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INFLUENCE OF SLEEP DISRUPTION AND CALORIE RESTRICTION ON BIOLOGICAL MARKERS FOR DEPRESSION
Saturday 8 November
INFLUENCE OF SLEEP DISRUPTION AND CALORIE RESTRICTION ON BIOLOGICAL MARKERS FOR DEPRESSION PAUL E. MULLEN CHRISTOPHER R. LINSELL DAVID PARKER
Department of Psychological Medicine and the Neuroscience Centre, University of Otago, Dunedin, New Zealand The
hypothesis that the dexamethasone suppression test (DST) and rapid-eyemovement (REM) latency test are not biological markers for depressive illness but artifacts arising from dietary and sleep disturbances that accompany depression was examined in Summary
28 normal volunteers. The restriction of calorie intake with moderate weight loss reproduced a pattern of response to dexamethasone closely resembling that claimed to be diagnostic of depressive illness. The shortened REM latencies claimed as a diagnostic marker were replicated in volunteers by mimicking the sleep pattern commonly found in depression. These changes could not be explained by the induction of mood disorder in the subjects. The results put in question the diagnostic value of the DST and REM latency tests in clinical practice, where sleep disorder and poor appetite, with reduced calorie intake, are the common accompaniments of depressive illness.
1986
patientshave established themselves as diagnostic aids in clinical practice despite the continuing debate on their utility. Fasting in normal volunteers, with an associated weight loss in excess of 8 kg, has been reported to increase the escape from dexamethasone suppression.7,8 Moderate calorie restriction with gradual weight loss has been investigated by two groups with opposite results.9,10 The studies in depressed patients, aimed at establishing the effects of weight loss on the dexamethasone response, have also produced conflicting results.1O,15 Feinberg and Carroll,16 in the most extensive study to date, claim that though DST results were related to weight loss the relation was indirect-ie, weight loss was more frequent in older and more severely depressed patients. In their view, weight loss did not account for the higher post-dexamethasone cortisol levels found in endogenous depression. Progressive sleep reduction has been reported to produce shortened REM latencies,17 but no-one has examined the influence on REM latency of sleep disruption comparable with that encountered in depressed patients. We have reproduced in normal volunteers the patterns of sleep and reduced calorie intake characteristic of depressive illness and have examined their influence
on
the DST and REM
latency. Methods
Subjects Introduction THE quest for biological markers of depression has been a central theme in psychiatric research for over a decade. The hope was that a valid marker would provide both an aid to diagnosis and a signpost to the underlying pathophysiology. The dexamethasone suppression test (DST) has been the most extensively investigated and canvassed of the putative biological markers for depressive disorders,l and has been claimed to be comparable in diagnostic specificity to serum acid phosphatase levels in carcinoma of the prostate.2,3 The sleep disturbance found in depression has several characteristic polysomnographic abnormalities, the most consistent being a decreased rapid-eye-movement (REM) latency’ and this decreased REM latency has also been put forward as a diagnostic marker for depression.5 These two procedures, which are reported to identify a similar group of
The study, which was approved by the hospital and university ethical committee, involved 28 normal volunteers aged from 18 to 25 years (mean 20 SE 0-3 years). They had no history of
psychiatric disorders, eating disorders, recent dieting, sleep disturbance, or serious ill-health. Subjects with mental disorder in first-degree relatives were excluded. To ensure homogeneous populations two groups were selected, one of 14 males with a body weight within 5 % of ideal normal, and a second of 14 females who were between 5% and 10% over ideal body weight.18 The subjects were non-smokers and did not have a history of alcohol or drug abuse. The only history of taking drugs in the previous 6 months was in 3 subjects who had taken an oral contraceptive. All were university students capable of understanding the nature of the procedures. The 28 volunteers maintained a regular sleep-wake cycle for two weeks, retiring between 2200 h and 2300 h and rising between 0700 h and 0800 h, and were restricted to a diet of 2000-3000 kcal (8-4-12-6 MJ) a day. 8515
1052
Sleep Disruption Procedure The 14 male volunteers
were
admitted
to a
research ward
equipped for sleep monitoring. They were fed on a regular schedule and had no food or caffeine-containing drinks after 1800 h. Daytime naps were not allowed. The first two nights were used to acclimatise the subjects to the ward and the sleep-recording procedures. On the third and fourth nights baseline sleep recordings were obtained. On the fifth and sixth nights a baseline DST was performed. The volunteers were then subjected to seventeen nights’ partial sleep deprivation. The sleep disruption was designed to mimic the pattern of sleep disturbance characteristic of major depressive disorders. The subjects’ time of retiring to bed was delayed by an hour to 2400 h and they were awoken 2 h earlier than usual at 0500 h. The subjects spent the time between 0500 h and 0700 h watching videos or taking an early morning swim according to their inclinations. The timing of their first refreshment of the day remained the same at 0730 h. Sleep recordings were recommenced with an acclimatisation night after thirteen nights of sleep deprivation and then definitive recordings on the fifteenth and sixteenth nights. A DST was performed on the subsequent night. Subjects were then discharged from the ward and allowed to return to their regular sleep-wake pattern. The subjects were readmitted after six months and, after two nights’ acclimatisation, the sleep recordings and DST were repeated.
sleep as if they had occurred spontaneously. A modified score was also obtained for the Beck inventory and the Carroll scale by deletion of questions pertaining to lack of sleep and tiredness for the sleep-deprived group, and deletion of questions concerning weight loss for the restricted-diet group. The mood scales and general assessments were monitored regularly to ensure that the procedures were not precipitating clinical depression in our subjects. Results 1 subject was withdrawn from the study after six nights’ sleep distruption because of the development of irresistible napping and sleep paralysis with hypnagogic hallucinations. The remaining 27 subjects completed the protocol without difficulty. REM latencies were not measured on three nights owing to technical problems.
