# Testing hypotheses

Introduction

My ME/CFS improves during summer, in the period of the year that goes from May/June to the end of September. I don’t know why. I have several hypotheses. One possible reason for the improvement in summer is an interaction between the light from the Sun and some parts of my physiology, the immune system for instance. We know that ME/CFS tends to have an oscillating course in most of the patients (Chu L. et al. 2019), but the presence of a seasonal pattern in this patient population has not been investigated so far, to my knowledge. And yet, if you ask directly to patients, many of them say that they feel better in summer. Unfortunately, we don’t have scientific data on that, this is an area worth investigating with some carefully done survey.

Seasonal variation of the immune system

The immune system has a high degree of variation for several reasons (Brodin P et Davis MM 2017). In particular, there are studies about the seasonal fluctuations in the expression of some crucial genes of the immune response (Dopico XC et al. 2014).

How does this regulation happen? Different mechanisms are possible, some of them might be related to changes in the light we receive from the Sun as the Earth rotates around it. We know that the length of the day has an effect on innate immunity: the more the hours of light, the lower the power of the innate immune system (Pierre K. et al. 2016). We also know that ultraviolet radiation, particularly UVB, is an agonist for the aryl hydrocarbon receptor (AhR) (Navid F. et al. 2013). This receptor seems to reduce the expression of the major histocompatibility complex II (MHC II) in dendritic cells (DCs), thus reducing their antigen-presenting activity (Rothhammer V. et Quintana F.J. 2019). UVB might be able to reach dendritic cells when they are circulating near the skin, during summer, thus inhibiting their antigen-presenting activity. Infrared radiation, on the other hand, seems to have an effect on energy metabolism: in Fall we lose a significant amount of infrared radiation in a wavelength range (0.7-1.0 nm) that is known to have an effect on mitochondrial activity (Nguyen L.M. et al. 2013) and it might perhaps have an indirect effect on immunity too.

As further proof of seasonal fluctuation in immunity, some immunological diseases have this kind of seasonality: Rheumatoid arthritis (Nagamine R. et al. 2014) and Rheumatic fever (Coelho Mota C.C. et al. 2010) are two examples. Moreover, the prevalence of Multiple Sclerosis is directly proportional to the latitude (Simpson S. et al. 2011). We also know that there is seasonal fluctuation in serum autoantibodies (Luong T.H. et al. 2001).

Of course, sunlight might be just one of the variables into play. The other aspect I am considering is the seasonal distribution of some common pathogens. Streptococcus, Enteroviruses and Fungi of the genus Penicillium are known to have a seasonal distribution with a peak in Fall and/or Winter (Ana S.G. et al. 2006), (Salort-Pons M et al. 2018), (Coelho Mota C.C. et al. 2010). Common influenza has this pattern too. Rheumatic fever, a disease due to an abnormal immune response to Streptococcus, has its flares in Fall because Streptococcus is more common in that period of the year (Coelho Mota C.C. et al. 2010). Even the composition of the gut microbiota has a seasonal pattern (Koliada A. et al. 2020). I am currently investigating my immunosignature, measured with an array of 150.000 random peptides, to see if I can find some relevant pathogen in my case. You can find this study here.

(A few months after I wrote these notes a pivotal study has been published on these same topics, avalilable here).

An experiment

I moved from Rome (Italy) to Rosario (Argentina) at the beginning of January. I was very sick and I steadily improved after about 40 days. I became a less severe ME/CFS patients and I could work several hours a day and care for myself, granted that I did not exceed with aerobic exercise. At the end of March, I started deteriorating as it usually happens at the end of September, when I am in Rome. In order to study this phenomenon, I have built a complete model of solar radiation at sea level, which considers the inclination of sunrays in function of the latitude and of the day of the year. It takes into account the effect of the atmosphere (both diffusion and absorption) and the eccentricity of the orbit (Maccallini P. 2019). If you look at the figure below (a byproduct of my mathematical model) you can see that when I started deteriorating in Rosario, the power of sunrays at noon in that city was still as high as it is in Rome during the summer solstice (this is due to the fact that the Earth is closer to the Sun in this period and to the fact that Rosario is closer to the Equator than Rome is).

