HD is a rare autosomal dominant neurodegenerative disease that causes devastating disorders. It affects principally people above the age of forty. The disease inalterably proceeds towards a multi-faceted cognitive deterioration, motor disorder-associated chorea and bradykinesia as well as psychiatric disturbances such as depression and irritability. The clinical symptoms - at least in the early stages of the disease - are related primarily with a hypo-functioning and a degeneration of the medium spiny GABAergic neurons in the striatum. In the later stages of the disease, cortical and sub-cortical structures, anatomically connected with the striatum, become affected too. The disease is fatal within 15 to 20 years of its onset in most patients (Bird and Coyle 1986) and has no cure or any effective treatment. Besides the search for therapeutic agents like neurotrophic factors which act against the molecular mechanisms of neurodegeneration in HD, therapeutic research also focuses on GABAergic nerve cell supplementation therapy.
Intrastriatal implantation of (striatal) fetal ganglionic eminence tissue was able to reverse a large number of the motor and cognitive deficits brought about by striatal lesions of various kinds in animal affected by HD (cf. Peschanski et al. 1995). Several indicators suggest that implanted neurons do mature normally, are mainly GABAergic and express both the expected corresponding neuropeptides (substance P, met-enkephalin, somatostatin or neuropeptide Y) and the dopaminergic and muscarinic receptors. Host afferent axons both grow into the grafts and connect to grafted neurons (cf. Wictorin 1992), and functional reconnection of grafted GABAergic cells to the experimentally denervated target neurons of the globus pallidus also develops. Grafted neurons do not reach more remote projection zones such as the substantia nigra, pars reticulata, but the globus pallidus is by far the most important projection zone of striatal neurons in primates and in humans. Behavioural analysis of grafted animals to a large extent confirms the rewiring of cortical output circuits in which striatal neurons normally act as first relay cells (Dunnett et al. 1988; Kendall et al. 1998; Palfi et al. 1998; Hantraye et al. 1990).
The converging evidence in animal studies, outlined above, has led to trials of intracerebral grafting in patients with HD. Except for the study carried out by Hauser et al. (2002), all studies involved patients at an early stage of the disease. The safety and feasibility of the grafting procedures appeared almost unquestioned in all studies (Kopyov et al. 1998; Bachoud-Levi et al. 2000b; Fink et al. 2000; Rosser et al. 2002) except in the Hauser et al. (2002) study where patients at a more advanced stage of the disease, and patients with a history of neurological problems, were included and some subjects developed subdural hemorrhages or required surgical drainage. The latter may indicate that patients at an advanced stage of the disease are particularly sensitive to medical interventions. No noticeable side effects were reported in the other studies except for difficulties encountered in obtaining a good compliance of patients for drug treatment and, in particular, for immuno-suppressive drugs (Bachoud-Levi et al. 2000b; Rosser et al. 2002). An autopsy in one patient, who died of causes unrelated to the transplant 18 months after surgery, revealed the presence of a large graft that contained a large number of neurons phenotypically similar to GABAergic medium-spiny striatal neurons (Freeman et al. 2000). Moreover, the grafted cells did not exhibit any signs of the disease, e.g. nuclear inclusions, in contrast with the host neurons in the surrounding striatum.