Weight The mean increase in weight of 06 (SE 03) kg following sleep deprivation compared with baseline was not significant. In contrast the 14 subjects on a restricted diet recorded a mean weight loss of 4-3 (SE 0-2) kg. Dexamethasone
Calorie Restriction Procedure The 14 female subjects were admitted to the research ward. After nights’ acclimatisation, baseline sleep recordings were performed on the third and fourth nights and the DST was begun on the next evening. The period of calorie restriction was then started with the subjects placed on a diet designed and supervised by the hospital dietitian of between 1000 and 1200 kcal (4-2-5-1MJ) per day. The subjects remained on this diet for the next 18 days. During the last four days of this period the sleep recordings and DST were repeated. The subjects were then discharged from the ward and returned to their usual dietary habits. The subjects were readmitted six months later and the sleep recordings and DST were two
repeated. Dexamethasone
Suppression Test There was a significant change in the response to dexamethasone after the period of restricted calorie intake (fig 1). Whether the threshold cortisol values for nonsuppression were taken as 3, 5, or 7 g per dl, the DSTs at completion of calorie restriction showed a significant switch from suppression to non-suppression: at 7 g/dl there were 2 non-suppressors at baseline increasing to 8 nonsuppressors after dieting (p=0-016, binomial test); at 5 ug/dl 2 subjects were non-suppressors at baseline increasing to 10 non-suppressors (p = 0004); and at 3 g/d1 4 non-suppressors at baseline increased to 11 non-suppressors after calorie restriction (p 0008). 2 subjects did not attend the six-month follow-up. The DST results for the 12 subjects studied did not differ significantly from baseline, with 4 non-suppressors by all criteria. The 4 non-suppressors at follow-up had maintained the weight loss established during the experiment with a weight gain of only 1-7 (SE 1) kg. The suppressors, in contrast, had gained an average 6-1(0-6) kg from the end of the experiment. The difference between the weight gain of the suppressors and the non-suppressors was significant =
Suppression
Test
Dexamethasone in a dose of 1 mg was administered orally at 2330 was sampled by venepuncture at 1600 h and 2300 h the following day and immediately centrifuged, the plasma being frozen at - 40°C until assay. Cortisol was measured blind and in duplicate by direct radioimmunoassay with the Amerlex radioimmunoassay kit. Intra-assay and inter-assay coefficients of variation were 4-2% and 6-8%, respectively. We used values of 3, 5, and 7 ug per dl to compute for a threshold for non-suppression.
h. Blood
Sleep Recording Sleep was recorded with two channels of the electroencephalogram (C3-A2 and 01-A2), two channels of the electrooculogram, and one channel of the electromyogram from the submental muscles. Each 20-second epoch was scored by three independent raters according to the standard criteria of Rechtschaffen and Kales.19 The definition of REM latency used was the time spent asleep from the first epoch of stage 2 until the first REM period of at least 3 min.
Psychometric Measures Mood was assessed every three days with the Nowlis Mood Adjective Checklist .21 All other psychometric measures were assessed on four occasions-at baseline, mid-point, and end-point of sleep deprivation or restricted diet, and at follow-up. State and trait anxiety were measured with the State-trait Anxiety Inventory21 (general population state mean =31-9 [SE 04] and trait mean 349 [SE 0-4]). Symptoms of depressive illness were assessed with the Beck Depression Inventory22 (general population mean == 109 [SE 0.8]) and the Carroll Rating Scale23 (general population mean = 4-6 [SE 0.4]). Subjects were instructed to score weight loss and lack of =
(p < 0-001, t test). In addition to looking at the DST results in their usual form we examined the post-dexamethasone cortisol levels. The log mean of the 1600 h and 2300 h cortisol levels increased significantly after calorie restriction from their baseline values (p < 0-01, pairedt test). This is in line with the
increase
in
the
number
of
non-suppressors.
Interestingly, the post-dexamethasone cortisol values remained significantly higher than baseline at follow-up (p<0-05, paired t test) although the difference in the number of non-suppressors did not reach statistical significance. There was a significant correlation (?-=073, p < 001) between the post-dexamethasone cortisol levels at follow-up and the loss of weight from baseline. The pre-dexamethasone log cortisol levels did not differ significantly between baseline and after calorie restriction or between baseline and follow-up. Thus the significant differences in post-dexamethasone cortisol levels after dieting and at follow-up reflect a changed response to dexamethasone. These changes at follow-up, though not sufficient to affect the number of non-suppressors
1053
Fig I-Effects of sleep deprivation and
calorie restriction
on
distribution of suppressors and non-suppressors for three criteria values of the DST.
Fig 2-Effects of sleep deprivation and calorie restriction on REM latencies.
significantly, do suggest a continuing difference in response to dexamethasone. The 3 subjects who had taken oral contraceptives recently showed no significant differences either in the log mean cortisol levels or DST results at baseline or after dieting. The number of subjects exhibiting non-suppression of cortisol levels in response to dexamethasone did not change significantly after sleep disruption (fig 1). When the log means of the 1600 h and 2300 h cortisol values were examined, pairedt tests showed no significant differences between baseline and sleep deprivation or between baseline and follow-up. REM Latency The REM latency shortened significantly after the sleep disruption procedure (fig 2). The mean REM latencies at baseline were 78 8 (SE 6-8) min and at follow-up 88-1(8) min. These values do not differ significantly. The REM latency at the end of the sleep disruption phase was significantly decreased to 55-9 (7-9) min (p < 0-01, Wilcoxon matched- pairs signed-ranks test.) After sleep disruption, six nights’ sleep recordings began with sleep-onset REM compared with none at baseline or follow-up. REM latency did not change significantly in response to calorie restriction and
loss (fig 2). The mean REM latency for the baseline was 87-4 (7-4) min which did not differ group significantly from the 95 -3 (7-4) min at the completion of the’
weight at
dieting phase. There were no occurrences of sleep REM in the calorie-restricted group of subjects.