So I have to discard the original idea that the power within the infrared range, or the ultraviolet radiation, or the visible one is responsible for my improvement in summer. If I still have to consider that sunlight has something to do with my improvement, I must conclude that it is the length of the day the relevant parameter: I may need more than 12 hours of light to feel better. Why? Because the longer the day, the lower the strength of the innate immunity. This is now my working hypothesis and I will start from the following mathematical model to pursue this research: (Pierre K. et al. 2016).

I will also use full-spectrum lamps early in the morning and in the evening to reproduce a 15 hours day, so to dampen down my innate immune system in a safe, drug-free way. I have to reproduce a day of 15 hours and see what happens. In the figure below the hours of the day at dawn and at dusk and the length of the day for Rome, for each day of the year (this is also a plot from my model).

What follows is the script I have coded in order to plot the first figure of this post. More details on this model of solar radiation are here: (Maccallini P. 2019). As a further note, I would like to acknowledge that I started pursuing this avenue in the summer of 2009: I was building the mathematical model of solar radiation (see figure below, made in 2009) but as the summer finished, I turned into a statue and I had to stop working on it. When I improved, about a year later I started working on the systematic analysis of the mechanical equilibrium of planar structures (it is a chapter of this book). I am proud of that analysis, but it has not been very useful for my health…

% file name = sun emissive power sea level Rosario vs Roma
% sun emissive power per unit area, per unit wavelength at sea level
clear all
% three parameters of the orbit
A = 6.69*( 10^(-9) ); % 1/km
B = 1.12*( 10^(-10) ); % 1/km
delta = pi*313/730;
% the two parameters of Plunk's law
C_1 = 3.7415*( 10^(-16) ); % W*m^2
C_2 = 1.4388*( 10^(-2) ); % mK
% Stefan-Boltzmann parameter ( W/( (m^2)*(K^4) ) )
SB = 5.670*( 10^(-8) );
% radius of the photosphere (m)
R_S = 696*(10^6); % m
% temperature of the photosphere (K)
T_S = 5875;
% conversion of units of measurments
N = 20; % dots for the equator
R = 3.8; % radius of the orbit
ro_E = 1.3; % radius of the earth
lambda_Rosario = -32*pi/180; % latitude of Rosario (radiants)
lambda_Roma = 41*pi/180; % latitude of Rome (radiants)
delta = 23.45*pi/180; % tilt angle
% the array of theta
theta(1) = 0; % winter solstice (21/22 December)
i_ws = 1;
day = 2*pi/365;
days = [1:1:366];
for i = 2:366
theta(i) = theta(i-1) + day;
if ( abs( theta(i) - (pi/2) ) <= day )
i_se = i; % spring equinox (20 March)
endif
if ( abs( theta(i) - pi ) <= day )
i_ss = i; % summer solstice (20/21 June)
endif
if ( abs( theta(i) - (3*pi/2) ) <= day )
i_ae = i; % autumn equinox (22/23 September)
endif
endfor
% the array of the radius (m)
for i=1:1:366
o_omega (i) = (10^3)/[ A + ( B*sin(theta(i) + delta ) ) ]; % m
endfor
% the array of the wavelength in micron
N = 471;
L(1) = 0.3;
L(N) = 5.0;
delta_L = ( L(N) - L(1) )/(N-1);
for j = 2:N-1
L (j) = L(j-1) + delta_L;
endfor
% the array of beta*L
% the array of L in metres
L_m = L*( 10^(-6) );
% angle psi
psi(1) = 0;
minute = pi/(12*60);
for i = 2:(24*60)+1
psi(i) = psi(i-1) + minute;
endfor
% -----------------------------------------------------------------------------
% Rosario
lambda = lambda_Rosario
% angle between n and r at noon in Rosario
for i= [i_ws, i_ae, i_ss, i_se]
for j=1:(24*60) + 1
% scalar product between n and r
scalar_p(j) = [cos(lambda)*sin(psi(j))*cos(delta) + sin(lambda)*sin(delta)]*( -cos(theta(i)) )+ [(-1)*cos(lambda)*cos(psi(j))]*( -sin(theta(i)) );
endfor
% value of psi at noon
for j=1:(24*60) + 1
if ( ( scalar_p(j) ) == ( max( scalar_p ) ) )
j_noon = j;
psi_noon (i) = psi(j);
endif
endfor
% angle between n and r at noon
cos_gamma (i) = scalar_p(j_noon);
endfor
% the array of the emissive power (W/(m^2)*micron) in Rosario
for i = i_se:i_se
for j=1:N
num = C_1*( (R_S)^2 );
den = ( (L_m(j)^5)*( (e^(C_2/( L_m(j)*T_S ))) - 1)*( (o_omega(i))^2 ) )*10^6;
power(j,i) = ( num/den )*( e^(-S(j)/cos_gamma (i)) );
endfor
% plotting
plot (L (1:N), power(1:N,i), '-r', "linewidth", 2)
xlabel('wavelenght ({\mu})');
ylabel('W/m^{2}{\mu}');
axis ([0.3,5,0,1500])
grid on
endfor
hold on
% -----------------------------------------------------------------------------
% Rome
lambda = lambda_Roma
% angle between n and r at noon in Rosario
for i= [i_ws, i_ae, i_ss, i_se]
for j=1:(24*60) + 1
% scalar product between n and r
scalar_p(j) = [cos(lambda)*sin(psi(j))*cos(delta) + sin(lambda)*sin(delta)]*( -cos(theta(i)) )+ [(-1)*cos(lambda)*cos(psi(j))]*( -sin(theta(i)) );
endfor
% value of psi at noon
for j=1:(24*60) + 1
if ( ( scalar_p(j) ) == ( max( scalar_p ) ) )
j_noon = j;
psi_noon (i) = psi(j);
endif
endfor
% angle between n and r at noon
cos_gamma (i) = scalar_p(j_noon);
endfor
% the array of the emissive power (W/(m^2)*micron) in Rosario
for i = [i_ae, i_ss]
for j=1:N
num = C_1*( (R_S)^2 );
den = ( (L_m(j)^5)*( (e^(C_2/( L_m(j)*T_S ))) - 1)*( (o_omega(i))^2 ) )*10^6;
power(j,i) = ( num/den )*( e^(-S(j)/cos_gamma (i)) );
endfor
endfor
hold on
plot (L (1:N), power(1:N,i_ae), '-k', "linewidth", 2)
plot (L (1:N), power(1:N,i_ss), '--k', "linewidth", 2)
legend ('spring equinox in Rosario', 'autumn equinox in Rome', 'summer solstice in Rome', "location",'NORTHEAST')
hold on
plot ([0.4,0.4], [0,1500], '--k', "linewidth", 1)
plot ([0.7,0.7], [0,1500], '--k', "linewidth", 1)

