CT38

CT38

La casa farmaceutica Cortene INC. sembra stia sviluppando un farmaco per la ME/CFS (e patologie connesse in qualche modo, come sindrome del Golfo, Lyme cronica etc). Il farmaco si chiama CT38 e agisce su un recettore (il CRF 2) della corticotropina. La corticotropina è l’ormone che stimola l’ipofisi a produrre ACTH (che a sua volta stimola i surreni a produrre cortisolo).

Nel sito della Cortene INC. viene detto che è stato già fatto uno studio di sicurezza del farmaco su esseri umani sani e diversi studi su animali che suggerirebbero che la ME/CFS sia dovuta a una espressione eccessiva di questo recettore nel sistema limbico. Cito il sito:

I nostri studi suggeriscono che la sovraregolazione di un recettore fra quelli che controllano la risposta allo stress, il CRF2 (espresso nel sistema limbico e nei nuclei del rafe),  spiegherebbe i sintomi e le anomalie dei pazienti ME/CFS. Infatti la sovrastimolazione di questo recettore induce molti dei sintomi della ME/CFS in animali sani, e noi abbiamo un potenziale farmaco in grado di inibire questo recettore.

Ma questi studi non li trovo su pubmed e non ho potuto reperire ancora nessuna documantazione su questi dati. Inoltre la validità di un modello animale di ME/CFS è quantomeno discutibile, e ad oggi un modello animale di questa patologia non è stato mai proposto, che io sappia.

Il sito comunica che è pianificato un trial di fase 2 su pazienti ME/CFS.

Annunci

We finally have the technology

We finally have the technology

I have always loved this speech from the movie Armageddon (see video below), but now I realize that there is an interesting parallelism between this sci-fi movie and what we are living right now with the quest for the solution of ME/CFS and related diseases. Just as the US president says in the movie (where a menacing asteroid is travelling toward the Earth), for the first time in the history of the planet we have the technology to understand what is going on in the bodies and in the brains of ME patients and hopefully the tools to end this tragic loss of lives. And just as it happens in the movie, we have to join our efforts in order to fight this global threat.

ME/CFS: un manuale per la pratica clinica

ME/CFS: un manuale per la pratica clinica

Sono felice di poter finalmente condividere la traduzione italiana del volume “ME/CFS: A Primer for Clinical Practiniores” della IACFS, realizzata da Giada Da Ros, con la consueta perizia e dedizione. Ricordo benissimo quando il documento originale fu reso disponibile nel 2014, e da allora l’ho sfogliato numerose volte, vuoi per verificare i dosaggi di un farmaco, vuoi per consultare le tabelle sulla diagnosi differenziale, vuoi ancora per cercare degli articoli scientifici nella bibliografia.

Il manuale ha un taglio pratico, con una attenzione particolare alla diagnosi a esclusione e al trattamento di alcuni aspetti della patologia, come ad esempio la intolleranza ortostatica, i disturbi cognitivi, i problemi gastrointestinali, eventuali processi infettivi etc. Eppure non manca di una agile trattazione teorica che include nozioni sulla epidemiologia della ME/CFS, sulla possibile eziologia e sulle anormalità riscontrate in questi pazienti negli ambiti immunitario, neurologico, metabolico.

Tra gli autori del manuale si annoverano nomi noti del mondo ME/CFS, come Lucinda Bateman e Leonard Jason. Questo volume è indicato dai CDC di Atlanta come come uno dei principali punti di riferimento per i medici.

Il PDF della traduzione italiana di Giada Da Ros è disponibile qui.

Mark Davis and the search for the universal immune test

Mark Davis and the search for the universal immune test

1. Introduction

These are some notes about the talk that Mark Davis gave during the Community Symposium held in August at Stanford (video). I will introduce some basic notions about T cell receptors (TCR) in paragraphs 2, 3, 4, and 5. Paragraphs 6 is a description of an innovative technology developed by Mark Davis and his colleagues, based on information gathered from the video itself and three research papers published by Davis and others in the last 4 years. This background should be hopefully enough to allow a good understanding of the exciting pilot data presented by Mark Davis on T cell activity in ME/CFS (paragraph 7), and in chronic Lyme (paragraph 8), and to realize why this technology promises to be some sort of universal test for any kind of infectious and autoimmune diseases, known or unknown.

2. T cells

T cells are a type of leukocytes (also known as white blood cells), the cellular component of our immune system. Most of our circulating T cells are represented by T helper cells (Th cells) and cytotoxic T lymphocytes (CTL). While the function of Th cells is to regulate the activity of other leukocytes through the production of a wide range of chemicals (cytokines), CTLs are directly involved in the killing of host cells infected by pathogens. T cells belong to the adaptive arm of the immune system, along with B cells (the factories of antibodies), and as such, they are meant to provide a defence tailored to specific pathogens: our immune system can provide not only antibodies specific for a given pathogen but also specific T cells (both Th cells and CTLs). The innate arm of the immune system (which includes natural killer cells, macrophages, dendritic cells, mast cells…) on the other hand can provide only a one-fits-all type of defense, which represents the first line of immune response, during an infection.

