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  • 1. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE JOURNAL OF MEDICINE Copyright © 2000 by PJD Publications Limited Research Communications Westbury, NY 11590-0966 USA ENHANCEMENT OF NORADRENERGIC NEURAL TRANSMISSION: AN EFFECTIVE THERAPY OF MYASTHENIA GRAVIS A REPORT ON 52 CONSECUTIVE PATIENTS Fuad. Lechin1,2, Bertha van der Dijs1,2, Betty Pardey-Maldonado1, Eduardo Jahn1, Vladimir Jimenez1, Beatriz Orozco1, Scarlet Baez2 and Marcel E. Lechin3 Sections of 1Neuropharmacology and 2Clinical Neurochemistry, Instituto de Medicina Experimental, Universidad Central de Venezuela. 3 Texas A & M University, School of Medicine, College Station, TX, USA Key Words: Myasthenia gravis and Th2 immune disorders, myasthenia gravis treatment, noradrenergic neural transmission, sympathetic activity and autoimmune disorders, serotonin and autoimmune disorders. Subjects: Patients with myasthenia gravis. Abbreviations: f5HT = free-serotonin, LC = locus coeruleus, MG = myasthenia gravis, NA = noradrenergic, SLE = systemic lupus erythematous. Abstract Neurochemical, neuroautonomic and neuropharmacological assessments carried out on all our myasthenia gravis (MG) patients showed that they presented a neural sympathetic deficit plus excessive adrenal-sympathetic activity. These abnormalities were registered during 77
  • 2. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE the basal (supine-resting) state, as well as after several stress tests (orthostasis, exercise, oral glucose and buspirone). In addition, MG patients showed increased levels of free-serotonin (f5HT) in the plasma, supposedly associated with the increased platelet aggregability which we found in all MG patients. As the above trio of neurochemical disorders (low noradrenergic-activity + high adrenergic-activity + increased f-5HT plasma levels) is known to favor Th-1 immunosuppression + Th-2 predominance, we outlined a neuropharmacological strategy for reverting the above neurochemical disorder. This treatment provoked sudden (acute), and late sustained improvements. Acute effects have been attributed to the increase of alpha-1 activity at the spinal motoneuron level. Late improvements always paralleled a significant normalization of immunological disorders. Complete normalization was registered only in non-thymectomized MG patients. Introduction In a preliminary, report we published the successful therapeutic results obtained in eight myasthenia gravis (MG) patients receiving neuropharmacological treatment (Lechin, van der Dijs et al., 1997a). In the present study, we presented the results obtained in a series of 52 consecutive MG patients given the same therapy aimed at enhancing noradrenergic (NA) neural transmission. The rationale of our neuropharmacological approach rests on two fundamental findings: first, all our MG patients showed a neurochemical deficiency of central and peripheral noradrenergic (NA) activity (Lechin, van der Dijs et al., 1985a, 1985b, 1990a, 1990b, 1995a, 1995b, 1996a, 1996b, 1996c, 1996e), and second, those MG patients who had not previously been submitted to surgical and/or chemical immunosuppression all showed an immunological Th-2 disorder, confirming data obtained by many other researchers (Barnard, Mahon et al., 1996; Faxvaag, Espevik et al., 1995; Romagnani, 1996). We decided, therefore, to attempt a two-pronged neuropharmacological therapy addressed to increasing NA neural, but not adrenal, sympathetic activity. This decision was adopted for the following reasons: it has been exhaustively demonstrated that NA neural activity stimulates the immune system, in particular macrophages and Th-1 cytokines, whereas adrenal glands sympathetic activity displays opposite effects and, in addition, is associated with excessive humoral immunity (Th-2 profile) and Th-2 cytokines (Kouassi, Li et al., 1990; Madden, Moynihan et al., 78
  • 3. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE 1994b; Spengler, Allen et al., 1990). Neuropharmacological manipulations were designed to stimulate the locus coeruleus (LC) NA neurons. Further-more, considering that all our MG patients showed high free serotonin in plasma rather than in platelets (raised f-5HT plasma levels), possibly associated with the increased platelet aggregability that all them presented, we assumed that eliminating the well-known Th-1 immunosuppressant effects of f-5HT would aid in reverting the autoimmune disorders underlying MG disease (Fuchs, Campbell et al., 1988; Jackson, Walker et al., 1998; Paegelow, Werner et al., 1985; Sternberg, Wedner et al., 1987; Walker and Codd, 1985; Warren, Kane et al., 1990) also included into the Th-2 autoimmune diseases (MokhtarianShirazian et al., 1993). It has been exhaustively demonstrated that peripheral neural sympathetic activity reflects, and is closely and positively correlated with, central noradrenergic (NA) neural transmission, which is ruled by the locus coeruleus (LC) or