Thursday, 8 December 2016

#ResearchSpeak: treating PML in the context of persistent lymphopaenia

PML may not be an intractable problem; could immunotherapy be the solution? #ResearchSpeak #MSBlog

At present we don't have an anti-viral agent that works against the JC virus that causes PML (progressive multifocal leukoencephalopathy). Therefore we have to rely on your own immune system to fight and clear the virus if you develop PML. This is why we wash out natalizumab with plasma exchange, or stop the immunosuppressive, when somebody develops PML. A problem arises when we can't reconsititute CNS immunosurveillance or your immune system. The latter can happen with PIRTs (pulsed immune reconstitution treatments) or in people with persistent lymphopaenia. This is where immunotherapies are needed. 

One strategy is to give unfortunate people with PML in this situtation donor anti-JCV lymphocytes that are matched to their HLA (human leukocye antigens) to fight the infection. In short this is a immune transplant, i.e. giving them donor-matched T-lymphocytes to fight JCV. The study below presented at last year's American Society of Haematology (ASH) meeting tested this approach in two patients. They used T-cells that were designed to attack BK virus that cross-reacts with JCV. In both these patients the approach worked. 

Logistics of this kind of therapy make it difficult. I advised lymphocyte donation a few year's ago in patient with lupus with persistent lymphopaenia who had developed PML. By the time her team had mobilised an HLA-matched bone marrow donor who was JCV-positive the patient was in extremis and died before she could receive the donor lymphocytes. The logistics of immunotherapy for PML are not insignificant and ideally need to be established before hand so as not to delay treatment. In other words we need to screen potential lymphocyte donors beforehand so that we can call them in at short notice to harvest their lymphocytes. The other option is to rely on Pharma to create a bank of HLA-specific cytotoxic T-lymphocytes that are frozen and cane be mobilised within 24-48 hours as a licensed treatment for PML. The cost of the latter will probably be in the $100,000s compared to the former that will only cost £1,000s and can be bolted onto a bone marrow donation register. The latter can be funded by charities and former will need large investment from Pharma and regulatory approval. There is clearly an unmet need in the management and treatment of PML; we probably need to explore both approaches.

If I had more energy and time I would try and set this up. London would be a good place to do this as we have the largest bone marrow donor register in the UK and the highest population density in the UK. Any volunteers? 

Muftuoglu et al. Use of Expanded Allogeneic Third Party BK Virus Specific Cytotoxic T Cells to Target Progressive Multifocal Leukoencephalopathy. ASH Session: 703. Adoptive Immunotherapy: Poster II. Sunday, December 4, 2016,

Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal demyelinating disorder of central nervous system caused by JC virus reactivation in patients with severe defects of cellular immunity. JC virus is genetically similar to BK virus. Both JC and BK virus express large tumor antigen (LT), small tumor antigen (ST), and the capsid proteins, VP1, VP2 and VP3 during replication. Due to antigenic epitope homology, ex vivo expanded BK virus specific T cells can also target JCV, especially for VP1 and LT. We developed a rapid, effective and GMP-compliant BK virus specific cytotoxic T lymphocyte (CTL) expansion method from peripheral blood mononuclear cells. Donor mononuclear cells were stimulated with a BK virus peptide mix in the presence of IL-2, IL-7 and IL-15 for 14 days. At the end of culture, the cells were harvested and cryopreserved until use.

Here, we report the first two cases of PML treated with BK virus specific CTL from the most closely HLA-matched donor.