3. T cell receptor

T cells search for their specific pathogens thanks to a molecule expressed on their surface, called T cell receptor (TCR). In figure 1 you can see a schematic representation of the TCR and of the mechanism by which T cells recognize their targets. Antigens (proteins) from pathogens are presented to T cells by other cells of our body: they are displayed on molecules called major histocompatibility complex (MHC), expressed on the outer membrane; if the antigen fits the TCR of a specific T cell, then this T cell is activated and proliferates (clonal expansion). The two chains (α and β) are assembled using the transcription of gene segments with several copies each: in other words, TCRs are assembled with peptides chosen randomly within a set of several possible choices. This leads to a repertoire of 10^15 possible different TCRs (Mason DA 1998). Each T cell displays only one type of TCR.

TCR
Figure 1. Upper half. Th cells and CTLs share the same TCR: in both cases this molecule is the assembly of two peptides (chain α and chain β), but while the TCR of Th cells (on the right) is expressed next to the molecule CD4 (which binds to class II MHC), the TCR of CTL is associated with the molecule CD8 (on the left), which is specific for MHC I. Black bars represent four chains (a complex called CD3) that are involved in the signaling of the TCR with the nucleus of the cell (by Paolo Maccallini). Lower half. A beautiful structural representation of the TCR, bound to the peptide-MHC complex (pMHC), from (Gonzàlez PA et al. 2013). In green the peptide, in blue the β chain, in dark green the α chain. CDRs (complementarity determining regions, orange) are composed of those residues of the α and β chains that directly bind the pMHC.

4. T helper cells

Th cells can recognize only antigens presented by class II MHC: this class of MHC is expressed on the outer membrane of some leukocytes, mainly dendritic cells, B cells, and macrophages (referred to as antigen presenting cells, APCs). MHC II engages the TCR of Th cells thanks to peptide CD4 (expressed exclusively by Th cells). The antigen presented by MHC II is a peptide with a length of 13-17 amino acids (Rudensky, et al., 1991) (figure 2).

MHC II.JPG
Figure 2. The TCR expressed by a Th cell binds an epitope presented by a class II MHC expressed on the plasma membrane of an APC. Chains α and β of MHC II are also represented (by Paolo Maccallini).

5. Cytotoxic T lymphocytes

TCRs expressed by CTLs can bind only antigens displayed by class I MHC, which can be found on the outer membrane of any cell of our body. CD8 is the molecule that makes the TCR expressed by CTLs specific for MHC I. While antigens presented by APCs belongs to pathogens that have been collected on the battlefield of the infection, peptides displayed by class I MHC of a specific cell belong to pathogens that have entered the cell itself, therefore they are the proof of an ongoing intracellular infection (figure 3). When a CTL recognizes an antigen that fits its TCR, then the CTL induces apoptosis (programmed death) of the cell that displays it. Antigens presented by MHC I are peptides in the range of 8 to 10 amino acids (Stern, et al., 1994).

MHC I.JPG
Figure 3. An infected cell displays a viral antigen on its MHC I. The TCR of a CTL binds this peptide and send a signal to the nucleus of the CTL itself, that responds with the induction of apoptosis (releasing granzymes, for instance) of the infected cell (by Paolo Maccallini).  

6. The universal immune testing

In his talk, Mark Davis presents an overview of some basic concepts about the immune system, before introducing his exciting new data about ME/CFS and post-treatment Lyme disease syndrome (PTLDS, also known as chronic Lyme). But he also describes a few details of a complex new assay that is theoretically able to read all the information packed in the repertoire of TCRs present – in a given moment – in the blood of a human being. As such, this test – that I have named the universal immune testing – seems to have the potential to determine if a given patient has an ongoing infection (and the exact pathogen) or an autoimmune disease (and the exact autoantigen, i.e. the tissue attached by the immune system). To my understanding, this assay requires three steps, described in the following sections.

6.1. First step: TCR sequencing

As said in paragraph 3, when T cells encounter their specific peptide presented by MHC, they proliferate so that in blood of patients with an ongoing infection (or with a reaction against self, i.e. autoimmunity) we can find several copies of T cells expressing the same TCR: while in healthy controls about 10% of total CD8 T cells is represented by clones of a few different T cells (figure 4, first line), in early Lyme disease – an example of active infection – and in multiple sclerosis (MS) – an example of autoimmune disease – we have a massive clonation of a few lines of CTLs (figure 5, second and third line, respectively). The first step of the universal immune testing is represented by the identification of the exact sequence of TCRs expressed by T cells in blood, as reported in (Han A et al. 2014) where it is described how to sequence genes for the α and the β chain of a given T cell. This approach allows to build graphs of the kind in figure 4, and therefore to determine whether the patient has an abnormal ongoing T cell activity or not. If a clonal expansion is found, then we can speculate that either an active infection is present or some sort of autoimmune condition.