A6-NA-neuronal pontine group (Bamshad, Kay Song et al., 1999; Elam, Thoren et al., 1986; Esler, Wallin et al., 1991; Lake, Ziegler et al., 1976; Lambert, Kaye et al., 1998; Thompson Wallin et al., 1998; Ziegler, Lake et al., 1977). During normal basal conditions, adrenal glands supply some 20% to the circulating pool of plasma NA, as expressed by the NA/Ad ratio ≥ 5 (Lechin, van der Dijs et al., 1985a, 1985b, 1990a, 1990b, 1995a, 1995b, 1996a, 1996b, 1996c, 1996e). Thus, increases or decreases of this NA/Ad ratio parallel enhancement or reduction of the contribution by noradrenergic neural transmission to the global sympathetic activity. According to the above, the neuropharmacological strategy we outlined in order to enhance central NA and peripheral neural sympathetic activities was based on three types of central acting drugs: 1) amino acid precursors of NA (Musgrave, Bachmann et al., 1985; Musso, Brenci et al., 1997); 2) drugs which are able to trigger enhancement of the LC-NA neuronal firing as, for instance, alpha2 antagonists (Rodriguez-Manso, 1999; Verwaerde, Tran et al., 1997; Yan, Jobe et al., 1993), and drugs like buspirone that trigger the firing of NA neurons and, in addition, inhibit serotonergic (5-HT) neurons and disinhibit NA neurons from 5HT-bridling axons (Lechin, van der Dijs et al., 1997b, 1998a; Piercey, Smith et al., 1994); 3) drugs which are able to inhibit the re-uptake of NA by axon terminals and thus prolong the action of NA at the synaptic level (desipramine, protryptiline, nortryptiline, doxepin) (Esler, Wallin et al., 1991; Lechin, van der Dijs et al., 1979b; Sugrue, 1983; Yan, Jobe et 79
  • 4. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE al., 1993). Finally, during the last 6 months we frequently prescribed adrafinil or modafinil (alpha-1 agonists) which are very useful substitutes for NA (Anderson and Harvey, 1988; Binder and Powers, 1999; Brustein and Rossignol, 1999; Duteil Rambert et al., 1990; Gerin and Privat, 1998; Jabre and Salzsieder, 1999; Fishman, Feigembaum et al., 1983; Sokoloff, Siegel et al., 1999; Wada, Hasegawa et al., 1997) particularly in some cases where central NA neurons are diminished because of aging. It is very widely known that LC-NA neurons diminish with aging (Arranz, Blennow et al., 1996) and so elder patients may have a limited response capacity to stimulating drugs. Although other pharmacological manipulations would include drugs that reduce free serotonin in the plasma (f-5HT) like tianeptine, (Lechin, van der Dijs et al., 1998b; 1998c) the results obtained with this tool are not presented in this report. In effect, f-5HT has been shown to block muscular ACh-receptors and so it should play some etiopathogenic role in MG disease, not only by suppressing Th-1 immunity but also by interfering with neuromuscular transmission (Grassi, 1999). Patients and Methods Patients The protocol used in this study was approved by the Ethical Committee of Funda-IME) (Fundación de apoyo al Instituto de Medicina Experimental), Universidad Central de Venezuela, Caracas, Venezuela. All patients signed an informed consent. This was an open study carried out on MG patients recruited from subjects arising from neurology departments in hospitals in Venezuela and other countries. All these patients were referred to our institute because they either did not obtain significant improvement or show progressive impairment. Up to the present, we have enrolled 61 MG patients. However, in this study we included 52 patients only. The remaining nine patients started our therapeutic trial but did not follow it for various reasons: two because of emergency hospitalization early in the trial; five out-of-town patients because of financial difficulties (due to travel expenses despite the fact that our trial, blood tests and drugs were free of charge); and two patients who only attended our institute once. Six patients had received prior treatment in other countries (Columbia Presbyterian Hospital in New York, Jackson Memorial Hospital in Miami, John's Hopkins Hospital in Baltimore, Posbus Hospital in the Republic of South Africa, Mexico City Hospital and the San José Hospital in Costa Rica). Forty-six patients 80
  • 5. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE were diagnosed and previously treated in Venezuela: Hospital Universitario in Caracas (24), Hospital Vargas in Caracas (4), Hospital Ruiz y Paez in Ciudad Bolivar (7), Hospital Central in Valencia (4), Hospital Universitario in Maracaibo (2), Hospital Guerra Méndez in Valencia (2), Hospital Militar in Caracas (3). All patients were diagnosed according to clinical features, electromyography findings and edrophonium testing. Data referring to clinical features are shown in Tables I to V. Methods Neurochemical Investigation Plasma neurotransmitters were dosified during basal (supine- resting) condition and after several stress tests: orthostasis, exercise (Lechin, van der Dijs et al. 1995a, 1995b, 1996b, 