerebrospinal fluid too) gives a low signal (black strips) while marrow bone gives a high signal (bright regions) (R).

  • Clival point (CP). It is the most dorsal extension of the cortical bone of the clivus at the level of the sphenooccipital suture. This suture can’t be seen clearly in some cases (figure 3 is one of these cases). So another definition can be used for CP: it is the point of the dorsal cortical bone of the clivus at 2 centimetres above the Basion (see next point).
  • Basion (B). It is the most dorsal extension of the cortical bone of the clivus. This is the easiest one to find!
  • Ventral cervicomedullary dura (vCMD). This is the most dorsal point of the ventral margin of the dura at the level of the cervicomedullary junction. I find this point the most difficult to search for and somehow poorly defined, but this is likely due to my scant anatomical knowledge.
  • Posteroinferior cortex of C-2 (PIC2). It is the most dorsal point of the inferior edge of C2.
sagital-section-2-1.jpg
Figure 2. This is the median slice from the sagittal sections of a T1-weighted magnetic resonance of the head. In blue, the three points used for the measure of the Grabb’s line and of the clival-canal angle. In red, the definitions of rostral, caudal, ventral and dorsal. By Paolo Maccallini.

4. Lines

Connecting the three points found in the previous paragraph allows us to define four lines (figure 3) that will be then used to calculate the Grabb’s measure and the clival-canal angle.

  • Clival slope (CS). It connects CP to vCMD. It is also called the Wackenheim Clivus Baseline (Smoker W.R.K. 1994).
  • Posterior axial line (PAL). It connects vCMD to PIC2.
  • Basion-C2 line (BC2L). It connects B to PIC2.
  • Grabb’s line (GL). It is the line from vCMD that is orthogonal with BC2L.

We now know all we need in order to take two of the most important measures for the assessment of craniocervical junction abnormalities.

Sagital section 4.jpg
Figure 3. On this middle sagittal section, the points CP, B, vCMD and PIC2 have been reported. Since the sphenooccipital suture is not clearly visible, the point CP has been identified measuring 2 centimetres from B, along the dorsal cortical bone. The lines CS, PAL, BC2L and GL have then been identified. The Grabb’s measure is of 0.8 centimetres while the clival-canal angle is 142°. By Paolo Maccallini.

5. The clival-canal angle and its meaning

The clival-canal angle (CXA) is the angle between CS and PAL. The value of this angle for the individual whose scan is represented in figure 4 is 142°. This angle normally varies from a minimum of 150° in flexion to a maximum of 180° in extension (Smoker WRK 1994). Ence, what we should normally see in a sagittal section from an MR scan of the brain is an angle between these two values. A value below 150° is often associated with neurological deficits according to (VanGilder J.C. 1987) and it is assumed that a CXA below 135° leads to injury of the brainstem (Henderson F.C. et al. 2019). A clival canal angle below 125° is considered to be predictive of CCI according to (Joaquim A.F. et al. 2018). In a study on 33 normal subjects employing standard MRI, CXA was measured in the sagittal section of each subject: this group had a mean value of 148° with a standard deviation of 9.88°; the minimum value was 129° and the maximum one was 175° (Botelho R.V. et al. 2013). The reader may have noted that the mean CXA in this study is below the cutoff for neurological deficits according to the 1987 book. This might be due to the fact that there is a difference between the measure taken on an MRI sagittal section and the one taken on radiographic images.

It has been demonstrated with a mathematical model that a decrease in the clival-canal angle produces an increase in the Von Mises stress within the brainstem and it correlates with the severity of symptoms (Henderson FC. et al. 2010). Von Mises stress gives an overall measure of how the state of tension applied to the material (the brainstem in this case) causes a change in shape. For those who are interested in the mathematical derivation of this quantity (otherwise, just skip the equations), let’s assume that the stress tensor in a point P of the brainstem is given by

stress tensor.JPG

Then it is possible to prove that the elastic potential energy due to change in shape stored by the material in that point is given by

deviatoric elastic energy.JPG

where E and ν are parameters that depend on the material. Since in monoaxial stress with a module σ the formula above gives

monoaxial.JPG

by comparison, we obtain a stress (called Von Mises stress) that gives an idea of how the state of tensions contributes to the change of shape of the material:

Von Mises.JPG

In the brainstem, this parameter – as said – appears to be inversely proportional to the clival-canal angle and directly proportional to the neurological complaints of patients, according to (Henderson FC. et al. 2010). For a complete mathematical discussion of Von Mises stress, you can see chapter 13 of my own handbook of mechanics of materials (Maccalini P. 2010), which is in Italian though.

total.jpg
Figure 4. Left. ME/CFS patient, female. The clival-canal angle is 146° while the Grabb’s measure is 0.8 cm. Right. ME/CFS patient, male. The clival-canal angle is 142° while the Grabb’s measure is about 0.6 cm. By Paolo Maccallini.