Case 1. A 32-year-old female with a diagnosis of FLT3+ acute myeloid leukemia underwent double cord blood transplantation. Her post-transplant course was complicated by acute graft versus host disease involving skin and gastrointestinal tract, HHV-6 infection and BK virus related hemorrhagic cystitis. Twenty months after transplantation she presented with left-sided extremity weakness, slurred speech and mental confusion. The physical examination revealed ataxic gait and weakness of left lower extremity. MRI revealed abnormal pattern of parenchymal enhancement and signal abnormality in the posterior fossa, predominantly involving cerebellum and brain stem. Lumbar puncture revealed low levels of JC virus DNA (130 copies/ml). Repeat MRI three weeks later showed progression in the peduncle and right cerebellum with an increase in the CSF JC virus load to 700 copies/ml. At this point the patient received 105/kg BKV-specific CTLs expanded from a 3/6 HLA-matched allogeneic donor. There was no infusion-related toxicity. Two weeks later, there was a significant reduction in the JC virus titer to 78 copies/ml (limit of detection 72 copies/mL). HLA BW6+ donor CD4+ and CD8+ T-cells could be detected in the CSF 2 weeks after infusion, confirming that BK virus CTLs can home to inflammatory sites in the central nervous system. The lymphocytes in CSF showed a distinct phenotypic profile, mainly composed of recipient CD56bright NK cells and a mixture of donor and recipient CD4+ and CD8+ T cells. Donor T cells in CSF expressed very high levels of PD1 and CXCR3 compared to peripheral blood T-cells. Four weeks from the infusion, the neurological symptoms have resolved and repeat MRI confirmed near complete resolution of the lesions. The patient received a second CTL infusion 3 weeks after the first for persistent low positive JCV in the CSF. The patient remains essentially asymptomatic 4 months after BK virus specific CTL infusion with intermitted low positive JC virus DNA titers in the CSF. Donor T cells continue to be present in the CSF.

Case 2. A 73-year-old female with JAK2 positive myeloproliferative disorder on treatment with ruxolitinib for 8 years presented with altered mental state, blurred vision and unsteady gait. MRI revealed parieto-occipital subcortical signals in left hemisphere extending to the posterior temporal lobe suggestive of PML. JC virus load in the CSF was 230,000 copies/ml. She received a 2/6 HLA-matched allogeneic BK virus specific CTLs. Three weeks after infusion JC virus titer in the CSF decreased to 5,200 copies/ml and her condition stabilized. The MRI remained stable. Donor-derived CD4+ and CD8+ T cells were detected in the CSF, with high expression of PD1 and inflammatory chemokines on T cells. The patient received a second dose of BK virus specific CTL from the same donor one month after the first infusion. The CSF viral load reduced further to 800 copies/ml but the there was no clinical or radiological improvement, suggestive of irreversible CNS damage. The patient remains alive.

In this proof-of-principle study, we have used ex vivo expanded BK virus specific CTL to target PML, a disease without viable treatment strategies and with a universally fatal outcome. Both patients showed a remarkable reduction in the viral load and there was near complete resolution of symptoms and MRI findings in one case. Use of third party partially HLA-matched BK virus specific CTLs to treat PML holds promise.

CoI: multiple

Full Filling your Wishes. CD52 depletion and maybe reasons for autoimmunity

Depletion of CD52 positive cells inhibits the development of CNS autoimmune disease, but deletes an immune-tolerance promoting CD8 T cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis

Stephanie von Kutzleben, Gareth Pryce, Gavin Giovannoni, David Baker  Immunology doi: 10.1111/imm.12696 Accepted manuscript online: 7 December 2016

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction via intravenous antigen administration such that it could be used to either: improve efficiency of inhibition of MS or to inhibit secondary autoimmunities that may occur following alemtuzumab use in multiple sclerosis. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses assessed clinically. A dose-response of CD4 mAb depletion indicated that 60-70% functional CD4 T cell depletion achieved in perceived failed trials in MS, was perhaps too low to even stop disease in animals. However, more marked (~75-90%) physical depletion CD4 T cells via CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness via a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rational for the rapid B cell hyper-repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T cell regulatory mechanisms that promote immune tolerance may account for the secondary B cell autoimmunities, which occur following alemtuzumab treatment of multiple sclerosis.

Recently someone on the blog was complaining about TeamG research output when we posted on our paper about what to call some one with MS

You said

"What causes progression and how can it be treated, making Alemtuzumab safer and more effective, encouraging them brain to kick-start repair.... so far Team G is in wizard of Oz land - big voice, until you pull back the curtain. Time for some real research Team G!"