Clonal expansion CD8.png
Figure 4. Each circle represents a patient. In the first line, we have four healthy controls, with no clonal expansion of CD8 T cells (the first one, left) or with only a low-level clonal expansion (slices in blue, white, and grey). In the second line, we have four patients with active Lyme disease (early Lyme) and all of them present a massive expansion of only three different T cells (slices in red, blue and orange). In the third line, we have four MS patient with most of their CD8 T cells represented by clones of a bunch of T cells. From the talk by Mark Davis.

6.2. Second step: TCR clustering

Mark Davis and his group have been able to code a software that allows to identify TCRs that share the same antigen, either within an individual or across different donors. This algorithm has been termed GLIPH (grouping of lymphocyte interaction by paratope hotspots) and has been found capable – for instance – to cluster T CD4 cell receptors from 22 subjects with latent M. tuberculosis infection into 16 distinct groups, each of which comprises TCRs from at least 3 different donors (Glanville J et al. 2017). Five of these groups are reported in figure 5. The idea here is that TCRs that belong to the same cluster, react to the same peptide-MHC complex (pMHC).

GLIPH.jpg
Figure 5. Five group of TCRs from 22 different donors with latent tuberculosis, clustered by GLIPH. The first column on the left has TCRs IDs, the second one reports donors IDs. Complementarity determining regions (CDR) for the β and the α chains are reported in the third and fifth column, respectively. From (Glanville J et al. 2017).

6.3. Third step: quest for the epitope(s)

As we have seen, this new technology allows to recognize if T cell clonal expansion is an issue in a given patient, by sequencing TCRs from his peripheral blood. It also allows to cluster TCRs either within an individual or across different patients. The next step is to identify what kind of antigen(s) each cluster of TCRs reacts to. In fact, if we could recognize these antigens in a group of patients with similar symptoms, with T cell clonal expansion and similar TCRs, we would be able to understand whether their immune system is fighting a pathogen (and to identify the pathogen) or if it is attacking host tissues and, if that was the case, to identify what tissue. As mentioned, the number of possible TCR gene rearrangement is supposed to be about 10^15, but the number of possible Th cell epitope is about 20^15 which is more than 10^19. This implies that TCRs have to be cross-reactive to some extent, in order to recognize all possible peptides presented by MHCs (Mason DA 1998). The exact extent of this cross-reactivity and the mechanism by which it is obtained has been elucidated by Mark Davis and his colleagues in a recent paper (Birnbaum ME et al. 2014) that gives us the third step of the universal immune testing. The aim of this phase is to take a given TCR and to find the repertoire of his specific antigens (as said, one TCR reacts to several antigens). In order to understand how this is possible let’s consider one of the experiments described in the paper mentioned above. The researchers considered two well-defined TCRs (named Ob.1A12 and Ob.2F3), cloned from a patient with MS and known to recognize peptide 85-99 (figure 6) of myelin basic protein (MBP) presented by HLA-DR15. They then prepared a set of yeast cells expressing HLA-DR15 molecules, each presenting a different peptide of 14 amino acids, with fixed residues only at position 1 and 4, where the peptide is anchored to MHC (figure 6, left). When copies of Ob.1A12 are added to this culture of yeast cells expressing pMHC complexes, they bind only some of them, and as you can see in the right half of figure 6, for each position of the epitopes bound by Ob.1A12, there is an amino acid that is more likely: for instance, the typical epitope of Ob.1A12 preferentially has alanine (A) at position -4, histidine (H) at position -3, arginine (R) at position -2, and so forth. As you can see, histidine (H) at position 2 and phenylalanine (F) at position 3 are obligate amino acids for a Ob.1A12 epitope.

ob-1a121.jpg
Figure 6. On the left: peptide 85-99 of myelin basic protein (first row) is known to be an epitope for Ob.1A12. At position 1 and 4 it has two residues that allow its binding to the MHC molecule. At position -2, -1, 2, 3, and 5 we find those residues that bind the TCR. The second row represents the generic epitope of the peptide library used to identify the degree of crossreactivity between all the possible Ob.1A12 specific epitopes. On the right: the likelihood of amino acids for each position of Ob.1A12 specific epitopes is represented by shades of violet. As you can see, histidine (H) at position 2 and phenylalanine (F) at position 3 are obligate amino acids for a Ob.1A12 epitope. From (Birnbaum ME et al. 2014).