onset
Mood The subjects on a calorie-restricted diet did not show any clinically obvious changes in affective state during the experiment and this was confirmed by the psychometric assessments (table I). Scores at the completion of the period of dieting showed a significant change only in the Carroll Rating Scale (p < 002, paired t test). When questions relating to weight loss were removed there was no longer a significant rise for the group as a whole though 3 subjects scored 12, 13, and 14 which is marginally above the cut-off of 10 for this screening instrument. There was, however, no significant correlation between the post-dexamethasone log mean cortisol levels and the scores on either the Carroll Rating Scale (Kendall’s=0 13) or the Beck Depression
Inventory (Kendall’s = - 0 -10). There was an apparent increased irritability and lability of mood in the sleep-deprived subjects towards the end of the experiment. These clinical impressions were supported by the systematic psychometric assessments (table i). The subjects showed a significant decline in mood as measured by the Nowliss Scale (p < 001), Beck Depression Inventory (p < 0-001), and the Carroll Rating Scale (p < 001) and the measure of state anxiety increased significantly (p < 0-02). These mood scales depend in part on questions relating to sleep disturbance and tiredness. When such questions were
1054 TABLE I-PSYCHOMETRIC SCORES EXPRESSED AS MEAN
(STANDARD ERROR)
subjects who had maintained a lower body weight during follow-up. Studies in depressed patients to date have been claimed to indicate that mild to moderate weight loss is neither sufficient for the
occurrence of nonof weight loss in depressed suppression.i5 is difficult. Virtually all severely notoriously patients of individuals loss appetite, though far depressed report fewer give a clear account of weight loss. Excluding an episode of decreased calorie intake with mild to moderate weight loss over the previous six months in depressed patients would present formidable practical problems. We contend, however, that unless this can be done with confidence the DST results become unreliable as a diagnostic marker for depression. The shortening of sleep time in our normal volunteers, to reproduce that reported in endogenous depression, caused a significant reduction in REM latency. The pattern of REM latency changes that results from this manipulation of the sleep-wake cycle in normal volunteers closely parallels that reported in depression.24 This does not conclusively demonstrate that the REM latency changes reported in depression are simply a reflection of reduced sleep time. What it does suggest is that diagnostic use of REM latency changes must be confined to depressed subjects without current sleep disturbance. This would remove any practical clinical applicability. This study cannot unravel the causal connections between the syndrome of major depression and both alterations in REM latency and response to dexamethasone. It remains possible that the biology of depression and calorie restriction have the same effects on the cortisol response to dexamethasone but mediated independently. Abnormal plasma cortisol control may be a symptom of depressive illness,25 but an unreliable marker where weight loss cannot be excluded. The REM latency changes found in depression may similarly reflect a fundamental neurophysiological change in depression and just happen to be reproduced by quite other mechanisms in sleep disruption. REM latency and sleep time may well vary independently in depression26 but its utility as a marker is vitiated in the presence of coexisting disruption of the sleep wake cycle. This study suggests that these frequently employed diagnostic tests will usually offer no more information to the clinician than carefully conducted histories that elicit the details of the patients’ appetite and weight loss as well as their patterns of sleep.
necessary
nor
The
*p
tp < 001;
#p < 0001.
TABLE II—COMPARISON OF RESULTS OF DST REPORTED BY
CARROLLAND THOSE PRODUCED BY CALORIE RESTRICTION
excluded the changes in the Beck Depression Inventory no longer reached statistical significance but the increase in the Carroll Score remained significant (p < 0-05). The scores, though they did indicate a deterioration in mood state, did not move into the range found in depressive disorders even when the questions relating directly to sleep disturbance were retained. This was true for all individuals as well as the group as a whole. The scores were consistent with increased anxiety but not the onset of clinically significant depression. Discussion In normal subjects, by restricting their calorie intake, we have reproduced the pattern of response to dexamethasone said to characterise depressive illness. The shortened REM
latencies claimed as a diagnostic indicator were replicated by mimicking the sleep pattern of depressives. These changes cannot be explained by the induction of a mood disorder since no subject became depressed either clinically or according to the rating scales. The sleep-disrupted group showed a greater level of psychological stress as measured by anxiety scores but their response to dexamethasone was unaffected; therefore a simple stress explanation for the positive DST results is unlikely. On calorie restriction for two weeks with a weight loss of 4-3 (SE 0-2) kg, over 70% of our subjects registered a positive DST at the 5 g/d1 criterion employed in our laboratories with this assay in the diagnosis of depressive illness. The results for criteria values of 3-7 g/dl can be expressed in the form of the sensitivity and specificity of the DST for detecting calorie restriction in our population of volunteers. When the data were expressed in this manner a close correspondence emerges between our findings and those of Carroll and his group2 for the DST as a diagnostic marker for depressive illness (table II). The effects of dieting on cortisol responses to dexamethasone in our subjects continued to be apparent at follow-up six months later, despite the subjects’ return to normal diets. Post-dexamethasone levels remained raised in the group as a whole but the rise was more pronounced in
assessment
This research was supported by a Grant from the Ashbum Hall Research Fund. C. R. L. is supported by the Hazel Buckland Trust.
Correspondence should be addressed to P. E. M., PO Box 913, Dunedin, New Zealand. REFERENCES 1. Carroll BJ. Dexamethasone suppression test m depression. Lancet 1980; ii: 1249. 2. Carroll BJ, Feinberg M, Greden JF, et al. A specific laboratory test for the diagnosis of melancholia. Arch Gen Psychiatry 1981, 38: 15-22. 3. Carroll BJ. The dexamethasone suppression test for melancholia. Br J Psychiatry
1982; 140: 292-304.
Kupfer DJ, Foster FG. EEG sleep and depression. In: Williams RL, Karacon I, eds. Sleep disorders: diagnosis and treatment New York: Wiley, 1978: 163-209. 5. Kupfer DJ Neurophysiological ’markers’—EEG sleep measures. Psychiatr Res 1984; 4.
18: 467-75. GM, Halbreich U, Sachar EJ, et al. Plasma cortisol section and REM penod latency in adult endogenous depression. Am J Psychiatry 1983; 140: 750-53. 7. Edelstein CE, Roy-Byme P, Fawzy FI, Domfeld L. Effects of weight loss on the dexamethasone suppression test. AmJ Psychiatry 1983; 140: 338-41. 8. Fichter MM, Pirke KM. Hypothalamic pituitary function in starving healthy subjects. In: Pirke KM, Ploog D, eds. The psychobiology of anorexia nervosa. Berlin: Springer Verlag, 1984: 124-35. 6. Asnis
1055
EFFECT OF CYCLOPHOSPHAMIDE THERAPY ON ONCOGENE EXPRESSION IN ANGIOIMMUNOBLASTIC LYMPHADENOPATHY DENNIS M. KLINMAN ALFRED D. STEINBERG J. FREDERIC MUSHINSKI Cellular Immunology Section, Arthritis Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, and Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Md 20892, USA
Summary
The was
expression of four cellular oncogenes studied by means of northern blot
of messenger RNA in peripheral-blood mononuclear cells from patients with angioimmunoblastic lymphadenopathy (AILD). On average, cells from patients with AILD and from those with systemic lupus erythematosus (SLE) expressed significantly more N-ras and significantly less c-fos mRNA than did cells from healthy controls. Expression of these cellular oncogenes was most abnormal in patients with the most severe disease. In contrast, increased levels of c-myc mRNA were found in patients with SLE but not in those with AILD. Administration of cyclophosphamide to patients with AILD was followed by return to normal of both N-ras and
analysis
c-fos expression. Introduction
ANGIOIMMUNOBLASTIC
lymphadenopathy with dysrelatively uncommon disease characterised by lymphadenopathy, hepatosplenomegaly, fever, chills, malaise, and rash.1-3 It can be rapidly fatal4 and is diagnosed histologically by the presence of immunoblasts and arborised capillaries in lymphoid tissue.5 The aetiology of AILD is unknown, but there are indications that it arises from an abnormality of the immune system. A lymphoid malignancy develops in approximately a third of all AILD proteinaemia is
9. Berger M, Pirke
cyclophosphamide. Patients and Methods
Patients All patients were treated at the Clinical Center of the National Institutes of Health. The 4 patients with AILD presented with typical clinical and laboratory features of the disease, and the diagnoses were confirmed by means of lymph-node biopsy.s Patients with a diagnosis of SLE met the American Rheumatism Association criteria for that disease.18 Cyclophosphamide, 0,75 mg/m2, was given as an intravenous bolus.