1996d, 1996e, 1997c), oral glucose (Lechin, van der Dijs et al., 1991, 1992, 1993), and buspirone (Lechin, van der Dijs et al., 1997b, 1998a) according to procedures explained in previously published papers. Blood samples for immunological investigations were processed and transferred to Laboratorios BIOCELL 220, C.A (Caracas, Venezuela) and for some special tests to SPECIALTY LABORATORIES, (Michigan Av., Santa Monica, CA, USA). These routine investigations included: antinuclear antibodies (ANA), cellular immune dysfunction evaluation, CFIDS, AH5O, CH5O, dsDNA autoantibodies, F-actin autoantibodies, humoral immune evaluation, NK cell evaluation, platelet auto-antibodies, Scl-70 autoantibodies, AChR autoantibodies, striational autoantibodies, anti- JO-1, C3/C4, and lymphocyte subpopulations (CD3, CD4, CD5, CD8, CD19, CD2O, CD45RA, CD45RO), immune-globulins (IgA, IgG, IgM, IgE). Clinical Assessment We modified the MG scoring system by Osserman and Genkins, (1966) by inverting average muscle scoring (AMS) (Mantegazza Antozzi et al., 1988; Mendell and Florence, 1990) which provided the numerical score for muscular strength (ocular, facial, abdominal and extremity muscles), and chewing, swallowing, phonation and respiration. Electromyographical parameters plus clinical ratings are expressed together. Scores were verified fortnightly and graded in percentages. 81
  • 6. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE Neurophamacological Therapy Restoration of central and peripheral NA (alpha-adrenergic) activity: a) Aminoacid precursors (L-phenylalanine, L-tyrosine), 50 mg before breakfast. b) Inhibitor of NA re-uptake (desipramine), 25 mg before breakfast. c) NA-releasing agent + suppressor of serotonergic activity (buspirone) l0-20 mg at 10 am + beta-adrenergic blocking agent (propranolol 10-20 mg). d) Alpha-1 agonists: adrafinil (150-300 mg) or modafinil (100-200 mg) at 10 a.m. Because this noradrenergic (alpha-adrenergic) activation usually induces insomnia, the treatment was not administered in afternoon hours. When this side effect was observed, a small dose of 5- hydroxytryptophan (25 mg) was administered before supper, in order to counterbalance excessive hyper-noradrenergic induced activity. Five- hydroxytryptophan, a serotonin precursor, can be added to a small dose (15 mg) of mirtazapine (Remeron), administered before bed. This drug stimulates the release from central neuronal circuits of NA and 5HT, neurotransmitters both needed to facilitate a normal slow wave sleep (SWS). All benzodiazepines and/or gaba-mimetic drugs were banned because they provoke strong suppression of noradrenaline + dopamine (DA) + serotonin release (Lechin, van der Dijs et al., 1994). Considering that late-onset MG and thymoma patients show antibodies to striated muscle antigens (Aarli, 1999), a condition frequently leading to muscular atrophy, we administered growth hormone (4 IU weekly) to some of these patients in order to provoke muscle fiber regeneration (Daugaard, Laustsen et al., 1998). This additional therapeutic tool proved be highly effective in two cases. Taking into account that f-5HT blocks the fetal more potently than the adult muscle acetylcholine receptor (Grassi, 1999), we administered small doses of tianeptine (a serotonin enhancer of platelet uptake - 6.75 mg each other day) (Lechin, van der Dijs et al., 1998b; 1998c). Analytical Methods Platelet aggregation was determined at 0 min and 5 min only, employing the adenosine diphosphate method described by Born (1962). 82
  • 7. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE For plasma NA, Ad, DA, and 5HT (p-5HT + f-5HT) readers are referred to papers previously published by our research group. These references give details dealing with orthostasis, exercise, (Lechin, van der Dijs et al., 1995a, 1995b, 1996e), oral glucose (Lechin, van der Dijs et al., 1992, 1993) and buspirone (Lechin, van der Dijs et al., 1997b, 1998a) stress tests. Results Assessment of the therapeutic effects resulting from our neuropharmacological approach should properly be based on clinical, EMG and immunological parameters. However, we will also comment on some neurochemical and neuroautonomic induced effects (see Tables I to V). Clinical Findings: All MG patients showed a highly significant and sustained reduction of symptoms during acute (≤ 6 days) and late (weeks and months) periods after starting the neuropharmacological therapy. Patients registered no relapses, no MG crises, nor new plasmapheresis after initiation of our treatment. Immunosuppressant drugs were totally eliminated. Suppression of steroids was acomplished progressively. Mestinon was also progressively omitted attaining until reaching complete or highly significant reduction. The total suppression of Mestinon was effected in all non-thymectomized patients and in three thymectomized cases. In the other thymectomized patients, Mestinon was reduced to a minimum. Immunological Findings Increased CD5+B self-reactive and CD8 (suppressor) lymphocytes were registered in all patients. They also showed immunological abnormalities consistent with the Th-2 profile (increased NK-cells, decreased NK-cell cytotoxicity, increased IgG and/or IgM plasma levels), increased CD45RO (memory T cells). These immunological abnormalities disappeared in all non-thymectomized patients after sustained neuropharmacological treatment (± 3 months). Thymectomized patients and some who had received prolonged immunotherapy presented an atypical profile of the immune disorder (see Tables IV and V). Most such abnormalities did not disappear despite 83
