INVITED COMMENTARY: from The Harvard Mahoney Neuroscience Institute Letter

Volume 3 Number 1 1994. Copyright The Fellows of Harvard College

by Ole Isacson, M.D.-Ph.D.
from the Neuroregeneration Laboratory

• Can we stop or halt the progression of nerve cell death in neurodegenerative diseases?
• Grafting adrenal medulla or genetically engineered cells .
• Are aborted human fetal cells needed for nerve cell transplantation?
• References on transplantation therapy for Parkinson's disease
• For a video on the transplantation of fetal pig cells into people with neurodegenerative diseases [1.1 Mb .mov format, courtesy of ABC News].

There are billions of nerve cells in a human brain. In neurodegenerative disorders, such as Parkinson's and Huntington's disease, selective loss of some 500,000 cells in critical brain regions can lead to devastating symptoms. Nerve cell death in these diseases occurs over years or decades and results in specific signs and symptoms, such as lack of movements (in Parkinson's) or excess movements (in Huntington's disease). Although several theories have been presented for the causes of neurodegenerative disease, the exact pathological mechanisms involved are not known.

Treatment alternatives are few and limited in effect and duration. Neuroscientists and neurologists working in this field have attempted to replace neurotransmitters lost in the disease process by pharmacological treatments. L-dopa or related agents bring relief to many Parkinsonian patients, but L-dopa becomes ineffective over time and debilitating side-effects develop with prolonged use. No equivalent drug alternative exists for patients with Huntington's disease.

Because standard therapies for these patients are largely ineffective, alternative strategies are being developed. Intense research efforts are directed towards drugs that may block nerve cell death and novel cell-based therapies, which replace defective nerve cells.

Can we stop or halt the progression of nerve cell death in neurodegenerative diseases?

Since we do not know the exact way in which specific nerve cell groups die in the brains of Parkinson or Huntington patients, it is difficult to devise successful strategies to treat these slowly developing diseases. However, we have hints of the general mechanisms involved in the neuronal cell death.

Toxins, either produced within the body or introduced from outside, may be involved. In Parkinson's disease it was recently suggested that the initiation of L-dopa therapy could be delayed for a year or so if the patients were treated with the inhibitor of the enzyme called Mono-Amine-Oxidase B (MAO-B) (Deprenyl). The idea for this treatment came from observations of people who developed a form of Parkinson's disease after illicit use of a drug called MPTP. MPTP had caused selective degeneration of a set of neurons in a small region of the mid-brain, neurons--called Ôdopaminergic'--that produce the neurotransmitter dopamine. This type of nerve cell death is also seen in most forms of Parkinson's disease.

Because MAO-B inhibitors can prevent the formation of toxic compounds from MPTP in the brain, it was reasoned that, if similar substances were responsible for injuring nerve cells in the more common forms of Parkinson's disease, then MAO-B inhibition could be beneficial. Initial, highly publicized clinical trials suggested that MAO-B inhibitors could slow down the progression of the disease, but more extensive clinical trials suggest that these changes are probably due to effects other than prevention of dopaminergic nerve cell death.

While one can be hopeful about future treatments to prevent neuro-degenerative diseases, current drug therapies or neuroprotective methods do not provide sufficient help to patients. Another rational approach to treat Parkinson's disease is therefore to replace damaged brain cells with new functional cells and thereby compensate for the loss of essential nerve cell groups.

Such cell transplantation to the brain is a relatively novel therapeutic method (compared to drugs) and pre-clinical attempts have been made to replace missing neurotransmitters with cells capable of producing dopamine from a gland above the kidney, the adrenal medulla, fetal neurons or genetically engineered cells.

In clinical trials, the difficulty lies not only in choosing the best cellular replacement for the lost neurons, but is further complicated by ethical controversies and profound technical problems associated with potential cell sources such as human fetal cells or genetically engineered cells.

Grafting adrenal medulla or genetically engineered cells.

Each cell source used for transplantation has advantages and disadvantages. A cell source that initially looked somewhat promising for cell replacement therapy in Parkinson's disease was adrenal medulla cell implants (grafts). As the patient's own adrenal medulla could be used, this transplantation procedure circumvented many donor issues. The therapeutic idea came from the belief that sufficient amounts of dopamine could be produced by such intracerebral grafts to compensate for the loss of this neurotransmitter in Parkinson patients.

