A direct ligand-binding radioassay for the measurement of methotrexate in tissues and biological fluids.

A direct ligand-banding radioassay for methotrexate (MTX) has been developed using dihydrofolate reductase, contained in the lysate of L1210 leukemia cells, as the binding determinant. The procedure is a two-phase reaction system where standard MTX concentrations or the sample being assayed in incubated with the reagent lysate in the first phase, and [3H]MTX is then added in the second phase to titrate the remaining unoccupied binding sites on the enzyme. This method eliminates the need for measuring the residual catalytic activity of the enzyme. The sensitivity of the radioassay is limited only by the specific activity of the [3H]MTX and how approximates 10 pg of the drug. Folic acid, methyltetrahydrofolate, formyltetrahydrofolate, and dehydrofolate in concentrations that are physiological do not interfere in the radioassay. Both mercaptoethanol and reduced nicotinamide andnine dinucleotide phosphate increase the binding capacity of the lysate for MTX; but the reduced nucleotide also increases the affinity of the enzyme for the inhibitor. MTX added to serum can be assayed without extraction if the concentration is greater than 500 pg/ml and recovery of the drug added to serum is about 92%. MTX has been assayed in serum, spinal fluid, and urine of patients who were treated with this drug. It has also been assayed in the lysates of L1210 cells from C57BL X DBA/2 F1 mice treated with MTX. The procedure is simple, rapid, and accurate and should permit better correlation of the therapeutic and toxic effects of MTX with blood concentrations over long-term treatment periods.

Phasic activities of rapid eye movement sleep in vegetative state patients.

STUDY OBJECTIVES: To assess the phasic components of rapid eye movement (REM) sleep in patients in vegetative state and to evaluate the possible relationship of these activities to patient outcome. SETTING: Sleep disorders unit at a major rehabilitation hospital. DESIGN: Comparative control study. PATIENTS: Eleven patients in vegetative state (10 males and 1 female) aged 17-53 years. INTERVENTIONS: Continuous 24-hour polysomnographic recording. MEASUREMENTS AND RESULTS: All the patients had REM sleep periods during the 24-hr recording session. Mean total REM sleep time for the whole session was 66.5 +/- 34.9 min, and for the nocturnal hours only, 37.3 +/- 19.7 min. Comparison with the control group (79.2 +/- 11.5 min) yielded a significant difference only for nocturnal REM sleep time (p<0.0003). The duration of the REM sleep periods was significantly shorter in the patients than the controls for the whole 24-hr session (10.9 +/- 6.0 vs.19.6 +/- 4.9 min, p<0.008), but not for the nocturnal period alone. Compared to controls, the density of rapid eye movements (REMs) (p=0.001), chin twitches (p=0.002), and leg muscle twitches (p=0.023) was significantly lower in the patient group. The density of the sawtooth waves was also lower in the patients, but the difference did not reach significance (p=0.069). Similar results were obtained when the comparison was done only for the nocturnal period. There was no significant difference for any of the REM sleep characteristics or REM sleep phasic activities (24-hr, nocturnal and diurnal periods) between the patients who recovered consciousness and those who did not. CONCLUSIONS: The present study shows that patients in vegetative state have a significant reduction in the phasic activities of REM sleep. However, the amount of these activities is unrelated to recovery from the clinical condition. These findings may reflect possible damage to the pedunculopontine tegmentum cholinergic mechanisms in vegetative state.

Sleep-related erections in vegetative state patients.

STUDY OBJECTIVES: To determine whether sleep-related erections occur in vegetative state and if so, to investigate their relationship with rapid eye movement (REM) sleep. DESIGN: N/A. SETTING: Major rehabilitation hospital. PATIENTS: Nine male patients in vegetative state aged 17-40 years. INTERVENTIONS: Continuous 24-hour polysomnographic recordings including penile circumferencial changes. MEASUREMENTS AND RESULTS: Sleep-related erection episodes (SREe's) were noted in all nine patients, ranging in number from 1-7/24 hr (average 4.4+2.4) and lasting 6 to 50 min (average 22.0+5.7 min). The number of REM periods (REMp's) ranged from 4-11 (average 6.6+2.5) and lasted for 1.0 to 44.0 min (average 16.0+6.6 min). Ninety-five percent of the SREe's recorded were associated with REMp's, usually (76.3%) appearing simultaneously with the REMp or soon thereafter; 64.6% of the REMp's were associated with SRE's. For both the nocturnal and diurnal periods, there were more REMp's with SREe's than without, and the REMp's associated with SREe's were of longer duration (by 25.5% and 28.4%, respectively). There were no statistically significant differences for any of the REMp or SREe parameters between the recovered and nonrecovered patients. CONCLUSIONS: The sleep-related erection characteristics of patients in vegetative state are similar to those of normal individuals. These findings may have implications for the assessment of the reorganization of REM sleep during recovery from vegetative state and may further help in our understanding of the pathophysiology of vegetative state. More studies are needed in larger groups of patients.

