miR-146a, miR-155, miR-370, and miR-708 Are CFTR-Dependent, Predicted FOXO1 Regulators and Change at Onset of CFRDs.

CONTEXT: Cystic fibrosis-related diabetes (CFRD) is the most frequent and severe co-morbidity in cystic fibrosis (CF). Presentation and severity are quite variable. OBJECTIVE: To investigate changes in microRNAs (miRNAs) due to CF transmembrane conductance regulator malfunctioning in vitro, to study the circulating levels of selected miRNAs in serum samples from patients, and to assess their relationships in different age groups with genotype, glucose tolerance state, and at onset of CFRD. Design/Setting/Patients/Interventions: Transcriptional profiling of all known miRNAs in CFBE41o- cells, in their normal counterparts (16HBE14o- cells), and in IB3 cells was performed. A set of miRNAs was differentially expressed in the CF cells. By in silico analysis, four miRNAs (miR-146a, miR-155, miR-370, and miR-708) were selected as potential regulators of the FOXO1 gene. Seventy-four CF patients and 50 healthy subjects whose glucose tolerance was characterized by an oral glucose tolerance test were enrolled in the study, and the identified miRNAs were quantified in serum by quantitative RT-PCR. Main Outcome Measurements/Results: A total of 111 miRNAs were differentially expressed in the two CF cell lines. miR-155, miR-370, and miR-708 were up-regulated and miR-146a was down-regulated in vitro, whereas in vivo, miR-146a, miR-155, and miR-370 were up-regulated, and miR-708 was down-regulated. These changes showed relationships with genotype, glucose tolerance state, and onset of CFRD. CONCLUSIONS: The data showed significant changes in miRNAs dependent on genotype and glucose tolerance state in CF patients and highlighted some miRNAs of importance in CFRD at onset. miRNAs could explain some of the variability observed in CF.

Comparison of Environmental Scanning Electron Microscopy in Low Vacuum or wet mode for the investigation of cell biomaterial interactions.

AIM: The aim of the present study was to investigate the efficacy of environmental scanning electron microscopy (ESEM), in low vacuum mode (LV-ESEM) and in wet mode (wet-ESEM) in the assessment of cell-material interactions. METHODS: Mouse calvaria MC3T3 cells (ATCC) were seeded on commercially pure machined titanium discs of 10 mm diameter in Dulbecco modified MEM, 10% Fetal Bovine Serum, 1% Penicillin and Streptomycin and 1% Glutamine. Samples were then processed for microscope observation by rinse in Phosphate Buffer saline and fixation in 4.5% Glutaraldehyde. Samples were then rinsed in Sodium Cacodylate buffer and observed or dehydrated in alcohol prior to LV-ESEM observation. Fresh samples in 0.9% NaCl solution were observed in wet- ESEM. RESULTS: No significant loss of detail was observed when dehydrated or non dehydrated samples were analysed at LV-ESEM.The observation of fresh samples in wet-ESEM however proved difficult for the need to eliminate water which forms a layer covering the sample, thus hiding cell surface details. When reducing the vapor pressure in the chamber, the layer evaporated and NaCl immediately started to precipitate and cells collapsed, thus no further investigation was possible. CONCLUSIONS: The use of low vacuum-ESEM after cell fixation, but without dehydration or gold sputter coating proved a viable alternative to traditional high vacuum SEM observation.

HMGB1 Is Increased by CFTR Loss of Function, Is Lowered by Insulin, and Increases In Vivo at Onset of CFRD.