# Back home

Continuation of this post.

Forty-four hours of travelling, in total, from Rosario to Rome, by pullman, by plane, and by train. With 40 kilos of books and papers.

I had a flight for Rome that was programmed to take off from Ezeiza, the International airport of Buenos Aires, on April 13th, but I decided to take the one organized by the Italian government for March 23th, a special flight set up to bring back home Italian citizens abroad, before a complete shut down of international flights from Argentina to our country. There were no flights from Rosario, my city, to Buenos Aires, though, but I managed to find a company that organizes transportations by pullman from one city to the other, in Argentina: Tienda Leon.

So, on March 22nd, I moved to Ezeiza where I waited several hours before sitting on my chair, on a brand new Boeing 787 bearing the colours of the Italian company Neos.

While at the airport, I met most of the Italians that were going to get the same flight, all wearing their masks. Some of them with some very fancy models, that made them look like a Star Wars character. I was there, well aware that I was going far beyond the limit set by my disease. I had to lay down continuously and I could see how frail I was in comparison with the other passengers waiting for the flight. No one knew how sick I was, I told nobody. No one knew that I have been living in my bedroom for most of the last 20 years. And that this was the very first long travel abroad for me.

I have just received the notification that my flight for April 13th has been cancelled, so my choice to come back as soon as possible has been a wise one. I took that decision also because of the advice from the diplomatic offices of the Italian Consulate in Rosario.

