Sunday, 26 February 2017

ACTRIMS 2017 further points to Memory B cells

This week ACTRIMS has been going. The academics have been microbioming and even got salt in the act as salt affects gut bacteria, but in terms of pharma news it was the reports of the phase III new sphingosine-1-phposhate modulator that is going to make the next new drug on the block.

However, a couple of weeks ago we suggested that drugs that inhibit MS, target memory B cells.

The data published with dimethyl fumarate was not so great, but it is nice to see data replicated and so more evidence to support the idea comes from ACTRIMS. The effect on B cells was not dependent on whether DMF was associated with leucopaenia and not related to antibody levels. However we know leucopaenia puts you at extra risk from infection.

Dimethyl fumarate effects on circulating B-cell phenotype
Erin E. Longbrake, Claudia Cantoni, Francesca Cignarella, Anne H. Cross MD, & Laura Piccio. ACTRIMS 2017.

Background: The efficacy of anti-CD20 monoclonal antibodies in clinical trials of relapsing multiple sclerosis (MS) confirmed that B-cells play an important role. Little is known about how dimethyl fumarate (DMF) affects B-cells. Objectives: To determine how the composition and function of circulating B-cell populations is affected by exposure to DMF.

Methods: Peripheral blood mononuclear cells (PBMC) and serum were collected from MS patients stably treated with DMF, patients on no immunomodulatory therapy and healthy volunteers. B-cell subpopulations were analyzed by flow cytometry.

Results: The proportions of circulating class-switched memory B-cells (CD20+ IgD- CD27+), non-class-switched memory B-cells (CD20+, IgD+ CD27+) and double negative memory B-cells (CD20+ IgD- CD27-) were significantly decreased among patients taking DMF relative to healthy and untreated MS controls. Concurrently, there was an increase in the proportion of circulating naïve B-cells (CD20+ IgD+ CD27-). Expression of the activation marker CD80 was also reduced on circulating B-cells from DMF-treated patients. Quantitative serum immunoglobulins did not change with DMF treatment.

Conclusions: DMF reduced the expression of circulating memory B-cells. A similar pattern has been observed among other disease modifying therapies, and this may underlie some of the drugs’ efficacy against relapsing MS. More study is needed to determine whether changes in B-cell phenotypic markers are paralleled by changes in B-cell function

Animals in Research

Why do we talk about Animal Research on the Blog?
Signatories (including Queen Mary and 111 other signatories) to the Concordat of animals in research (Click here) have agreed to be more open about their use of animals in research, and to abide by the following four commitments.
Commitment 1: We will be clear about when, how and why we use animals in research

Commitment 2: We will enhance our communications with the media and the public about our research using animals

Commitment 3: We will be proactive in providing opportunities for the public to find out about research using animals

Commitment 4: We will report on progress annually and share our experiences
All signatories agree that they will work to fulfil the four Commitments, initiating projects and strategies that are relevant and appropriate to their organisations to be more open about their use of animals in research. The signatories will be asked to report on their progress in taking these steps

Saturday, 25 February 2017

#ClinicSpeak & #ResearchSpeak: chubby, female and young are not a good mix

Can you reduce the chances of your children getting MS? #ClinicSpeak #ResearchSpeak #MSBlog

Obesity, in particular adolescent obesity, is a risk factor for developing MS. The study below shows that this risk, not surprisingly, extends into childhood or paediatric MS. 

Is obesity simply associated with MS due to another factor or does obesity act in the MS causal pathway? An example of an association would be that its actually low vD levels, or lack of outdoor activity and less sun exposure, that is the causal factor. People who have less outdoor activity tend to be more sedentary and hence more likely to be obese. The risk factor here is less outdoor activity and not the associated obesity. 

Obesity could be causal if some part of adipose tissue biology interacts with the MS causal pathway. An example of this is could be one of pro-inflammatory mediators that adipose tissue produces, and there are many such mediators, prime the immune system to develop autoimmunity. In other words if there was less adipose tissue, and as a result less adipose tissue induced systemic inflammation, then the risk of autoimmunity will drop.

Another way adipose tissue may interfere with the causal pathway is actually via vD metabolism. Adipose tissue may lower systemic vD levels by consuming vD as part of its metabolism. The low vitamin D level, and not the obesity, that is the risk factor here.

The only way to answer the association vs. causation question is to do a randomised controlled trial of a dietary, or pharmacological intervention, which reverses or prevents adolescent obesity, and to see if the intervention reduces the risk of developing MS. This type of trial would be very difficult to do and in my opinion is not feasible now. It may become feasible when we have a pharmaceutical that effectively treats obesity; with drugs it is easier to do randomised controlled trials. With lifestyle interventions, particularly weight loss and obesity, this is even more difficult when the outcome needs to be looked at a population level.