CONTEXT: Cystic fibrosis-related diabetes (CFRD) is associated with worsening of inflammation and infections, and the beginning of insulin treatment is debated. OBJECTIVES: To verify high-mobility group box 1 protein (HMGB1) levels in CF patients according to glucose tolerance state, and analyze relationships with insulin secretion and resistance. To verify, in an in vitro model, whether HMGB1 gene expression and protein content were affected by insulin administration and whether these changes were dependent on CF transmembrane conductance regulator (CFTR) loss of function. PATIENTS AND METHODS: Forty-three patients in stable clinical conditions and 35 age- and sex-matched controls were enrolled. Glucose tolerance was established in patients based on a 5 point oral glucose tolerance test (OGTT). Fasting glucose to insulin ratio (FGIR), HOMA-IR index, whole-body insulin sensitivity index (WIBISI), and the areas under the curve for glucose (AUCG) and insulin (AUCI) were calculated. HMGB1 was assayed in serum, in cell lysates and conditioned media using a specific ELISA kit. For the in vitro study we used CFBE41o- cells, homozygous for the F508del mutation, and 16HBE14o- as non-CF control. HMGB1 gene expression was studied by real-time RT-PCR. Cells were stimulated with insulin at 2.5 and 5 ng/mL. The CFTR inhibitor 172 and CFTR gene silencing were used to induce CFTR loss of function in 16HBE14o- cells. RESULTS: HMGB1 levels were increased at onset of CFRD (5.04 ± 1.2 vs 2.7 ± 0.3 ng/mL in controls; P < .05) and correlated with FGIR (R = +0.43; P = .038), and AUCI (R = +0.43; P = .013). CFTR loss of function in the 16HBE14o- cells increased HMGB1 and was lowered by insulin. CONCLUSION: HMGB1 was increased in CF patients with deranging glucose metabolism and showed relationships with indexes of glucose metabolism. The increase in HMGB1 was related to CFTR loss of function, and insulin lowered HMGB1. Further research is required to verify whether HMGB1 could potentially be a candidate marker of onset of CFRD and to establish when to start insulin treatment.

Di-(2-ethylhexyl) phthalate metabolites in urine show age-related changes and associations with adiposity and parameters of insulin sensitivity in childhood.

OBJECTIVES: Phthalates might be implicated with obesity and insulin sensitivity. We evaluated the levels of primary and secondary metabolites of Di-(2-ethylhexyl) phthalate (DEHP) in urine in obese and normal-weight subjects both before and during puberty, and investigated their relationships with auxological parameters and indexes of insulin sensitivity. DESIGN AND METHODS: DEHP metabolites (MEHP, 6-OH-MEHP, 5-oxo-MEHP, 5-OH-MEHP, and 5-CX-MEHP), were measured in urine by RP-HPLC-ESI-MS. Traditional statistical analysis and a data mining analysis using the Auto-CM analysis were able to offer an insight into the complex biological connections between the studied variables. RESULTS: The data showed changes in DEHP metabolites in urine related with obesity, puberty, and presence of insulin resistance. Changes in urine metabolites were related with age, height and weight, waist circumference and waist to height ratio, thus to fat distribution. In addition, clear relationships in both obese and normal-weight subjects were detected among MEHP, its products of oxidation and measurements of insulin sensitivity. CONCLUSION: It remains to be elucidated whether exposure to phthalates per se is actually the risk factor or if the ability of the body to metabolize phthalates is actually the key point. Further studies that span from conception to elderly subjects besides further understanding of DEHP metabolism are warranted to clarify these aspects.

FOXO1 content is reduced in cystic fibrosis and increases with IGF-I treatment.

Cystic fibrosis-related diabetes is to date the most frequent complication in cystic fibrosis (CF). The mechanisms underlying this condition are not well understood, and a possible role of insulin resistance is debated. We investigated insulin signal transduction in CF. Total insulin receptor, IRS1, p85 PI3K, and AKT contents were substantially normal in CF cells (CFBE41o-), whereas winged helix forkhead (FOX)O1 contents were reduced both in baseline conditions and after insulin stimulation. In addition, CF cells showed increased ERK1/2, and reduced ß2 arrestin contents. No significant change in SOCS2 was observed. By using a CFTR inhibitor and siRNA, changes in FOXO1 were related to CFTR loss of function. In a CF-affected mouse model, FOXO1 content was reduced in the muscle while no significant difference was observed in liver and adipose tissue compared with wild-type. Insulin-like growth factor 1 (IGF-I) increased FOXO1 content in vitro and in vivo in muscle and adipose tissue. In conclusion; we present the first description of reduced FOXO1 content in CF, which is compatible with reduced gluconeogenesis and increased adipogenesis, both features of insulin insensitivity. IGF-I treatment was effective in increasing FOXO1, thereby suggesting that it could be considered as a potential treatment in CF patients possibly to prevent and treat cystic fibrosis-related diabetes.

