Human cell based assays to study the immunopathology of the inflammatory neuropathies

The inflammatory neuropathies occur when a person's immune system mistakenly attacks their own peripheral nerves. These nerves carry instructions to the muscles and sensations from the skin. When they are damaged weakness and numbness result. In severe cases a patient can lose their ability to walk, swallow and breathe. Guillain Barré syndrome (GBS) is the most common acute inflammatory neuropathy. It affects around 1200 people in the UK each year, and each of us has a 1 in 1000 chance of developing the disease during our lifetime. GBS tends to follow an infection such as a cold or tummy bug, and has recently been associated with the Zika virus. The disease comes on over a few days, but patients can require intensive care treatment for weeks, and often need to stay in hospital for months. About 1 in 20 people die during their illness, and many do not fully recover their previous level of function even years later. Chronic types of inflammatory neuropathy come on over longer periods (typically months), frequently relapse, and often require long term treatment with steroids, infusions or chemotherapy. Treatments for these diseases are only partly effective and have not improved in over 20 years. Part of the problem is that it is still not known exactly how the immune system damages nerves. Previously, researchers have used chemical tests and animals to try and detect and study antibodies which might be attacking the nerves. There are several reasons why these approaches could be misleading. Even if antibodies are detected, they may not bind to nerves in the human body. Even if they do, they may not injure the nerves. Animal nerves, processed tissue samples and extracted chemical are different to human nerves themselves.

Recently, scientists have learned how to generate stem cells from human skin. Stem cells have a special ability to turn into all of the different cell types in the body, including those of the nervous system. I have used stem cells to grow human peripheral nerve cells in dishes in the laboratory. These cells offer a great opportunity to improve the way we study the inflammatory neuropathies. They are from human rather than animal tissue, and form the structures found in normal nerves. They can also be produced in large numbers for multiple experiments without the need to use animals.

In the proposed study I will collect blood samples from patients with different neuropathies and apply these to the dishes of human nerve cells. I will be able to see if antibodies bind to the nerves and look at how the nerves are injured. I will look at different nerve cells alone in in combination, to see if and how some are protected from or sensitised to injury. Using specially coated magnetic beads, the chemical target being attacked by the antibody will be identified. The nerve cells can also be used to see the extent to which antibodies and patients' blood samples prevent or slow nerve regrowth.

The proposed study has a number of potential benefits. Understanding how the nerves are injured might help develop new treatments, and the nerve cell cultures would also be a useful way to test these treatments in future. Identifying antibodies would allow new blood tests to be developed to improve diagnosis. Detecting specific antibodies may also indicate that a particular treatment is more (or less) likely to be effective. The ability to detect whether blood samples continue to injure nerves or prevent repair after initial therapy might indicate that more intensive treatment should be given.

The inflammatory neuropathies are significant causes of morbidity and mortality with sub-optimal diagnostic algorithms, biomarkers and treatments. Previous research has relied heavily on solid phase assays and animal models, with limited success. The proposed study aims to extend understanding of inflammatory neuropathy pathogenesis, improve diagnostic assays, and better guide future treatment decisions. This objective will be achieved by identifying pathogenic mechanisms and antibodies using human induced pluripotent stem cell (hiPSC) derived sensory neurons, spinal motor neurons, Schwann cells and myelinating co-cultures. The topographical pattern of antibody binding will then be assessed without a priori specification of putative molecular targets. Pathological processes and regeneration will be studied over longer time periods than is possible in other systems. Co-cultures will allow assessment of the deleterious effects of patient derived serum on axo-glial interactions and myelination. Factors modulating or protecting from injury, such as antibody internalisation, and complement activation and regulation, will be evaluated. Antigen targets will be identified by immunoprecipitation and mass spectrometry. Detected antibody specificities and neuropathological activity will then be linked back to key clinical features such as prognosis and the response to particular treatments. Specific hypotheses that will be addressed in the proposed research are; i) comparing sensory and motor neurons, and control and disease-associated cells, will identify reasons for the selective and regional sensitivity of specific peripheral nerve tissue to immune mediated injury, ii) cell based assays will offer improved diagnostic sensitivity and specificity, iii) patients whose serum show persistent neuropathic potential in culture are likely to continue to progress and/or recover more slowly, providing a biomarker to identify those most likely to benefit from additional treatment.

Who will benefit from this research?

The research will benefit my professional development, and also provide post-doctoral training in stem cell, immunological and peripheral nerve research for another individual. Other beneficiaries of this work will be investigators working in related fields, but also those with interests in a range of other areas, such as;

a) other researchers studying immune mediated neuropathies
b) investigators studying other neurological and non-neurological antibody mediated, or potentially antibody mediated, diseases
c) scientists interested in other neuropathies, including those which are genetically mediated
d) researchers looking at the biology of peripheral nerves, their repair processes, and strategies for enhancing these
e) those studying other non-antibody lectin-glycolipid interactions (eg. bacterial toxins and sialic-acid binding immunoreceptors)

Within the commercial sector potential beneficiaries include;

a) companies developing diagnostic immunoassays
b) pharmaceutical companies developing
i. new immunotherapies
ii. neuroprotective agents in a more general sense
iii. agents promoting nerve repair/regeneration

Within the health service and amongst regulators, potential beneficiaries include;

a) those involved in the planning of neurology inpatient and neurorehabilitation provisions
b) those involved in approving and rationing expensive treatments (eg. intravenous immunoglobulin (IVIg))

Charitable beneficiaries include;
a) The GBS and Associated Inflammatory Neuropathies (GAIN) charity

Ultimately, neuropathy patients will also benefit.

How will they benefit from this research?
I will gain personal experience in leading an independent research programme, and managing other staff. I will supervise a post-doctoral researcher, who will themselves gain experience of stem cell, peripheral nerve and neuroimmunological research. We will also both benefit from exposure to the wider programme of research activity within the department. My continued involvement in inflammatory neuropathy research will allow me to continue to provide up to date expert medical advice to the GAIN charity. The specific research proposed addresses some questions which have been relatively neglected to date - namely, what factors modulate antibody-antigen interactions within the living membrane, and what sensitises or protects nerves from antibody mediated injury? It will also result in fully humanised assay platforms which should offer enhanced sensitivity and specificity for detecting disease associated antibodies and assessing neuropathological effects. The development and subsequent clinical application of serological tests may improve diagnosis and prognostication, reduce the need for invasive tests such as nerve biopsy, allow the selection of patients who will benefit from treatments such as IVIg, avoid the need for a treatment trial in those who will not benefit, or provide impetus to initiate or escalate treatment in others. The research focus on factors modulating antibody mediated injury may suggest new strategies for abrogating this. The use of human cultures as a model system for inflammatory neuropathies offers a more cost effective way of trialling new immunomodulatory or neuroprotective therapies, unreliant on experimental animals. The efficiency and cost-effectiveness of screening such therapies would be improved. Furthermore, the core methodology could be used to enhance understanding of other causes of neuropathy. It would provide genetic neuropathy researchers with a relevant platform in which to assess the pathogenic mechanisms and importance of detected mutations, something which is not easily assessed by sequencing data alone. The cell based assays would provide a powerful tool for researchers to study some of the process of peripheral nerve regeneration and allow multiple strategies for enhancing this to be evaluated in parallel.