The table on the right side of figure 6 is, in fact, a substitution matrix with dimension 14×20, a tool that can be used to scan the peptide database in order to find, among all the known peptides expressed by living creatures, all the possible Ob.1A12 specific epitopes. Substitution matrices are commonly used for the so-called peptide alignment, a technique that aims at the identification of similarities between peptides. These matrices are based on evolutionary considerations (Dayhoff, et al., 1978) or on the study of conserved regions in proteins (Henikoff, et al., 1992). But the search for specific epitopes of a given TCR requires (as we have seen here for Ob.1A12) a substitution matrix built ad hoc for that TCR: each TCR requires its own substitution matrix that is obtained adding clones of that TCR on a culture of yeast cells presenting a huge peptide library on their MHCs, and analyzing data from this experiment. So, quite a complex process! In the case of Ob.1A12, this process led to 2330 peptides (including MBP), while the Ob.2F3 specific substitution matrix found 4824 epitopes within the whole peptide database. These peptides included both non-human proteins (bacterial, viral…) and human peptides. For 33 of them (26 non human and 7 human proteins), this group of researchers performed a test in order to directly verify the prediction: 25/26 of environmental peptides and 6/7 of the human peptides induced proliferation of T cells expressing Ob.1A12 and/or Ob.2F3, and this is a huge proof of the validity of this analysis! These 33 peptides are reported in figure 7. This is the last step of the universal immune testing, the one that from the TCR leads to the epitopes. As you have seen, a huge set of different peptides from different sources is linked to each single TCR, in other words, crossreactivity is an intrinsic property of TCR. This also means that lymphocyte transformation tests (LTTs), widely used in Europe for the detection of infections like Borrelia burgdorferi and others, bear a high risk of false-positive results and require a process of experimental and theoretical validation, that is currently lacking (see also this post on this issue).

Crossreactive epitopes.JPG
Figure 7. A set of 33 peptides (both human and environmental) predicted to be specific epitopes for both Ob.1A12 and Ob.2F3. From (Birnbaum ME et al. 2014).

We are now ready to fully appreciate the pilot data that Mark Davis presented at the Symposium on CD8 T cell clonal expansion in ME/CFS and in chronic Lyme.

7. We have a hit!

Mark Davis, along with Jacob Glanville and José Montoya, has sequenced TCRs from the peripheral blood of 50 ME/CFS patients and 49 controls (first step of the universal immune testing, remember?), then they have clustered them using the GLIPH algorithm (second step). They have found 28 clusters with more than 2500 similar sequences each, where each cluster collects multiple sequences from the same individual as well as (which is perhaps more important) sequences from different patients (figure 8). The cluster that I have circled in red, for instance, is a collection of more than 3000 similar TCRs. The presence of this wide clusters in ME/CFS patients, compared to healthy controls, represents an indirect proof of a specific T cell response to some common trigger in this group of patients, which might be a pathogen or a tissue of the body (or both).

Clustered TCR
Figure 8. In ME/CFS, TCRs sequences from 50 patients form 28 clusters with more than 2500 similar sequences, and this is clearly not the case in healthy controls. This point to some specific immune response to a pathogen or to a human tissue (or both). This slide is from the talk by Mark Davis.

Among these 50 ME/CFS patients, Davis and colleagues selected 6 patients with similar HLA genes (figure 9, left), 5 females among them, and studied their TCRs deeper. In the right half of figure 9, you can see for each patient the degree of CTL clonal expansion. Remember that in healthy controls only about 10% of CTLs is composed by clones of a few cells (figure 4, first raw), while here we see that about 50% of all CTLs is composed by clones. So, a “marked clonal expansion” of CD8 T cells, as Davis said.

ME subjects CD8
Figure 9. On the left: 6 MECFS patients with similar HLA genes have been selected. Patient ID is reported in the first column on the left, the second column indicates the age of each patient, the third indicates the gender, the fourth column is about exposure to cytomegalovirus, the third one is on MHC I genes. On the right: analysis of clonal expansion of CD8 T cells for each of the six patients. There is a marked clonal expansion (about 50%) compared to healthy controls (about 10%).

Sequences of α and β chains of TCRs from three of the six patients (patients L4-02, L4-10, and L3-20) are reported in figure 10 (I have verified that in fact these are sequences of α and β chains of human TCRs using them as query sequences in standard protein BLAST).

TCR epitope.png
Figure 10. Beta chains (first column) and respective α chains (fifth column) from 3 ME/CFS patients (L4-02, L4-10, and L3-20, last column).