The first small clinical study (4 patients) by an experienced research team showed only modest improvements in the implanted patients. However, this was followed by uncontrolled clinical studies outside the U.S., claiming that Parkinson patients had been cured by adrenal medulla grafts. These claims were subsequently determined to be wildly exaggerated, later clinical trials in the U.S. and elsewhere have failed to show any systematic or lasting benefit from adrenal medulla grafts and no evidence exists that such grafts can produce sufficient amounts of dopamine to overcome the deficit produced by the disease.

Furthermore, brain circuitry works mainly by nerve cells with fibers contacting other nerve cells at fine multiple sites called synapses. Synapses provide conditions of specific communication between nerve cells and also allow for control of neurotransmitter release and levels. Implantation of adrenal medulla cells cannot achieve these conditions since synapses are not created to any significant degree by these cells.

Using existing techniques in molecular biology we can insert genes into some types of cells. These novel methods can be employed to generate cell sources that release dopamine. Such cells are advantageous compared to adrenal medulla cells because they can be made to produce large amounts of dopamine. However, available genetically engineered cells are non-neuronal and therefore lack the ability to form synapses.

If dopamine producing neuronal cells are constructed that can give rise to synapses and function optimally in existing host brain circuitry, such genetically engineered cells may also become clinically useful. Currently, the only way to obtain such brain circuitry reconstruction in animal experiments, and probably in patients, is through the use of fetal neurons.

Are aborted human fetal cells needed for nerve cell transplantation?

Numerous animal experiments over the last decade provide evidence that implanted fetal neurons can replace dead host neurons, form effective synapses with host neurons and produce necessary neurotransmitters. In this way, minute amounts of implanted immature rat, mouse, pig or human dopamine cells have been shown to grow in rat animal models of Parkinson's disease and effectively reduce most symptoms. The fetal nerve cells can be injected into a desired brain location as a liquid containing cells. The number of implanted dopaminergic cell needed for recovery of movement represents only about one- tenth of a million of the total number of nerve cells in the brain.

Over the last 4 years, studies with implantation of human fetal dopaminergic cells into patients suffering from Parkinson's disease indicate that patients can improve substantially by this treatment. However, the experience so far is limited to a few patients, and the pioneering clinical teams differ markedly in their success and transplantation methods used.

The controversy regarding this method to treat Parkinson's disease is mainly an ethical debate about abortion of human fetuses. This linkage with the abortion issue is unfortunate since the use of human cells is probably not necessary, or even desirable, for applying this transplantation method to patients.

First, development of a major medical treatment that will rely on the event, or availability, of aborted human fetal donor tissue is undesirable. Second, the use of human fetal tissue may be associated with infection risks to the patients with implants. Third, techniques for coordinating and handling aborted human fetal brain tissue have proved to be difficult, and may not be provide a large enough number of surviving dopamine cells for patients to recover from the disease.

To overcome these problems, fetal cells from non-human fetuses (such as porcine) or other biotechnology derived nerve cells can likely be developed as safe and effective alternative cell sources for transplantation to patients with neurodegenerative diseases.

Dr. Isacson is Associate Professor in the Program in Neuroscience at Harvard Medical School and Massachusetts General Hospital and Director of the Neuroregeneration Laboratory at McLean Hospital, Belmont, MA 02178. This article was prepared as an invited commentary on controverisies in transplantation for Parkinson's diease.

Bibliography on Transplantation for Parkinson's Disease

AN 95089583

AU Kupsch A. Oertel WH.

IN Klinikum Grosshadern, Neurological Unit, Ludwig-Maximilians-University, Munchen, FRG.

TI Neural transplantation, trophic factors and Parkinson's disease. [Review] SO Life Sciences. 55(25-26):2083-95, 1994.

AB Part 1 of this update on new restorative therapeutic strategies against Parkinsons's disease focuses on transplantation of dopamine-secreting tissue. Special emphasis is put on clinical trials with fetal mesencephalic cells. Problems and potential alternative approaches are discussed. Part 2 emphasizes progress in the related field of neurotrophic factors for dopaminergic midbrain neurons. [References: 78]

AN 95066035

AU Aminoff MJ.