6. The Grabb’s measure and its meaning

The Grabb’s measure is the length of the segment on the Grabb’s line whose extremes are vCMD and the point in which the Grabb’s line encounters the Basion-C2 line. In figure 4 this measure is 0.8 centimetres. This measure has been introduced for the first time about twenty years ago with the aim of objectively measuring the compression of the ventral brainstem in patients with Chiari I malformation. A value greater or equal to 9 mm indicates ventral brainstem compression (Grabb P.A. et al. 1999). In a set of 5 children with Chiari I malformation and/or basal invagination (which is the prolapse of the vertebral column into the skull base) a high Grabb’s measure was associated with a low clival canal angle (Henderson FC. et al. 2010). When using MRI, it is assumed that values above 9 mm is abnormal (R) but I have not been able to find statistical data on this measure in MRIs of healthy individuals. Moreover, the study by Grabb was mainly on a pediatric population (38 children and two adults) with Chiari malformation. So it is unclear if these measures can be used to assess the CCJ in adults. The measure was made on sagittal sections of MRIs.

The CXA only takes into account osseous structures (it depends on the reciprocal positions between the body of the axis and the clivus), so it can potentially underestimate soft tissue compression by the retro-odontoid tissue. This problem can be addressed with the introduction of the Grabb’s measure (Joaquim A.F. et al. 2018). Nevertheless, we can assume that they both measure the degree of ventral brainstem compression, and if you look at figure 3 you realize that as the angle opens up, the Grabb’s measure becomes shorter. Points and lines described in these paragraphs for two more patients are represented in figure 4.

7. Horizontal Harris measure

Another measure that has been introduced to check the anatomical relationship between the skull and the Atlas is the distance between PAL and point B (figure 5). This measure has been introduced in (Harris J.H. e al. 1993) where it was performed in 400 adults and with a normal cervical spine and in 50 healthy children. In the first group, 96% of the individuals had a distance of the basion from PAL longer than 1-4 mm and shorter than 12 mm. All the children had a distance below 12 mm. This measure has been used recently to assess craniocervical instability in hypermobile patients (Henderson F.C. et al. 2019), along with the Grabb’s measure and the clival-canal angle. We will refer to this measure as HHM. It is important to mention that the study by Harris was based on radiographs, so it is unclear if they can be used for a comparison of measures taken from MRI sagittal sections. Yet a measure below 12 mm was considered normal in a study employing MRI (Henderson F.C. et al. 2019).

HHM.JPG
Figure 5. The Horizontal Harris measure in the sagittal section of the MRI of a patient. Highlighted by the red arrow. By Paolo Maccallini.

8. Distance between Chamberlain’s line and the odontoid process

Another measure that has been introduced to determine whether occipitovertebral relationship is normal or not is the distance between the Chamberlain’s line and the closest point of the tip of the odontoid process (also called dens) (figure 6). The Chamberlain’s line extends between the posterior pole of the hard palate and the posterior margin of the foramen magnum (called opisthion) (Smoker W.R.K. 1994). In a study on 200 healthy European adults employing standard MRI, this measure was taken from the T1 weighted sagittal section of each subject. Measures start from the cortical bone, i.e. from the dark signal. The mean was -1.2 mm with a standard deviation SD = 3 mm (Cronin C.G. et al. 2007). The minus before the number indicates that the mean position of the selected point of the dens is below the line.

Andrea Catenacci Chamberlain.JPG
Figure 6. Chamberlain’s line (in red). The distance between the Chamberlain’s line and the closest point of the tip of the dens is – 4 mm, in this subject. The minus before the number indicates that the selected point of the dens is below the line. By Paolo Maccallini.