a
KM, Doerr P, Kreig C, von Zerssen D. influence of weight loss on the dexamethasone suppression test. Arch Gen Psychiatry 1983; 40: 585-86. 10. Yerevanian BI, Baciewicz GJ, Iker HP, Prirtera MR. The influence of weight loss on the dexamethasone suppression test. Psychiatr Res 1984; 12: 155-60. 11. Kline MD, Beeber AR. Weight loss and the dexamethasone suppression test. Arch Gen Psychiatry 1983; 40: 1034-35. 12. Targum SD. Reported weight loss and the dexamethasone suppression test. Psychiatr Res 1983; 9: 173-74. 13. Coppen A, Harwood J, Wood K. Depression, weight loss and the dexamethasone test. 1984; 145: 88-90. Br J 14. Zimmerman J, Pfohl BM, Coryell WH. Appetite and weight change and the dexamethasone suppression test. Biol Psychiatry 1984; 19: 923-28. 15. Keitner GI, Brown WA, Qualls CB, Haier RJ, Bames KT. Results of the dexamethasone suppression test in psychiatric patients with and without weight loss. Am J Psychiatry 1985; 142: 246-48. 16. Feinberg M, Carroll BJ. Biological markers for endogenous depression. Arch Gen Psychiatry 1984; 41: 1080-85. 17. Mullaney DJ, Johnson LC, Naitoh P, Friedman JK, Globus GG. Sleep during and after gradual sleep reduction. Psychobiology 1977; 14: 237-44. 18. Metropolitan Life Insurance Company. New weight standards for men and women. Statist Bull Metropolitan Life Insurance Co 1959; 40: 1-5. 19. Rechtschaffen A, Kales A. A manual of standardised terminology techniques and scoring system for sleep stages of human subjects. Washington, DC: USPHS, 1968. 20. Nowliss V. Research with the mood adjective check list. In: Tomkins SS, Izard CE, eds. Affect, cognition and personality. New York. Springer, 1965. 21. Spielberger CD, Gorsuch RL, Luchenev R. State trait anxiety inventory manual. Palo Alto: Consulting Psychiatrists Press, 1970. 22. Beck AT, Ward CH, Mendelson M, Mock J, Erbough J. An inventory for measuring depression. Arch Gen Psychiatry 1961; 4: 561-71. 23. Carroll BJ, Feinberg M, Smouse PE, Roswon SG, Greden JF. The Can-oll rating scale for depression. BrJ Psychiatry 1981; 136: 194-200. 24. Coble PA, Kupfer DJ, Shaw DH. Distribution of REM latency in depression. Biol Psychiatry 1981; 16: 453-66. 25. Braddock L. The dexamethasone suppression test. Fact and artefact. Br J Psychiatry 1986; 148: 363-74. 26. Kupfer DJ, Foster FG, Detre TP. Sleep continuity changes in depression. Dis Nerv System 1973; 34: 192-95.
Psychiatry
the course of the disease. Lymphomas of cell types10 have been described. Patients T both B6-9 and also with AILD present with many features of autoimmune diseases including hypergammaglobulinaemia and production of various autoantiboclies .2 .. There is a clear association between abnormal expression of certain cellular oncogenes by mononuclear cells and the development of specific neoplastic and autoimmune diseases.14 To understand AILD better, we examined the messenger RNA levels of those c-onc genes whose expression was known to vary in lymphoproliferative disorders.15-17 Healthy controls and patients with systemic lupus erythematosus were analysed for comparison. Abnormal c-onc gene mRNA levels were found in the peripheral-blood mononuclear cells (PBMC) of patients with AILD and those with SLE when their disease was clinically active. These abnormalities became less pronounced in patients with AILD after treatment with
patients during
mRNA
Samples
After written informed consent had been obtained continuousflow centrifugation leukapheresis was used to collect 3-5 x 109 PBMC from patients and controls. In AILD patients leukapheresis was done at times when the white cell count was not measurably suppressed by cyclophosphamide (see table). Messenger RNA was isolated from these samples by selection over oligo (dT)-cellulose columns.19,20
Northern Blot Analysis
of Oncogene Expression
samples of poly(A) RNA were electrophoresed on 12% agarose-methylmercury hydroxide gels (northern blot analysis).21 Nitrocellulose blots obtained from these gels were hybridised with various nick-translated oncogene probes, washed, and then exposed to X-ray film.21 Standard controls were included on all gels for comparison of oncogene expression." Hybridisation intensity was calculated with a Hoeffer GS 300 scanning densitometer at a constant setting. 10 pg
Results
Oncogene Expression in AILD The expression of four cellular oncogenes was compared in 4 patients with biopsy-proven AILD and 3 controls (fig 1). In patients with AILD expression of the c-fos oncogene (which encodes a phosphoprotein localised primarily in the nucleus) was diminished, whereas that of the N-ras oncogene (which encodes a plasma-membrane protein with GTP-ase activity) was enhanced. However, not all c-onc genes were abnormally expressed: the levels of c-myb and c-myc RNA (both of which encode proteins primarily found in the nucleus) did not differ between patients and controls (fig 1). Although AILD patient 2 expressed abnormally high levels of c-myb, as reported previously,ls this was not a feature of AILD patients in general. The B-actin "housekeeping" gene was also expressed at nearly identical levels in all PBMC samples. Cellular
INFLUENCE OF SLEEP DISRUPTION AND CALORIE RESTRICTION ON BIOLOGICAL MARKERS FOR DEPRESSION PAUL E. MULLEN CHRISTOPHER R. LINSELL DAVID PARKER
Department of Psychological Medicine and the Neuroscience Centre, University of Otago, Dunedin, New Zealand The
hypothesis that the dexamethasone suppression test (DST) and rapid-eyemovement (REM) latency test are not biological markers for depressive illness but artifacts arising from dietary and sleep disturbances that accompany depression was examined in Summary
28 normal volunteers. The restriction of calorie intake with moderate weight loss reproduced a pattern of response to dexamethasone closely resembling that claimed to be diagnostic of depressive illness. The shortened REM latencies claimed as a diagnostic marker were replicated in volunteers by mimicking the sleep pattern commonly found in depression. These changes could not be explained by the induction of mood disorder in the subjects. The results put in question the diagnostic value of the DST and REM latency tests in clinical practice, where sleep disorder and poor appetite, with reduced calorie intake, are the common accompaniments of depressive illness.