IN Department of Neurology, University of California, San Francisco, School of Medicine 94143-0114.

TI Treatment of Parkinson's disease. [Review]

SO Western Journal of Medicine. 161(3):303-8, 1994 Sep.

AB Pharmacotherapy with levodopa for Parkinson's disease provides symptomatic benefit, but fluctuations in (or loss of) response may eventually occur. Dopamine agonists are also helpful and, when taken with low doses of levodopa, often provide sustained benefit with fewer side effects; novel agonists and new methods for their administration are therefore under study. Other therapeutic strategies are being explored, including the use of type B monoamine oxidase inhibitors to reduce the metabolic breakdown of dopamine, catechol-O-methyltransferase inhibitors to retard the breakdown of levodopa, norepinephrine precursors to compensate for deficiency of this neurotransmitter, glutamate antagonists to counteract the effects of the subthalamic nucleus, and various neurotrophic factors to influence dopaminergic nigrostriatal cells. Surgical procedures involving pallidotomy are sometimes helpful. Those involving cerebral transplantation of adrenal medullary or fetal mesencephalic tissue have yielded mixed results; benefits may relate to the presence of growth factors in the transplanted tissue. The transplantation of genetically engineered cell lines will probably become the optimal transplantation procedure. The cause of Parkinson's disease may relate to oxidant stress and the generation of free radicals. It is not clear whether treatment with selegiline hydrochloride (a type B monoamine oxidase inhibitor) delays the progression of Parkinson's disease, because the drug also exerts a mild symptomatic effect. Daily treatment with vitamin E (a scavenger of free radicals) does not influence disease progression, perhaps because of limited penetration into the brain. [References: 57]

AN 95007800

AU Koutouzis TK. Emerich DF. Borlongan CV. Freeman TB. Cahill DW. Sanberg PR.

IN Department of Surgery, University of South Florida College of Medicine, Tampa 33612.

TI Cell transplantation for central nervous system disorders. [Review] SO Critical Reviews in Neurobiology. 8(3):125-62, 1994.

AB Initially, the specific aim of transplantation studies was to investigate the regenerative capabilities of the mammalian nervous system. From this underlying impetus, a myriad of knowledge, spanning from molecular biology to neurobiology, has enhanced our understanding of regeneration and the applicability of fetal tissue transplantation in treating various neurodegenerative diseases. Current evidence suggests that transplantation of fetal neural tissue ameliorates the neurobiological and behavioral changes observed in animal models of central nervous system (CNS) disorders. In light of numerous basic science studies, clinical trials have begun to evaluate the potential of neural transplantation in treating human diseases. Indeed, modest progress has been reported in the treatment of Parkinson's disease. However, whereas fetal tissue transplantation has reached considerable success, it has also been observed to produce either no beneficial effects, magnify existing behavioral abnormalities, or even produce a unique constellation of deficits. Thus, while the prospects are promising, further investigations aimed at improving and refining existing transplantation paradigms are warranted before neural transplantation techniques can be of widespread value. This review article attempts to provide an overview of the neuroanatomical, neurochemical, and behavioral effects produced by transplanted fetal tissue in several animal models of CNS disorders. We have attempted to present both positive and adverse effects and to critically analyze the suitability of neural transplantation as a therapy for the various neurological disorders. In addition, alternative approaches, including the use of encapsulated neural tissue implants and genetically engineered cell lines along with their clinical potential, are discussed when appropriate. [References: 275]

AN 93283757

AU Widner H. Rehncrona S.

IN Department of Neurology, University Hospital, Lund, Sweden. TI Transplantation and surgical treatment of parkinsonian syndromes. [Review]

SO Current Opinion in Neurology & Neurosurgery. 6(3):344-9, 1993 Jun.

AB Neurosurgical attempts to correct parkinsonism use strategies aimed either at alleviating the underlying dopamine deficiency or at correcting abnormal compensatory effects in neural circuits within the basal ganglia. During the review period, clinical trials of four different neurosurgical approaches were reported. These approaches are intracerebral transplantation of fetal dopamine neurons, intracerebral transplantation of adrenal medullary tissue, tremor-reducing surgical lesions in the ventrolateral thalamus, and ventroposterior pallidotomy aimed at reducing akinesia and rigidity. Experimental studies in rats and monkeys designed to explore mechanisms of graft actions were also reported. [References: 33]

AN 93063066

AU Widner H. Tetrud J. Rehncrona S. Snow B. Brundin P. Gustavii B. Bjorklund A. Lindvall O. Langston JW.