  • 8. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE the significant and sustained clinical improvement shown by these patients. 84
  • 9. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE TABLE I. Clinical characteristics and therapeutical responses to neuropharmacological-induced noradrenergic stimulation in 52 Myasthenia Gravis patients (Cases 1 to 17). Patient Sinc Osserman Crisis Thymectomy Mestinon mg/d Plasmapher. Improv. % e Class. Date M/Y Nr Sex Age Pre Post Pre Post Pre Post Acute Late 1. M 71 11/96 II-a 0 0 no 0 0 0 0 100 100 2. F 58 07/90 II-b 3 0 no 360 0 3 0 100 80 3. F 65 08/91 II-b 4 0 no 420 0 6 0 80 90 4. F 36 09/93 II-a 0 0 1995 180 0 0 0 100 100 5. F 66 06/97 II-a 0 0 no 180 0 0 0 100 100 6. M 63 08/89 II-a 0 0 no 240 0 0 0 100 100 7. M 61 05/97 II-b 1 0 no 240 0 1 0 100 100 8. M 70 01/95 II-a 0 0 no 180 0 0 0 100 100 9. F 50 05/91 II-a 0 0 1992 180 60 0 0 80 80 10. F 35 05/93 II-b 1 0 1995 180 0 4 0 100 100 11. F 41 02/94 I 0 0 no 60 0 0 0 100 100 12. M 31 07/87 IV 13 0 1989 240 60 15 0 70 50 13. M 13 04/96 II-a 0 0 no 0 0 0 0 80 80 14. F 53 06/92 II-b 3 0 1994 360 60 6 0 60 60 15. F 50 10/93 III 0 0 1994 180 0 2 0 100 80 16. F 32 07/69 III 2 0 1992 240 0 0 0 100 70 17. F 31 01/84 II-b 3 0 1984 600 0 7 0 100 80 Mestinon doses: pre = at the first visit; post = after late improvement. Improvement: acute = at the second visit (15-day); late = > 3-month. No relapse was observed in any case. 85
  • 10. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE TABLE II. Clinical characteristics and therapeutical responses to neuropharmacological-induced noradrenergic stimulation in 52 Myasthenia Gravis patients (Cases 18 to 34). Patient Since Osserman Crisis Thymectomy Mestinon mg/d Plasmapher. Improv. % M/Y Class. Date Nr Sex Age Pre Post Pre Post Pre Post Acute Late 18. F 20 03/79 IV 24 0 1980 300 0 22 0 90 90 19. M 27 05/98 II-a 0 0 no 210 0 0 0 100 100 20. F 18 11/96 I 0 0 no 0 0 0 0 100 100 21. M 38 03/95 II-a 0 0 1993 180 0 0 0 100 70 22. F 39 02/86 I 0 0 no 240 0 0 0 100 100 23. F 18 03/99 II-b 0 0 no 180 0 0 0 100 100 24. F 27 10/94 IV 14 0 1996 360 120 5 0 70 70 25. F 56 09/99 II-b 2 0 1990 240 0 4 0 100 80 26. F 62 07/81 II-b 1 0 no 480 0 0 0 80 100 27. F 44 10/85 II-b 0 0 no 1020 240 0 0 50 90 28. F 32 02/92 II-a 0 0 no 180 60 0 0 70 70 29. F 64 08/75 II-a 0 0 1976 0 0 0 0 80 80 30. F 40 07/83 II-b 1 0 no 240 120 2 0 60 60 31. F 32 09/94 III 12 0 1995 360 180 12 0 80 80 32. F 16 06/97 II-b 0 0 no 360 0 0 0 100 100 33. F 36 06/79 III 15 0 1979 300 0 18 0 70 50 34. M 21 04/94 III 6 1 1995 360 120 3 0 40 80 Mestinon doses: pre = at the first visit; post = after late improvement. Improvement: acute = at the second visit (15-day); late = > 3-month. No relapse was observed in any case. 86
  • 11. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE TABLE III. Clinical characteristics and therapeutical responses to neuropharmacological-induced noradrenergic stimulation in 52 Myasthenia Gravis patients (Cases 35 to 52). Patient Since Osserman Crisis Thymectomy Mestinonmg/d Plasmapher. Improv. % M/Y Class. Date Nr Sex Age Pre Post Pre Post Pre Post Acute Late 35. F 24 03/96 II-a 1 0 1997 480 0 2 0 100 80 36. F 50 04/71 III 12 0 1993 1260 630 19 0 50 50 37. M 36 04/98 IV 4 0 no 600 300 4 0 70 50 38. F 46 03/93 II-a 1 0 1999 360 120 0 0 80 80 39. F 15 03/94 II-b 0 0 1994 0 0 0 0 100 90 40. F 51 07/74 II-a 8 0 1995 720 120 0 0 80 70 41. F 41 04/83 II-a 1 0 no 240 60 0 0 80 80 42. F 35 06/86 II-b 0 0 1988 480 120 0 0 90 70 43. F 36 09/98 III 1 0 1999 360 0 1 0 100 80 44. M 47 07/97 II-a 2 0 no 120 0 10 0 70 80 45. F 44 02/92 II-b 0 0 1992 360 90 3 0 80 90 46. F 36 03/98 II-a 1 0 no 480 90 1 0 70 80 47. M 76 10/91 II-b 2 0 no 240 0 1 0 80 100 48. F 59 04/96 II-b 0 0 no 360 120 0 0 80 80 49. M 62 06/89 II-b 0 0 1993 240 0 0 0 70 70 50. M 64 11/97 II-a 1 0 no 240 0 1 0 90 100 51. M 65 04/95 II-a 0 0 no 240 120 0 0 90 90 52. M 48 08/97 II-a 3 0 1998 300 60 2 0 80 70 Mestinon doses: pre = at the first visit; post = after late improvement. Improvement: acute = at the second visit (15-day); late = > 3-month. No relapse was observed in any case. 87