So, we have seen so far the first two steps of the universal immune testing in ME. What about the third step? In his talk, Mark Davis didn’t present any particular epitope, he just showed a slide with what likely is the selection of the epitopes from the peptide library (see paragraph 6.3) by one of the TCRs reported in figure 10. This selection is reported in figure 11, but from that picture, it is not possible to gather any information about the identity of these epitopes. As you probably remember from paragraph 6.3, the analysis of the peptides selected by a TCR among the peptide library allows the identification of a substitution matrix that can be used to select all the possible epitopes of that specific TCR, from the peptide database. This last crucial step has to be performed yet, or it has been already performed, but Davis has not communicated the preliminary results during his talk. Recently new resources have been made available by Open Medicine Foundation, for this promising research to be further pursued, among other projects (R). The aim here, as already said, is to find the antigen that triggers this T cell response. As Mark Davis said, it might be an antigen from a specific pathogen (perhaps a common pathogen that comes and goes) that elicits an abnormal immune response which ends targeting some host tissue (microglia, for instance), thus leading to the kind of immune activation that has been recently reported by Mark Davis himself and others in ME/CFS (Montoya JG et al. 2017). The idea of a common pathogen triggering a pathologic immune response is not new in medicine, and rheumatic fever (RF) is an example of such a disease: RF is an autoimmune disease that attacks heart, brain and joints and is generally triggered by a streptococcal throat infection (Marijon E et al. 2012). The other possible avenue is, of course, that of an ongoing infection of some kind, that has yet to be detected. As said (see par. 6.1), CD8 T cell clonal expansion is present in both acute infections (like early Lyme disease) and autoimmune diseases (like MS) (figure 4), so we have to wait for the antigen identification if we want to understand if the CTLs activity is against a pathogen and/or against a host tissue.

peptide-library.png
Figure 11. In this picture, we can see the selection, through several rounds, of a bunch of peptides by a particular TCR from a ME patient. The selection takes place among a huge collection of peptides presented by HLA-A2 (MHC I) expressed by yeast cells. At each round the number of possible peptides is smaller.

8. Chronic Lyme does exist

It has probably been overlooked that in his talk, Mark Davis reported also very interesting data on post-treatment Lyme disease syndrome (PTLDS, also known as chronic Lyme disease). In particular, he found a marked clonal expansion in CD8 T cells of 4 PTLDS patients (about 40% of total CTLs) as reported in figure 12: consider that in this case, blue slices represent unique T cells, while all the other slices represent clones! All that has been said about CD8 clonal expansion in ME/CFS does apply in this case too: it might be the proof of an ongoing infection – perhaps the same B. burgdorferi, as suggested by several animal models (Embers ME et al. 2017), (Embers ME et al. 2012), (Hodzic E et al. 2008), (Yrjänäinen H et al. 2010) – or a coinfection (a virus?) or it could be the expression of an autoimmune reaction triggered by the initial infection. This has still to be discovered, running the complete universal immune testing, but what is already clear from figure 12 is that PTLDS is a real condition, with something really wrong going on within the immune response: chronic Lyme does exist.

ptlds-cd8.jpg
Figure 12. CD8 T cells clonal expansion in four chronic Lyme patients: there is a marked clonal expansion that stands for T cell activity against a pathogen or against host tissue.

9. Conclusions

Mark Davis and other researchers have developed a complex assay that is able to sequence TCRs from patients, cluster them into groups of TCRs that react to the same antigens, and discover the antigens that triggered that particular T cell response. This assay is a kind of universal immune testing that is theoretically able to recognize if a person (or a group of patients) presents an immune response against a pathogen or against one of his own tissues (or both). This approach has already given pilot data on an ongoing CD8 T cell activity in ME/CFS patients and in chronic Lyme patients and will hopefully identify the trigger of this immune response in the near future. Whether ME/CFS is an ongoing infection, an autoimmune disease or both, the universal immune testing might be able to tell us. This new technology is for immunology, what whole genome sequencing is for genetics, or metabolomics is for molecular diseases: it doesn’t search for a particular pathogen or a particular autoimmune disease. No, it searches for all possible infections and immune disorders, even those that have yet to be discovered.


As mentioned, the Open Medicine Foundation is funding Mark Davis’ research, among other research projects. Please consider a donation to the Open Medicine Foundation: donate.

Severely ill big data study, a first glance

Severely ill big data study, a first glance

A first glance at the results of the “severely ill big data study” that was launched two years ago and that have been presented during the Community Symposium, held in August at Stanford (pics and contents are from this video and this other one).

There is an alteration in cortisol production, with a low level early in the morning and an higher than normal level as time passes by (figure 1).

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Figure 1. There is a distruption in cortisol secretion in patients vs controls, with low cortisol early in the morning and a slight increase later on.

There is an increase in leptin and a decrease in brain-derived neurotrophic factor (BDNF) (figure 2), an increase in lysine and a decrease in indolepropionate (which is involved in tryptophan metabolism) (figure 3).

20818900_10212561169240657_639960759344760665_o-e1512989057505.jpg
Figure 2. Leptin is increased while BDNF is decreased.
20776677_10212561284203531_3801233735178140551_o
Figure 3. Indolepropionate is significantly reduced while lysine is increased in patients vs controls.

In microbiota we see an increase in Firmicutes at the expense of Bacterioides, with a huge increase in Verrucomicrobia in two patients (figure 4).