The in vivo enhancement of IL-1alpha receptor expression on polyoma virus transformed tumor derived cells.

The aim of this study was to evaluate expression of receptor for IL-1 on tumor-derived cells. The in vivo acquisition of an expression of a receptor for Fc ç immunoglobulin on polyoma virus transformed cells has been established by us. We investigated whether a receptor for the IL-1 cytokine, like that for Fc gamma immunoglobulin, could also contribute to the heterogeneity of tumor cell population, as well as to its tumorigenic phenotype. Various clones of polyoma virus transformed 3T3 cells were passaged once in syngeneic mice and resulting tumors explanted and recultured. The expression of receptor for IL-1 was tested on in vitro maintained clones (designated C for culture) and on tumor derived clones (designated CTC - culture-tumor-culture). Expression was determined using a 125I radiolabeled ligand and confirmed by flow cytometry with anti-mouse IL-1 receptor (IL-1R) antibodies. Some CTC clones expressed a higher level of IL-1 receptor than others. A positive correlation between the level of IL-1R and a metastatic phenotype was established with some tumor derived cells. A high IL-1R expressing tumor cell population, sorted by flow cytometry, was considerably more metastatic than the sorted low IL-1 expressing cells. IL-1R expression by tumor derived cells may, contribute to the metastatic phenotype of a tumor cell population.

The sleep supine position has a major effect on optimal nasal continuous positive airway pressure : relationship with rapid eye movements and non-rapid eye movements sleep, body mass index, respiratory disturbance index, and age.

STUDY OBJECTIVES: To evaluate the impact of sleep position on optimal nasal continuous positive airway pressure (nCPAP [op-nCPAP]) in obstructive sleep apnea (OSA) patients and to investigate how rapid eye movements (REM) and Non-REM (NREM) sleep, body mass index (BMI), respiratory disturbance index (RDI), and age are related to this effect. DESIGN: Retrospective analysis. SETTING: Sleep Disorders Unit at Loewenstein Hospital Rehabilitation Center. PATIENTS: Eighty-three consecutive adult OSA patients who underwent a complete nCPAP titration. From this group, 60 patients who spent at least 30 min in both the supine (Sup) and lateral (Lat) positions and 46 patients who had data on both positions during REM and NREM sleep were included in the analysis. RESULTS: In most OSA patients (52; 86.7%), the recommended op-nCPAP was obtained when the patients slept in the Sup posture. The mean op-nCPAP was significantly higher in the Sup posture (10.00 +/- 2.20 cm H(2)O) than it was in the Lat posture (7.61 +/- 2.69 cm H(2)O). The op-nCPAP was significantly higher in the Sup position than it was in the Lat position in both REM and NREM sleep, as well as in the severe BMI group (BMI >/= 30) and in the less obese group (BMI < 30). Similarly, in the severe (RDI >/= 40) and less severe groups (RDI < 40), as well as in both age groups (< and > 60 years of age), the op-nCPAP was significantly higher in the Sup posture than it was in the Lat posture. Irrespective of the four parameters mentioned, the actual differences in op-nCPAP between the two body postures were almost identical, ranging between 2.31 and 2.66 cm H(2)O. CONCLUSIONS: For most OSA patients, the op-nCPAP level is significantly higher in the Sup position than it is in the Lat position. This is true for REM and NREM sleep, for obese and nonobese patients, for patients with different degrees of severity, and for young and old OSA patients. Since the op-nCPAP was highest in the Sup posture during REM sleep, no nCPAP titration should be considered complete without the patient having slept in the Sup posture during REM sleep.

Positional vs nonpositional obstructive sleep apnea patients: anthropomorphic, nocturnal polysomnographic, and multiple sleep latency test data.

