Immunotherapy in autoimmune neuromuscular disorders

Review

Neuromuscular immunotherapy

Immunotherapy in autoimmune neuromuscular disorders Ralf Gold, Marinos C Dalakas, and Klaus V Toyka Important progress has been made in our understanding of the cellular and molecular processes underlying autoimmune neuromuscular diseases that has led us to identify targets for rational therapeutic intervention. Although antigen-specific immunotherapy is not yet available, old and new immunomodulatory treatments, alone or in combination, provide effective immunotherapy for most autoimmune disorders. In parallel, the achievements of molecular medicine provide more specific yet largely experimental therapeutic tools that need to be tested in the human diseases. Here we review the principles and targets of immunotherapy for autoimmune neuromuscular disorders, address applications and practical guidelines, and give an outlook on future developments. Lancet Neurology 2003; 2: 22–32

The pathogenesis of autoimmune inflammatory disorders of the neuromuscular system is multifactorial. In the past decade, substantial progress in understanding the basic cellular and molecular processes has opened the way for more target-oriented immunotherapy. The ways by which traditional immunomodulatory drugs, which are still the mainstay of treatment, exert their beneficial effect have also been elucidated. In parallel, new agents have been developed that effectively inhibit the signalling pathways of T-cell activation, proliferation, and antigen recognition in animal models and, in some cases, in human beings. We review principles and targets of immunotherapy for autoimmune neuromuscular disorders and address applications and, briefly, practical guidelines. We also discuss the prospects for immunotherapy with agents that are currently in advanced clinical trials. With the exception of self-limiting acute Guillain-Barré syndrome and its variants, the common autoimmune disorders of the peripheral nerve, the neuromuscular junction, and skeletal muscle generally start slowly or subacutely and follow a chronic or chronic undulating course with persistent deficit or even death if untreated. The main autoimmune disorders we discuss are listed in table 1.

Breakdown of tolerance Normally, autoreactive T and B lymphocytes are present in the immune system of healthy individuals. These cells are RG and KVT are at the Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany. MCD is at the National Institutes of Health, NINDS, Neuromuscular Diseases Section, Bethesda, MD, USA Correspondence: Dr Ralf Gold, Department of Neurology, University of Würzburg, D-97080 Würzburg, Germany. Tel +49 931 201 23755; fax +49 931 201 23488; email [email protected]

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Table 1. Outcomes of neuromuscular disorders Disease (target of autoimmune reaction)*†

Outcome

Guillain-Barré syndrome (P2?, P0?, gangliosides, others?)

In 70%, remission occurs after intravenous immunoglobulin or plasmapheresis (half remain mildly affected); remission is better in younger patients; 20% are severely disabled; 3% die

CIDP (P0, PMP-22, gangliosides, sulphatides, others)

In 70%, recovery is good and is better in younger patients; permanent disability is more common if axonal loss is substantial

Multifocal motor neuropathy (GM1, other gangliosides?)

Improvement is seen with intravenous immunoglobulin in about 80%; persistent atrophy, weakness, and fatigue; possibly normal life span; some benefit from cyclophosphamide and monoclonal anitibodies to CD20

Neuropathy with paraproteinaemia (MAG, gangliosides [eg, GD1a, GD1b] or other glycolipids)

Minimum response to plasmapheresis and intravenous immunoglobulin; some promising early results with monoclonal anitibodies to CD20. Outcome less favourable with malignant plasma-cell dyscrasia

Vasculitic neuropathy (ANCA, others?)

About 65% respond favourably, but up to 30% may worsen despite optimum treatment; mortality up to 10% in necrotising vasculitis; concomitant systemic disease affects outcome

Myasthenia gravis (nAChR [common], MuSK [rare] others?)

About 80% go into remission; myasthenic crisis in 1–2% of treated patients. Normal life span except in presence of malignant thymoma. Overall response to treatment excellent, but 5–10% may be more difficult to treat; some residual weakness

Lambert-Eaton myasthenic syndrome (VGCC)

Very good response to treatment; transient improvement is seen with tumour removal if disorder is paraneoplastic; crisis rare. Normal life span except in cases of underlying malignant disease.

Dermatomyositis (endothelial cell antigens?)

Good overall response to immunosuppression, intravenous immunoglobulin, or both, but up to 30% may have difficult disease; mortality depends on underlying malignant disease

Polymyositis (?)

Good response to immunosuppression; about 30% reach remission, and in 40% working status preserved; 30% may be difficult to treat effectively

Inclusion body myositis (amyloid?)

No definitive treatment; intravenous immunoglobulin may halt progression of bulbar symptoms and of proximal leg weakness but benefit is not long lasting

ANCA=cytoplasmic antibodies to neutrophil; CIDP=chronic inflammatory demyelinating polyradiculoneuropathy; nAChR=nicotinic acetylcholine receptor; VGCC=voltage-gated calcium channel; MuSK=muscle specific kinase. *Association of more than one disease in individual patients is probably linked to underlying malignant disease. †In many disorders, potential molecular targets of the autoimmune reaction have been identified but the pathogenic role has not been defined beyond doubt except for nAChR130 and MuSK in myasthenia gravis,21 VGCC in Lambert-Eaton myasthenic syndrome,131,132 and possibly complex gangliosides and sphingolipids in some forms of immune mediated neuropathies.133–135

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Review

Neuromuscular immunotherapy

fundamental for immune surveillance and are normally in a state of tolerance. The salient feature of autoimmunity is the breakdown of immunological tolerance. Tolerance may fail in the presence of an infective organism that shares epitopes with endogenous proteins (molecular mimicry) or when the ensuing systemic infection or other immunogenic stimuli, such as immunisation with live vaccine,1 induce profound immune stimulation. This effect is often seen in Guillain-Barré syndrome—disease onset is associated with a defined infection in up to 70% of cases2—but may also initiate relapses in other disorders such as chronic inflammatory demyelinating polyradiculoneuropathy or myasthenia gravis. Molecular mimicry of epitopes between malignant tumours and antigens in the nerve or end plate may be established for some paraneoplastic cases of chronic inflammatory demyelinating polyradiculoneuropathy,3 Lambert-Eaton myasthenic syndrome,4 and myasthenia gravis with thymoma.5,6 With recovery from the infection, or eradication of the antigen pool contained in the tumour in paraneoplastic diseases, the autoaggressive state does not necessarily stop. This indicates that a selfsustaining autoimmune process has been generated, which maintains a chronic autoimmune reaction to the antigenic epitopes on the target organ rather than the original source.