A friend has crafted the picture above, not knowing how much Indiana Jones has meant for me when I was a teenager. But, even though an appealing adventure, the tragedy behind it is real, it is not a movie. Once in Milan, I could start seeing the effects of the pandemic in the eyes of the staff of the airport of Malpensa: the fear and the concern. Then I moved from Milan to Fiumicino, where I found a train for Rome, my city. A city that I left two and a half months ago full of life and noise, now empty as in a dream.

# From Argentina to Italy, during a pandemic

It has been a great ride, my almost-three-month period here in Rosario, next to the huge slowly flowing river of Paranà. This is a city full of life, embraced by the warmest summer I have ever seen. Populated by wonderful citizens.

I have been living in an apartment where the sun awakes me very early in the morning, through a wide window next to my bed. I could see the roofs of the centre of the city as I opened my eyes, including the top of the Monumento National a la Bandera, a gigantic building that celebrates this great nation. For the second half of the day, I had the light from the opposite window and I could follow its changes, while I was working, as the hours passed by; I saw every day the same magic ritual: as the photons from our star went through thicker layers of the atmosphere, they changed their frequency, turning redder and redder, culminating in a warm explosion, just before the night.

And in the meanwhile, news from Italy was scarier and scarier and the hypothesis that the new coronavirus could reach this continent was more obvious as the weeks passed by. Now we have the virus here, and president Alberto Fernandez has declared the state of quarantine from March 20th.

At that point, the connection between Argentina and other countries (including Italy) has become uncertain; my flight planned for March 28th has been cancelled and I have decided to get one of the special flights organized by the Italian government to bring back its citizens from Argentina, before a complete shut down of international travels. So I had about 48 hours to find a means of transport from Rosario to the airport in Buenos Aires, where the flight will take off tomorrow, at 1:00 AM.

But there was no way I could find a flight from Rosario to Buenos Aires in such a short time, also because of the shut down of Argentina, and no trains were available. Fortunately enough I have found a Pullman, and I am going to leave this apartment in a few hours.

My come back to Italy is becoming more and more adventurous also because I will land in Milan, one of the places most hit by the infection in the whole planet. There I have to reach Rome. I have been able to find a plane from Milan to Rome, so it will be possible to be at home on the evening of March 23rd. I have to avoid to get the virus though, during this travel. I will be exposed to it for sure, so I am taking any possible measure to ensure my safeness.

This travel to Argentina has been a success. My health has improved, even though now I am deteriorating again, as was expected, as the light of the summer of the southern hemisphere becomes weaker. But I have been able to use my new energies to write and submit a paper on the cingulate cortex in ME/CFS, I have gone further with my studies on the analysis of the immunosignature (measured using random peptides) in my own serum (R), I have started the study of a mathematical model for the diffusion of Coronavirus 19 among the Italian population (R). I have learnt a great deal about computational neuroanatomy (R) and neurosciences in general. I have finished a complete model for solar radiation at sea level (R) and I might have found one of the environmental parameters that determine my improvement during summer. And yes, I have also been able to draw a portrait.

The adventure in the realm of science and art has been great, now I have to live the adventure of coming back home going through a world that is facing one of the greatest health challenges of the last century.

# A Mathematical model for the diffusion of Coronavirus 19 among the Italian population

Abstract

In this document, I propose a distribution for the number of infected subjects in Italy during the outbreak of Coronavirus 19. To do that I find a logistic curve for the number of deaths due to this virus, in Italy. I also use a density of probability for the fatality rate and one for the number of days from infection to death. These functions have been built using recently published statistical data on the Chinese outbreak.

Go to the article

# Brain Normalization with SPM12

Introduction

Brain normalization is the rigid rotation and/or deformation of a 3D brain scan so that it can match a template brain. This is a necessary step for the analysis of brain magnetic resonance data (whether it is morphological or functional) as well as of brain PET data. It allows introducing a set of spatial coordinates such that each triplet (x,y,z) identifies the same anatomical region both in the brain we are studying and in the template (Mandal P.K. et al. 2012). So, for instance, once the normalization has been performed on an fMRI of the brain of a patient and on a set of fMRIs from a suited control group, we can compare the BOLD activation of each anatomical region of the patient’s brain with the activation of the corresponding anatomical region of the control group.