20728801_10212561307084103_1868596309719464780_o-e1512989135810.jpg
Figure 4. There is an increase in Firmicutes and a decrease in Bacteriodes in patients vs healthy controls.

If we consider gene expression in ME/CFS and compare it with what we have in other diseases, we find a strong similarity with systemic inflammatory response syndrome, and with diseases caused by parasites, gram negative bacteria, trypanosoma, lentivirus. Enchondromatosis was another disease with similar gene expression (figure 5).

20819054_10212561315564315_5018893509325920693_o
Figure 5. Gene expression of ME/CFS compared to other diseases.
20728709_10212561362525489_2553184777059756306_o
Figure 6. Cell free DNA is normal in all ME/CFS patients enrolled in this study with the exception of two of them.

Cell free DNA, which is often high in diseases with tissue damage, is normal in patients (figure 6). DNA from several viruses has not been found in blood (figure 7). HHV7 and EBV copies are found more frequently in peripheral blood from healthy individuals than from patients (figure 8).

20776631_10212561364725544_682927784255997997_o-e1512953828433.jpg
Figure 7. Several common viruses have been searched for in blood, wuth negative results.
viruses
Figure 8. Positive PCR for EBV and HHV/ is more frequent in controls than in patients.

Cytokines are high in patients vs control, and the worse the clinical picture the higher they are (figure 9).

20746378_10212561368005626_4552494947980509062_o
Figure 9. In this set of 63 cytokines, the worse the symptoms, the higher the cytokines in blood.

CMRC conference 2017, altri interventi

CMRC conference 2017, altri interventi

Spettroscopia di Raman

In questo intervento di Karl Morten (Oxford University) viene discussa la applicazione della spettroscopia di Raman nella ME. In questa tecnica si esamina il cambiamento nella frequenza della emissione luminosa che attraversa delle molecole e si risale alla natura delle molecole stesse. In particolare, qui si discute l’applicazione di questo metodo allo studio dei metaboliti contenuti nei leucociti dei pazienti ME vs controlli sani. In figura 1 il contenuto di fenilalanina nei leucociti di 5 controlli sani (a sinistra) e in quelli di 5 pazienti (a destra). Si vede una tendenza a una riduzione nel livello di fenilalanina nei pazienti.

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Figura 1. Contenuto di fenilalanina nei leucociti di 3 controlli sani (a sinistra) e di 5 pazienti (a destra).

Ricordo che nello studio di Fluge e Mella sui metaboliti nel sangue periferico (spettroscopia di massa), la fenilalanina era fra i metaboliti ridotti nei pazienti di sesso femminile (vedi qui). Quindi si avrebbe un accordo fra ciò che è stato rilevato nel sangue e ciò che si trova dentro le cellule.

Ipoperfusione cerebrale in ortostatismo

Frans Visser ha presentato uno studio del flusso sanguigno cerebrale nei pazienti ME/CFS durante il tilt table test. L’apparato sperimentale è abbastanza semplice: il flusso sanguigno cerebrale è assunto proporzionale alla portata della arteria carotidea interna e della arteria vertebrale (collo); le portate si misurano con un ecografo (figura 2).

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Figura 2. Misura del flusso sanguigno cerebrale durante il tilt table test (TTT).

Durante l’ortostatismo si ha una fisiologica riduzione del flusso sanguigno cerebrale così misurato, ma nei pazienti la riduzione è significativamente più consistente (figura 3).

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Figura 3. Il flusso sanguigno cerebrale è ridotto nei pazienti vs i controlli durante il TTT.

Un aspetto innovativo di questo studio è che ci si è premurati di fare la stessa misura anche mettendo i pazienti seduti per mezzora. Anche in questo caso la riduzione del flusso sanguigno cerebrale è più consistente nei pazienti vs i controlli (figura 4).

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Figura 4. Il flusso sanguigno cerebrale è ridotto nei pazienti vs i controlli sani anche considerando la posizione da seduti.

 

Sembra una banalità, ma in tutti gli studi sulla intolleranza ortostatica che ho letto finora si usa il tilt table test, o lo standing test e quindi la posizione seduta non è contemplata. Un anno fa ebbi la curiosità di confrontare i miei parametri cardiaci dello standing test con quelli da seduto e ho trovato che mentre nello standing test si apprezza una classica POTS, nella posizione seduta (sitting test?) si rileva una ipotensione ortostatica (se si usano i parametri diagnostici classici della ipotensione ortostatica) dopo 20-30 minuti. Non so se questo risultato sia estendibile ad altri pazienti.

Tornando alla presentazione di Visser, la conclusione è che tanto in ortostatismo che da seduti, il 71% pazienti ha una anomala riduzione del flusso sanguigno cerebrale, se confrontati con i controlli sani (figura 5). E questo potrebbe giustificare il fatto che molti pazienti semplicemente non tollerano né la posizione seduta, né l’ortostatismo e sono costretti per la maggior parte del tempo in clinostatismo. Il sangue che arriva al cervello è inadeguato e questo letteralmente li costringe a stendersi.