1986
patientshave established themselves as diagnostic aids in clinical practice despite the continuing debate on their utility. Fasting in normal volunteers, with an associated weight loss in excess of 8 kg, has been reported to increase the escape from dexamethasone suppression.7,8 Moderate calorie restriction with gradual weight loss has been investigated by two groups with opposite results.9,10 The studies in depressed patients, aimed at establishing the effects of weight loss on the dexamethasone response, have also produced conflicting results.1O,15 Feinberg and Carroll,16 in the most extensive study to date, claim that though DST results were related to weight loss the relation was indirect-ie, weight loss was more frequent in older and more severely depressed patients. In their view, weight loss did not account for the higher post-dexamethasone cortisol levels found in endogenous depression. Progressive sleep reduction has been reported to produce shortened REM latencies,17 but no-one has examined the influence on REM latency of sleep disruption comparable with that encountered in depressed patients. We have reproduced in normal volunteers the patterns of sleep and reduced calorie intake characteristic of depressive illness and have examined their influence
on
the DST and REM
latency. Methods
Subjects Introduction THE quest for biological markers of depression has been a central theme in psychiatric research for over a decade. The hope was that a valid marker would provide both an aid to diagnosis and a signpost to the underlying pathophysiology. The dexamethasone suppression test (DST) has been the most extensively investigated and canvassed of the putative biological markers for depressive disorders,l and has been claimed to be comparable in diagnostic specificity to serum acid phosphatase levels in carcinoma of the prostate.2,3 The sleep disturbance found in depression has several characteristic polysomnographic abnormalities, the most consistent being a decreased rapid-eye-movement (REM) latency’ and this decreased REM latency has also been put forward as a diagnostic marker for depression.5 These two procedures, which are reported to identify a similar group of
The study, which was approved by the hospital and university ethical committee, involved 28 normal volunteers aged from 18 to 25 years (mean 20 SE 0-3 years). They had no history of
psychiatric disorders, eating disorders, recent dieting, sleep disturbance, or serious ill-health. Subjects with mental disorder in first-degree relatives were excluded. To ensure homogeneous populations two groups were selected, one of 14 males with a body weight within 5 % of ideal normal, and a second of 14 females who were between 5% and 10% over ideal body weight.18 The subjects were non-smokers and did not have a history of alcohol or drug abuse. The only history of taking drugs in the previous 6 months was in 3 subjects who had taken an oral contraceptive. All were university students capable of understanding the nature of the procedures. The 28 volunteers maintained a regular sleep-wake cycle for two weeks, retiring between 2200 h and 2300 h and rising between 0700 h and 0800 h, and were restricted to a diet of 2000-3000 kcal (8-4-12-6 MJ) a day. 8515
1052
Sleep Disruption Procedure The 14 male volunteers
were
admitted
to a
research ward
equipped for sleep monitoring. They were fed on a regular schedule and had no food or caffeine-containing drinks after 1800 h. Daytime naps were not allowed. The first two nights were used to acclimatise the subjects to the ward and the sleep-recording procedures. On the third and fourth nights baseline sleep recordings were obtained. On the fifth and sixth nights a baseline DST was performed. The volunteers were then subjected to seventeen nights’ partial sleep deprivation. The sleep disruption was designed to mimic the pattern of sleep disturbance characteristic of major depressive disorders. The subjects’ time of retiring to bed was delayed by an hour to 2400 h and they were awoken 2 h earlier than usual at 0500 h. The subjects spent the time between 0500 h and 0700 h watching videos or taking an early morning swim according to their inclinations. The timing of their first refreshment of the day remained the same at 0730 h. Sleep recordings were recommenced with an acclimatisation night after thirteen nights of sleep deprivation and then definitive recordings on the fifteenth and sixteenth nights. A DST was performed on the subsequent night. Subjects were then discharged from the ward and allowed to return to their regular sleep-wake pattern. The subjects were readmitted after six months and, after two nights’ acclimatisation, the sleep recordings and DST were repeated.
sleep as if they had occurred spontaneously. A modified score was also obtained for the Beck inventory and the Carroll scale by deletion of questions pertaining to lack of sleep and tiredness for the sleep-deprived group, and deletion of questions concerning weight loss for the restricted-diet group. The mood scales and general assessments were monitored regularly to ensure that the procedures were not precipitating clinical depression in our subjects. Results 1 subject was withdrawn from the study after six nights’ sleep distruption because of the development of irresistible napping and sleep paralysis with hypnagogic hallucinations. The remaining 27 subjects completed the protocol without difficulty. REM latencies were not measured on three nights owing to technical problems.