Mathematical notes

This part can be skipped, it is not necessary to read these passages for the understanding of the following paragraphs. If we assume that P is a point of the brain before normalization and we call S(P) its position after the normalization, we can consider the vectorial function:

which gives the new position of each point of the brain, after normalization. If P_0 is a point of the brain before the normalization, then we can write:

and remembering the expression of the differential of a vectorial function, we have

With a few passages we can write:

From the above formula, we realize that in order to define the configuration of the brain after normalization, we have to define, for each point P, a set of 12 parameters. Of these parameters, 6 describe the rigid movement and can be considered the same for each point. The other 6 (the coefficients of the matrix) are those that describe the change of shape and size and, in general, they are different for each point. The job of SPM is to calculate these parameters.

Brain atlases

There are several templates (also called brain atlases) that have been developed across the years. The very first one was published in 1957 (Talairach J. et al. 1957). The same researcher then participated in building one of the most used brain atlas ever, based on a single female brain, published in 1988 (Talairach J. et Tournoux P. 1988). Another widely used template is the so-called MNI-152. It was built adapting the 3D MRI brain scans of 152 healthy individuals to the Talairach and Tournoux template. The adaptation was achieved using both a rigid roto-translation and a deformation (Maintz J.B. et Viergever M.A. 1988). The second step is required for overcoming the problem of differences in brain shape and dimension that we encounter within the human population.

Limitations

Available brain atlases have some limitations. One of them being the fact that despite diseased brains are the most widely studied, they are also the most difficult to register to a template built from healthy individuals, because of usually marked differences in shape and/or size. This is true for instance for brains of patients with Alzheimer’s disease (Mandal P.K. et al. 2012). Another important limitation is that registration algorithms perform poorly for the brainstem (particularly for pons and medulla) (Napadow V. et al. 2006). This might have represented a problem for the study of diseases where a possible involvement of the brainstem is suspected, like for instance ME/CFS (VanElzakker M. et al. 2019).

SPM12

The SPM software package is one of the most widely used instruments for the analysis of functional brain imaging data (web page). It is freely available for download, but it requires that you have a MatLab copy in your computer. Those who don’t have a MatLab license can request and install a standalone version of SPM12 by following the instructions of this page.

Importing a DICOM file

Once you have installed SPM12 in your computer, the first step in order to register a brain is to convert the format the series of images are written in, to a format that SPM12 can read. MRI images are usually in .dcm format (DICOM) while SPM12 reads .nii files. In order to do that, click DICOM import (figure below, on the left, red ellipse), then click on DICOM files (on the right, red ellipse), then select your .dcm file and click DONE (below, red ellipse). If you then click DISPLAY (blue ellipse, left) you will see your MRI scan in another window (see next paragraph). A video tutorial on these operations is available here.

Setting the origin

To start the normalization process, it is highly recommended to set manually the origin of the coordinates. If this is done properly, the registration will not only take less time but, even more importantly, the chances of a successful normalization will increase. The origin is set at the level of the anterior commissure (figure below). To find this anatomical structure, you can follow this video. Once you have put the cross on the right place in the sagittal, coronal and axial windows, just click SET ORIGIN (red ellipse) and then save your work clicking REORIENT (blue ellipse).

Normalization estimate

In this step, SPM12 calculates the set of distortions that have to be applied to the source brain to adapt it to the template in MNI space. On the main menu select NORMALIZE (ESTIMATE) (figure, on the left, red). This will open the batch editor where you are asked to load the subject you want to apply normalization to (figure, right, red). You have also a set of estimation options (blue), that we leave as they are.  Then you click the RUN button, the arrow on the top of the batch editor.

At this point, your PC will perform a set of calculations that will require a few minutes. At the end of this process, a new .nii file will be saved in the spm12 folder. This is the set of distortions that will allow your subject’s brain to be registered to the template.

Normalization writing

Now click on NORMALIZE (WRITE) on the main menu. The batch editor will then ask you for the deformation field, which is the file generated in the previous step, and for the images to write, which is the scan of your subject (figure below). Select them, then press the RUN button on the batch editor. A new .nii file will be written in the spm12 folder. This is the normalized brain!