The effect of age, gender, and insertion site on marginal bone loss around endosseous implants: results from a 3-year trial with premium implant system.

OBJECTIVES: The goal of this study was to evaluate bone changes around endosseous implants in partially edentulous patients. MATERIALS AND METHODS: A total of 632 two-stage implants were placed in 252 patients. The implants had straight emergence profile, ZirTi surface, 3.3 to 5 mm diameter, and 8.5 to 13 mm length. Bone levels were assessed on orthopantomography immediately after surgery and after 36 months and marginal bone loss (MBL) was calculated from their difference. RESULTS: Cumulative survival rate was 98.73%. Overall MBL was 0.8 mm ± 0.03 (mean ± SEM). Higher MBL was observed around implants in the maxilla than in the mandible (P < 0.007). A relation between implant diameter and MBL (P < 0.0001) was observed in male and, more limitedly, female patients. Older patients had higher MBL in the maxilla, but not in the mandible (P < 0.0001). MBL progressively increased with age in male patients, but reached a peak already in the 50-60 years age group in the female subset (P < 0.001). CONCLUSIONS: The overall MBL is consistent with the available literature. Site difference and patient age and gender appear to significantly affect MBL, representing important factors to be considered during implant placement.

Artificial Neural Networks, and Evolutionary Algorithms as a systems biology approach to a data-base on fetal growth restriction.

One of the specific aims of systems biology is to model and discover properties of cells, tissues and organisms functioning. A systems biology approach was undertaken to investigate possibly the entire system of intra-uterine growth we had available, to assess the variables of interest, discriminate those which were effectively related with appropriate or restricted intrauterine growth, and achieve an understanding of the systems in these two conditions. The Artificial Adaptive Systems, which include Artificial Neural Networks and Evolutionary Algorithms lead us to the first analyses. These analyses identified the importance of the biochemical variables IL-6, IGF-II and IGFBP-2 protein concentrations in placental lysates, and offered a new insight into placental markers of fetal growth within the IGF and cytokine systems, confirmed they had relationships and offered a critical assessment of studies previously performed.

Effects of cord serum insulin, IGF-II, IGFBP-2, IL-6 and cortisol concentrations on human birth weight and length: pilot study.

BACKGROUND: The IGF system is recognised to be important for fetal growth. We previously described increased Insulin-like growth factor binding protein (IGFBP)-2 cord serum concentrations in intra-uterine growth retardation (IUGR) compared with appropriate for gestational age (AGA) newborns, and a positive relationship of IGFBP-2 with Interleukin (IL)-6. The role of cortisol in the fetus at birth is largely unknown, and interactions among peptides are their real effect on birth size is unknown. Furthermore, almost all studies have previously assayed peptides in serum several years after birth, and follow-up data from pregnancy are always lacking. This study aimed at establishing and clarifying the effect of cord serum insulin, IGF-II, IGFBP-2, cortisol and IL-6 concentrations on birth length and weight. METHODS: 23 IUGR and 37 AGA subjects were followed up from the beginning of pregnancy, and were of comparable gestational age. Insulin, IGF-II, IGFBP-2, cortisol and IL-6 concentrations were assayed in cord serum at birth, and a multiple regression model was designed and applied to assess which were the significant biochemical determinants of birth size. RESULTS: Insulin, cortisol, and IL-6, showed similar concentrations in IUGR and AGA as previously described, whereas IGF-II was lower, and IGFBP-2 increased in IUGR compared with AGA. IGF-II serum concentration was found to have a significant positive effect on both birth length (r:(:)0.546; p: 0.001) and weight (r:0.679; p: 0.0001). IGFBP-2 had a near significant negative effect on both birth weight (r:-0.342; p: 0.05) and length (r:-0.372; p:0.03). CONCLUSION: IGF-II cord serum concentration was shown to have a significant positive effect on both birth length and weight, whereas IGFBP-2 had a significant negative effect. Insulin, cortisol, and IL-6 cord serum concentrations had no significant effect on birth size.