Conclusive clinical benefits so far have only been shown in the Creteil clinical trial by Bachoud-Levi et al. (2000b; 2002). The other study, whose clinical data has now been published (Tampa trial; Hauser et al. 2002), was inconclusive. This was possibly due to the type of patients included or the fact that it allowed too short a follow-up time (Peschanski and Dunnett 2002). In Creteil, an improvement in motor, cognitive and functional abilities became apparent only at about twelve months in three of five HD patients, and remained so in the subsequent two years (Bachoud-Levi et al. 2000b). These clinical results matched with the reduction of both the striatal and the frontal hypometabolism as measured by positron emission tomography using 18F-fluorodeoxyglucose (Gaura et al. 2004). In a fourth patient, this improvement was transient, starting around nine to ten months after a first right-side unilateral graft, and lasted up to five months after a second left-sided graft. In this patient, the secondary loss of all improvements coincided with the disappearance of the grafted tissue as evaluated with MRI (Bachoud-Lévi et al. 2002), indicating a link between graft survival and clinical benefits. In the fifth patient, the graft was never active for reasons that remain unknown, and MRI scans still shows declining signals for the striatal metabolic activity (Bachoud-Lévi et al. 2002). Therefore, despite the absence of a control group, the coincidence of results acquired in various domains (clinical, images, electrophysiology) and analysed blindly, strongly point to the efficacy of neurografting. The positive treatment result in a very small population of HD patients obtained in a single centre trial initiated in 2001, initiated a large, controlled randomised trial on 60 patients at the early stages of HD in France and Belgium (Multicenter Intracerebral Grafting in HD, MIG-HD). For control purposes, and to avoid the use of sham surgery, 30 patients randomly received transplants either after 13-14 months or after 33-34 months, with a follow-up of all patients towards 52 months. Currently, this strategy is being replicated in a separate study in which the results of grafts conducted in Belgium, Germany, Switzerland and Italy will be compared with a large cohort of non-treated patients in the UK. Therefore, the efficacy of fetal neural grafts as putative therapy for HD will be fully known in the next three to five years.
The sustainability of the positive effect resulting from grafts is currently being assessed in the patients from the Créteil's pilot study (Bachoud-Lévi et al. 2006). The gene defect is still present and the patients' condition is expected to deteriorate at some point in the future. This secondary deterioration has appeared heterogeneous so far, starting at 4-5 years in the case of motor symptoms and after 6 years in the case of cognitive functions. Thus, the potential therapeutic effects of fetal striatal grafts possibly fade away due to a process of remission. This indicates that a neuroprotective treatment of the graft is needed as an unavoidable complement to the initial surgery. However, the graft will remain the only therapy able to restore lost functions and, therefore, will be indicated in patients exhibiting the symptoms of HD.
A number of experimental studies conducted on animals affected by HD striatal lesions have demonstrated that various neurotrophic factors can provide neuroprotection. Among these factors CNTF appeared to offer the most effective protection. However, the short half life of the CNTF in plasma, its inability to cross the blood brain barrier and its severe side effects (inflammation, cachexia) in a phase I/III clinical trial in patients with ALS (Cedar-baum et al. 1995), precludes its systemic administration. Following the positive results of striatal protection in rats and non-human primates using CNTF-delivering mini-pumps (Anderson et al. 1996) or gene therapy approaches (Emerich et al. 1996; 1997; Mittoux et al. 2000; 2002), an intraventricular implant of encapsulated CNTF-producing cells was chosen for a phase I trial (Bachoud-Levi et al. 2000a). Cells were taken from a baby hamster cell line engineered to synthesise and release large amounts of CNTF which were subsequently introduced into semi-permeable tubes with pores i) permitting CNTF and all nutrients to cross the membrane, and ii) excluding larger proteins (e.g. antibodies) and cell processes to traverse. The cell encapsulation method has the advantage that in the clinical situation it immuno-isolates the cells, whereas removal of the device can stop the treatment whenever needed. The capsule was inserted into the lateral ventricle of six HD patients using stereotactic neurosurgery and was retrieved and exchanged every six months during a two year period. Little, if any, 18F-fluo-rdeoxyglucose-determined metabolic change was observed in the ipsilateral striatum, but significant recovery of normal electrophysiological values was associated with active CNTF-releasing tubes in three patients (Bloch et al. 2004). There were no adverse effects related to the procedure. However, secondary adverse effects (mainly depression) related to the interruption of the procedure were observed a few months after the extraction of the last tube, showing the symbolic and emotional aspect of such therapy.