  • 12. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE TABLE IV. Electromyographic, neurochemical, serological and immunological parameters registered before (B) and after (A) (acute or late) neuropharmacologically-induced improvement in 52 thymectomized* and non-thymectomized Myasthenia Gravis patients (Cases 1 to 26). Immunological Findings Patient EMG NA/Ad AChR/Ab NK % CD8(s) % CD5+B % IgG (mg/dl) IgM (mg/dl) Nr % ratio nmol/L activity B A B A B A B A B A B A B A 1 100 0.7 7.9 6.9 1.2 18.7 32.4 47.1 26.2 55.3 22.1 2321 1055 204 75 2 80 0.6 5.8 8.5 0.8 15.5 27.7 43.2 29.6 57.4 27.3 1924 1133 117 86 3 80 0.7 5.6 1.7 1.2 22.4 31.6 39.8 30.4 48.7 32.5 2006 1146 99 87 4* 80 1.3 6.4 12.4 5.3 15.6 28.4 31.4 33.6 33.9 29.4 777 961 185 118 5 100 0.8 5.9 9.6 3.2 21.1 32.6 44.7 28.4 56.3 24.2 948 973 337 217 6 100 1.2 8.7 34.3 8.8 20.1 32.7 45.3 30.0 59.1 26.3 1018 711 549 220 7 100 0.9 7.5 18.7 0.4 19.3 30.4 41.0 25.2 58.3 27.2 888 642 425 212 8 100 1.0 15.3 7.6 7.6 22.5 32.7 39.7 26.3 51.3 26.2 1037 954 399 146 9* 80 0.8 7.4 7.2 8.1 24.7 29.7 35.3 37.4 39.7 31.5 932 847 351 347 10* 70 0.9 6.4 8.8 9.3 26.6 27.8 37.4 30.0 36.3 24.2 947 718 269 217 11 100 1.3 7.7 4.5 1.7 22.5 35.5 49.6 25.8 58.7 23.3 1055 546 348 222 12* 50 0.9 6.8 5.2 4.8 27.5 29.9 46.7 36.4 50.7 49.8 887 795 366 279 13 80 0.4 6.8 25.2 17.4 19.5 34.7 43.2 25.2 57.3 21.1 1998 1000 209 147 14* 60 0.7 5.3 32.7 30.7 23.7 28.9 39.4 40.1 45.5 38.7 1777 1200 297 181 15* 80 0.9 6.6 13.2 9.7 25.0 29.9 38.7 36.0 41.1 34.8 998 743 156 148 16* 70 1.1 6.4 18.9 10.1 21.4 28.2 41.4 33.3 43.6 32.2 886 654 229 114 17* 80 0.8 5.8 9.4 6.3 18.9 23.8 39.7 35.6 48,6 37.8 1002 954 195 137 18* 90 1.5 7.6 4.5 2.2 23.3 29.0 40.0 31.8 49,3 36.6 976 759 201 185 19 100 0.8 5.9 15.2 6.6 18.8 36.1 48.1 29.7 43,8 29.7 1489 678 327 89 20 100 1.1 6.8 6.9 0.8 21.1 34.8 55.9 31.7 49,8 27.7 885 533 289 96 21 70 0.8 5.5 14.3 10.0 19.7 26.4 59.3 45.8 53.5 45.2 1002 995 314 253 22 100 1.7 6.4 9.5 2.2 25.7 34.1 46.3 33.8 50.4 31.3 948 523 270 114 23 100 0.9 5.8 4.7 2.1 21.2 34.7 40.0 32.7 47.7 34.0 936 670 159 58 24* 70 1.3 7.4 14.7 16.7 17.9 26.8 47.8 39.6 41.9 35.8 889 579 223 146 25* 80 0.8 6.3 31.7 26.8 25.6 30.0 43.6 35.7 44.4 36.8 1230 997 246 200 26 100 1.3 7.2 9.5 2.2 21.1 29.7 39.7 30.2 43.7 30.1 966 668 223 146 EMG% = percentage of improvement (decrement + jitter); NA = noradrenaline; Ad = adrenaline; CD8(s)% = CD8 suppressor cells, percentage of total CD3 lymphocytes; CD5+B% = percentage of total B lymphocytes; NK% activity against K563 target cells. 88
  • 13. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE TABLE V. Electromyographic, neurochemical, serological and immunological parameters registered before (B) and after (A) (acute or late) neuropharmacologically-induced improvement in 52 thymectomized* and non-thymectomized myasthenia gravis patients (Cases 27 to 52). Immunological Findings Patient EMG NA/Ad AChR/Ab NK % CD8(s) % CD5+B % IgG (mg/dl) IgM (mg/dl) Nr % ratio nmol/L activity B A B A B A B A B A B A B A 27 90 0.7 6.8 8.5 1.4 25.5 32.7 38.4 29.7 40.8 31.6 895 534 179 98 28 70 1.5 6.6 0.8 0.8 30.8 29.7 42.5 38.7 35.2 30.0 1089 876 227 159 29* 80 1.7 6.7 3.3 2.9 22.7 29.6 43.7 31.2 46.5 33.0 976 579 234 178 30 60 0.8 5.9 4.7 5.7 20.0 24.4 48.7 43.7 49.0 36.7 897 767 268 176 31* 80 1.1 6.8 2.8 1.4 25.3 34.6 43.5 34.7 41.8 37.5 995 689 170 154 32 100 1.4 7.7 4.5 0.8 22.1 31.4 46.7 30.0 49.6 31.2 897 678 212 153 33* 50 0.9 6.3 15.5 18.7 20.0 30.1 49.3 47.2 46.7 44.4 932 956 214 210 34* 80 1.1 5.8 9.7 6.6 21.3 32.4 44.7 32.0 41.9 30.0 897 534 187 155 35* 80 0.9 6.6 4.7 1.7 23.3 30.0 43.7 35.1 40.9 33.6 914 637 212 176 36* 50 1.5 5.7 13.2 9.7 21.0 25.2 49.7 41.1 38.6 35.7 985 764 233 214 37 50 0.8 6.5 34.7 29.9 19.3 22.4 48.7 45.4 42.7 40.1 879 766 225 215 38* 80 1.1 7.7 26.6 14.3 20.1 26.4 50.5 40.4 46.8 30.7 999 657 198 179 39* 90 0.9 6.5 9.2 4.9 23.8 32.3 54.7 36.7 45.9 32.7 874 623 201 177 40* 70 1.0 7.6 0.8 0.8 22.7 31.1 46.4 32.8 44.0 33.7 895 768 234 198 41 80 0.9 6.3 0.7 0.7 26.8 35.1 39.7 31.4 40.6 30.9 963 567 199 146 42* 70 1.3 7.1 0.8 0.8 20.1 30.2 43.6 37.7 39.8 33.7 888 652 201 177 43* 80 0.7 6.4 13.2 5.4 23.1 33.7 44.9 35.6 38.7 30.1 943 789 199 163 44 90 0.9 7.5 7.4 1.5 21.5 30.4 41.7 33.3 39.7 30.0 899 567 201 144 45* 90 0.9 6.8 3.4 1.0 21.2 32.3 43.8 32.7 44.4 32.0 923 766 214 167 46 80 1.1 6.8 12.2 6.4 23.5 33.7 40.1 33.6 47.9 36.2 897 653 198 179 47 100 0.8 6.5 10.5 2.3 20.8 31.2 46.4 31.5 48.8 31.3 954 763 200 167 48 80 0.9 6.3 14.5 5.3 21.7 30.6 45.9 33.7 43.7 32.0 897 675 233 156 49* 70 1.1 5.8 10.7 13.4 22.0 29.7 43.4 32.8 41.7 35.9 768 563 195 149 50 100 0.9 7.3 2.3 0.8 19.6 32.5 43.4 31.7 42.7 30.7 943 567 186 194 51 90 1.3 6.2 0.7 0.7 20.0 30.5 53.2 33.8 56.8 31.1 879 654 200 178 52* 70 1.2 5.9 13.4 12.7 21.3 33.5 46.5 32.6 48.7 45.7 997 898 234 248 EMG% = percentage of improvement (decrement + jitter); NA = noradrenaline; Ad = adrenaline; CD8(s)% = CD8 suppressor cells, percentage of total CD3 lymphocytes; CD5+B% = percentage of total B lymphocytes; NK% activity against K563 target cells. 89