A cheaper, and cleverer, way to do randomised-controlled trial to prove causation is to use the Mendelian randomization method and to see if the genetic variants that are linked to obesity are risk factors for developing MS. 

An earlier study using people from California registered with the Kaiser Permanente HMO and a replication sample from Sweden showed just this. The investigators constructed a weighted genetic risk score using genetic variants previously established to predict obesity. Subjects with higher genetically-induced obesity scores had a higher risk of developing MS. Although the investigators controlled for birth year, sex, education, smoking status, ancestry, and genetic predictors of MS they clearly couldn't control for other important con-founders that are very relevant to this analysis, for example dietary factors, exercise - in particular out-door activity - and vD levels. Despite this that study suggested that obesity is probably part of the MS causal pathway and that if we want to reduce the incidence of MS in the population we need to tackle the problem of adolescent obesity. 

Tackling adolescent obesity is much easier said than done! However, if you have MS and you children are already at higher risk of developing MS you may want to think about this more seriously. 

In high prevalence areas such as Western Europe the lifetime risk of a woman developing MS is ~ 1 in 500 (background population risk). If you are a woman with MS and have children you need o be aware that the chance of your daughter(s) getting the disease is ~ 1 in 40 and your son(s) ~1 in 80. Would you want to do everything you can to lower that risk? 

Chitnis et al. Distinct effects of obesity and puberty on risk and age at onset of pediatric MS. Ann Clin Transl Neurol. 2016 Nov 4;3(12):897-907.

OBJECTIVE: The aim of this study was to examine the relative contributions of body mass index (BMI) and pubertal measures for risk and age of onset of pediatric MS.

METHODS: Case-control study of 254 (63% female) MS cases (onset<18 years of age) and 420 (49% female) controls conducted at 14 U.S. Pediatric MS Centers. Sex- and age-stratified BMI percentiles were calculated using CDC growth charts from height and weight measured at enrollment for controls, and within 1 year of onset for MS cases. Sex-stratified associations between MS risk and age at symptom onset with both BMI and pubertal factors were estimated controlling for race and ethnicity.

RESULTS: Only 11% of girls and 15% of boys were prepubertal (Tanner stage I) at MS onset. 80% of girls had onset of MS after menarche. BMI percentiles were higher in MS cases versus controls (girls: P < 0.001; boys: P = 0.018). BMI was associated with odds of MS in multivariate models in postpubertal girls (OR = 1.60, 95% confidence interval [CI]: 1.12, 2.27, P = 0.009) and boys (OR = 1.43, 95% CI: 1.08, 1.88, P = 0.011). In girls with MS onset after menarche, higher BMI was associated with younger age at first symptoms (P = 0.031). Younger menarche was associated with stronger effects of BMI through mediation and interaction analysis. In pubertal/postpubertal boys, 89% of whom were obese/overweight, earlier sexual maturity was associated with earlier onset of MS (P < 0.001).

INTERPRETATION: Higher BMI in early adolescence is a risk factor for MS in girls and boys. Earlier age at sexual maturity contributes to earlier age at MS onset, particularly in association with obesity.

Secondary Progressive EAE. Is it the astrocyte?

Some body asked me to comment on this paper.

Rothhammer V, Kenison JE, Tjon E, Takenaka MC, de Lima KA, Borucki DM, Chao CC, Wilz A, Blain M, Healy L, Antel J, Quintana FJ. Sphingosine 1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS inflammation. Proc Natl Acad Sci U S A. 2017;114(8):2012-2017.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the CNS that causes disability in young adults as a result of the irreversible accumulation of neurological deficits. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, these therapies show limited efficacy in secondary progressive MS (SPMS). Thus, there is an unmet clinical need for the identification of disease mechanisms and potential therapeutic approaches for SPMS. Here, we show that the sphingosine 1-phosphate receptor (S1PR) modulator fingolimod (FTY720) ameliorated chronic progressive experimental autoimmune encephalomyelitis in nonobese diabetic mice, an experimental model that resembles several aspects of SPMS, including neurodegeneration and disease progression driven by the innate immune response in the CNS. Indeed, S1PR modulation by FTY720 in murine and human astrocytes suppressed neurodegeneration-promoting mechanisms mediated by astrocytes, microglia, and CNS-infiltrating proinflammatory monocytes. Genome-wide studies showed that FTY720 suppresses transcriptional programs associated with the promotion of disease progression by astrocytes. The study of the molecular mechanisms controlling these transcriptional modules may open new avenues for the development of therapeutic strategies for progressive MS.

I said that I was reluctant to do this.....The human data is out there for every one to see.

However, it did reach the news and so here is my take, which you asked for.

I've been told I have been abit grumpy. Maybe I should enthuse more, but research is about questioning

If you are not interested in understanding science papers and their problems, then this is not for you.

If you think every thing in science is clear cut then read on... as we make black and white become grey.
For science readers it is food for thought.

