Wednesday, 10 February 2016

ThinkSpeak: do we really need a wrecking ball to crack a nut?

Wrecking-ball or molecular tweezers; choose your poison. #ThinkSpeak #MSBlog #MSResearch

ThinkSpeak = thinking aloud

"I have said many time before the autoimmune hypothesis of multiple sclerosis may be wrong. This is why I refer to black swans, field hypotheses and viral causes of MS. I am not alone in the world when it comes to taking an alternative view of MS. Many of you will argue that I am wrong and that this position, or hypothesis, is incongruent with the effectiveness of induction treatment strategies such as HSCT, alemtuzumab, cladribine/fludarabine, etc. When you look at all the highly-effective therapies common to them all is an anti-B-cell effect, except for daclizumab which is an outlier. In other words HSCT may be simply be working via its effect on B-cells. If this is the case then the T-cell depletion, general bone marrow suppression and other off-target toxicities are simply collateral damage. Do we really need a wrecking-ball to crack a nut? Likewise, if alemtuzumab is working via B-cell depletion do we really need to collateral damage that comes with alemtuzumab's depletion and immunological changes that come from  its specific immune system reconstitution pattern (secondary autoimmunity). Do we really need a sledgehammer to crack a nut? Cladribine and fludarabine may be safer to use than either HSCT or alemtuzumab, but we would still be using a hammer to crack a nut."

"With a short-term efficacy profile that is looking very good anti-CD20 therapies must be the way to go; they work like heat-seeking missiles to take-out their target and the collateral damage from depleting B-cells seems relatively contained. This view may change with longer follow-up. What population of B cell is anti-CD20 therapies targeting? Could it be the EBV infected pool? If this is the case then we need better drugs; drugs that target EBV hence the need for the Charcot Project. More importantly, are anti-CD20 therapies enough? I suspect not. Stephen Hauser dropped a bomb on the stage at ECTRIMS when he mentioned that many of their patients with active RRMS treated on rituximab for many years have gone onto to develop secondary progressive MS. Why? It may be that anti-CD20 therapies do not target long-lived plasma cells within the central nervous system and these plasma cells, which produce OCBs may be driving progressive disease. This is why I am so disappointed that MedImmune have put their anti-CD19 MS programme on ice; CD19 is at least expressed on plasma cells and anti-CD19 may be one way of targeting this population of long-lived plasma cells."

"People are often surprised  that I am so interested in daclizumab as a treatment for MS. Why? Daclizumab is one highly-effective DMT  that does not have an obvious upstream impact on B-cell biology. If I am right about the B-cell depletion hypothesis then how is daclizumab working in MS? If we can work out daclizumab's exact mode of action in MS we will be much further along the path of finding out the cause of MS. At present we know that daclizumab as a monotherapy seems not to be immunosuppressive in the classic sense and appears to working as IL-2 modulator. By directing IL-2 away from its high-affinity receptor to its intermediate affinity receptor it expands the population of CD56-bright NK cells. The expansion of this population of cells correlates with daclizumab's efficacy. The function of CD56-bright NK cells is complex; they are part of the innate immune response and have regulatory functions in relation to activated effector T-cells. The latter fits with the autoimmune dogma. Their other role is anti-viral. Could CD56-bright NK cells be controlling the virus that causes MS? I therefore can't wait to study the impact of daclizumab on HERV and EBV biology in pwMS. This is why I am hoping the FDA and EMA, and NICE,  give daclizumab a greenlight. Not only will it provide people with MS another option to treat their MS, but it will allow us to test the daclizumab antiviral hypothesis."

"I am sure many readers will disagree with me on many of the points above. If you do lets have a conversation or debate about the issues. I really don't believe we need wrecking balls, sledgehammers, hammers or heat-seeking missiles to treat MS. I would prefer a precision tool; a set of molecular tweezers that cause no collateral damage."

CoI: multiple

How to Read an EAE paper

Gibson-Corley KN, Boyden AW, Leidinger MR, Lambertz AM, Ofori-Amanfo G, Naumann PW, Goeken JA, Karandikar NJ. A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis. PeerJ. 2016 ;4:e1600. eCollection 2016.

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions
Recently when we wrote a grant and we said that we would assess neurodegeneration using a quantitative biochemical method , which we have validatd it against conventional histology, which assesses the total nerve content within the nervous system using a technology, where you can test hundreds of samples in one day.
However we were chastised and told to go away and come back with a proposal that used histology to show nerve lost!

However, I said this was not properly qualitative because with histology you would usually only count nerves in a few places and not the whole nerve content in a spinal cord section and maybe only look in one area in the whole of the spinal cord and maybe only give is a +, ++ scoring which is not really quantitative. Furthermore you can select areas to show what you like. Next it may take a few weeks to process the tissues, section then and stain them. 

However, there was a blinkered response and you just have to waist your time and do it to keep the i dotters and t crossers happy.

So this paper needs to be read by these people and if you read animal study papers then you need to think about this also because you can show what you like withhistology by being selective. What you need to do is to show pictures that are representative.

How many times have you seen a picture of drug X showing no infiltrates compared to the controls that are jam packed with infiltrates yet when we look at the clinical scores, the drug treatment only drops the severity of disease by a small fraction. 

If there is clinical disease then you must expect to see infiltrates. If there are none and there is clinical disease the pictures are selective and not representative.

So in this paper they get the whole cord processed within the spinal column. They treat the bone to make it soft, they then section and stain the spinal cord.

So here you see if you look for infiltrates (clusters of the blue dots, which are the nuclei of cells and are circled) you can see in some areas there are none and in other areas there is alot. So if you cut at B there is nothing and cut at C there is alot, yet they ar occuring at the same time. Likewise if you looked at C maybe a week or two later the inflitrates may have gone as the clinical signs abate and so waiting until the end of the experiment may give you meaningless results.