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Figura 5. Il 71% dei pazienti presenta un ridotto flusso cerebrale in ortostatismo, rispetto ai controlli sani.

Una cosa interessante di questo studio è che i pazienti ME non erano stati selezionati in base alla presenza di classica intolleranza ortostatica (POTS o ipot. ortos.). Quindi la riduzione del flusso sanguigno cerebrale nella ME potrebbe essere indipendente dalla positività al tilt table test.

Montoya alla CMRC conference 2017

Montoya alla CMRC conference 2017

Quello che segue è un resoconto dell’intervento di Josè Montoya durante la CMRC conference 2017, tenutasi a settembre. Il video dell’intervento si trova qui.

Montoya presenta un caso di ME associato a positività a HHV6-A (PCR) nel sangue periferico e nel liquido spinale, e a una lesione nella materia bianca nella fase iniziale della malattia, che poi è regredita. Il paziente ha risposto a una terapia di valgancyclovir e idrossiclorochina (usata come anti infiammatorio) (figura 1).

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Figura 1. Un caso di ME/CFS associata con HHV6 e con risposta a valganciclovir.

Montoya non crede che la “Lyme cronica” sia causata da una infezione cronica da Borrelia burgdorferi.

In seguito ricorda lo studio di brain imaging del 2015 in cui si era riscontrato un ispessimento della parte anteriore del fascicolo arcuato e del fascicolo inferiore longitudinale destro (figura 2). Montoya osserva che il fascicolo arcuato anteriore non è presente negli altri primati e quindi deve giocare qualche ruolo nelle funzioni cognitive superiori della nostra specie. In effetti le due strutture citate collegano l’area temporo-parietale della corteccia con la corteccia frontale, ovvero quelle strutture anatomiche dove risiede – direi – ciò che siamo, il tessuto che ci rende umani. Contestualmente si ha una perdita di volume della materia bianca.

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Figura 2. Evidenziate le strutture anatomiche in cui si è riscontrata una differenza morfologica nei pazienti vs i controlli.

Montoya confronta il loro studio del 2015 con quello giapponese sulla attivazione della microglia (studio PET del 2014) in cui la maggiore area di infiammazione era la parte sinistra della base del cervello. Perché l’infiammazione dovrebbe essere a sinistra e la anomalia anatomica invece si trova a destra? Montoya sostiene – citando uno studio sulla epilessia – che l’ispessimento dei fascicoli a destra sia una compensazione della infiammazione a sinistra.

Del recente studio sulle citochine, Montoya evidenzia la proporzionalità diretta tra il livello di 17 citochine (tra cui 13 pro-infiammatorie) e il grado di severità dei pazienti. Queste citochine sono associate tanto alla immunità innata che a quella adattiva. Montoya riporta l’elevazione del TGF beta, già riscontrata in alcuni studi precedenti. Il TGF beta è elevato nel linfoma, e sappiamo che la ME è un fattore di rischio per il Linfoma non-Hodgkin, dunque Montoya si chiede se ci sia un legame fra questi dati. Montoya sorvola sul fatto che altri studi non abbiano riscontrato un aumento del TGF beta e che lo studio di Hornig M. del 2015 ha addirittura trovato che il TGF beta è ridotto nei pazienti di lunga durata. Il TGF beta è elevato anche nella sindrome di Ehlers-Danlos, che sembra avere in alcuni casi una presentazione simile alla CFS, e – aggiungo io – nella sindrome di Marfan, che è una malattia affine alla Ehlers-Danlos. Montoya descrive come il suo gruppo abbia trovato un difetto (unspecific binding) del test per le citochine, e come sia stato considerato nella elaborazione statistica dei dati.

Così la ME si profilerebbe come una malattia infiammatoria, e a prescindere da cosa la causi, dovrebbe essere possibile trattarla, come in effetti si fa con l’artrite reumatoide o la sclerosi multipla. Per questo confida nel fatto che si possano iniziare trial con farmaci anti-infiammatori nella ME.

Hanno trovato profili HLA più comuni nei malati che nei controlli sani. E tra i malati, alcuni profili HLA sembrano più comuni nelle persone con gravità maggiore. Dati non pubblicati.

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Figura 3. I sintomi che permettono a Montoya di scegliere i pazienti ME/CFS candidati per una terapia antivirale.