IN Department of Neurology, University Hospital, Lund, Sweden. TI Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [see comments].

SO New England Journal of Medicine. 327(22):1556-63, 1992 Nov 26. AB BACKGROUND. Intracerebral transplantation of fetal dopaminergic neurons is a promising new approach for the treatment of Parkinson's disease. Patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have a relatively stable lesion limited to the nigrostriatal system, rendering them ideal candidates for transplantation. Improvement of motor function after neural grafting has previously been observed in nonhuman primates with MPTP-induced parkinsonism. METHODS. We grafted human fetal tissue from the ventral mesencephalon (obtained six to eight weeks after conception) bilaterally to the caudate and putamen in two immunosuppressed patients with severe MPTP-induced parkinsonism, using a stereotaxic technique. The patients were assessed regularly with clinical rating scales, timed tests of motor performance, and [18F]fluorodopa positron-emission tomography during the 18 months before the operation and the 22 to 24 months after the operation. RESULTS. Both patients had substantial, sustained improvement in motor function and became much more independent. Postoperatively, the second patient's maintenance dose of levodopa was decreased to 150 mg daily, which was 30 percent of the original dose. Striatal uptake of fluorodopa was unchanged 5 to 6 months postoperatively but was markedly and bilaterally increased at 12 to 13 and 22 to 24 months in both patients, closely paralleling the patients' clinical improvement. There were no serious complications. CONCLUSIONS. Bilateral implantation of fetal mesencephalic tissue can induce substantial long-term functional improvement in patients with parkinsonism and severe dopamine depletion and is accompanied by increased uptake of fluorodopa by the striatum. The results in these patients resemble those obtained in MPTP-treated primates and suggest that this will be a useful model for the assessment of transplantation therapies in Parkinson's disease.

AN 93063065

AU Freed CR. Breeze RE. Rosenberg NL. Schneck SA. Kriek E. Qi JX. Lone

Zhang YB. Snyder JA. Wells TH. et al.

IN University of Colorado Health Sciences Center, Denver 80262. TI Survival of implanted fetal dopamine cells and neurologic improvement 12 to 46 months after transplantation for Parkinson's disease [see comments].

SO New England Journal of Medicine. 327(22):1549-55, 1992 Nov 26.

AB BACKGROUND AND METHODS. Patients with Parkinson's disease tend to have a reduced response to levodopa after 5 to 20 years of therapy, with "on-off" fluctuations consisting of dyskinesia alternating with immobility. In an effort to modify the motor disability of advanced Parkinson's disease, we implanted embryonic mesencephalic tissue containing dopamine cells into the caudate and putamen of seven patients. Two patients received unilateral grafts in the caudate and the putamen on the side opposite the side with worse symptoms. Five patients received bilateral grafts implanted in the putamen only. In six of the seven patients, the fetal tissue was obtained from a single embryo with a gestational age of seven to eight weeks. The tissue was injected by means of 10 to 14 needle passes. There were no surgical complications. Four of the seven patients underwent immunosuppression with cyclosporine and prednisone. RESULTS. All patients reported improvement according to the Activities of Daily Living Scale when in the on state 3 to 12 months after surgery (P < 0.01). Neurologic examination according to the Unified Disease Rating Scale showed that five of the seven patients improved when in the on state six months after surgery. The mean group Hoehn-Yahr score improved from 3.71 to 2.50 (P < 0.01). Computer and videotape testing in the home supported these findings. Fluctuations in clinical state were moderated, and periods of dyskinesia and off episodes were shorter and less severe than before implantation. Drug doses were reduced by an average of 39 percent (P < 0.01; maximum, 58 percent). The results of cliniical evaluation and fluorodop positron-emission tomography in one patient were compatible with transplant survival for as long as 46 months. Both immunosuppressed and nonimmunosuppressed patients improved. CONCLUSIONS. Fetal-tissue implants appear to offer long-term clinical benefit to some patients with advanced Parkinson's disease.