To address your comment how do you make "Alemtuzumab safer and more effective"  

You have got your wish.

To ask how you make alemtuzumab safer, you first have to understand why it is not safe and to have ideas. 

You have to have the data, but it does not always easily come to hand or do you have to do some detective work?

The safety issues relating to alemtuzumab are: 

  • infusion reactions, 
  • consequences of long term blanket immunosuppression and
  • secondary autoimmunities that occur in about 50% of people within five years. 
How do you get rid of all those problems?...Simple!.

Use another drug that does not have these problems...

However, to be serious....Ask the many people who have had great benefit for the alemtuzumab and I am sure they will say it was worth it.

However to get rid of the safety issues you first need to understand what they are and how are they caused.

We know how to turn off an autoimmune response within a day in the beasties. To do this we simply reduce the T cell number and then re-induce immune tolerance by intravenously injecting the target antigen. Sounds is.

The way we do this is deplete CD4 T cells but we asked the question is could we use CD52 depletion instead?

Why? Because we could not get anybody to give us access to a CD4 depleting 

However, the answer was surprsingly no.

We found essentially three things

(a) CD52 depletes CD4 T cells and inhibits relapsing EAE

(b) CD52 depletion did not deplete B cells in lymph glands as much as it did in the blood 

(c) It blocked the development of unresponsiveness in our hands and this was probably because it depleted a CD8 regulatory T cell population that was inducing tolerance.

What does this mean?

(a) If MS is caused by CD4, Th1/TH17 T cells then these will be inhibited by alemtuzumab...

(b) The problem with alemtuzumab is that it causes B cell autoimmunities like Graves Disease (hyperactive thyroid disease), ITP (blood clotting problem). This means you have have blood tests every month for 4 years......

So if the antibody that does not clear B cells out lymph glands/bone marrow it means that once the antibody disappears ,which takes about a month then B cell, that have escaped destruction can rush out of the lymph glands and they can then fill up the blood with new cells and they can overshoot...This is the elephant in the Room.

When the B cells repopulate they are doing so when there is a limited T regulation, in this case a CD8 T cell population, which is destroyed by the CD52 depletion. These used to be called CD8 T suppressor cells. So is this the reason why people with MS get lots of secondary B cell autoimmunities...Prompted some delving and it was found that not only was B cell autoimmunites a problem but anti-drug responses occured with staggeringly high frequency. 

Do they affect treatment response?

However, it pointed us to places that allowed us to understand how alemtuzumab-probably induces autoimmunities and works in MS,
But that's another story to be reported soon

Advent Calendar 8

Wednesday, 7 December 2016

Last chance for Free Download

Baker D, Anandhakrishnan A, Tuite-Dalton KA, Lockart-Jones H, Middleton RM, Ford DV, Crowe C, Giovannoni G. Mult Scler Relat Disord. 2016 Nov;10:127-133. doi: 10.1016/j.msard.2016.09.007

Last chance for free copy
How do you describe someone you study.
Please Retweet. May help you get a grant


#ClinicSpeak: the rise of the bots is inevitable

Let's automate the neurological examination ASAP and give you the power to challenge your neurologist. #ClinicSpeak #ResearchSpeak #MSBlog

Every now again there is a research paper that has nothing to do with MS, but its findings have broader implications that are so important for the field that I feel obliged to discuss them and put them in context for pwMS. 

Google are the current masters of artificial intelligence (AI) and they are heavily into healthcare. Google taught one of its AI bots (robots) how to read retinal photographs so that it could diagnose diabetic retinopathy. Surprise, surprise, once the bot had learnt it was able to detect with remarkable accuracy, in fact better than most doctors, referable diabetic retinopathy. For the geeks reading this post the sensitivity and specificity were both well over 90% and the area under the ROCs (receiver operating curves) were over 99%. These numbers are quite staggering. 

What this mean for medicine? If you have diabetes you can simply get a picture taken of your retina using relatively cheap technology, which could be made available to you in pharmacies or supermarkets, have the images uploaded to the cloud automatically and have Google assess whether or not you need to see an ophthalmologist. This technology is simply going to revolutionise the way people with diabetes are monitored and it is going to free up doctor-time for more useful things.