9. Distance between McRae’s line and the odontoid process

McRae’s line is drawn from the anterior margin of the foramen magnum (basion) to its posterior border (opisthion). It was introduced in 1953 to assess normality at the level of the CCJ (McRae D.L. et Barnum A.S. 1953). The distance between McRae’s line and the closest point of the tip of the dens can be used, as in the case of Chamberlain’s line, to assess abnormality of the CCJ along the z-axis (figure 7). In a study on 200 healthy European adults employing standard MRI, this measure was taken from the T1 weighted sagittal section of each subject. Measures start from the cortical bone, i.e. from the dark signal. The mean was -4.6 mm with a standard deviation SD = 2.6 mm (Cronin C.G. et al. 2007). The minus before the number indicates that the mean position of the selected point of the dens is below the line. In normal individuals, the dens is always below the McRae’s line (McRae D.L. et Barnum A.S. 1953), (Cronin C.G. et al. 2007).

Andrea Catenacci McRae
Figure 7. McRae’s line (in green). The distance between the McRae’s line and the closest point of the tip of the dens is – 6 mm, in this subject. The minus before the number indicates that the selected point of the dens is below the line. By Paolo Maccallini.

10. Distance between basion and odontoid process

It is the distance between the basion and the tip of the dens. It is also called basion-dental interval (BDI) and it has been proposed that a value greater of 10 mm is abnormal and predicts occipito-atlantal instability. Moreover, the average value is 5 mm, according to (Handerson F. 2016). I have not been able to find statistical data for BDI measured in MRI sagittal sections of healthy subjects. Moreover, I do not have a cutoff for the minimum value.

11. Craniocervical instability

According to some authors, the craniocervical junction is considered to be unstable (craniocervical instability, CCI) in the case of “any anomaly that leads to neurological deficits, progressive deformity, or structural pain”. A clival canal angle below 125° and/or a Grabb’s measure above 9 mm are considered to be predictive of CCI (Joaquim A.F. et al. 2018). Craniocervical instability has been described in congenital conditions like Down syndrome (Brockmeyer D 1999), Ehlers-Danlos syndrome (Henderson F.C. et al. 2019), and Chiari malformation (Henderson FC. et al. 2010) as well as in rheumatoid arthritis (Henderson F.C. et al. 1993).

In one study on craniocervical junction stabilization by surgery in five patients with Chiari I malformation or basal invagination (Henderson FC. et al. 2010), inclusion criteria, beside abnormal Grabb’s measure and CXA, were:

  • signs of cervical myelopathy (sensorimotor findings, hyper-riflexia);
  • signs of pathology at the level of the brainstem, collected in this table;
  • severe head and/or neck pain, improved by the use of a neck brace for at least a 2 weeks period.

The same inclusion criteria were adopted in another similar study on patients with hereditary hypermobile connective tissue disorders (Henderson F.C. et al. 2019).

Several mechanisms are believed to play a role in the genesis of the clinical picture described in CCI: stretch of the lower cranial nerves (vagus nerve is among them) and of the vertebral arteries; deformation of the brainstem and of the upper spinal cord (Handerson F. 2016).

12. Craniocervical instability and ME/CFS

CCI has been described in Ehlers-Danlos syndrome hypermobile type (Henderson F.C. et al. 2019), although the prevalence of CCI in EDSh has not been established, yet (to my knowledge). At the same time, an overlapping between EDSh and ME/CFS has been reported in some studies: most of EDSh patients met the Fukuda Criteria, according to (Castori M. et al. 2011) and it has been proposed that among patients with ME/CFS and orthostatic intolerance, a subset also has EDS (Rowe P.C. et al. 1999), (Hakim A. et al. 2017). So, it might seem not unreasonable to find CCI in a subgroup of ME/CFS patients.

Moreover, both in CCI and in ME/CFS there is an involvement of the brainstem. Briefly, hypoperfusion (Costa D.c: et al. 1995), hypometabolism (Tirelli U. et al. 1998), reduced volume (Barnden L.R. et al. 2011), microglial activation (Nakatomi Y et al. 2014), and loss of connectivity (Barnden L.R. et al. 2018) have been reported in the brainstem of ME/CFS patients. Basal ganglia dysfunction has also been documented in ME/CFS (Miller AH et al. 2014), and this could be an indirect measure of midbrain abnormal functioning, given the connection between substantia nigra (midbrain) and basal ganglia, via the nigrostriatal tract. It is worth mentioning here that vagus nerve infection has been proposed as a feasible cause of ME/CFS (VanElzakker MB 2013) and vagus nerve (the tenth cranial nerve) has its origin in the lower part of the brainstem. Recently, brainstem pathology in ME/CFS (midbrain serotoninergic neurons alteration, in particular) has been theorized as part of a mathematical model on disrupted tryptophan metabolism (Kashi A.A. et al. 2019), (R). So, one might argue that CCI could in some cases lead to a clinical picture similar to the one described in ME/CFS because in both these conditions there is a pathology in the same anatomical district (figure 8).