AN 93063064

AU Spencer DD. Robbins RJ. Naftolin F. Marek KL. Vollmer T. Leranth C. Roth RH. Price LH. Gjedde A. Bunney BS. et al.

IN Neural Transplant Program, Yale University School of Medicine, New Haven, Conn. 06510.

TI Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson's disease [see comments].

SO New England Journal of Medicine. 327(22):1541-8, 1992 Nov 26. AB BACKGROUND. Parkinson's disease is characterized by the loss of midbrain

dopamine neurons that innervate the caudate and the putamen. Studies in animals suggest that fetal dopaminergic neurons can survive transplantation and restore neurologic function. This report compares the clinical results in four case patients with severe Parkinson's disease who underwent stereotaxic implantation of human fetal ventral mesencephalic tissue in one caudate nucleus with the results in a control group of similar subjects assigned at random to a one-year delay in surgery. METHODS. Each case patient received cryopreserved tissue from one fetal cadaver (gestational age, 7 to 11 weeks). Before implantation, adjacent midbrain tissue underwent microbiologic, biochemical, and viability testing. Cyclosporine was administered for six months postoperatively. RESULTS. The procedure was well tolerated. Three case patients showed bilateral improvement on motor tasks, as assessed on videotape, and were more functional in the activities of daily living, as assessed by themselves and neurologists, during both optimal drug therapy and "drug holiday" periods. One case patient, who died after four months from continued disease progression, had striatonigral degeneration at autopsy. In the patients who received transplants, optimal control was achieved with a lower dose of antiparkinsonian medications, whereas the controls required more medication. Positron-emission tomography with [18F]fluorodopa before and after surgery in one patient revealed a bilateral restoration of caudate dopamine synthesis to the range of normal controls, but continued bilateral deficits in the putamen. CONCLUSIONS. Although the case patients continued to be disabled by their disease, unilateral intracaudate grafts of fetal tissue containing dopamine diminished the symptoms and signs of parkinsonism during 18 months of evaluation.

AN 93044295

AU Bjorklund A.

IN Department of Medical Research, University of Lund, Sweden. TI Dopaminergic transplants in experimental parkinsonism: cellular mechanisms of graft-induced functional recovery. [Review]

SO Current Opinion in Neurobiology. 2(5):683-9, 1992 Oct.

AB The ability of intrastriatal grafts of fetal mesencephalic dopamine neurons to ameliorate the symptoms of experimental and clinical parkinsonism has raised the question of the mechanisms underlying the transplant-induced functional effects. Recent studies have taken advantage of quantitative cytochemical and in situ hybridization techniques to study functional graft-host interactions at the cellular level in the rat Parkinson model. The results provide evidence that behaviorally functional grafts restore dopaminergic neurotransmission and normalize dopamine receptor function in the denervated striatum, and that these effects are likely to depend on both synaptic and extrasynaptic mechanisms. [References: 51]

AN 92246461

AU Sawle GV. Bloomfield PM. Bjorklund A. Brooks DJ. Brundin P. Leenders KL. Lindvall O. Marsden CD. Rehncrona S. Widner H. et al.

IN Medical Research Council Cyclotron Unit, Hammersmith Hospital, London, England.

TI Transplantation of fetal dopamine neurons in Parkinson's disease: PET [18F]6-L-fluorodopa studies in two patients with putaminal implants.

SO Annals of Neurology. 31(2):166-73, 1992 Feb.

AB Two patients with Parkinson's disease who underwent implantation of fetal mesencephalic tissue into the putamen were serially studied using positron emission tomography and [18F]6-L-fluorodopa ([18F]dopa). The uptake of [18F]dopa is related to the functional integrity of the presynaptic dopaminergic system. Preoperative studies revealed a marked decrease in putamen [18F]dopa uptake, with lesser involvement of the caudate. Two and 4 months, respectively, after operation, both patients demonstrated functional improvement, as described elsewhere. One patient was scanned 5, 8, and 13 months after the operation and the other was scanned 7 and 12 months after the operation. In both patients, [18F]dopa uptake increased within the operated putamen despite a progressive decrease in tracer uptake in the unoperated striatal structures. We believe that this increased uptake of [18F]dopa at the implantation site represents functional integrity within a surviving neural graft. While there has been little further clinical improvement beyond the fifth postoperative month, the uptake of [18F]dopa at the operation site in both patients has progressively increased. The kinetic data provide evidence of disease progression in the unoperated striatum, which, balanced against increasing graft function, may explain why clinical improvement reached a plateau within months after surgery.