In the above paper they say "Following immunization......NOD (non-obese diabetic) mice initially develop an acute neurological event, which is followed by a chronic progressive phase that starts at approximately day 25"
When they looked in the CNS of animals treated for ages with fingolimod they found reduced activation of the innate immune system 

Then the study looks at the effect of the drug on isolated astrocytes and importantly human astrocytes and finds a number of pro-inflammatory genes down regulated, some are upregulated like IL-10 and neurotoxicity goes down, through blockade of one of the central transcription factors controlling inflammatory signalling. 
So they suggest that fingolimod is going to protect nerves and fingolimod will stop secondary progressive MS. 

It is however pointed out that fingolimod actually failed to affect primary progressive MS, but there was some effect in secondary progressive MS with siponimod. This study sponsored by Novartis, makes it look promising. Maybe Siponimod works via blocking astrocytes

However, whilst the message in the paper is crystal clear, the supporting EAE is perhaps not quite as clear cut as it seems.

We have a read round the subject because if this study highlights a quick way to find treatments for progressive MS, we need to be doing this.

However, NOD mice develop diabetes and so are classed as a harmful mutants, creating more problems to use them. Generally however the adjuvants used to cause EAE, block NOD mice from becoming diabetic. Anyway back to the study.

First thing to be said, in contrast to the suggestion tin the paper, there were studies looking at chronic EAE, years before this current report.

It was not mentioned...wonder why I get grumpy:-)

Al-Izki S, Pryce G, Jackson SJ, Giovannoni G, Baker D. Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmune encephalomyelitis. Mult Scler. 2011;17(8):939-48

In a similar experiment in ABH mice treated with fingolimod, there was a marked effect on disease with treated animals. So the results look the similar but perhaps the treatment effect was much more marked.

There was less nerve damage and less myelin loss, however              the interpretation was somewhat different. 

It was not that this was about astrocytes, they were not looked at, but it was that fingolimod was blocking relapses.

This is because the model in relapsing progressive, where relapses occur but animals recover with increasing deficit. The relapses burn out after 3-4 attacks and you get a very slow worsening of disease (secondary progression starting months after disease onset) that does not respond to T cell inhibition and incidentally did not respond to fingolimod treatment, so suggesting that fingolimod may fail in progressive MS, which it was subsequently shown to do.

ProfG has mentioned the importance of "Thinkhand" as a treatment option and has reported that ocrelizumab and natalizumab have protected hand function in the none hole peg test. However the trials with S1P1 modulators have not reported effects on hand function. Why not?

It must tell us something but what?

Anyway in the first study (above) the beasties were treated from day 40 and "FTY720 ameliorated progressive NOD EAE without significant effects on the peripheral T-cell response".  So we have a difference in World View.

However, this was measured by monitoring T cell responses from the spleen. However, as the response of the drug is to keep cells in lymphoid organs, would we be expecting a decrease in T cell function..maybe if we looked at the blood there may had been an effect. However the inference is clear this is workig via blockade of astrocytes and not T cells  

However, we need to look at the history of the generation of the progressive model.

We studied EAE in NOD mice many years ago, where it was clear that this was a low-EAE susceptibly strain
Genetic analysis of experimental allergic encephalomyelitis in mice. Baker D, Rosenwasser OA, O'Neill JK, Turk JL. J Immunol. 1995;155(8):4046-51.

We also showed that the mice would respond to MOG.

Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice. Amor S, Groome N, Linington C, Morris MM, Dornmair K, Gardinier MV, Matthieu JM, Baker D. J Immunol. 1994;153(10):4349-56.

The NOD mice and ABH mice share a transplantation antigen called H-2Ag7 that is used to recognise the myelin oligodendrocyte glycoprotein. The MOG 35-55 epitope is a minor disease causing epitope. Similar it may not be the major target in C57BL/6 mice (type of mouse) either.

Delarasse C, Smith P, Baker D, Amor S.Novel pathogenic epitopes of myelin oligodendrocyte glycoprotein induce experimental autoimmune encephalomyelitis in C57BL/6 mice. Immunology. 2013;140(4):456-64.

Yes I know I'm just gettin gthe altmetrics up:-)

Anyway back to NOD mouse EAE and the suggestion that is a progressive EAE model.

Basso AS, Frenkel D, Quintana FJ, Costa-Pinto FA, Petrovic-Stojkovic S, Puckett L, Monsonego A, Bar-Shir A, Engel Y, Gozin M, Weiner HL. Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis. J Clin Invest. 2008;118:1532-4
They said when 10-week-old NOD mice were immunized with MOG35–55 ..., the first signs of disease appeared at days 11–12 (peaking at days 16–18), and after partial recovery from this initial acute attack, there was a progressively worsening relapse without full remission. This was then followed by a secondary progressive course characterized by chronic clinical impairment. 