Model.JPG
Figure 8. In this model, it is assumed that CCI may produce a clinical picture similar to ME/CFS because in both the conditions there is a pathology of the brainstem. By Paolo Maccallini.

We know that in most of the cases ME/CFS starts after an infection (Chu L. et al. 2019). That said, how could CCI be linked to this kind of onset? The presence of CCI in rheumatoid arthritis (Henderson F.C. et al. 1993) might be a clue for a causal role of the immune system in this kind of hypermobility. In fact, a link between hypermobility and the immune system has been found also in a condition that is due to the duplication/triplication of the gene that encodes for tryptase (a proteolytic enzyme of mast cells) (Lyons JJ et al. 2016).

A piece of evidence against a link between CCI and ME/CFS is perhaps represented by the results of a study on EDSh patients with CCI who underwent surgery for their craniocervical junction abnormalities. Before surgery, all the 20 patients reported fatigue among their symptoms and two yers after surgery the improvement in this symptom was not statistically significant, despite improvement in the craniocervical joint measures (CXA and Grabb’s measure) and improvement in overall functioning (Henderson F.C. et al. 2019). This seems to be a clue against the role of CCI in fatigue, at least in this patient population.

13. Craniocervical measures in a few ME/CFS patients

I have collected standard MRIs of the head of seven ME/CFS patients and I have performed the measures described in this article, using the sagittal section of T1 weighted series. Data are collected in table 1.

GM stands for Grabb’s measure and the cutoff for this value has been taken from an MRI study on children with Chiari malformation (Grabb P.A. et al. 1999). I have not been able to find a study on adult normal subjects, so I don’t have any reliable statistical data on that measure. Yet, the reported cutoff of 9 mm is what is commonly indicated for GM (R), (Handerson F. 2016), (Joaquim A.F. et al. 2018). HHM stands for horizontal Harris measure and the cutoff was deduced from (Henderson F.C. et al. 2019), but again, I have not found statistical data on this measure from MRIs sagittal sections of an adult healthy population. BDI is the basion-dens interval and the cutoff comes from (Handerson F. 2016) and no statistical data available on a suitable population. CDD and MDD are the distances of the tip of the dens from the Chamberlain’s line and the McRae’s line, respectively and I got the statistical data from an MRI study on adult healthy subjects (Cronin C.G. et al. 2007). CXA is the clival-canal angle: statistical data were from an MRI study on 33 healthy adults (Botelho R.V. et al. 2013), while the cutoff was indicated in (Henderson F.C. et al. 2019).

The only abnormal values found are the distance between the tip of the dens and both Chamberlain’s line and McRae’s line in P2 and the Grabb’s measure in P7, with the caveat that I don’t have suitable statistical data for comparison, in the latter case. And of course, I don’t know what the meaning of these slightly abnormal values is. Of notice, none of these patients would fit the criteria proposed in (Henderson F.C. et al. 2019) for surgery of the craniocervical junction.

Table.JPG
Table 1. GM: Grabb’s measure; HHM: horizontal Harris measure; BDI: basion-dens interval; CDD: distance between dens and Chamberlain’s line; MDD: distance between dens and McRae’s line; CXA: clival-canal angle; POTS: postural orthostatic tachycardia syndrome; PEM: post-exertional malaise; μ: mean value; σ: standard deviation. By Paolo Maccallini.

Patient 4 should probably be excluded from this table: she had a documented B12 deficiency at the onset of her disease; she was treated with vitamin B12 injections. After some months she has substantially improved. So it might have been a case of vitamin B12 deficiency. She also has a problem with iron, which tends to be low and has to be supplemented; since vitamin B12 and iron are both absorbed in the small intestine, this patient may have some pathology in that area. In fact, signs of inflammation were found in a sample of her duodenum, but it was not possible to define a specific diagnosis (celiac disease was ruled out, as well as Crohn’s disease).  Interesting enough, this patient had a diagnosis of POTS (by tilt table test) and vitamin B12 deficiency has been linked to POTS (Öner T. et al. 2014). As mentioned, she is in remission now.