Sleep phenotypes of intellectual disability: a polysomnographic evaluation in subjects with Down syndrome and Fragile-X syndrome.

OBJECTIVE: To analyze sleep architecture and NREM sleep alterations by means of the Cyclic Alternating Pattern (CAP) in children with Down syndrome (DS) and Fragile-X syndrome (fraX), the two most common causes of inherited mental retardation, in order to find out eventual alterations of their sleep microstructure related to their mental retardation phenotypes. METHODS: Fourteen patients affected by fraX (mean age 13.1 years) and 9 affected by Down syndrome (mean age 13.8 years) and 26 age-matched normal controls were included. All subjects underwent overnight polysomnography in the sleep laboratory, after one adaptation night and their sleep architecture and CAP were visually scored. RESULTS: FraX subjects showed a reduced time in bed compared to DS subjects, whereas DS subjects showed a lower sleep efficiency, a higher percentage of wakefulness after sleep onset, and a reduced percentage of stage 2 NREM compared to the other groups. Furthermore, DS and fraX subjects, compared to normal controls, showed a higher percentage of stage 1 NREM and a lower percentage of REM sleep. FraX subjects showed the most disrupted sleep microstructure with low total CAP rate and CAP rate in S2 NREM. Both patient groups showed a lower percentage of A1 and higher percentage of A2 and A3 compared to normal controls. CONCLUSIONS: The analysis of CAP might be able to disclose new important findings in the sleep architecture of children with mental retardation and might characterize sleep microstructural patterns of the different phenotypes of intellectual disability. SIGNIFICANCE: The NREM sleep microstructure alterations found in our subjects, associated with the reduction in REM sleep percentage, seem to be distinctive features of intellectual disability.

Polysomnographic study of intermittent zolpidem treatment in primary sleep maintenance insomnia.

BACKGROUND AND PURPOSE: Treatment of chronic insomnia with nightly hypnotics is efficacious, but discontinuation is recommended after 1 month, less than the average disease duration. This study was undertaken to determine the efficacy of intermittent administration. PATIENTS AND METHODS: A double-blind study was carried out on 8 patients (age, 32.8 +/- 9 years; 3 men) with primary sleep maintenance insomnia longer than 1 month. Polysomnography of conventional sleep parameters, cyclic alternating patterns (CAPs), and arousals was performed. Perception of sleep quality was assessed on a visual analog scale. After an adaptation night, baselines were recorded followed by 6 consecutive nights of alternating treatment with zolpidem (10 mg) or placebo. RESULTS: Significant improvements on baseline values (P < 0.0001) were observed on all 3 active treatment nights for total sleep time, sleep efficiency, CAP time, CAP rate, subtype A2, arousals, and arousal index. Deep non-rapid eye movement sleep increased with the second and third doses of active treatment (P < 0.0001). Rapid eye movement sleep increased during the last 3 polysomnographic recordings (P < 0.014). Sleep quality (visual analog scale) improved on all nights after the initial dose of active treatment (P < 0.0001). There was no evidence of rebound insomnia with placebo. CONCLUSIONS: Intermittent treatment with zolpidem in primary insomnia patients improves CAP parameters and arousals, as well as sleep duration and quality, in the absence of rebound insomnia.

Sleep in children with autistic spectrum disorder: a questionnaire and polysomnographic study.

OBJECTIVE: To evaluate sleep in children with autistic spectrum disorder (ASD) by means of sleep questionnaires and polysomnography; moreover, to analyze their cyclic alternating pattern (CAP). METHODS: Thirty-one patients with ASD (28 males, 3 females, aged 3.7-19 years) and age-matched normal controls were included. ASD children were evaluated by a standard sleep questionnaire that consisted of 45 items in a Likert-type scale covering several areas of sleep disorders and by overnight polysomnography in the sleep laboratory after one adaptation night. RESULTS: The questionnaire results showed that parents of ASD children reported a high prevalence of disorders of initiating and maintaining sleep, enuresis, repetitive behavior when falling asleep, and daytime sleepiness. Polysomnographically, ASD children showed reduced time in bed, total sleep time, sleep period time and rapid eye movement (REM) latency. ASD subjects had a CAP rate during slow-wave sleep (SWS) lower than normal controls, together with a lower percentage of A1 subtypes. CONCLUSIONS: ASD children questionnaires showed a higher percentage of disorders of initiating and maintaining sleep than normal controls; this was not completely confirmed by sleep staging. CAP measures showed subtle alterations of NREM sleep which could be detected with an appropriate methodology of analysis. The reduction of A1 subtypes during SWS might play a role in the impairment of cognitive functioning in these subjects.