  • 14. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE Moreover, the thymectomized and immunosuppressed patients registered a lower recovery than that shown by the non-thymectomized or non-immunosuppressed patients. Twenty-four out of the 27 non-thymectomized patients showed increased levels of ACh-receptor autoantibodies before neuropharmaco- logical treatment. After therapy, 20 of those 24 patients showed normalization or a great reduction of this parameter. On the other hand, an erratic response was observed in MG patients previously submitted to surgical or drug-induced immunosuppression. Seven out of the 25 thymectomized patients showed Scl-70 auto- antibodies [usually registered in systemic lupus erythematous (SLE) patients]. Only one patient showed increased anti-striated muscle autoantibodies plus increased levels of ACh-R autoantibodies, before and after therapy. He was a 38-year-old male whose myasthenia gravis symptoms began in 1985. He was affected by hepatitis during childhood. He was rated as a II-a MG patient. The electromyography test was positive for MG. Thymectomized on Sept. 20, 1993, he was prescribed Mestinon therapy which he took for six years. He accepted neither steroids nor immunosuppressant drugs. We performed immunological investigations on March 3/99, May 18/99, and June 22/99 with similar immune abnormalities (reduced CD4/CD8 ratio plus increased T virgin cells). These abnormalities were found on all three occasions. Neither immunological nor serological abnormalities disappeared despite his clinical improvement with neuropharmaco-logical therapy. Neuropharmacological, neurochemical and autonomic test results were normalized in all patients after completing our therapy. These tests paralleled clinical improvement. Neurochemical Findings All patients showed very low NA/Ad ratio plus high f-5HT plasma levels before treatment. After treatment, all neurochemical abnormalities were reverted (see Tables IV and V). The NA/Ad plasma ratio reached normal or greater than normal values (≥ 5). This NA/Ad ratio showed further increases after orthostasis, exercise, oral glucose and/or buspirone challenge. Noradrenaline but not adrenaline increased during these tests. The abnormal diastolic blood pressure (DBP) and heart rate (HR) responses returned to normal after one month of neuropharmacological therapy. Normalization of these autonomic parameters depends on the disappearance of adrenal gland 90
  • 15. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE hyperresponsiveness + recovery of central sympathetic (neural) activity (Lechin, van der Dijs et al., 1995a, 1995b, 1996e). Discussion The results obtained from the present study ratified findings showing that myasthenia gravis is an immunologic disorder presenting a Th-2 profile of immunity (Jaretzki, 1997; Karussis, Lehmann et al., 1994; Kouassi, Li et al., 1990; Mokhtarian, Shirazian et al., 1993; Ragheb and Lissak, 1993; Talal, Dauphinee et al., 1998; Yi and Lefvert, 1993; Zhang, Yu et al., 1997). The results also confirmed that thymectomy is followed by an undefined disturbance of the immune system (Gerli, Paganelli et al., 1999). This finding has been demonstrated by many researchers who reported that a high percentage of thymectomized patients developed any of several types of autoimmune diseases, frequently systemic lupus erythematous (SLE) (Jaretzki, 1997; Karussis, Lehmann et al., 1994; Talal, Dauphinee et al., 1998; Yi and Lefvert, 1993; Zhang, Yu et al., 1997). Finally, the present study showed that MG patients present a deficiency of central and peripheral noradrenergic (NA) activity and that neuropharmacological manipulations addressed to enhancing neural transmission of this system are able to attain clinical remission as well as reversion of the Th-1 < Th-2 imbalance (Nicholson and Kuchroo, 1996). Our results showed that although the onset of clinical improvement occurred rapidly (< one week), immunological normalization took months (2-3 months). When this stage of late improvement was obtained, doses of drugs were reduced. With regard to this, we observed that MG patients taking high Mestinon doses responded slowly to our neuropharmacological therapy. This finding may be associated with the fact that acetylcholinesterase inhibitors provoke down-regulation of ACh-receptors (Tiedt, Albuquerque et al., 1978). We also found that MG patients taking Mestinon at night responded more slowly than those taking only diurnal Mestinon. We inferred that these patients do not sleep well because of a lack of muscular relaxation induced by the sustained enhanced neuromuscular nocturnal ACh-activity. This factor would contribute to aggravating the diurnal muscular fatigue they usually presented. Furthermore, considering that normalization of immunity as well as central noradrenergic activity occurs during sleep, mainly during phases III and IV of slow wave sleep (SWS) when growth hormone secretion peaks, it 91