Let’s try now a statistical analysis for the values of the clival canal angle reported in Table 1, using as control group the one published in (Botelho R.V. et al. 2013). We can use Cantelli’s inequality (see Eq. 2, paragraph 15) and extend it to a random vector. We get for the p value:

Cantelli.JPGIn our case m = 8, µ = 148, σ = 9.88. By using the following very simple code we calculate a p value < 0.03, which is statistically significant.  The problem here is that the measure of the CXA in the control group has been made by someone else than me, so this might be a source of error. Moreover, the sample is very small. All that said, a tendency towards a reduction of the clival canal angle among ME/CFS patients might be further proof of increased mobility of the cranio-cervical joint in this patient population, in agreement with previous studies on other joints (Rowe P.C. et al. 1999), (Hakim A. et al. 2017).

clear all
mu = 148
ds = 9.88
m = 8
p = 1.;
x = [142, 146, 142, 142, 135, 140, 140, 139];
for i=1:m
  p = p*( 1/( 1 + ( ( (mu-x(i))/ds )^2 ) ) );
endfor
p

14. Craniocervical instability and Euler’s angles

A more sound definition of CCI might perhaps be obtained with the introduction of the angles that are used to describe the orientation of a rigid body with respect to a fixed coordinate system. To simplify our analysis, we assume here that atlas (C1) and axis (C2) are fixed one to the other. Then, consider the coordinate system (O; x, y, z) in figure 1 to be fixed to C1-C2 and then let’s introduce a second coordinate system (Ω; ξ, η, ζ), fixed to the skull. The orientation of (Ω; ξ, η, ζ) with respect to (O; x, y, z) is given by the angles ψ, φ, θ, called Euler’s angle (figure 7). The angle θ is the one between z and ζ. In order to define the other two angles, we have to introduce the N axis, known as line of nodes, which is the intersection between plane xy and plane ξη. That said, ψ is the angle between x and N, while φ is the angle between ξ and N.

coordinate systems.JPG
Figure 7. The orientation of  (Ω; ξ, η, ζ) with respect to (O; x, y, z) is univocally determined by the angles ψ, φ, θ. N is the line of nodes, defined as the intersection between plane xy and plane ξη. By Paolo Maccallini.

All that said, craniocervical hypermobility may be defined as follows.

Def. We have CCI when there is an increase in the physiological range of Euler’s angles and/or when |ΩO|≠0.

In this definition, we have assumed that in physiological conditions the length of the vector ΩO is nought. The length of ΩO is indicated as |ΩO|. The condition |ΩO|≠0 means that at least one of the components of ΩO along the axises x, y, z is different from zero

The reader can easily recognize now that:

  • the clival-canal angle is a measure of instability in the angle θ; we can also say that clival-canal angle measures instability around N;
  • Grabb’s measure and Horizontal Harris measure both indicate instability along the x-axis; they are a measure of the x component of vector ΩO;
  • Chamberlain’s line gives a measure of instability along the z-axis; the same applies to McRae’s line and to BDI.

15. Cantelli’s inequality

To assess the statistical significance of the experimental data in Table 1 we have used Cantelli’s inequality, also known as one-tailed Chebyshev’s inequality. Given the random variable X whose distribution has mean E[X] and variance Var[X], then Cantelli’s inequality states that:

eq-1-e-2-e1564518778287.jpg

for any η>0. The importance of these two inequalities is that they are true whatever the distribution is. In the case of our patient’s MRS data, we only knew mean values and standard deviations (which is the square root of variance) of the distributions of the metabolic values of the control group. So one way to assess significance was to use this inequality (the other way would be to use the less precise Chebyshev’s inequality). To prove Eq. 1 and Eq. 2 we have first to prove Markov’s inequality, which states that

Eq 3

for any a>0. In order to prove that, consider that for the probability on the left of the inequality we can write

Eq a.JPG

At the same time, the expectation (or mean) of the distribution can be written

Eq b.JPG

Thus we haveEq c.JPG

and Markov’s inequality is proved. Let’s now come back to the proof of Cantelli’s inequality. If we consider the random variable Y = X – E[X] we have that P(Y≥η) = P(Y+t≥η+t) and assuming that η+t > 0 we have

Eq d.JPG

That said, Markov’s inequality gives

Eq e.JPG

For the expectation on the right we have

Eq f.JPG

and knowing that E[Y²] = Var[X] and that E[Y] = 0, we can write

Eq 4.JPG

The function on the right of the inequality is represented in Figure 4. It is easy to recognize that it assumes its lower value for t = Var(X)/η and this proves Eq. 1. The other inequality (Eq. 2) can be proved in the same way, considering the random variable Z = E[X] – X.

plot.JPG
Figure 8. This is the qualitative plot of the function in the second term of the inequality in Eq. 4. In this diagram, we have written the variance through the standard deviation σ. Remember that σ² = Var[X].