Venule 2

BNB

Resident macrophage

T-helper cell Axon

4

C'

TCR

B cell

3

Platelets

TNF OH– PGE

LTC4

1

MMP ROS

Myelin antibodies

T cell Schwann cell

C

T cell Apoptosis/ phagocytosis

Fas

MMP FasL

OH– CD8 T cell

6 Bcl2 BDNF?

5

Figure 1. Hypothetical sequence of events during the induction and effector phases of the immune response in the peripheral nervous system.129 (1) Circulating T cells are activated by unknown antigen, eg, P0 or P2. Platelets may provide a co-stimulatory signal via CD40L. T cells then adhere to the endothelial layer and migrate intraneurally, a process that is controlled by a number of cell adhesion molecules. (2) T cells are activated by their specific antigen presented by professional antigen presenting cells or by non-professional antigen presenting cells, such as glia. (3) T-cell activation leads to cytokine secretion that activates macrophages, which release proinflammatory cytokines such as TNF, OH -, complement factors, and PGE, and may directly damage the myelin sheath by antibody-dependent cellular toxicity. (4) T cells activate B cells, which produce myelin-specific antibodies that attach to the myelin sheath, damage nerve fibres by complement mediation, and may also lead directly to functional blockade. (5) Schwann cells armed with Fas and FasL are susceptible to inflammatory T-cell attack but can also counterattack. Local phagocytes clear apoptotic Schwann cell fragments and T cells. Survival of Schwann cells may be modulated by upregulation of Bcl-2 or neurotrophic cytokines. (6) Demyelinated axons may present antigen to invading CD8 T cells and become the target of a perforinmediated cytotoxic effect, which may result in axonal damage as observed in some subtypes of chronic inflammatory demyelinating polyradiculoneuropathy. BNB=blood–nerve barrier; TCR=T-cell receptor; TNF=tumour necrosis factor; C=complement; PGE=prostaglandins; BDNF=brain-derived neurotrophic factor; ROS=reactive oxygen species; MMP=matrix metalloproteinase; LTC4=leukotriene C4.

Role of animal models Animal models have been crucial for our understanding of the major mechanisms of immune-mediated damage in human disorders, although they do not always mimic all the features of the human disease. Animals immunised with nicotinic acetylcholine receptor (nAChR) develop experimental autoimmune myasthenia gravis, which is clinically, electrophysiologically, and histologically identical to the human form. The disorder can also be passively induced by transfer of immunoglobulins or nAChR IgG from patients with myasthenia gravis to mice.7 nAChR is the crucial autoantigen,8 and the antibodies to this receptor cause the defect in postsynaptic neuromuscular transmission, thereby fulfilling all four criteria of the Koch-Witebsky postulates as applied to an autoimmune disorder.9 In Lambert-Eaton myasthenic syndrome, all features of this presynaptic transmission disorder can be reproduced by passive transfer of immunoglobulins from patients with idiopathic or paraneoplastic disease, who have antibodies to the voltage-gated calcium channel,10 yet a model disorder induced by active immunisation with the respective

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Peripheral nerve

Antigen presenting cell

autoantigen has not been produced. Experimental autoimmune neuritis induced in different species by immunisation with myelin proteins serves as a model to characterise pathomechanisms of autoimmunity mediated by T and B cells; this model has also helped us to formulate the following hypothetical sequence of events (figure 1). After activation, T cells invade the peripheral nerve and migrate intraneurally and damage nerve fibres by cellular cytotoxic effects (natural killer cells, CD8 cells, and antibodydependent cellular cytotoxic effects), through cytotoxic proinflammatory mediators, and through B-cell activation leading to antibody production and complement attack on nerve fibres.11,12 For experimental autoimmune myositis, the models have not been satisfactory since most of them do not lead to overt muscular weakness, although inflammatory mechanisms can be well studied in situ.13 Only through animal models can we experimentally delineate the sequence of events in immune-mediated attacks, and derive a stepwise approach. Moreover, genetic mutants and antigen-driven models allow the study of modifier genes that code for factors relevant in disease manifestation or clinical course, and which may help understanding of key

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Antigen-presenting cell

Antigen/ MHC T-cell receptor

Antigen-presenting cell

B7 CD28

Antigen/ MHC T-cell receptor

Signal 1 Signal 2 T cell

Proliferation Interleukin-2 production Protection from apoptosis

Signal 1 T cell

Anergy or apoptosis

Figure 2. Two-signal model of T-cell activation. T cells recognise their specific antigen by T-cell-receptor binding in the context of MHC molecules on antigen-presenting cells. If no co-stimulatory signal is provided, this may lead to anergy or even T-cell death via apoptosis. Only co-stimulation, in this case delivered via B7/CD28 interaction, activates the full signal that is needed for cytokine production and proliferation.

molecular events.14,15 The disease-modulating role of ciliary neurotrophic factor and leukaemia inhibitory factor— important neurotrophic factors that act on oligodendrocyte precursors—has been shown in experimental autoimmune encephalomyelitis, a model of multiple sclerosis, induced by myelin oligodendrocyte glycoprotein in mice.16,17 Another example is the secondary immune-mediated events that are typically superimposed on dysmyelinating hereditary motorsensory neuropathies in mice with targeted mutations of genes coding for the myelin proteins P0 and PMP22 and for the gapjunction protein connexin 32.18 In the respective human disorders, mild inflammatory changes have been seen but their impact was not understood. Experimental abrogation of the immune system strikingly improves the pathology of these Charcot-Marie-Tooth models.19 These observations have potential relevance for the treatment of the human disease.