STUDY OBJECTIVES: To compare anthropomorphic, nocturnal polysomnographic (PSG), and multiple sleep latency test (MSLT) data between positional (PP) and nonpositional (NPP) obstructive sleep apnea (OSA) patients. DESIGN: This is a retrospective analysis of anthropomorphic, PSG, and MSLT data of a large group of OSA patients who underwent a complete PSG evaluation in our sleep disorders unit. The patients were divided in two groups: the PP group, those patients who had a supine respiratory disturbance index (RDI) that was at least two times higher than the lateral RDI, and the NPP group, those patients in whom the RDI in the supine position was less than twice that in the lateral position. SUBJECTS: From a group of 666 consecutive OSA patients whose conditions were diagnosed in our unit from September 1990 to February 1995, 574 patients met the following criteria and were included in the study: RDI > 10; age > 20 years, and body mass index (BMI) > 20. RESULTS: Of all 574 patients, 55.9% were found to be positional. No differences in height were observed but weight and BMI were significantly higher in the NPP group, these patients being on the average 6.5 kg heavier than those in the PP group. The PP group was, on average, 2 years younger than the NPP group. Nocturnal sleep quality was better preserved in the PP group. In this group, sleep efficiency and the percentages of deep sleep (stages 3 and 4) were significantly higher while the percentages of light sleep (stages 1 and 2) were significantly lower than in the NPP group. No differences for rapid eye movement (REM) sleep were found. In addition, wakefulness after sleep onset and the number of short arousals (< 15 s) were significantly lower in the PP group. Apnea index and total RDI were significantly higher and the minimal arterial oxygen saturation in REM and non-REM sleep was significantly lower in the NPP. No differences in periodic limb movements data were found between the two groups. The average MSLT was significantly shorter in the NPP group. Univariate and multivariate stepwise logistic regression analysis showed that the most dominant variable that correlates with positional dependency in OSA patients is RDI, followed by BMI which also adds a significant contribution to the prediction of positional dependency. Age, although significant, adds only a minor improvement to the prediction of this positional dependency phenomenon. A severe, obese, and older OSA patient is significantly less likely to be positional than a mild-moderate, thin, and young OSA patient. In four obese OSA patients who lost weight, a much more pronounced reduction was seen in the lateral RDI than in the supine RDI, and three of these cases who were previously NPP became PP. CONCLUSIONS: In a large population of OSA patients, most were found to have at least twice as many apneas/hypopneas in the supine than in the lateral position. These so-called "positional patients" are on the average thinner and younger than "nonpositional patients." They had fewer and less severe breathing abnormalities than the NPP group. Consequently their nocturnal sleep quality was better preserved and, according to MSLT data, they were less sleepy during daytime hours. RDI was the most dominant factor that could predict the positional dependency followed by BMI and age. RDI showed a threshold effect, the prevalence of PP in those with severe RDI (RDI > or = 40) was significantly lower than in those OSA patients with mild-moderate RDI. BMI showed a major significant inverse relationship with positional dependency, while age had only a minor although significant inverse relationship with it. Body position during sleep has a profound effect on the frequency and severity of breathing abnormalities in OSA patients.

Avoiding the supine position during sleep lowers 24 h blood pressure in obstructive sleep apnea (OSA) patients.

Obstructive sleep apnea (OSA), is a common clinical condition affecting at least 2-4% of the adult population. Hypertension is found in about half of all OSA patients, and about one-third of all patients with essential hypertension have OSA. There is growing evidence that successful treatment of OSA can reduce systemic blood pressure (BP). Body position appears to have an important influence on the incidence and severity of these sleep-related breathing disturbances. We have investigated the effect of avoiding the supine position during sleep for a 1 month period on systemic BP in 13 OSA patients (six hypertensives and seven normotensives) who by polysomnography (PSG) were found to have their sleep-related breathing disturbances mainly in the supine position. BP monitoring was performed by 24-h ambulatory BP measurements before and after a 1 month intervention period. We used a simple, inexpensive method for avoiding the supine posture during sleep, namely the tennis ball technique. Of the 13 patients, all had a reduction in 24-h mean BP (MBP). The mean 24-h systolic/diastolic (SBP/DBP) fell by 6.4/2.9 mm Hg, the mean awake SBP/DBP fell by 6.6/3.3 mm Hg and the mean sleeping SBP/DBP fell by 6.5/2.7 mm Hg, respectively. All these reductions were significant (at least P < 0.05) except for the sleeping DBP. The magnitude of the fall in SBP was significantly greater in the hypertensive than in the normotensive group for the 24 h period and for the awake hours. In addition, a significant reduction in BP variability and load were found. Since the majority of OSA patients have supine-related breathing abnormalities, and since about a third of all hypertensive patients have OSA, avoiding the supine position during sleep, if confirmed by future studies, could become a new non-pharmacological form of treatment for many hypertensive patients.