2.4.3 Alzheimer's Disease
AD is a neurodegenerative disease associated with the formation of tangles and plaques in the brain, resulting in neuronal atrophy leading first to mild forgetfulness (which can be confused with age-related memory change) and an inability to solve simple mathematical problems and followed later by severe cognitive deficits and problems in speaking, understanding, reading and writing. In the final stages of the disease, patients often exhibit anxious-ness or aggressiveness and become in need of total care. The precise cellular or molecular origin of the disease is not known, so that there is no clearly definable "point of attack" at which to fight the cause of the disease nor its progress. Yet certain symptoms can be traced back to changes in particular brain nuclei. For instance, cholinergic neurons of the basal forebrain atrophy and die in the brains of those affected with AD. This process has been correlated with attention deficits and an overall cognitive decline. As the application of NGF in this area has shown to protect cholinergic cell loss following their axotomy (cf. Lad et al. 2003), the chronic delivery of NGF in the human basal forebrain to reduce, or prevent, the loss of cholinergic nerve cells could possibly result in the relief of these symptoms (Tuszynski 2002).
In order to investigate the effect of NGF in AD patients, local application is needed as infusion of NGF in the ventricles of the brain results in intolerable side effects. For instance, rats and monkeys undergoing cholinergic cell rescue procedures using NGF lost their appetite leading to severe weight losses, and this was also observed in a clinical trial of NGF infusion involving three AD patients (which had to be stopped because of painful side effects were experienced by the patients; Eriksdotter Jonhagen et al. ). These negative effects were not observed in subsequent studies in which autolo-gous NGF-secreting cells were implanted into the cholinergic basal forebrain of aged monkeys in which a substantial reversal of age-related neuronal atrophy was achieved (Tuszynski et al. 1998; Tuszynski and Blesch 2004). This had led to a phase I clinical trial of ex vivo NGF gene delivery through the implantation of transduced fibroblasts isolated from the skin of the AD patient, grown and transduced ex vivo (Tuszynski et al. 2005). In this study, after a mean follow-up of 22 months in six AD subjects, no long-term adverse effects resulting from the NGF occurred. Preliminary outcomes showed the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent to be improved suggesting cognitive decline have decelerated. Serial PET scans showed significant increases in cortical 18F-fluorodeoxyglucose after treatment, indicating a return of brain activity at pre-disease stages. The brain autopsy from one subject suggested robust neurite growth responses to NGF.
Amyotrophic lateral sclerosis (ALS) causes the progressive degeneration of motoneurons of the CNS. If the motor neurons die, the ability of the brain to initiate and control muscle movement is lost. The course of the symptoms starts with muscle weakness in one or more muscles of the hands, arms, legs or of the muscles involved in speech, swallowing or breathing. This then develops into twitching and a cramping of muscles, impairment in the use of the arms and legs (paralysis), "thick speech" and, in advanced stages, difficulty in breathing and swallowing, eventually leading to death. Yet, for the vast majority of ALS patients, their minds remain unaffected throughout. Currently there is no treatment for this disorder starting with loss of function of the motoneurons in the spinal cord.
Ciliary neurotrophic factor (CNTF) has been shown to protect motoneu-rons from deterioration. Thus, subsequent patient studies with this peptide were initiated. Systemic delivery of hCNTF in ALS patients, however, had no beneficial effect on the primary (limb strength and pulmonary function) or secondary end points (individual function tests and activities-of-daily-living outcome measures and survival; Miller et al. 1996), but has been frustrated by peripheral side effects, as well as the molecule's short half life, and its inability to cross the blood-brain barrier. Aebischer and his collaborators (Sagot et al. 1995; Tan et al. 1996) have conducted experiments in mice with symptoms of ALS which showed that encapsulated baby hamster kidney cells, genetically engineered to make CNTF and placed intracerebroventri-cally or intrathecally, also reduced the degeneration of these neurons. Trials in human ALS patients in 1996, using the same procedure described above in the treatment of HD but involving the implantation of the CNTF-releasing tube intrathecally in the lumbar CNS (Aebischer et al. 1996; Zurn et al.