Potential targets of immunotherapy Induction phase, antigen presentation, and co-stimulatory molecules

Different components of the cellular and humoral immune system generally interact in the immunopathological process. The T-cell receptor of naïve or memory T lymphocytes recognises (auto)antigens when presented by molecules of the MHC class I or class II on antigen-presenting cells (figures 1 and 2). Only in myasthenia gravis is the principal target autoantigen defined as being the nAChR.8,20 A muscle specific kinase MuSK has been identified as relevant in some patients with myasthenia gravis who do not have antibodies agsinst nAChR.21 The situation is more complex in immune disorders of the peripheral nervous system; an array of putative protein autoantigens has been described in experimental autoimmune neuritis,22 as is also the case in multiple sclerosis. In addition,

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several gangliosides may serve as non-protein autoantigens.23–25 Limited information is available for inflammatory myopathies. Although restricted use of the T-cell-receptor gene segments and clonal expansion of T lymphocytes in polymyositis and inclusion body myositis also support a role for selected autoantigens, their identification has not yet been achieved in these disorders.26,27 In addition to classic MHC antigens, another surface molecule located in this region may be of major interest: HLA-G expression parallels upregulation of MHC class I,28 and it may be involved in triggering a CD8 T-lymphocyte response after viral or bacterial infection.29 Antigen-presenting cells belong mostly to the dendritic cell lineage (so-called professional antigen-presenting cells), but may also be recruited from resident cells of the nervous system that upregulate expression of immune molecules during the inflammatory process (non-professional antigenpresenting cells). Professional antigen-presenting cells have the complete repertoire of co-stimulatory molecules of the immunoglobulin superfamily, such as CD28/B7 and ICOS, or of the tumour necrosis factor family, such as OX40/OX40L and 4-1BB/4-1BBL, which contribute to the formation of the immunological synapse (figure 2).26,30 This term was coined for the specialised junction between T cells and antigenpresenting cells. In addition, antigen-presenting cells can secrete cytokines that contribute to T-lymphocyte activation and differentiation into the T-helper 1 or T-helper 2 phenotypes.31 These phenotypes, as defined by molecular markers, are associated with well-characterised intracellular signalling pathways.26 In particular, T-helper 2 lymphocytes activated by interleukins 2, 4, 5, 6, and 10 can stimulate B cells in an antigen-specific way supported by CD40/CD40L interaction. The process initiates a cascade of B-lymphocyte maturation processes, resulting in specific humoral immunity from the antibody-producing cells of the B-lymphocyte lineage.32 These antibodies can induce tissue damage via local complement activation or by functionally blocking signal transduction at the neuromuscular endplate.33 However, T-helper 1 lymphocytes, activated by interferon
and interleukins 2, 12, and 18, mediate CD8-positive T-cell toxicity restricted by MHC class I antigen, as seen in polymyositis and inclusion body myositis.27,34 Treatment strategies directed at the induction phase of immune stimulation involve structurally modified antigen analogues35 (altered peptide ligands) that bind to the T-cell receptor yet lead to downregulation of the autoimmune reaction. An alternative approach is induction of oral tolerance, for example by excessive antigen doses, leading to antigen-specific hyporesponsiveness. Another strategy is vaccination with a dominant pathogenic T-cell receptor to induce regulatory T cells. In addition, modulation of costimulation by use of monoclonal antibodies to molecules of the immunological synapse that induce tolerance by interfering with CD28/B7–CTLA-4 interaction36 has been used successfully in experimental models. No agent has been tested in a human neuromuscular disorder, but advances in biotechnology and molecular immunology suggest feasibility. The prototype of an autoantigen-derived selective agent developed through studies of experimental autoimmune encephalomyelitis is copolymer 1 (now called glatiramer

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acetate because of its polytetrapeptide nature). This is the only antigen-specific substance that has successfully progressed from animal models to clinical use, showing therapeutic benefit in multiple sclerosis.37 Transmigration, local antigen presentation, and cytotoxic effects Transmigration of activated T lymphocytes across endothelial

barriers that separate specialised compartments, such as the nervous system from the bloodstream (figure 1),38 is mediated by upregulation of enzymes and adhesion molecules in both lymphocytes and endothelial cells. During recruitment and transmigration, T lymphocytes anchor to the vessel wall via interaction of adhesion molecules with their ligands on endothelial cells.39 The expression of 41 integrin (VLA-4; CD49d) on leucocytes is fundamental for adhesion and transmigration of T cells, especially in the nervous system, and matrix metalloproteinases are also critical for this process. Matrix metalloproteinases are calcium-dependent zinc endopeptidases that facilitate T-cell adhesion to endothelial cells and matrices and aid transmigration of lymphoid cells towards targeted tissues. Matrix metalloproteinases types 2 and 9 are upregulated in muscle or nerve samples from patients with autoimmune myopathies or neuropathies,24,40,41 as shown by immunocytochemistry, reverse-transcriptase PCR, and zymography. By transmigration and damage to the separating endothelial barrier, T cells enable passage of other humoral and cellular components of the inflammatory cascade. Subsequently, CD4 cells or CD8-positive T cells meet their target antigen presented by MHC class II or class I molecules, respectively, on non-professional antigenpresenting cells such as muscle fibres42 or Schwann cells.43,44 This action leads to T-cell re-activation, which in turn augments secretion of proinflammatory molecules by T lymphocytes. Finally, death of the target cell is induced by cytotoxic T cells via two pathways. First, expression of perforin granules is upregulated in CD8-positive T cells and vectorially directed to the target cell. When released, perforin causes pore formation in the plasma membranes of muscle and nerve causing osmotic cell lysis.45 Second, Fas or FasL and related death molecules are expressed on autoinvasive T lymphocytes and their local constituents, such as damaged and regenerating muscle fibres26 or Schwann cells.46 The precise manner of cell death involving muscle fibres is currently unclear. Signs of apoptosis have not been seen; local upregulation of death suppressor molecules such as Bcl-247 or inhibitor of apoptotic proteases48 may help to counteract apoptosis. In antibodymediated disorders, cytotoxic effects are mediated by: complement activation and formation of a membrane-attack complex (eg, in dermatomyositis, Guillain-Barré syndrome, and myasthenia gravis); macrophages, such as in antibodydependent cellular cytotoxic effects; or cross linking with antigens, which results in internal isation and proteolytic degradation (eg, in myasthenia gravis).49 Therapeutic strategies directed at the transmigration phase involve blockade of adherence or diapedesis of inflammatory cells by monoclonal antibodies50 or small cyclic molecules51 directed against integrins or selectins on the wall of microvessels. Disruption of the blood–nerve barrier and