AU Lindvall O. Widner H. Rehncrona S. Brundin P. Odin P. Gustavii B. Frackowiak R. Leenders KL. Sawle G. Rothwell JC. et al.

IN Department of Neurology, University Hospital, Lund, Sweden. TI Transplantation of fetal dopamine neurons in Parkinson's disease: one-year

clinical and neurophysiological observations in two patients with putaminal implants.

SO Annals of Neurology. 31(2):155-65, 1992 Feb.

AB Ventral mesencephalic tissue from aborted human fetuses (age, 6-7 weeks' postconception) was implanted unilaterally into the putamen using stereotaxic surgery in 2 immunosuppressed patients (Patients 3 and 4 in our series) with advanced idiopathic Parkinson's disease. Tissue from 4 fetuses was grafted to each patient. Compared with our previous 2 patients, the following changes in the grafting procedure were introduced: the implantation instrument was thinner, more tissue was placed in the operated structure, and the time between abortion and grafting was shorter. There were no postoperative complications. Both patients showed a gradual and significant amelioration of parkinsonian symptoms (most marked in Patient 3) starting at 6 and 12 weeks after grafting, respectively, reaching maximum stability at approximately 4 to 5 months; patients remained relatively stable thereafter during the 1-year follow-up period. Clinical improvement was observed as a reduction of the time spent in the "off" phase and the number of daily "off" periods; a lessening of bradykinesia and rigidity during the "off" phase, mainly but not solely on the side contralateral to the graft; and a prolongation and change in the pattern of the effect of a single dose of L-dopa. Neurophysiological measurements revealed a more rapid performance of simple and complex arm and hand movements bilaterally, but primarily contralateral to the graft. The results indicate that patients with Parkinson's disease can show significant and sustained improvement of motor function after intrastriatal implantation of fetal dopamine-rich mesencephalic tissue. The accompanying paper by Sawle and colleagues describes the results of repeated positron emission tomography scans in these patients.

AN 89272713

AU Lindvall O. Rehncrona S. Brundin P. Gustavii B. Astedt B. Widner H. Lindholm T. Bjorklund A. Leenders KL. Rothwell JC. et al.

IN Department of Neurology, University of Lund, Sweden.

TI Human fetal dopamine neurons grafted into the striatum in two patients with severe Parkinson's disease. A detailed account of methodology and a 6-month follow-up.

SO Archives of Neurology. 46(6):615-31, 1989 Jun.

AB By using stereotaxic surgical techniques, ventral mesencephalic tissues from aborted human fetuses of 8 to 10 weeks' gestational age were implanted unilaterally into the striata in two patients with advanced Parkinson's disease. The patients were treated with a cyclosporine, azathioprine, and steroid regimen to minimize the risk for graft rejection. They were examined for 6 months preoperatively and 6 months postoperatively and continued to receive the same doses of antiparkinsonian medication. There were no significant postoperative complications. No major therapeutic effect from the operation was observed. However, in the clinical tests, both patients showed small but significant increases of movement speed for repeated pronation-supination, fist clenching, and foot lifting. The rate of walking also increased in the one patient tested. For both patients, there was an initial worsening postoperatively, followed by improvement vs preoperative performance at 1 to 3 months. Both patients also showed significant improvement in the magnitude of response to a single dose of levodopa (L-dopa), but there was no increase in the duration of drug action. The motor readiness potential increased in both patients postoperatively, primarily over the operated hemisphere. Neurophysiological measurements also showed a more rapid performance of simple and complex arm and hand movements on the side contralateral to transplantation in one patient at 5 months postoperatively. Positron emission tomography demonstrated no increased uptake of 6-L-(18F)-fluorodopa in the transplanted striatum at 5 and 6 months. Taken together, these results suggest that the fetal nigral implants may have provided a modest improvement in motor function, consistent with the presence of small surviving grafts. Although our results support further scientific experimentation with transplantation in Parkinson's disease, widespread clinical trials with this procedure are probably not warranted at this time.