Cosa fa Montoya quando un paziente arriva al suo ambulatorio? Misura sierologia e PCR per virus erpetici, fa una ricerca per Coxiella burnetii (febbre Q), Borrelia burgdorferi (malattia di Lyme), Babesia, epatiti e HIV. Funzione delle NK (figura 3). I pazienti positivi per Coxiella burnetii vengono trattati da Montoya con doxiciclina e idrossiclorochina e hanno miglioramenti sostanziali. Qui ci tengo ad osservare che la Coxiella burnetii – da quanto ho potuto verificare con altri pazienti negli ultimi 3 anni – è poco considerata in Italia e non viene cercata nei malati, sebbene sia presente nelle zecche italiane. C. burnetii può essere trasmessa anche dal consumo di latte non pastorizzato (per ulteriori informazioni leggi questo post).

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Figura 4. Alcuni pazienti con sintomi CFS presentano una perdita di liquido spinale che può essere trattata con sollievo dei sintomi.

Alcuni pazienti hanno perdite di liquido spinale (figura 4), quindi non hanno la CFS, ma hanno sintomi analoghi. Questi pazienti hanno mal di testa che peggiora quando sono in ortostatismo e intolleranza ortostatica. Possono essere trattati con un intervento chirurgico (blood patch). La diagnosi viene fatta con il CT myelogram (una mielgramma tomografico computerizzato?). Questi pazienti hanno spesso un fenotipo riconducibile alla Ehlers-Danlos di tipo ipermobile.

Il trattamento si avvale anche di antivirali (figura 5), immunomodulanti, anti-infiammatori, supplementi di vario tipo e una gestione oculata delle energie, per prevenire la PEM. Il trattamento antivirale è mirato soprattutto ai virus erpetici, e Montoya cita i sui studi sul valgancyclovir. Si tratta di trattamenti molto lunghi. Trattamenti brevi non fuzionano.

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Figura 5. Gli antivirali usati da Montoya in quei pazienti ME/CFS in cui sospetta che la malattia sia sottesa da un processo infettivo di natura virale.

Montoya parla anche di HHV6 integrato nei cromosomi, che risponde bene agli antivirali, e di herpes genitale e presenta un caso di una donna con HHV2 con manifestazione cutanea su una natica (insolita). Pazienti con ME e evidenza di attività di HHV2, se trattati per 2-5 anni, tendono a guarire. Se si trova evidenza della attività di un qualunque virus erpetico (da HHV1 a HHV8) o anche il virus della varicella zoster, Montoya suggerisce un trattamento anti-virale. I trattamenti antivirali proposti per i virus erpetici sono: PO acyclovir, PO famciclovir, PO valacyclovir, PO valgancyclovir. Non vengono usati IV foscarnet e IV cidofavir per via della tossicità. (PO: per os, cioè per bocca dal latino os, oris; IV: per via endovenosa). Il trattamento anti-infiammatorio di scelta è la idrossiclorochina (Plaquenil). Montoya è ancora restio ad usare farmaci immunomodulanti più impegnativi, come il Rituximab, a meno che non lo si faccia nell’ambito di trial clinici ben progettati.

Montoya ritiene che la scoperta della causa eziologica della ME porterà a un cambiamento per tutte le malattie infiammatorie e autoimmuni. Da quello che posso capire su questo punto, integrando anche con la recente intervista a Montoya fatta da Charles Ortleb, Montoya ritiene che alla base della ME e di altre malattie ‘simili’, come artrite reumatoide, lupus e sclerosi multipla, ci sia un processo infettivo (o pseudo-infettivo, vedi oltre). Questo non toglie che il trattamento possa legittimamente essere un farmaco immunosoppressivo o anti-infiammatorio, perché ad oggi non abbiamo di meglio. Questa idea di Montoya secondo me viene in parte dal fatto che lui sia infettivologo (e dunque ha un bias infettivologico, come si dice) e anche perché è obiettivamente difficile pensare che il sistema immunitario concerti una risposta infiammatoria ”a vuoto”. Ma questo punto è controverso, ed è tuttavia un nodo centrale, secondo me, nella moderna reumatologia e immunologia.

Ciò che sostiene la malattia – conclude Montoya – potrebbe essere la riattivazione di una infezione latente intracellulare (ad esempio un virus erpetico), oppure un agente non-patogeno, di cui non dice nulla, ma attingendo ancora una volta alla intervista di Charles Ortleb, credo si riferisca a un retrovirus endogeno, cioè parte del nostro DNA da migliaia di generazioni. Dunque un non-patogeno, appunto.

Una nota personale. Confrontando l’esposizione di un medico come Montoya e di scienziati provenienti da altre discipline, sul medesimo argomento, trovo che Montoya si affida forse troppo a credenze non provate e a casi aneddotici, come se volesse raccontare una storia di cui conosce già il finale. Mi trovo a disagio oggi davanti a questo tipo di atteggiamento, mentre in passato forse lo trovavo confortante. I pazienti non hanno bisogno di essere confortati, non quanto hanno bisogno della verità. E in questo senso Montoya desta qualche sospetto, se paragonato ai biochimici, gli informatici e alle altre professionalità intervenute ad esempio nel simposio di Stanford.