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cellular invasion of muscle and nerve cells is prevented by matrix-metalloproteinase inhibitors52 or recombinant interferon .53 Minocycline hydrochloride, a tetracycline-like antibiotic, inhibits matrix-metalloproteinase activity in experimental models of inflammatory CNS disease 54 and may be useful in the peripheral nervous system. Amplification of local immune response

In addition to direct local cytotoxic action, CD8 T cells drive the inflammatory cascade by release of soluble cytokines and chemokines.55 Interferon
may directly exert a cytotoxic effect in synergy with tumour necrosis factor , which is generated from its inactive preform via the action of matrix metalloproteinases. The same cytokines also upregulate local chemokine expression, which leads to attraction and local activation of monocytic cells and amplification of the immune response.55 Moreover, macrophages, the principal antigen-presenting cells in the peripheral nervous system, are armed with various inflammatory mediators for the production of free oxygen and nitric-oxide radicals. They also release free nitric oxide, which further augments tissue damage and blocks impulse propagation.56 In addition, macrophages bind via Fc receptors to sites of immunoglobulin and complement deposition.57 Thus, they serve as phagocytic cells and receive stimulatory signals that augment their proinflammatory function and thereby initiate a cycle of immune activation. Most macrophages in an inflammatory focus derive from the blood pool, but some represent activated resident cells that can have important early effects.58 Endogenous tissue components may, however, lessen the proinflammatory and destructive properties of activated complement factors. For example, Schwann cells have several regulatory complement proteins on their membrane, such as CR1 (CD35), decay-accelerating factor CD55, and membrane cofactor proteins CD46 and CD59.57 Systemic and tissue-specific counter-regulatory mechanisms are key to limiting the extent of the autoimmune inflammatory cascade. Potential mediators are immunoregulatory T-helper 2 cytokines. For example, the pleiotropic transforming growth factor  may have a pivotal role in inflammatory muscle diseases since it stimulates fibromatous transformation of inflamed muscle in dermatomyositis26 and may be equally important in Guillain-Barré syndrome.59 Successful immunotherapy is associated with downregulation of transforming growth factor  and its mRNA,60 but the extent to which this reflects the pathogenic relevance of this immunoregulatory circuit is unknown. Treatment strategies directed at cytokines and chemokines include neutralisation by monoclonal antibodies, soluble recombinant receptor molecules, and cytokine modulators such as interferon .61 Several approaches have been studied in multiple sclerosis, but for disorders of the peripheral nervous system experience is limited. Monoclonal antibodies against tumour necrosis factor  or soluble tumour-necrosis-factor receptors have some therapeutic effect in rheumatoid arthritis62 and are being tried in some neuromuscular disorders. In multiple sclerosis, a treatment trial was started after encouraging effects were seen in experimental autoimmune encephalomyelitis, but did not prove to be effective.63 These negative results highlight the differences

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Table 2. Immunosuppressive and immunomodulatory treatments in neuromuscular disorders Treatment* Glucocorticosteroids

Typical dose 1 mg/kg bodyweight daily and subsequent tapering (high-dose IV pulses optional)

Azathioprine

2·5–3·0 mg/kg bodyweight daily (initially 3·0–4·0 mg/kg in severe disease)

Side-effects Diabetes, thrombosis, nervousness, hypertension, gastric ulcer, osteoporosis, glaucoma Nausea, raised liver enzymes, allergic reaction

Ciclosporin

100–150 mg twice daily; trough concentration 70–100 g/L

Hirsutism, tremor, hypertension, renal function

Cyclophosphamide

Pulse therapy with booster infusion every 6–12 weeks

Alopecia, nausea, bone-marrow suppression, late malignant disease, male infertility

Methotrexate

10–20 mg orally once weekly

Pulmonary fibrosis, headache, raised liver enzymes

Mycophenolate mofetil

20 mg/kg daily; trough concentration 1 mg/L Haemolysis, diarrhoea

Intravenous immunoglobulins Plasmapheresis

2 g/kg bodyweight within 2–5 days Traditionally five exchanges at 50 ml/kg body weight within 2 weeks

Headache, skin rash; rarely, anaphylaxis, stroke, heart or kidney failure Hypocalcaemia, infection, allergic reaction, thrombosis of venous catheter

Indications for use CIDP, MGUS, myasthenia and LEMS, polymyositis, dermatomyositis, neurosarcoidosis, vasculitis CIDP, MGUS, myasthenia and LEMS†, polymyositis and dermatomyositis†, neurosarcoidosis, vasculitis; steroid sparing Second line or add on in CIDP; add on to azathioprine in myasthenia and LEMS†; steroid sparing Induction of remission in severe myasthenia gravis and CIDP, MGUS neuropathy, sarcoidosis, vasculitis; add on in MMN Second line in polymyositis, dermatomyositis; in others if sideeffects of azathioprine interolerable If side-effects of azathioprine intolerable; second or third line in myasthenia, CIDP GBS, CIDP, MMN, second line in myasthenia, LEMS, dermatomyositis GBS, CIDP, myasthenia, and LEMS

IV=intravenous; CIDP=chronic inflammatory demyelinating polyradiculoneuropathy; MGUS=monoclonal gammopathy of undetermined significance; LEMS=Lambert-Eaton myasthenic syndrome; MMN=multifocal motor neuropathy; GBS=Guillain-Barré syndrome. *Many of these drugs have not been tested by appropriate clinical trials. †Drugs are contraindicated in the presence of underlying malignant disease.

between animal models of a human disease, such as multiple sclerosis, and the disease itself and warn that therapeutic interventions that alter the balance of the complex cytokine network should be tested with caution in patients with neuromuscular disorders. By contrast to these more selective approaches, pleiotropic agents such as intravenous immunoglobulin or plasmapheresis probably act at various effector levels, such as by neutralisation or removal of pathogenic antibodies25 or complement,64 and by activation of inhibitory Fc receptors on monocytes.65 Elimination of immune cells by apoptosis