In the next figure, you have the normalized brain on the left and the initial scan of the same subject on the right. As you can see, there is an overall change of shape.

Anatomical areas

Now that we have normalized our brain in the MNI space, we can easily find anatomical regions within its sections. We can, for instance, load the normalized brain with MRIcron and overlay a template with Brodmann’s areas highlighted in various colours (figure below).

.

# I cannot die

“I can’t say if it is titanic patience or endless desperation. What are they waiting  for? Have they given up waiting? Which is the present they are immersed in?”

Italo Calvino, Palomar

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 one whole 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

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. Three phages of bacterial human pathogens were identified, 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.

(a commentary in Dutch is available here)

1. The quest for a pathogen

Scientists have been speculating about an infectious aetiology of ME/CFS for decades, without ever 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 prokaryotic and eukaryotic organisms reported that most of the ME/CFS patients have DNA belonging to the eukaryotic genera Perkinsus and Spumella and to the prokaryotic 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.

Recently an array of overlapping peptides that cover the proteins for many know viruses has been successfully used for the study of acute flaccid myelitis (AFM). This technology, called VirScan, has succeeded in linking AFM to enteroviruses where metagenomic of the cerebrospinal fluid has failed (Shubert R.D. et al. 2019). This kind of approach is probably better than the one employing arrays of random peptides, for pathogen hunting. The reason being that a set of only 150.000 random peptides is unlikely to collect a significant amount of B cell epitopes from viruses, bacteria etc. Random peptides are more suited for the establishment of immunosignatures.

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.

 1 ALHHRHVGLRVQYDSG 2 ALHRHRVGPQLQSSGS 3 ALHRRQRVLSPVLGAS 4 ALHRVLSEQDPQLVLS 5 ALHVRVLSQKRPLQLG 6 ALHLHRHVLESQVNSL

Table 1. My immunosignature, as detected 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.

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. 2016), (Maccallini P. et al. 2018) led me to the conclusion that cross-reactive 7-amino-acid long peptides might often have 100% identity.

So, to recap, I use the following method: BLAST search (blastp algorithm) against viral proteins (taxid 10239), a perfect match (100% identity) of at least 7-amino-acid peptides (≥43% query cover), max target sequences: 1000, substitution matrix: BLOSUM62.

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 ALHRHRVGPQLQSSGS (102_2_F_viruses) 2-LHRHRVG-8 YP_009619965.1(63-69) Stenotrophomonas phage vB_SmaS_DLP_5 Stenotrophomonas maltophilia (HP) ALHRRQRVLSPVLGAS (102_3_F_viruses) 2-LHRRQRV-8 QHN71154.1 (288-294) Mollivirus kamchatka Protozoa (R) 8-VLSPVLG-14 QDB71078.1 (24-30) Serratia phage Moabite Serratia marcescens (HP) ALHRVLSEQDPQLVLS (102_4_F_viruses) 7-SEQDPQL-13 BAR30981.1 (151-157) uncultured Mediterranean phage uvMED Archea and Bacteria, Med. sea 3-HRVLSEQ-9 AXS67723.1 (494-500) Cryptophlebia peltastica nucleopolyhedrovirus invertebrates 2-LHRVLSE-8 YP_009362111.1 (74-80) Marco virus Ameiva ameiva 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 matches 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

There are no human viruses detected by this search. There are some bacteriophages and three 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 prokaryotic 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.

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: Stenotrophomonas maltophilia (HP), Serratia marcescens (HP), Mycobacterium smegmatis mc²155 (HP). In brackets, there are links to research about the pathogenicity for humans of each species. M. smegmatis belongs 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).

Mollivirus kamchatka is a recently discovered giant virus whose hosts are presumed to be protozoa that inhabit the soil of subarctic environment (Christo-Fourox E. et al. 2020). Not sure what the meaning might be in this context.

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 eukaryotic microorganisms and for human proteins (in search for autoantibodies).

# 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).

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).

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).

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.

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 to communicate what was happening to me, to describe 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…

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.

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.

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.

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

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.

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?

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.

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.

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.

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.

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!

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!

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.