Weight The mean increase in weight of 06 (SE 03) kg following sleep deprivation compared with baseline was not significant. In contrast the 14 subjects on a restricted diet recorded a mean weight loss of 4-3 (SE 0-2) kg. Dexamethasone
Calorie Restriction Procedure The 14 female subjects were admitted to the research ward. After nights’ acclimatisation, baseline sleep recordings were performed on the third and fourth nights and the DST was begun on the next evening. The period of calorie restriction was then started with the subjects placed on a diet designed and supervised by the hospital dietitian of between 1000 and 1200 kcal (4-2-5-1MJ) per day. The subjects remained on this diet for the next 18 days. During the last four days of this period the sleep recordings and DST were repeated. The subjects were then discharged from the ward and returned to their usual dietary habits. The subjects were readmitted six months later and the sleep recordings and DST were two
repeated. Dexamethasone
Suppression Test There was a significant change in the response to dexamethasone after the period of restricted calorie intake (fig 1). Whether the threshold cortisol values for nonsuppression were taken as 3, 5, or 7 g per dl, the DSTs at completion of calorie restriction showed a significant switch from suppression to non-suppression: at 7 g/dl there were 2 non-suppressors at baseline increasing to 8 nonsuppressors after dieting (p=0-016, binomial test); at 5 ug/dl 2 subjects were non-suppressors at baseline increasing to 10 non-suppressors (p = 0004); and at 3 g/d1 4 non-suppressors at baseline increased to 11 non-suppressors after calorie restriction (p 0008). 2 subjects did not attend the six-month follow-up. The DST results for the 12 subjects studied did not differ significantly from baseline, with 4 non-suppressors by all criteria. The 4 non-suppressors at follow-up had maintained the weight loss established during the experiment with a weight gain of only 1-7 (SE 1) kg. The suppressors, in contrast, had gained an average 6-1(0-6) kg from the end of the experiment. The difference between the weight gain of the suppressors and the non-suppressors was significant =
Suppression
Test
Dexamethasone in a dose of 1 mg was administered orally at 2330 was sampled by venepuncture at 1600 h and 2300 h the following day and immediately centrifuged, the plasma being frozen at - 40°C until assay. Cortisol was measured blind and in duplicate by direct radioimmunoassay with the Amerlex radioimmunoassay kit. Intra-assay and inter-assay coefficients of variation were 4-2% and 6-8%, respectively. We used values of 3, 5, and 7 ug per dl to compute for a threshold for non-suppression.
h. Blood
Sleep Recording Sleep was recorded with two channels of the electroencephalogram (C3-A2 and 01-A2), two channels of the electrooculogram, and one channel of the electromyogram from the submental muscles. Each 20-second epoch was scored by three independent raters according to the standard criteria of Rechtschaffen and Kales.19 The definition of REM latency used was the time spent asleep from the first epoch of stage 2 until the first REM period of at least 3 min.
Psychometric Measures Mood was assessed every three days with the Nowlis Mood Adjective Checklist .21 All other psychometric measures were assessed on four occasions-at baseline, mid-point, and end-point of sleep deprivation or restricted diet, and at follow-up. State and trait anxiety were measured with the State-trait Anxiety Inventory21 (general population state mean =31-9 [SE 04] and trait mean 349 [SE 0-4]). Symptoms of depressive illness were assessed with the Beck Depression Inventory22 (general population mean == 109 [SE 0.8]) and the Carroll Rating Scale23 (general population mean = 4-6 [SE 0.4]). Subjects were instructed to score weight loss and lack of =
(p < 0-001, t test). In addition to looking at the DST results in their usual form we examined the post-dexamethasone cortisol levels. The log mean of the 1600 h and 2300 h cortisol levels increased significantly after calorie restriction from their baseline values (p < 0-01, pairedt test). This is in line with the
increase
in
the
number
of
non-suppressors.
Interestingly, the post-dexamethasone cortisol values remained significantly higher than baseline at follow-up (p<0-05, paired t test) although the difference in the number of non-suppressors did not reach statistical significance. There was a significant correlation (?-=073, p < 001) between the post-dexamethasone cortisol levels at follow-up and the loss of weight from baseline. The pre-dexamethasone log cortisol levels did not differ significantly between baseline and after calorie restriction or between baseline and follow-up. Thus the significant differences in post-dexamethasone cortisol levels after dieting and at follow-up reflect a changed response to dexamethasone. These changes at follow-up, though not sufficient to affect the number of non-suppressors
1053
Fig I-Effects of sleep deprivation and
calorie restriction
on
distribution of suppressors and non-suppressors for three criteria values of the DST.
Fig 2-Effects of sleep deprivation and calorie restriction on REM latencies.
significantly, do suggest a continuing difference in response to dexamethasone. The 3 subjects who had taken oral contraceptives recently showed no significant differences either in the log mean cortisol levels or DST results at baseline or after dieting. The number of subjects exhibiting non-suppression of cortisol levels in response to dexamethasone did not change significantly after sleep disruption (fig 1). When the log means of the 1600 h and 2300 h cortisol values were examined, pairedt tests showed no significant differences between baseline and sleep deprivation or between baseline and follow-up. REM Latency The REM latency shortened significantly after the sleep disruption procedure (fig 2). The mean REM latencies at baseline were 78 8 (SE 6-8) min and at follow-up 88-1(8) min. These values do not differ significantly. The REM latency at the end of the sleep disruption phase was significantly decreased to 55-9 (7-9) min (p < 0-01, Wilcoxon matched- pairs signed-ranks test.) After sleep disruption, six nights’ sleep recordings began with sleep-onset REM compared with none at baseline or follow-up. REM latency did not change significantly in response to calorie restriction and
loss (fig 2). The mean REM latency for the baseline was 87-4 (7-4) min which did not differ group significantly from the 95 -3 (7-4) min at the completion of the’
weight at
dieting phase. There were no occurrences of sleep REM in the calorie-restricted group of subjects.