  • 16. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE becomes clear why normalization of the sleep pattern is necessary to the recovery of all autoimmune diseases (Kelley, 1989). It has long been known that NA facilitates neuromuscular transmission and muscle contraction (Binder and Powers, 1999; Brustein and Rossignol, 1999; Bukcharaeva, Kim et al., 1999; Duteil, Rambert et al., 1990; Fishman, Feigembaum et al., 1983; Gerin and Privat, 1998; Jabre and Salzsieder, 1999; Kernell, Bakels et al., 1999; Sokoloff, Siegel et al., 1999; Sugrue, 1983). The sudden (acute) clinical improvement registered in all our MG patients during neuropharmacological therapy should be attributed to the effect of NA released at spinal motoneuron levels. At this level, NA excites alpha-1 post-synaptic receptors which facilitate muscular activity. As is amplied known, the CNS controls posture and movements through the activation of spinal motoneurons which receive noradrenergic excitatory and serotonergic modulatory axons from medullary and pontine nuclei. Spinal motoneurons receive thousands of presynaptic excitatory (NA) and inhibitory (5HT) axons which distribute throughout their dendritic trees. Noradrenaline excites alpha-1 receptors whereas serotonin, acting at 5HT-1a receptors, modulates NA effects at the spinal motoneurons level. In addition, NA synchronizes the evoked quantal release at neuromuscular junctions (Bukcharaeva, Kim et al., 1999). Noradrenergic axons innervating spinal motoneurons arise from the locus coeruleus (LC) also called A6-NA and A5-NA cell groups (Lechin, van der Dijs et al., 1989). Serotonergic axons innervating spinal motoneurons arise from the medullary raphe pallidus and raphe obscurus nuclei. Alpha-2 antagonist drugs excite motoneurons by increasing the firing activity of NA neurons which release noradrenaline from axons innervating spinal motoneurons. Similar effects are displayed by buspirone. This drug at low doses (similar to those employed in anxiety patients) may exert a dual effect on spinal motoneurons: an excitatory effect by increasing the release of NA from noradrenergic axons, and an indirect polysynaptic effect secondary to the reduction of activity of the medullary raphe nuclei which possess 5HT-1a inhibitory autoreceptors (Lechin, van der Dijs et al., 1979a). The short-lived effect of the alpha2 antagonists-induced release of noradrenaline can be prolonged by the addition of an inhibitor of NA re-uptake at synaptic level (desipramine, nortryptiline, protryptiline, doxepin, etc.) (Lechin et al., 1979b). According to all the above, it becomes clear why neuropharmacological therapy addressed to enhancing central NA neural transmission triggers 92
  • 17. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE an acute and sudden improvement of muscular strength in MG patients. This phenomenon has been known since the 1980s decade to neurophysiologists familiar with the findings obtained in spinal cats. Adrafinil and modafinil (two alpha1 agonists) trigger acetylcholine release from motor nerves and facilitate neuromuscular transmission by a selective action at presynpatic alpha1 receptors located at that level (Wessler, 1992). The late and sustained improvement registered in our study would reflect the role played by the excitatory effect of noradrenergic innervation at lymphoid organs and the immunosuppressant effects displayed by plasma serotonin (f-5HT). Scientific literature dealing with these two immunomodulating factors is too extensive to be covered in this discussion. By way of example, however, we will cite some studies: Ackerman, Felten et al., 1987; Bencsics, Sershen et al., 1997; Besedowsky, del Rey et al., 1979; Felten and Olschowka, 1987; Felten, Felten et al., 1987; Livnat, Felten et al., 1985; Lorton, Hewitt et al., 1990; Tang, Shankar et al., 1999; Thyaga-Rajan, Madden et al., 1999. It has been exhaustively demonstrated that the regions in which lymphocytes T cells reside, and through which they recirculate, receive direct sympathetic neural input. Therefore, the immune system can be considered “hard-wired” to the brain. Chemical sympathectomy of adult mice resulted in reduced antibody responses to T-dependent antigens. The interaction between sympathetic NA nerve fibers and cells of the immune system has been shown through the distribution of tyrosine hydrolase (TH+) nerve fibers among lymphocytes and macrophages in lymphoid organs, the expression of adrenoceptors on cells of the immune system, and the immunomodulatory effects of NA. In old rats, a conspicuous decline in NA innervation and NA contents is observed in the splenic white pulp as well as in the cell bodies in superior celiac- mesenteric ganglia that provide preganglionic sympathetic innervation to the spleen (Arnason, 1993; Carlson, Fox et al., 1997; Madden. Felten et al., 1994a; Roszman and Carlson, 1991). Paralleling these alterations in sympathetic NA neuronal activity is an age-related loss of T cell mediated immune responses, including reduced T cell proliferation and IL-2 production by antigen- and mitogen-stimulated lymphocytes. Treatment of these rats with drugs inducing noradrenergic regeneration and re-innervation reverted the rats’ immune abnormalities (Tang, Shankar et al., 1999; Thyaga-Rajan, Madden et al., 1999). Noradrenergic innervation of the spleen is responsible for a significant increase of 93