Cyclic alternating pattern sequences and non-cyclic alternating pattern periods in human sleep.

OBJECTIVE: The CAP cycle is a module of activation (phase A) and inhibition (phase B) which repeats itself in sequences. The study aims at testing the hypothesis that the duration of CAP sequences is determined primarily by the number and not by the length of CAP cycles. METHODS: The polysomnographic recordings of 24 normal subjects, 12 males and 12 females, ranging in age from 20 to 35 years (mean 27.8+/-7.2), were examined. RESULTS: A total of 1053 CAP sequences were counted with an average of 43.9 sequences per night. The mean duration of CAP sequences was 2 min and 33 s. Each CAP sequence was composed of an average of 5.6 CAP cycles. All subjects presented CAP sequences lasting at least 5 min and 30s. The mean duration of CAP cycles was 26.9+/-4.1s. CAP cycles including subtypes A1 presented the highest correlation with the CAP sequence length (r=0.92; p<0.0001). CONCLUSIONS: The progressive increase of CAP sequences length is linked to the progressive accumulation of CAP cycles. SIGNIFICANCE: CAP sequences can be considered as strings of time-constant modules, i.e., CAP cycles, which are involved in the dynamic tailoring of sleep structure.

Cyclic alternating pattern (CAP) alterations in narcolepsy.

BACKGROUND AND PURPOSE: Narcolepsy is a sleep disorder with clinical symptoms attributed to a reduced activation of the arousal system. Cyclic alternating pattern (CAP) is the expression of rhythmic arousability during non-rapid eye movement (NREM) sleep. CAP parameters, arousals and conventional sleep measures were studied in narcoleptic patients with cataplexy. PATIENTS AND METHODS: Data were collected from all-night polysomnographic (PSG) recordings and the multiple sleep latency test (MSLT) on the intervening day of 25 drug-naive patients (10 males and 15 females; mean age: 34+/-16 years) after adaptation and exclusion of other sleep disorders. A group of 25 age- and gender-matched normal sleepers were selected as controls. Each PSG recording was subdivided into sleep cycles. Analysis of CAP included classification of A phases into subtypes A1, A2, and A3. RESULTS: There was an increase in sleep period time mainly due to an increased wake time after sleep onset. REM latency was sharply reduced. The percentage of NREM sleep was slightly reduced and the balance between light sleep (S1+S2) and deep sleep (S3+S4) showed a curtailment of the former, while deep sleep was slightly increased. Excluding sleep cycles with sleep onset REM periods (SOREMPs), the duration of ordered sleep cycles was not different between narcoleptics and controls. The two groups showed similar values of arousal index, while CAP time, CAP rate, number of CAP cycles and of phase A subtypes (in particular subtypes A1) were significantly reduced in narcoleptic patients. CONCLUSIONS: The reduced periods of CAP in narcoleptic NREM sleep could be the electroencephalographic (EEG) expression of a generally reduced arousability or an increased strength of sleep-promoting forces in the balance between sleep and arousal systems. This can explain some of the clinical correlates of the disorder, i.e. excessive sleepiness, short sleep latency and impaired attentive performances, even without any sign of arousal-induced sleep fragmentation.

CAP and arousals are involved in the homeostatic and ultradian sleep processes.