onset
Mood The subjects on a calorie-restricted diet did not show any clinically obvious changes in affective state during the experiment and this was confirmed by the psychometric assessments (table I). Scores at the completion of the period of dieting showed a significant change only in the Carroll Rating Scale (p < 002, paired t test). When questions relating to weight loss were removed there was no longer a significant rise for the group as a whole though 3 subjects scored 12, 13, and 14 which is marginally above the cut-off of 10 for this screening instrument. There was, however, no significant correlation between the post-dexamethasone log mean cortisol levels and the scores on either the Carroll Rating Scale (Kendall’s=0 13) or the Beck Depression
Inventory (Kendall’s = - 0 -10). There was an apparent increased irritability and lability of mood in the sleep-deprived subjects towards the end of the experiment. These clinical impressions were supported by the systematic psychometric assessments (table i). The subjects showed a significant decline in mood as measured by the Nowliss Scale (p < 001), Beck Depression Inventory (p < 0-001), and the Carroll Rating Scale (p < 001) and the measure of state anxiety increased significantly (p < 0-02). These mood scales depend in part on questions relating to sleep disturbance and tiredness. When such questions were
1054 TABLE I-PSYCHOMETRIC SCORES EXPRESSED AS MEAN
(STANDARD ERROR)
subjects who had maintained a lower body weight during follow-up. Studies in depressed patients to date have been claimed to indicate that mild to moderate weight loss is neither sufficient for the
occurrence of nonof weight loss in depressed suppression.i5 is difficult. Virtually all severely notoriously patients of individuals loss appetite, though far depressed report fewer give a clear account of weight loss. Excluding an episode of decreased calorie intake with mild to moderate weight loss over the previous six months in depressed patients would present formidable practical problems. We contend, however, that unless this can be done with confidence the DST results become unreliable as a diagnostic marker for depression. The shortening of sleep time in our normal volunteers, to reproduce that reported in endogenous depression, caused a significant reduction in REM latency. The pattern of REM latency changes that results from this manipulation of the sleep-wake cycle in normal volunteers closely parallels that reported in depression.24 This does not conclusively demonstrate that the REM latency changes reported in depression are simply a reflection of reduced sleep time. What it does suggest is that diagnostic use of REM latency changes must be confined to depressed subjects without current sleep disturbance. This would remove any practical clinical applicability. This study cannot unravel the causal connections between the syndrome of major depression and both alterations in REM latency and response to dexamethasone. It remains possible that the biology of depression and calorie restriction have the same effects on the cortisol response to dexamethasone but mediated independently. Abnormal plasma cortisol control may be a symptom of depressive illness,25 but an unreliable marker where weight loss cannot be excluded. The REM latency changes found in depression may similarly reflect a fundamental neurophysiological change in depression and just happen to be reproduced by quite other mechanisms in sleep disruption. REM latency and sleep time may well vary independently in depression26 but its utility as a marker is vitiated in the presence of coexisting disruption of the sleep wake cycle. This study suggests that these frequently employed diagnostic tests will usually offer no more information to the clinician than carefully conducted histories that elicit the details of the patients’ appetite and weight loss as well as their patterns of sleep.
necessary
nor
The
*p
tp < 001;
#p < 0001.
TABLE II—COMPARISON OF RESULTS OF DST REPORTED BY
CARROLLAND THOSE PRODUCED BY CALORIE RESTRICTION
excluded the changes in the Beck Depression Inventory no longer reached statistical significance but the increase in the Carroll Score remained significant (p < 0-05). The scores, though they did indicate a deterioration in mood state, did not move into the range found in depressive disorders even when the questions relating directly to sleep disturbance were retained. This was true for all individuals as well as the group as a whole. The scores were consistent with increased anxiety but not the onset of clinically significant depression. Discussion In normal subjects, by restricting their calorie intake, we have reproduced the pattern of response to dexamethasone said to characterise depressive illness. The shortened REM
latencies claimed as a diagnostic indicator were replicated by mimicking the sleep pattern of depressives. These changes cannot be explained by the induction of a mood disorder since no subject became depressed either clinically or according to the rating scales. The sleep-disrupted group showed a greater level of psychological stress as measured by anxiety scores but their response to dexamethasone was unaffected; therefore a simple stress explanation for the positive DST results is unlikely. On calorie restriction for two weeks with a weight loss of 4-3 (SE 0-2) kg, over 70% of our subjects registered a positive DST at the 5 g/d1 criterion employed in our laboratories with this assay in the diagnosis of depressive illness. The results for criteria values of 3-7 g/dl can be expressed in the form of the sensitivity and specificity of the DST for detecting calorie restriction in our population of volunteers. When the data were expressed in this manner a close correspondence emerges between our findings and those of Carroll and his group2 for the DST as a diagnostic marker for depressive illness (table II). The effects of dieting on cortisol responses to dexamethasone in our subjects continued to be apparent at follow-up six months later, despite the subjects’ return to normal diets. Post-dexamethasone levels remained raised in the group as a whole but the rise was more pronounced in
assessment
This research was supported by a Grant from the Ashbum Hall Research Fund. C. R. L. is supported by the Hazel Buckland Trust.
Correspondence should be addressed to P. E. M., PO Box 913, Dunedin, New Zealand. REFERENCES 1. Carroll BJ. Dexamethasone suppression test m depression. Lancet 1980; ii: 1249. 2. Carroll BJ, Feinberg M, Greden JF, et al. A specific laboratory test for the diagnosis of melancholia. Arch Gen Psychiatry 1981, 38: 15-22. 3. Carroll BJ. The dexamethasone suppression test for melancholia. Br J Psychiatry
1982; 140: 292-304.
Kupfer DJ, Foster FG. EEG sleep and depression. In: Williams RL, Karacon I, eds. Sleep disorders: diagnosis and treatment New York: Wiley, 1978: 163-209. 5. Kupfer DJ Neurophysiological ’markers’—EEG sleep measures. Psychiatr Res 1984; 4.
18: 467-75. GM, Halbreich U, Sachar EJ, et al. Plasma cortisol section and REM penod latency in adult endogenous depression. Am J Psychiatry 1983; 140: 750-53. 7. Edelstein CE, Roy-Byme P, Fawzy FI, Domfeld L. Effects of weight loss on the dexamethasone suppression test. AmJ Psychiatry 1983; 140: 338-41. 8. Fichter MM, Pirke KM. Hypothalamic pituitary function in starving healthy subjects. In: Pirke KM, Ploog D, eds. The psychobiology of anorexia nervosa. Berlin: Springer Verlag, 1984: 124-35. 6. Asnis
1055
EFFECT OF CYCLOPHOSPHAMIDE THERAPY ON ONCOGENE EXPRESSION IN ANGIOIMMUNOBLASTIC LYMPHADENOPATHY DENNIS M. KLINMAN ALFRED D. STEINBERG J. FREDERIC MUSHINSKI Cellular Immunology Section, Arthritis Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, and Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Md 20892, USA
Summary
The was
expression of four cellular oncogenes studied by means of northern blot
of messenger RNA in peripheral-blood mononuclear cells from patients with angioimmunoblastic lymphadenopathy (AILD). On average, cells from patients with AILD and from those with systemic lupus erythematosus (SLE) expressed significantly more N-ras and significantly less c-fos mRNA than did cells from healthy controls. Expression of these cellular oncogenes was most abnormal in patients with the most severe disease. In contrast, increased levels of c-myc mRNA were found in patients with SLE but not in those with AILD. Administration of cyclophosphamide to patients with AILD was followed by return to normal of both N-ras and
analysis
c-fos expression. Introduction
ANGIOIMMUNOBLASTIC
lymphadenopathy with dysrelatively uncommon disease characterised by lymphadenopathy, hepatosplenomegaly, fever, chills, malaise, and rash.1-3 It can be rapidly fatal4 and is diagnosed histologically by the presence of immunoblasts and arborised capillaries in lymphoid tissue.5 The aetiology of AILD is unknown, but there are indications that it arises from an abnormality of the immune system. A lymphoid malignancy develops in approximately a third of all AILD proteinaemia is
9. Berger M, Pirke
cyclophosphamide. Patients and Methods
Patients All patients were treated at the Clinical Center of the National Institutes of Health. The 4 patients with AILD presented with typical clinical and laboratory features of the disease, and the diagnoses were confirmed by means of lymph-node biopsy.s Patients with a diagnosis of SLE met the American Rheumatism Association criteria for that disease.18 Cyclophosphamide, 0,75 mg/m2, was given as an intravenous bolus.