[The effect of memorization processes on the structural organization of natural human sleep]. / Vliianie protsessov zapominaniia na strukturnuiu organizatsiiu estestvennogo sna cheloveka.

The effect of sleep on learning was investigated, comparing results of memorizing and reproduction of an unknown text before and after subsequent sleep, with a detailed analysis of sleep patterns. Psychological tests excluded the possibility of emotional and stress factors. Presleep learning did not influence mean values of such sleep parameters as total sleep time or duration of different sleep stages. The main finding ot the present experiment was a redistribution of stage REM during nocturnal sleep following learning--its increase in the second sleep cycle with a corresponding decrease toward the end of night. Also, individual difficulties in learning were inversely related to REM latency. Changes in sleep patterns after learning didn't influence the total number of sleep cycles. It is suggested that the REM phase of sleep might be involved in the processing of information acquired during wakefulness.

Lensless imaging by spatial Fourier synthesis holography.

The technique of Fourier synthesis holography is extended to the spatial domain. A spatially extended source is decomposed into its Fourier components, and a hologram of an object distribution is formed at each spatial frequency and stored in a computer. Upon synthesis in the computer a clear image can be formed of the object without the use of lenses.

Improved imagery through scattering materials by quasi-Fourier-synthesis holography.

An improvement on the technique of Fourier-synthesis holography is proposed and demonstrated. Artifacts produced during the process of sampling are eliminated when the laser is swept over a continuous bandwidth between samples. The advantages of Fourier-synthesis holography, such as the ability to select the gating time delay and to shape the autocorrelation function after data acquisition, are retained.

Analysis of Fourier synthesis holography for imaging through scattering materials.

The technique of Fourier synthesis holography to image through scattering materials is analyzed in detail. A broad spectral source is decomposed into its Fourier components, and a hologram is formed at each wavelength and stored in the computer. Upon synthesis in the computer, a clear image can be formed of the obscured object. Post-data-acquisition processing such as selection of the gating time delay and autocorrelation shaping are also demonstrated.

Nonscanning confocal ranging system.

We demonstrate a nonscanning confocal ranging system based on spatially incoherent interferometry. Such a system has significant advantages over the conventional confocal imaging system and other interferometric systems. We develop the theory in terms of coherence cells and demonstrate the equivalence of our method to the conventional confocal methods. Experimental results are also provided.

Use of Fourier synthesis holography to image through inhomogeneities.

A method for image formation through inhomogeneities is demonstrated. A broad spectral source is decomposed into its Fourier components, and a hologram is recorded at each wavelength through a diffusing medium. When the holograms are synthesized in a computer, a clear image can be formed of the obscured object.

Organization and functional analysis of the mouse transporter associated with antigen processing 2 promoter.

In accordance with the key role of MHC class I molecules in the adaptive immune response against viruses, they are expressed by most cells, and their expression can be enhanced by cytokines. The assembly and cell surface expression of class I complexes depend on a continuous peptide supply. The peptides are generated mainly by the proteasome and are transported to the endoplasmic reticulum by a peptide transport pump consisting of two subunits, TAP1 and TAP2. The proteasome low molecular weight polypeptide (2 and 7), as well as TAP (1 and 2) genes, are coordinately regulated and are induced by IFNs. Despite this coordinate regulation, examination of tumors shows that these genes can be discordantly down-regulated. In pursuing a molecular explanation for these observations, we have characterized the mouse TAP2 promoter region and 5'-flanking sequence. We show that the 5' untranslated regions of TAP2 genes have a characteristic genomic organization that is conserved in both the mouse and the human. The mouse TAP2 promoter belongs to a class of promoters that lack TATA boxes but contain a MED1 (multiple start site element downstream) sequence. Accordingly, transcription is initiated from multiple sites within a 100-nucleotide window. An IFN regulatory factor 1 (IRF1)/IRF2 binding site is located in this region and is involved in both basal and IRF1-induced TAP2 promoter activity. The implication of the extensive differences found among the promoters of class I heavy chain, low molecular weight polypeptide, and TAP genes, all encoding proteins involved in Ag presentation, is discussed.