There is growing evidence that cyclic alternating pattern (CAP) and arousals are woven into the basic mechanisms of sleep regulation. In the present study, the overnight sleep cycles (SC) of 20 normal subjects were analyzed according to their stage composition, CAP rate, phase A subtypes and arousals. Individual SC were then divided into 10 normalized temporal epochs. CAP parameters and arousals were measured in each epoch and averaged in relation to the SC order. Subtypes A2 and A3 of CAP in non-rapid eye movement (NREM) sleep, and arousals, both in REM and NREM sleep when not coincident with a A2 or A3 phases, were lumped together as fast electroencephalographic (EEG) activities (FA). Subtypes A1 of CAP, characterized by slow EEG activities (SA), were analyzed separately. The time distribution of SA and FA was compared to the mathematical model of normal sleep structure including functions representing the homeostatic process S, the circadian process C, the ultradian process generating NREM/REM cycles and the slow wave activity (SWA) resulting from the interaction between homeostatic and ultradian processes. The relationship between SA and FA and the sleep-model components was evaluated by multiple regression analysis in which SA and FA were considered as dependent variables while the covariates were the process S, process C, SWA, REM-on and REM-off activities and their squared values. Regression was highly significant (P < 0.0001) for both SA and FA. SA were prevalent in the first three SC, and exhibited single or multiple peaks immediately before and in the final part of deep sleep (stages 3 + 4). The peaks of FA were delayed and prevailed during the pre-REM periods of light sleep (stages 1 + 2) and during REM sleep. SA showed an exponential decline across the successive SC, according to the homeostatic process. In contrast, the distribution of FA was not influenced by the order of SC, with periodic peaks of FA occurring before the onset of REM sleep, in accordance with the REM-on switch. The dynamics of CAP and arousals during sleep can be viewed as an intermediate level between cellular activities and macroscale EEG phenomena as they reflect the decay of the homeostatic process and the interaction between REM-off and REM-on mechanisms while are slightly influenced by circadian rhythm.

Sleep cyclic alternating pattern in normal preschool-aged children.

STUDY OBJECTIVES: To evaluate the characteristics of the cyclic alternating pattern (CAP) in the sleep of preschool-aged children in order to obtain normative parameters in this age range. DESIGN: Prospective study. SETTINGS: University sleep laboratory. PARTICIPANTS: Ten normal healthy subjects (6 girls and 4 boys, mean age 4.6 years; range 3-6 years) underwent polysomnography recordings for 2 consecutive nights in a standard laboratory setting. Sleep data were stored on a computer using a polysomnography digital system (Embla N7000, Medcare, Iceland). Sleep structure was visually scored according to the Rechtschaffen and Kales criteria. The cyclic alternating pattern was visually scored following the criteria by Terzano et al. These criteria were modified for the purposes of this study because it was noticed that, at the age of the group under analysis, most electroencephalogram arousals, often accompanied by electromyogram activation, are expressed at the level of the electroencephalogram by theta frequencies and not alpha or higher frequencies. MEASUREMENTS AND RESULTS: The CAP rate in preschool-aged children (25.93%) showed a progressive increase with the deepness of sleep, with highest values during non-rapid eye movement (NREM) stage 3 (44.0%) and 4 sleep (46.08%) and lowest values in NREM stage 2 (17.26%). The CAP time showed its longest duration during stage 2 sleep, followed by stage 4, stage 3, and NREM stage 1. The CAP cycle duration showed no differences across NREM stages. The cyclic alternating pattern phase A was longer and phase B was shorter during stage 1 than during stages 2, 3, and 4. A1 phases were the most numerous (63.2%), followed by A2 (21.5%) and by A3 (15.3%). The distribution of A-phase subtypes across NREM sleep stages (A index) showed significant differences for the A1 subtypes that occurred more frequently during stage 3 and 4 sleep than during stages 1 and 2. The A2 index showed no significant differences across NREM sleep stages, while the A3 index was significantly higher during stage 1 sleep than during stages 2, 3, and 4. The analysis of the A1 interval distribution showed a log-normal-like distribution with a peak around 25 seconds for the A1 phases and no clear peak for A2-A3 phases. CONCLUSIONS: The analysis of CAP in preschool-aged children is characterized by an increase of CAP rate during slow-wave sleep and a high percentage of A1 phases and A2 phases. However, the lower percentage of A1 paralleled by an increase of A2 could represent a signal of higher sleep instability in this age group as compared with prepubertal school-aged children.

Reorganization of sleep patterns in severe OSAS under prolonged CPAP treatment.