Apoptosis was brought to attention in neuroimmunology in the early 1990s.66,67 Since membrane integrity is well preserved during apoptosis, it could protect the target organ by avoiding release of proinflammatory intracellular enzymes. The diseaselimiting role of T-cell apoptosis is seen in experimental immune neuritis68 and has been implicated in Guillain-Barré syndrome, but not in chronic inflammatory disorders of nerve and muscle. In human myositis, T cells do not undergo apoptosis in the diseased muscle fibres, even in the presence of HIV infection.69 We speculate that this phenomenon contributes to chronic persistence of inflammation in muscle. Currently, not all mechanisms operative in the induction of apoptosis are understood. Tumour necrosis factor  has a crucial role in experimental autoimmune encephalomyelitis in transgenic mice deficient for tumour-necrosis-factor receptors 1 and 2,70 but nothing is known for neuromuscular disease models. Therapeutic strategies that increase apoptosis of infiltrating inflammatory cells include high-dose glucocorticosteroids71 and antigen-specific approaches.72 Since the latter, in experimental models, depend on identification of the candidate autoantigen, this remains a challenge in human disorders.

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Tissue-specific factors, trophic support, and survival

Knowledge of how local factors in the target tissue modulate recovery from an immune attack is as yet insufficient. In myasthenia gravis, resynthesis of nAChR may, to some degree, compensate for blockade and destruction of receptors by pathogenic autoantibodies.73,74 Downregulation of nAChR resynthesis by glucocorticosteroids is but one explanation for the transient aggravation of symptoms seen typically in patients with myasthenia gravis during early treatment.75 For the peripheral nervous system, as yet unknown genetic susceptibility factors may modulate the response of axons and myelinating Schwann cells to the inflammatory assault. In the CNS, we have identified the neurotrophic cytokine ciliary neurotrophic factor as a non-immunological disease modulator, and others have identified a similar role for leukaemia inhibitory factor.16,17 Neurotrophic factors may also be operative in disorders of the peripheral nervous system. In addition, the new concept of neuroprotective features of local inflammation via secretion of trophic factors76 may also apply to neuromuscular disorders. Two tissue-specific processes play major parts in the prognosis and treatment response in neuromuscular diseases: axonal degeneration in autoimmune neuropathies and fibrosis in muscles and nerves.77 Concomitant axonal degeneration secondary to primary demyelination occurs in chronic inflammatory demyelinating polyradiculoneuropathy and affects prognosis.77 We face similar difficulties with tissue fibrosis in myositis. Whether the release of neurotoxic cytokines, such as tumour necrosis factor , and toxic mediators such as matrix metalloproteinases, increases axonal loss and muscle fibrosis remains unclear.

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Principles of practical immunotherapy in neuromuscular diseases A wide gap still exists between the current knowledge of the basic mechanisms of autoimmunity and the availability of approved treatments. Therefore, we structure the discussion of treatments by disease and type of intervention rather than by mechanism of action and provide treatment approaches based on expert opinion and the understanding of pathogenetic mechanisms. The range of treatments available is summarised in table 2 and the panel. Only a few treatments have been tested, and no algorithm has been the subject of formal trials. Neuromuscular disorders have the advantage that clinical and laboratory monitoring of therapeutic efficacy is accessible and quantifiable. Efficacy is assessed by monitoring strength, sensory deficits, or autonomic dysfunction with appropriate clinical testing, and by established quantitative scores, based on validated scales supplemented by surrogate markers such as nerve-conduction studies, electromyography, and antibody titres. Safety can be monitored by measuring drug concentrations, if available.78–80 In the age of evidence-based medicine, treatment recommendations based on evidence from double-blind, randomised, placebo-controlled studies would be preferable. For most immunotherapies, especially established treatments, only data from cohort and case-control studies or case reports are available. Even without these studies, however, clinical efficacy of several drugs or procedures is widely accepted, such as plasmapheresis in myasthenic crisis or the use of glucocorticosteroids in common neuromuscular disorders. Given the ethical standards defined in the Helsinki declaration and the enormous costs involved, the prospects for doing placebo-controlled studies of these accepted treatments are poor. New agents are, however, being studied in randomised trials. Acute diseases or relapses

As soon as an autoimmune neuromuscular disorder is diagnosed and weakness is manifest, treatment aimed at terminating the inflammatory attack or at removal of blocking antibodies is started. The goal of treatment is to improve strength and activities in daily living. Given the lack of controlled studies, judgements are mostly based on case series and expert opinion. The use of treatments may vary between countries dependent on restriction of off-label use by regulatory agencies. With the exception of Guillain-Barré syndrome and

multifocal motor neuropathy, glucocorticosteroids are the mainstay, or an important component, of treatment. These drugs are given orally (1–2 mg prednisone or equivalent analogue per kg bodyweight in mild to moderate cases) or, less commonly, as intravenous pulse therapy, typically starting at 500–1000 mg methylprednisolone daily for 3–5 days.81 Concomitant infections have to be excluded by laboratory screening and chest radiography. Some non-genomic mechanisms of steroid action can operate in high-dose pulse therapy, which hasten clearance of inflammation through apoptosis.71 In myasthenia gravis, steroids may initially aggravate neurological symptoms, probably by nonimmunological mechanisms;73–75 therefore some physicians introduce treatment by gradually raising the daily dose. After improvement of clinical symptoms, steroid dose is tapered slowly in parallel with the initiation of long-term immunosuppressive or immunomodulatory treatment. In multifocal motor neuropathy steroids are ineffective and, for unknown reasons, may even temporarily worsen the disease. In Guillain-Barré syndrome, no benefit is seen from glucocorticosteroid monotherapy.82 Evidence from one randomised trial suggests that steroids offer an additional antiinflammatory effect when combined with intravenous immunoglobulins,83 but confirmation is required. In addition, in paraneoplastic neuropathies, immunosuppressive treatment is started with glucocorticosteroids but the response is often not satisfactory. In case of a therapeutic response, steroid-sparing immunosuppressive drugs may be considered. If circulating humoral factors have a predominant pathogenetic role, such as in myasthenia gravis and GuillainBarré syndrome, plasmapheresis is useful, starting at standard exchange rates (550 mL/kg bodyweight 2–3 times per week). The efficacy of plasmapheresis in Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy has been shown in controlled trials.84–86 Intravenous immunoglobulins are generally equally effective in adults87–90 and children,91 but might be less effective for myasthenic crisis.89 Intravenous immunoglobulin is given according to established, yet arbitrary, regimens (2 g/kg within 2–5 days).92 Immunoadsorption columns (tryptophanepolyvinylalcohol resins) or protein A columns semiselectively remove pathogenic antibodies without deprivation of most other plasma proteins, and, if available for human use, may be particularly useful in myasthenia gravis.93–95 Their effect in other disorders is unclear. In chronic inflammatory demyelinating polyradiculoneuropathy, use of plasma-