2000), showed no evidence that the CNTF alleviated motor neuron deterioration (P. Aebischer, personal communication). One of the reasons for this could be that insufficient amounts of CNTF were released or that the intervention was too late to be of use (Schorr et al. 1996).
Huang et al. reported significant improvements in ALS patients after the implantation of human fetal olfactory ensheathing glia cells in the motor cortex of the brain at an international conference in 2004. However, the rationale of this surgery, advertised as a therapy to both prolong the life span and improve the quality of life of patients, is not based on animal experimentation, and the study has still not been published in a peer-reviewed journal in which the methods they employed would be evaluated in detail.
Multiple sclerosis (MS) is an unpredictable, chronic disease of the CNS, whose symptoms can range from the relatively benign to the somewhat and potentially devastating. Pathologically, MS is characterised by the presence of areas of demyelination and predominant T-cell perivascular inflammation in the brain white matter, which disrupts efficient communication between the brain and other parts of the body. MS is believed to be an autoimmune disease that attacks the nerve-insulating myelin. Common symptoms of MS include fatigue, weakness, spasticity, balance problems, bladder and bowel problems, numbness, loss of vision, tremors and depression. Symptoms are determined by the location of the lesion and thus not all symptoms affect all MS patients. Symptoms may be continuous or may be sporadic. These periods of remission may be complete, leaving no residual damage or leaving only partial permanent impairment. A variety of medication can be used to treat the disease symptomatically, but there is, as yet, no cure for the demyelination in MS. New therapies, therefore, need to aim at reducing specific autoimmune responses and to assist in remyelination. It is the latter goal in which neurotransplantation may have potential.
Animal studies have shown remyelination processes following cellular therapies in experimental demyelination (Kocsis et al. 2002). The use of Schwann cells, glial cells that normally insulate axons in the PNS, were found to remyelinate fibers in the CNS of rats and reinstate message transmission (Kohama et al. 2001; Bachelin et al. 2005). In a 2001 pilot study Tomothy Vollmer and co-workers (http://www.myelin.org/schwannupdate.htm, accessed on December 7th, 2006) transplanted autologous Schwann cells in three patients with MS and found that the technique was safe. Further studies are needed to determine whether the cells can also repair myelin and aid functional improvement in patients. Other cells for remyelination are olfactory ensheathing cells that inhabit the nose but can also make myelin (Franklin et al. 1996; Lakatos et al. 2003) and neural stem cells, which assist to stimulate remyelination by endogenous oligodendrocyte precursor cells or mature themselves into oligodendrocytes and subsequently produce myelin in the CNS (Totoiu et al. 2004; Copray et al. 2006). In one recent study scientists found that in mice with an MS-like disease, transplants of stem cells travelled to multiple areas of damage and matured into myelin-forming cells. Animals undergoing such transplantation showed a decrease in myelin damage and nerve fiber destruction. Some animals also regained lost movement in their legs or tails (Pluchino et al. 2003; Pluchino and Matino 2005).
A large clinical trial using autologous SSCs from bone marrow or blood as peripheral implants not as brain implants, combined with high-dose immunosuppression, revealed slight neurological improvements in 21% of the MS patients and a stabilisation of the clinical condition in approximately 70% of the patients trialed by completely abrogating the inflammatory process in the brain as evidenced in magnetic resonance imaging (Fassas et al. 2002). However, the procedure is associated with a transplant-related mortality risk of around 3% to 8%. Therefore, it cannot be recommended for the treatment of a chronic, non-lethal disease like MS. However, the systemic or peripheral approach of cellular treatment has the advantage that the skull need not be opened up for surgery. On the other hand, a direct approach of the MS lesion area for any type of therapy may be more effective and reduce the chances of side effects due to maladaptive myelination in uninjured parts of the brain.
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