OBJECTIVE: To evaluate the immediate and long-term recovery processes of sleep and daytime vigilance in patients with sleep apnea syndrome (OSAS) after continuous CPAP treatment. METHODS: Five consecutive polysomnographic (PSG) studies were carried out on 10 male patients with severe OSAS. The first recording (baseline) was accomplished without ventilatory support (N0). The other 4 recordings were carried out during the CPAP titration night (N1), during the second night of treatment (N2), during the third night of treatment (N3), and after 30 days of regular CPAP use (N30). Ten age-balanced healthy male subjects were selected from the Parma Sleep Center database as controls. Respiratory variables, conventional PSG variables, arousals, CAP (cyclic alternating pattern) variables, and daytime function (including MSLT) were quantified. ANOVA followed by post-hoc tests explored the differences between controls and OSAS patients in the different recording conditions (N0, N1, N2, N3, N30). The PSG measures that showed significant ANOVA values were correlated with the MSLT scores. RESULTS: Values of control subjects were recovered by REM sleep, REM latency, subtypes A3 and arousal index during N1, by CAP rate and total arousals during N2, by deep sleep (stages 3 + 4) during N3, by light sleep (stages 1 + 2) during N30. The only measures which remained below control values even after 1 month of sustained treatment were the amount of CAP cycles and A1 subtypes. MSLT scores correlated significantly with CAP rate, deep sleep and arousals. CONCLUSIONS: The changes induced by CPAP treatment do not restore immediately a normal sleep structure, which is re-established with different time scales SIGNIFICANCE: The modifications of sleep patterns and the different adjustments of phase A subtypes allow us to monitor the reorganization of sleep in OSAS patients treated with CPAP and the hierarchy of the mechanisms involved in sleep regulation.

Inter-rater reliability of sleep cyclic alternating pattern (CAP) scoring and validation of a new computer-assisted CAP scoring method.

OBJECTIVE: To assess inter-rater reliability between different scorers, from different qualified sleep research groups, in scoring visually the Cyclic Alternating Pattern (CAP), to evaluate the performances of a new tool for the computer-assisted detection of CAP, and to compare its output with the data from the different scorers. METHODS: CAP was scored in 11 normal sleep recordings by four different raters, coming from three sleep laboratories. CAP was also scored in the same recordings by means of a new computer-assisted method, implemented in the Hypnolab 1.2 (SWS Soft, Italy) software. Data analysis was performed according to the following steps: (a) the inter-rater reliability of CAP parameters between the four different scorers was carried out by means of the Kendall W coefficient of concordance; (b) the analysis of the agreement between the results of the visual and computer-assisted analysis of CAP parameters was also carried out by means of the Kendall W coefficient; (c) a 'consensus' scoring was obtained, for each recording, from the four scorings provided by the different raters, based on the score of the majority of scorers; (d) the degree of agreement between each scorer and the consensus score and between the computer-assisted analysis and the consensus score was quantified by means of the Cohen's k coefficient; (e) the differences between the number of false positive and false negative detections obtained in the visual and in the computer-assisted analysis were also evaluated by means of the non-parametric Wilcoxon test. RESULTS: The inter-rater reliability of CAP parameters quantified by the Kendall W coefficient of concordance between the four different scorers was high for all the parameters considered and showed values above 0.9 for total CAP time, CAP time in sleep stage 2 and percentage of A phases in sequence; also CAP rate showed a high value (0.829). The most important global parameters of CAP, including total CAP rate and CAP time, scored by the computer-assisted analysis showed a significant concordance with those obtained by the raters. The agreement between the computer-assisted analysis and the consensus scoring for the assignment of the CAP A phase subtype was not distinguishable from that expected from a human scorer. However, the computer-assisted analysis provided a number of false positives and false negatives significantly higher than that of the visual scoring of CAP. CONCLUSIONS: CAP scoring shows good inter-rater reliability and might be compared in different laboratories the results of which might also be pooled together; however, caution should always be taken because of the variability which can be expected in the classical sleep staging. The computer-assisted detection of CAP can be used with some supervision and correction in large studies when only general parameters such as CAP rate are considered; more editing is necessary for the correct use of the other results. SIGNIFICANCE: This article describes the first attempt in the literature to evaluate in a detailed way the inter-rater reliability in scoring CAP parameters of normal sleep and the performances of a human-supervised computerized automatic detection system.