Step-by-step therapeutic practice* ●

● ● ● ●

● ●

Try glucocorticosteroids at an intermediate dose of 100 mg daily or initially intravenous pulse treatment (500–1000 mg methylprednisolone daily for 3–5 days). When improvement starts, taper dose gradually; be aware that some patients with myasthenia gravis may transiently worsen Add azathioprine for long-term treatment in more severe disease or if improvement is not satisfactory Switch to ciclosporin if response is not satisfactory or if steroids are contraindicated Add cyclophosphamide (pulse treatment) if disease does not respond sufficiently to the first three steps Use intravenous immunoglobulin for all rapidly progressive and critical patients or plasma exchange (except for polymyositis or dermatomyositis) in addition to medical immunosuppression In relapses or later deterioration, restart with steps one to four In myasthenia, thymectomy should be done in patients younger than 50 years if medically stable

*Relates to myasthenia gravis, Lambert-Eaton myasthenic syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, polymyositis, and dermatomyositis. Steps are carried through if no favourable treatment response is induced or if the disease is very severe. Note that more complex algorithms are needed to reflect disease variability and heterogeneity in treatment responses.78,79 Only few treatment modalities have been tested and none of the algorithms has been the subject of formal trials.

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Neuromuscular immunotherapy

2 Alefacept Hu1124 BB1 LFA-1 LFA-3

CD3 complex 


MHC-1

5 Interleukin -2 receptor

CD52 CD45

different mechanisms of action; however no evidence justifies such action. Clinical grounds, such as concomitant infection or difficulties in venous access in children, may favour intravenous immunoglobulin over plasmapheresis from onset. Long-term maintenance immunotherapy

V

In parallel to successful acute immunointervention, long-term treatment should be considered in all chronic or chronic relapsZAP-70 ing autoimmune neuromuscular disorders. ITAM Costimulation Rapid therapeutic response to glucocortiPTK's Cytoplasm costeroids or intravenous immunoglobulin p56Ick PLC may allow maintenance of remission by 4 P Steroids tapering of treatment to low doses in indi1 urin vidual patients. Given the pleiotropic sideNFAT e 3 n i Nucleus Calc effects of steroids, 10 mg of prednisone or P– Interleukin-2 Ciclosporin prednisolone daily, or 20 mg on alternate - gene promoter Tacrolimus days is generally judged the upper limit for a safe long-term steroid monotherapy if the Gene transcription dose is well tolerated. In most chronic neuFigure 3. Signalling pathways activated by T-cell receptor (TCR) engagement. TCR interaction romuscular diseases immunosuppressive with antigen/MHC activates intracellular phosphotyrosine kinases (p56, ZAP-70) that mediate agents are required to maintain remission signalling via phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). This or near remission. The rationale is norsignalling is sensitive to ciclosporin and tacrolimus (1). T-cell activation is increased via mally sparing of steroids, but intrinsic costimulatory factors delivered, for example, by LFA/ICAM interaction, and can be blocked by effects such as resetting the autoreactive antibodies to LFA3, such as alefacept (2). TCR engagement and co-stimulation in turn activate downstream events that, via phospholipase PLC and activation of the phosphatase calcineurin disease state may arise in some disorders, as (sensitive to the cyclophilin ciclosporin and the FK binding protein tacrolimus [3]), activate the suggested by the study in myasthenia nuclear factor of activated T cells, which is translocated into the nucleus where it induces specific gravis.98 Because immunosuppressive drugs gene transcription leading to cell proliferation and differentiation. Glucocorticosteroids mediate have a delayed onset of action (up to sevtheir inhibitory action via the cytosolic steroid receptor also at the level of gene transcription (4). Further modulation of T-cell activation can be achieved via blockade of the interleukin 2 receptor eral months), they should be started once a (5). LFA-1=lymphocyte function antigen 1; LFA-3=lymphocyte function antigen 3; steroid-sparing effect is clearly needed or a ITAM=immunoreceptor tyrosine-based activation motifs; ICAM=intercellular adhesion molecule; low-dose glucocorticosteroid treatment is ZAP-70=zeta-chain associated protein 70; PTK=protein tyrosine kinase; NFAT=nuclear factor of not sufficient. Severe or rapidly progressing activated T cells; CTLA-4=cytotoxic T lymphocyte antigen; PLC=phospholipase C. disease calls for an early combined regipheresis, intravenous immunoglobulin, and steroids are men. All cytotoxic substances require safe contraceptive methalmost equally effective but a few patients respond ods for both sexes, and at least a 6 month interval should be preferentially to one or other of these regimens.96 Treatment recommended before pregnancy after treatment is stopped. decisions must be made individually, taking into account For the chronic disorders that respond only to intravenous clinical availability, venous access, the patient’s age, and immunoglobulin (tables 1 and 2) the maintenance dose varies associated disorders. The multiple potential mechanisms of from patient to patient. We have been able to reduce the dose action for intravenous immunoglobulin92 may all be relevant to 1 g/kg in some patients, and in others 2 g/kg every 2–3 in functional terms, some acutely (eg, competition for months was sufficient. The high cost of intravenous immunoglobulin-Fc receptors on macrophages) and some in immunoglobulin encourages us to find the lowest dose for the long term (eg, downregulation of antibody synthesis). each patient. Only a few mechanisms have been clearly identified in defined Immunosuppressive drugs were introduced for the treatdisorders. In Guillain-Barré syndrome, intravenous ment of neurological diseases in the early 1960s and are now immunoglobulin may act partly by neutralisation of widely accepted as a therapeutic standard, despite few neuroelectric blocking antibodies,25 and in dermatomyositis prospective controlled trials having been done. Long-term use this treatment inhibits complement consumption and of immunosuppressive drugs has been associated with a slight intercepts the deposition of membrane attack complex.97 In risk of development of malignant disease, which is part of the Guillain-Barré syndrome, if secondary worsening of clinical labelled risk profile. Available observations in myasthenia signs occurs despite initial improvement with the first-line gravis and multiple sclerosis support the notion that the risk of treatment (plasmapheresis or intravenous immunoglobulin), malignant disease from azathioprine use is raised two to four the same treatment should be repeated. Some researchers times if taken over many years,99–101 although some workers recommend switching to an alternative treatment option at believe that the risk is lower. For cyclophosphamide, the risk that time or after 10–14 days of observation because of the for haematological malignant disease may be up to 1·5% per V