The implications of AI for the medical profession are profound and its coming to neurology and MS. I envisage us using the same technology to assess the retina and optic nerve in people with MS. This will allow you to know if your optic nerve is involved and will allow you to complete your online, or web, EDSS more accurately. We are currently working on a simple web APP that will allow you to assess your own visual function. May be we should simply ask Google to rent us one of their AI-bots and hand over the process to Google? I suspect that very soon I am going to need a new job; maybe I should go and work for Google and make this happen more quickly? 

I often lament about the automation of medicine, the demise of the traditional doctor-patient relationship and the dehumanising effect technology is having on our profession, but when technology does this to a field may be it is time to stopping fighting and to join them on the other side.  We are clearly living in a brave new world!

Gulshan et al. Development and Validation of a Deep Learning Algorithm for Detection of Diabetic Retinopathy in Retinal Fundus Photographs. JAMA. Published online November 29, 2016. doi:10.1001/jama.2016.17216

Importance: Deep learning is a family of computational methods that allow an algorithm to program itself by learning from a large set of examples that demonstrate the desired behavior, removing the need to specify rules explicitly. Application of these methods to medical imaging requires further assessment and validation.

Objective: To apply deep learning to create an algorithm for automated detection of diabetic retinopathy and diabetic macular edema in retinal fundus photographs.

Design and Setting:  A specific type of neural network optimized for image classification called a deep convolutional neural network was trained using a retrospective development data set of 128 175 retinal images, which were graded 3 to 7 times for diabetic retinopathy, diabetic macular edema, and image gradability by a panel of 54 US licensed ophthalmologists and ophthalmology senior residents between May and December 2015. The resultant algorithm was validated in January and February 2016 using 2 separate data sets, both graded by at least 7 US board-certified ophthalmologists with high intragrader consistency.

Exposure: Deep learning–trained algorithm.

Main Outcomes and Measures:  The sensitivity and specificity of the algorithm for detecting referable diabetic retinopathy (RDR), defined as moderate and worse diabetic retinopathy, referable diabetic macular edema, or both, were generated based on the reference standard of the majority decision of the ophthalmologist panel. The algorithm was evaluated at 2 operating points selected from the development set, one selected for high specificity and another for high sensitivity.

Results: The EyePACS-1 data set consisted of 9963 images from 4997 patients (mean age, 54.4 years; 62.2% women; prevalence of RDR, 683/8878 fully gradable images [7.8%]); the Messidor-2 data set had 1748 images from 874 patients (mean age, 57.6 years; 42.6% women; prevalence of RDR, 254/1745 fully gradable images [14.6%]). For detecting RDR, the algorithm had an area under the receiver operating curve of 0.991 (95% CI, 0.988-0.993) for EyePACS-1 and 0.990 (95% CI, 0.986-0.995) for Messidor-2. Using the first operating cut point with high specificity, for EyePACS-1, the sensitivity was 90.3% (95% CI, 87.5%-92.7%) and the specificity was 98.1% (95% CI, 97.8%-98.5%). For Messidor-2, the sensitivity was 87.0% (95% CI, 81.1%-91.0%) and the specificity was 98.5% (95% CI, 97.7%-99.1%). Using a second operating point with high sensitivity in the development set, for EyePACS-1 the sensitivity was 97.5% and specificity was 93.4% and for Messidor-2 the sensitivity was 96.1% and specificity was 93.9%.

Conclusions and Relevance:  In this evaluation of retinal fundus photographs from adults with diabetes, an algorithm based on deep machine learning had high sensitivity and specificity for detecting referable diabetic retinopathy. Further research is necessary to determine the feasibility of applying this algorithm in the clinical setting and to determine whether use of the algorithm could lead to improved care and outcomes compared with current ophthalmologic assessment.

CoI: We run an maintain a website called ClinicSpeak that aims to automate self-neurological assessment for pwMS. The objective is for pwMS to monitor and manage their own disease. I am also a Googophile (somebody who loves Google's technologies).