Is insomnia a neurophysiological disorder? The role of sleep EEG microstructure.

Unlike other sleep disorders, such as sleep-related breathing disorders and periodic limb movement (PLM), the nature and severity of which are quantified by specific respiratory and motor indexes, no apparent organ dysfunction underlies several cases of insomnia (in particular primary insomnia), which can be objectively diagnosed only through the structural alterations of sleep. Polysomnography (PSG) investigation indicates that insomnia is the outcome of a neurophysiological disturbance that impairs the regulatory mechanisms of sleep control, including sleep duration, intensity, continuity and stability. In particular, analysis of sleep microstructure has permitted to establish that etiologic factors of different nature (including depressive disorders) exert a common destabilizing action on sleep, which is reflected in an increase of cyclic alternating pattern (CAP) rate. These premises allow us to attribute a more objective identity to insomnia, which risks otherwise to be considered as an unexplainable mental complaint. In conclusion, PSG remains the "gold standard" for measuring sleep, and especially insomnia.

Quantitative analysis of sleep EEG microstructure in the time-frequency domain.

A number of phasic events influence sleep quality and sleep macrostructure. The detection of arousals and the analysis of cyclic alternating patterns (CAP) support the evaluation of sleep fragmentation and instability. Sixteen polygraphic overnight recordings were visually inspected for conventional Rechtscaffen and Kales scoring, while arousals were detected following the criteria of the American Sleep Disorders Association (ASDA). Three electroencephalograph (EEG) segments were associated to each event, corresponding to background activity, pre-arousal period and arousal. The study was supplemented by the analysis of time-frequency distribution of EEG within each subtype of phase A in the CAP. The arousals were characterized by the increase of alpha and beta power with regard to background. Within NREM sleep most of the arousals were preceded by a transient increase of delta power. The time-frequency evolution of the phase A of the CAP sequence showed a strong prevalence of delta activity during the whole A1, but high amplitude delta waves were found also in the first 2/3 s of A2 and A3, followed by desynchronization. Our results underline the strict relationship between the ASDA arousals, and the subtype A2 and A3 within the CAP: in both the association between a short sequence of transient slow waves and the successive increase of frequency and decrease of amplitude characterizes the arousal response.

CAP variables and arousals as sleep electroencephalogram markers for primary insomnia.

OBJECTIVE: Polysomnographic (PSG) measures consistently reflect poor sleep quality and effective treatment in insomniac patients. METHODS: The PSG findings of 47 patients (18 M and 29 F, 42.5+/-10 years) meeting the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for a diagnosis of primary insomnia were compared with those of 25 age- and gender-balanced healthy subjects (controls) without sleep complaints. After one adaptation night to the sleep lab, each patient underwent two randomized double-blind PSG recordings. Twenty-four patients followed a placebo-drug sequence and 23 a drug-placebo succession. Active treatment consisted of widely used hypnotic drugs, i.e. zolpidem, triazolam, zopiclone, brotizolam. Conventional PSG measures, electroencephalogram (EEG) arousals and CAP variables (including phase A subtypes) were quantified and statistically analyzed. RESULTS: Compared to controls, insomniac patients under placebo showed a significant increase of CAP rate, subtypes A1 and A2, EEG arousals, nocturnal wakefulness and stage 1, associated with reduced values of total sleep time and slow wave sleep (stages 3 and 4). In insomniac patients, sleep quality was significantly improved by hypnotic treatment. Compared to placebo, active medication significantly reduced CAP rate, subtypes A1 and A2, but had only marginal effects on subtypes A3 and on EEG arousals. Under hypnotic treatment total sleep time, nocturnal awakenings, stage 1 and slow wave sleep recuperated normal values. The most significant correlation between sleep quality and PSG variables was found for CAP rate (P<0.0001). CONCLUSIONS: PSG investigation extended to CAP variables and EEG arousals can be an important procedure for the diagnosis of primary insomnia and evaluation of treatment efficacy.