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Review

year and seems to be related to the cumulative 1 Anti-Interleukin-2 dose given.102 The data of the actual risk are, how[CD25] monoclonal ever, limited, and controlled studies of risk are antibodies rudimentary. Azathioprine, an inhibitor of different stages 
Interleukin-2 receptor  of purine metabolism, is generally well tolerated and its long-term side-effects have been characterised in different autoimmune diseases. Azathioprine catabolism includes enzymatic JAK3 JAK1 thiomethylation, which shows low activity in Cytoplasm PTK's 5–10% of the white and Asian population as a 103 genetic trait. This difference may be one reason STAT3-5 TOR for early idiosyncratic adverse reactions. If Sirolimus Ribosome patients do not tolerate the standard dose 2 FKBP G1 S (2·5–3·0 mg/kg), a lower starting dose can be used with division of the daily dose. Azathioprine is Nucleus Cell 3 Mycophenolate contraindicated in patients with concomitant use cycle Interleukin-2 mofetil, azathioprine of allopurinol. In case of severe gastrointestinal - gene promoter symptoms or allergic reactions, methotrexate or M G2 mycophenolate mofetil may be considered as Gene transcription alternatives to azathioprine. Methotrexate is an antineoplastic agent that Figure 4. Induction of cell cycle and T-cell proliferation by interleukin 2 receptor. Activated T acts as an antagonist of folate metabolism.104 In cells synthesise the T-cell growth factor interleukin 2 and its receptor. The interleukin 2 receptor of resting T cells is composed of a  chain and a
chain and binds interleukin 2 low doses, this drug has immunosuppressive with moderate affinity; on activation the  chain associates with the  chain and
chain, properties and may have additional immunomo- creating a high-affinity receptor. This receptor can be blocked by monoclonal antibody to dulatory effects, for example by adenosine accu- CD25 (1). Binding of interleukin 2 to the receptor in turn activates intracellular signalling mulation at sites of inflammation. It is given once cascade via janus kinases (JAK1, JAK3) and subsequent phosphorylation of signal weekly orally, subcutaneously, or intramuscu- transducer and activator of transcription (STAT) proteins. Signalling associated with interleukin 2 receptor is sensitive to the action of sirolimus, which mediates its action via larly, and only renal or hepatic dysfunction and FK-506 binding protein (FKBP) (2). Mycophenolate mofetil and azathioprine inhibit de novo pulmonary fibrosis are clear contraindications. In purine synthesis, which is required for T-cell activation (3). TOR=target of rapamycin neurological disorders, methotrexate is recom- protein; STAT=signal transducer and activator of transcription. mended as a possible first-line option to supplement steroid therapy in polymyositis or in case of intolerable thenia gravis and myositis have suggested an optimum blood side-effects of azathioprine. Although not widely used, this concentration range of 1–2 mg/L.106 Side-effects are rare; they drug may be useful in disorders such as myasthenia gravis or mainly occur at daily doses higher than 2 g107 and include gaschronic inflammatory demyelinating polyradiculoneuro- trointestinal symptoms and increased risk of developing pathy. The typical weekly dose of methotrexate is 10 to 20 mg. cytomegalovirus infection. Little is known about the risk proNeurological side-effects can be reduced by low-dose supple- file for associated malignant disease. Controlled trials of mycophenolate mofetil in myasthenia are currently being mentation of oral folate (eg, 2·5 mg daily). Ciclosporin is a member of the immunophilin-binding done. Because of their different site of action for T-cell activafamily of drugs with a mechanism of action similar to tion (figures 3 and 4), azathioprine or mycophenolate mofetil tacrolimus. By binding to cyclophilin, ciclosporin inhibits cal- can be combined with ciclosporin. Cyclophosphamide is an alkylating antineoplastic agent cineurin-dependent signal transduction, which leads to a reduced production of interleukin 2 and of other T-cell-asso- that belongs to the group of the strongest immunosuppressive ciated cytokines (figures 3 and 4).105 Side-effects have limited drugs. In fulminant vasculitis, mixed connective tissue disorthe use of ciclosporin to a second-line drug in myasthenia der, and in some subgroups of immune-mediated neugravis and severe chronic inflammatory demyelinating ropathies—including vasculitic neuropathy, multifocal motor polyradiculoneuropathy. It can also be used as add-on therapy neuropathy, and severe chronic inflammatory demyelinating with azathioprine. Trough concentrations of 70–100 g/L are polyradiculoneuropathy—immunosuppressive treatment achieved by twice daily intake of 100–150 mg. Because with cyclophosphamide is helpful. A switch to azathioprine or ciclosporin acts faster than azathioprine and methotrexate, it mycophenolate mofetil is possible after remission is achieved. is sometimes preferable as a second-line treatment if glucocor- Our preference is 0·5–1·0 g/m2 body surface given intravenously every 4–6 weeks. In very active disease, an initial ticosteroids are contraindicated or poorly tolerated. Mycophenolate mofetil (figure 4) modulates de novo induction pulse of three cycles at 350 mg/m2 body surface may pathways of purine biosynthesis, leading to depletion of be considered. Antiemetics are recommended to lessen nauguanosine nucleotides in T and B cells. It is generally well tol- sea, and uroprotective agents are essential. Cyclophosphamide erated at doses up to 2 g daily. As for ciclosporin, trough con- pulse therapy is generally better tolerated than long-term oral centrations of mycophenolate mofetil can be measured in treatment, and it takes longer until the critical cumulative dose whole blood. First reports on its therapeutic efficacy in myas- for an increased risk of developing malignant disease is