The implication is that the worsening occurs progressively from day 25 onwards.

This was also reported in another study. But in this case, disease was treated with myelin (MOG) to block the T cell immune response.

Levy Barazany H, Barazany D, Puckett L, Blanga-Kanfi S, Borenstein-Auerbach N, Yang K, Peron JP, Weiner HL, Frenkel D. Brain MRI of nasal MOG therapeutic effect in relapsing-progressive EAE. Exp Neurol. 2014;255:63-70.

However, remember secondary progressive MS doesn't respond too well to immunosuppression. So if this works in the secondary progressive model then it perhaps lacks predicitve value to that occuring after treatment of MS.

Now let's look at the line graph. Here's one of ours (below)
An attack and then it goes progressive?

Actually it does, but not at day 25 as the line graph suggests.

The above is all caused by relapsing-remitting disease driven by T cells. The relapses show increasingly poor recovery and so deficits accumulate with time. They are also asynchronous so they are not occurring at the same time and if you only look at the "line" you think the disease is secondary progressive. It is not.

We seldom get to see the individual data. I have repeated said people should publish the number of animals that get disease, the maximal severity of disease and timing of onset so it may allow you interpret these lines.

However, there appears to be one example, when the same people reporting the secondary progressive model, report in another paper and show individual scores. It suggests that EAE in NOD mice, is if fact not really an instant secondary progressive model starting at day 25 but it is in fact a relapsing-remitting disease with poor recovery, like the ABH mouse. This accumulates deficits related to relapsing attacks.

This is not a slow insidious progressive disease as implicated by looking at the line,which is an average of a number of animals

Levy H, Assaf Y, Frenkel D. Characterization of brain lesions in a mouse model of progressive multiple sclerosis.Exp Neurol. 2010 Nov;226(1):148-58.

So if disease is really relapsing in NOD mice it will respond to FTY720 by their suppression, causing less demyelination, nerve loss and cytokine X or Y and less astrocyte activation. 

What was found?

Rothhammer V, Kenison JE, Tjon E, Takenaka MC, de Lima KA, Borucki DM, Chao CC, Wilz A, Blain M, Healy L, Antel J, Quintana FJ. Sphingosine 1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS inflammation. Proc Natl Acad Sci U S A. 2017;114(8):2012-2017.

Why is this important because, if it is a true secondary progressive model with validity it would not respond to peripheral immunosuppression very well as occurs in MS and maybe we can use this (taking a couple of months) rather than our current model (Taking 6 months) to test agents.

You can make your own conclusions,

If we had the raw data we could see in an instant what is really going on.

If we did, there would be time to eat humble pie or is this a case of MD-investigative reporter?

We have been talking about animal work this week. We had the suggestion of multi-centre animal studies. However, for EAE research raw data files would be the type of data that would be worth more for drug development than consortia doing multi-centre studies.
Could blocking astrocytes result in blockade of progressive EAE and MS ...absolutely.

P.S. Dear "Headmaster" hope this style of walking you through data is informative..rather than giving a sound bite. This will be important when we do the clinical posts soon.

Friday, 24 February 2017

Dodgy EAE experiments means more wasted animals

Yesterday we say that there was a desire to do multi centre animal studies.

We said if you do rubbishy animal experiments, you get rubbish.
A well planned and undertaken experiment can mean more than ten rubbish experiments.

The funny thing is that the suggestion of mutli-centre animal trails was published in the Journal Nature .

This is a bit rich, because whilst we want to get our papers published, this it is also one of the homes of unreproducible pop science. 

A while ago it was suggested that salt intake makes autoimmunity worse. This got one of the biggest altmetrics on the plantet. It was covered by loads of media and every one lapped it up.

MD2 said hang on what are they saying and worked out the doses of animals and humans and the conclusion was what a load of.......

To MD2's credit they posted a comment on the Nature website that you should take it with a pinch. Nature removed the comment twice and MD2 complained and on the third time it is still there. 

The authors were livid. How dare MD2 question this?

Because it sounded dodgey?

However dissecting the science and looking at the dose of salt used to induce the issues you are looking at the dose equivalent to half a kilo of salt a day.

This will kill the average human and make people vomit. About 8g makes you puke. However, mice & rats can't vomit and so what was the equivalent 600g must have felt like I don't know.

So you get a paper in Nature and it spawns another ten or so experiments.

So something that you should take with a pinch.. means you are wasting loads of animals.

So what happens in EAE? 

It has been repeated and the original study was shown partially reproduced and shown to be weak. Here we have another paper in a different disease, we will have loads more.

Read the abstract if you want

High salt intake does not exacerbate murine autoimmune thyroiditis. Kolypetri P, Randell E, Van Vliet BN, Carayanniotis G.
Clin Exp Immunol. 2014;176(3):336-40.