a
KM, Doerr P, Kreig C, von Zerssen D. influence of weight loss on the dexamethasone suppression test. Arch Gen Psychiatry 1983; 40: 585-86. 10. Yerevanian BI, Baciewicz GJ, Iker HP, Prirtera MR. The influence of weight loss on the dexamethasone suppression test. Psychiatr Res 1984; 12: 155-60. 11. Kline MD, Beeber AR. Weight loss and the dexamethasone suppression test. Arch Gen Psychiatry 1983; 40: 1034-35. 12. Targum SD. Reported weight loss and the dexamethasone suppression test. Psychiatr Res 1983; 9: 173-74. 13. Coppen A, Harwood J, Wood K. Depression, weight loss and the dexamethasone test. 1984; 145: 88-90. Br J 14. Zimmerman J, Pfohl BM, Coryell WH. Appetite and weight change and the dexamethasone suppression test. Biol Psychiatry 1984; 19: 923-28. 15. Keitner GI, Brown WA, Qualls CB, Haier RJ, Bames KT. Results of the dexamethasone suppression test in psychiatric patients with and without weight loss. Am J Psychiatry 1985; 142: 246-48. 16. Feinberg M, Carroll BJ. Biological markers for endogenous depression. Arch Gen Psychiatry 1984; 41: 1080-85. 17. Mullaney DJ, Johnson LC, Naitoh P, Friedman JK, Globus GG. Sleep during and after gradual sleep reduction. Psychobiology 1977; 14: 237-44. 18. Metropolitan Life Insurance Company. New weight standards for men and women. Statist Bull Metropolitan Life Insurance Co 1959; 40: 1-5. 19. Rechtschaffen A, Kales A. A manual of standardised terminology techniques and scoring system for sleep stages of human subjects. Washington, DC: USPHS, 1968. 20. Nowliss V. Research with the mood adjective check list. In: Tomkins SS, Izard CE, eds. Affect, cognition and personality. New York. Springer, 1965. 21. Spielberger CD, Gorsuch RL, Luchenev R. State trait anxiety inventory manual. Palo Alto: Consulting Psychiatrists Press, 1970. 22. Beck AT, Ward CH, Mendelson M, Mock J, Erbough J. An inventory for measuring depression. Arch Gen Psychiatry 1961; 4: 561-71. 23. Carroll BJ, Feinberg M, Smouse PE, Roswon SG, Greden JF. The Can-oll rating scale for depression. BrJ Psychiatry 1981; 136: 194-200. 24. Coble PA, Kupfer DJ, Shaw DH. Distribution of REM latency in depression. Biol Psychiatry 1981; 16: 453-66. 25. Braddock L. The dexamethasone suppression test. Fact and artefact. Br J Psychiatry 1986; 148: 363-74. 26. Kupfer DJ, Foster FG, Detre TP. Sleep continuity changes in depression. Dis Nerv System 1973; 34: 192-95.
Psychiatry
the course of the disease. Lymphomas of cell types10 have been described. Patients T both B6-9 and also with AILD present with many features of autoimmune diseases including hypergammaglobulinaemia and production of various autoantiboclies .2 .. There is a clear association between abnormal expression of certain cellular oncogenes by mononuclear cells and the development of specific neoplastic and autoimmune diseases.14 To understand AILD better, we examined the messenger RNA levels of those c-onc genes whose expression was known to vary in lymphoproliferative disorders.15-17 Healthy controls and patients with systemic lupus erythematosus were analysed for comparison. Abnormal c-onc gene mRNA levels were found in the peripheral-blood mononuclear cells (PBMC) of patients with AILD and those with SLE when their disease was clinically active. These abnormalities became less pronounced in patients with AILD after treatment with
patients during
mRNA
Samples
After written informed consent had been obtained continuousflow centrifugation leukapheresis was used to collect 3-5 x 109 PBMC from patients and controls. In AILD patients leukapheresis was done at times when the white cell count was not measurably suppressed by cyclophosphamide (see table). Messenger RNA was isolated from these samples by selection over oligo (dT)-cellulose columns.19,20
Northern Blot Analysis
of Oncogene Expression
samples of poly(A) RNA were electrophoresed on 12% agarose-methylmercury hydroxide gels (northern blot analysis).21 Nitrocellulose blots obtained from these gels were hybridised with various nick-translated oncogene probes, washed, and then exposed to X-ray film.21 Standard controls were included on all gels for comparison of oncogene expression." Hybridisation intensity was calculated with a Hoeffer GS 300 scanning densitometer at a constant setting. 10 pg
Results
Oncogene Expression in AILD The expression of four cellular oncogenes was compared in 4 patients with biopsy-proven AILD and 3 controls (fig 1). In patients with AILD expression of the c-fos oncogene (which encodes a phosphoprotein localised primarily in the nucleus) was diminished, whereas that of the N-ras oncogene (which encodes a plasma-membrane protein with GTP-ase activity) was enhanced. However, not all c-onc genes were abnormally expressed: the levels of c-myb and c-myc RNA (both of which encode proteins primarily found in the nucleus) did not differ between patients and controls (fig 1). Although AILD patient 2 expressed abnormally high levels of c-myb, as reported previously,ls this was not a feature of AILD patients in general. The B-actin "housekeeping" gene was also expressed at nearly identical levels in all PBMC samples. Cellular