  • 18. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE gamma-interferon, IL-2 and tumor necrosis factor alpha, the three Th-1 cytokines, and a lowering of IL-4, IL-5 and IL-10 (TH-2 cytokines) production (Carlson, Fox et al., 1997; Madden, Moynihan et al., 1994b; Spengler, Allen et al., 1990). Other evidence showed that elevated plasma NA concentrations increased the level of Th-1 cytokines (Kappel, Poulsen et al., 1998; Ross, Williams et al., 1987). These and other findings demonstrate that the noradrenergic innervation of bone marrow is functionally dynamic and is responsive to central activation. Furthermore, these results lend credence to the premise that neural mechanisms participate in regulating lymphopoietic cellular events. Data demonstrates that adrenaline (Ad) suppresses Th-1 immunity and stimulates Th-2 cytokines (Cook-Mills, Cohen et al., 1995; Crary, Houser et al., 1983; Cunnick, Lysle et al., 1990; Ernstrom and Sandberg, 1973; Felsner, Hofer et al., 1995; Kouassi, Li et al., 1990; Tvede, Kappel et al., 1994; Van Tits, Michel et al., 1990). These effects are mediated by beta-adrenoceptors in human lymphocytes. Beta- adrenoceptors also mediate NK- activity (Kappel, Tvede et al., 1991; Schedlowski, Hosch et al., 1996; Vredevoe, Moser et al., 1995) The role of circulating free serotonin (f-5HT) has also been exhaustively investigated. For instance, macrophages possess a 5HT- uptake system, the kinetic properties of which make them sensitive to changes in plasma levels of 5HT (Fuchs, Campbell et al., 1988; Jackson, Walker et al., 1998; Sternberg, Wedner et al., 1987; Young and Matthews, 1995). In addition, it has been shown that serotonin stimulates Th-2 lymphokine secretion (Paegelow, Werner et al., 1985). It is known that NA alters the capacity of platelets to sequester and/or catabolize 5HT, thus regulating its physiologically active pool in the plasma (Fuchs, Campbell et al., 1988; Walker and Codd, 1985; Warren, Kane et al., 1990). Thus, through its ability to regulate plasma levels of 5HT, an immunosuppressive amine with access to macrophages, the nervous system can influence cells involved in antigen recognition. In aging rodents and humans, the central noradrenergic deficit is associated with a decreased platelet affinity for 5HT and an increased plasma content of 5HT. In addition to the above, the increased f-5HT we registered in our MG patients would also be associated with the raised adrenaline plasma levels they presented. This latter factor would be responsible for the increased platelet aggregability we registered in all our MG patients (Larsson, Hjemdahl et al., 1989). Finally, it has been known for several years that pharmacological enhancement of 5HT metabolism suppresses 94
  • 19. VOL. 31, NOS. 5 & 6, 2000 JOURNAL OF MEDICINE the immune response in vivo. This immunosuppression occurs peripherally, not centrally (Walker and Codd, 1985). Free serotonin in plasma should also play some important role in triggering myasthenic crisis since we were able to accelerate the recovery of several MG patients affected by this severe complication by administering tianeptine to them. This finding is consistent with the bronchial constriction effect exerted by f-5HT in asthmatic patients who were also dramatically improved by this serotonin-enhancing uptaker drug (Lechin, van der Dijs et al., 1996b, 1998b, 1998c). Acknowledgments This work was supported by grants from FundaIME and Fundaneuroinmunologia. References Aarli, J.A. (1999).: Late-onset myasthenia gravis. A changing scene. Arch. Neurol. 56: 25-27. Ackerman, K.D., Felten, S.Y., Bellinger, D.L., Livnat, S. and Felten, D.L. (1987).: Noradrenergic sympathetic innervation of spleen and lymph nodes in relation to specific cellular compartments. In: Progress in Immunology Edited by B. Cynader and R.G. Miller. Academic Press. Orlando, FL. 4: 588-600. Anderson, A. and Harvey, A. (1988).: Effects of the facilitatory compounds cathecol, guanidine, noradrenaline, and phencycli- dine on presynaptic currents of mouse motor nerve terminals. Naunyn-Schmiedeberg´s Arch. Pharmacol. 338: 133-137. Arnason, B.G.W. (1993).: The sympathetic nervous system and the immune response. In: Clinical autonomous disorders. Evaluation and management. Edited by P. Low. Little Brown, London. pp 143-165. Arranz, B., Blennow, K., Ekman, R., Eriksson, A., Mansson, J-E. and Marcusson, J. (1996).: Brain monoaminergic and neuropep- tidergic variations in human aging. J. Neural Transm. 103: 101-115. Bamshad, M., Kay Song, C. and Bartnes, T.J. (1999).: CNS origins of the sympathetic nervous system outflow to brown adipose tissue. Am. J. Physiol. 276(Regulatory Integrative Comp Physiol 45): R1569-R1578. 95
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