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Search strategy and selection criteria We based this review on our own experience and research connected with these disorders and on a comprehensive Medline search using the terms “immunotherapy” and “neuromuscular diseases”. We focused on peer-reviewed works published in English in major scientific journals in the past 20 years, and on reviews written by specialists in the field. All available articles were critically reviewed. Papers presenting the strongest evidence or providing important insights into the immunopathogenesis and immunotherapy of these disorders were referred and cited. When possible, randomised trials were cited for new agents or reference to the Cochrane database was made. Given the fact that a few thousand reports were published in the field, we had to be selective.

reached, which is estimated as being 50 g or to increase by 1·5% per year of exposure.102 In our experience, use of the proper combination of available drugs, starting at the lower end of the toxicity range and escalating to drugs with higher toxicity if necessary, is beneficial in many autoimmune neuromuscular disorders that are difficult to treat. Application of newer immunosuppressive agents, such as tacrolimus or sirolimus (figures 3 and 4), might be considered when other drugs have failed. In myasthenia gravis, a purely surgical approach may improve long-term prognosis.108 Most younger patients with this disorder have thymic hyperplasia or thymitis.109,110 Thymectomy is thought to facilitate the removal of a major source of autoantibody and T-cell production.109–111 Although the effect of this intervention has been challenged because of lack of controlled data, a large meta-analysis favours its use.108 A controlled study on the role of thymectomy in myasthenia gravis is being done. Thymectomy should be considered in younger patients (<50 years) with nAChR antibodies and generalised myasthenia after appropriate pretreatment (eg, plasmapheresis or immunosuppressive treatment) to induce temporary improvement. There is debate as to whether endoscopic (minimally invasive) surgery112 is equivalent to a transsternal approach. By contrast, a malignant thymoma or thymic carcinoma should be removed at any age and radiochemotherapy should be delivered for infiltrative growth.

New directions for immunotherapy In principle, all the different stages of cellular activation, transmigration, proinflammatory mediators, and effector mechanisms of the immune response can be manipulated. Much emphasis has been placed on the trimolecular complex, yet this has proved the most difficult of targets; for most diseases, target antigens have been poorly described or cannot be identified at an individual level. Even in myasthenia gravis, in which the antigen is known, this strategy was effective only in experimental autoimmune myasthenia gravis.113 Approaches such as modulation of intracellular signalling pathways seem currently to hold more promise (figures 3 and 4). Leflunomide, an inhibitor of tyrosine kinase signalling, is already licensed for chronic polyarthritis,114 has been successfully used in experimental autoimmune neuritis,115 and may be a promising alternative for human autoimmune neuropathies. Interferons certainly have pleiotropic effects,116 and interferon  and  are useful as downregulatory immunomodulating 30

cytokines. Although interferons are effective in relapsingremitting multiple sclerosis, their usefulness for autoimmune neuromuscular disorders is not yet established; however, some patients with cryoglobulinaemic neuropathy,117 chronic inflammatory demyelinating polyradiculoneuropathy,118 and multifocal motor neuropathy119 may respond favourably to interferon . Additional controlled studies with this drug are being done or are planned in chronic inflammatory demyelinating polyradiculoneuropathy and inflammatory myopathies. Another approach is to block cellular adhesion to the vessel wall and egression of inflammatory cells from the bloodstream to the target tissues. Notably, monoclonal antibody to VLA4 has quickly moved out of successful phase II studies in multiple sclerosis to a phase III trial. It may become a promising agent for the treatment of autoimmune diseases of the peripheral nervous system, as shown in experimental autoimmune neuritis,50 in which VLA4 blockade led to rapid termination of T-cell inflammation by apoptosis.120 Studies with modulators of immunological checkpoints such as CTLA-4121 are underway in multiple sclerosis. Immunomodulation of B cells may be achieved by antibody to CD20, which has been used successfully in neuropathies associated with paraproteinaemia.122,123 Monoclonal antibodies to molecules in T-cell activation pathways are promising and are involved in current or planned phase II trials. Antibodies to CD52124 or interleukin 2 receptors125 seem especially promising. Novel immunotherapies are frequently used first in general medicine or other specialties before being applied to neurology—eg, intravenous immunoglobulin in idiopathic thrombocytopenic purpura or pulsed cyclophosphamide in lupus erythematosus. Neuroimmunologists have, however, been introducing novel immunotherapies in autoimmune neurological disorders based on experience in animal models, especially experimental autoimmune neuritis. The application of new drugs for the treatment of multiple sclerosis37 has been instrumental for the treatment of autoimmune neuromuscular disorders. Care must be taken in the introduction of new cytokine-based therapies as main or add-on treatments. Even stronger caveats apply to the most radical immunosuppressive treatment—haemopoetic stem-cell transplantion—which is being studied in multiple sclerosis and has been reported in chronic inflammatory demyelinating polyradiculoneuropathy.126 The basic idea is to eradicate immune cells and replenish them with healthy stem cells. A large retrospective study of this intervention in multiple sclerosis showed a high mortality rate of 5–10%,127,128 which discourages the use of this approach in neuromuscular disorders until the procedure is improved.

Conclusion The study of modern immunotherapy is moving rapidly forward, especially as targets on T-cell signalling are being recognised and industry provides more specific tools. New compounds and new indications for established drugs await clinical testing. Vigorous clinical trials are needed, despite their high cost and uncertain safety profile. We are optimistic that tailored immunotherapies will become more successful as our understanding of molecular mechanisms deepens.

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Acknowledgment

Conflict of interest

We apologise to colleagues whose original contributions have not been referenced because of limitations in space.

There are no conflicts of interest.

Authors’ contributions

RG wrote a first draft of the review, which was circulated to the other authors. All authors checked the pertinent references and contributed to the scientific content of the review. References 1

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Role of the funding source

Cited work done in the authors’ laboratories was supported by the DFG, Gemeinnützige Hertie Stiftung, and IZKF funds from the state of Bavaria, and the National Institutes for Health. All authors have received institutional grants for their experimental research into the mechanisms of action of various treatments or in the process of drug trials.

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