Sex and Dosing-Time Dependencies in Irinotecan-Induced Circadian Disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50?mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers. (Author correspondence: francis.levi@inserm.fr)     

Prediction of Survival by Neutropenia According To Delivery Schedule of Oxaliplatin–5-Fluorouracil–Leucovorin for Metastatic Colorectal Cancer in a Randomized International Trial (EORTC 05963)

Circadian clocks control cellular proliferation and drug metabolism over the 24?h. However, circadian chronomodulated chemotherapy with 5-fluorouracil, leucovorin, and oxaliplatin (chronoFLO4) offered no survival benefit as compared with the non–time-stipulated FOLFOX2, in an international randomized trial involving patients with previously untreated metastatic colorectal cancer (EORTC 05963). The authors hypothesized that treatment near maximum tolerated dose could disrupt circadian clocks thus impairing the efficacy of chronoFLO4 but not of FOLFOX2. Patients with available data (N?=?556) were categorized into three subgroups according to the worst grade (G) of neutropenia experienced during treatment. Distinct multivariate models with time-dependent covariates were constructed for each treatment schedule. Neutropenia incidence (all grades) was 33% on chronoFLO4 and 61% on FOLFOX2 (p?<?.0001), and G3–4 were 7% and 25%, respectively (p < .0001). Neutropenia was significantly more frequent in women than men on either schedule (FOLFOX2, p = .003; chronoFLO4, p = .04). Median survival was 20.7 mo in patients with G3–4 neutropenia versus 12.5 mo in neutropenia-free patients on FOLFOX2 (p < .0001). Corresponding figures were 13.7 and 19.4 mo, respectively, on chronoFLO4 (p?=?.36). Multivariate analysis confirmed occurrence of severe neutropenia independently predicted for better overall survival on FOLFOX2 (HR?=?0.56; p = .015), and worse survival on chronoFLO4 (HR?=?1.77, p = .06), with a significant interaction test (p < .0001). Prediction of better survival in neutropenic patients on FOLFOX2 supports the administration of conventional chemotherapy near maximum tolerated dose. The opposite trend shown here for chronoFLO4 supports the novel concept of jointly optimized hematologic tolerability and efficacy through personalized circadian-timed therapy. (Author correspondence: francis.levi@inserm.fr)     

Sex and dosing-time dependencies in irinotecan-induced circadian disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.     

Low-Carbohydrate,High-Protein Diet Affects Rhythmic Expression of Gluconeogenic Regulatory and Circadian Clock Genes in Mouse Peripheral Tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues. (Author correspondence: k-ooishi@aist.go.jp)     

Memory for Time of Day (Time Memory) Is Encoded by a Circadian Oscillator and Is Distinct From Other Context Memories

We report that the neural representation of the time of day (time memory) in golden hamsters involves the setting of a 24-h oscillator that is functionally and anatomically distinct from the circadian clock in the suprachiasmatic nucleus (SCN), but is entrained by the SCN acting as a weak zeitgeber. In hamsters, peak conditioned place avoidance (CPA) was expressed only near the time of day of the learning experience (±2?h) for the first days after conditioning. On a 14:10 light:dark cycle, with conditioning at the end of the light period (zeitgeber time 11 [ZT11]), CPA behavior, including time of day memory, was retained for more than 18 d. With conditioning in the early day (zeitgeber time 03 [ZT03]), CPA was completely lost after 5 d but reemerged after an additional 6 d, with the peak avoidance time shifted to ZT11. When the entraining light cycle was shifted immediately following learning at either ZT11 or ZT03, with no additional experience in the training apparatus, peak CPA 18 d later was always found at ZT11 on the shifted light cycles. When conditioned at ZT03, then placed into constant dark for 18 cycles, the peak shifted to subjective circadian time 11 (CT11). In all experiments, the peak CPA time was set initially to the time of experience, and was reset subsequently to the end of the subjective day, without memory loss for other context associations. In the absence of an SCN, peak avoidance was not reset. Therefore, time memory is distinct from other context memories, and involves the setting of a non-SCN circadian oscillator. We suggest that circadian oscillators underlying time memory work in concert with the SCN to enable anticipation of critical conditions according to both immediate- and long-term probabilities of where and when important conditions could be encountered again. (Author correspondence: ralph@psych.utoronto.ca)     

The Effects of Thoracic and Cervical Spinal Cord Lesions on the Circadian Rhythm of Core Body Temperature

Individuals with a spinal cord injury (SCI) have compromised afferent and efferent information below the lesion. Intact afferent information regarding skin temperature and the ability to regulate skin blood flow lead to an altered heat balance, which may impact the circadian variation in core body temperature (Tcore) and sleep-wake cycle. The authors assessed the circadian variation of Tcore in SCI individuals and able-bodied controls matched for the timing of the sleep-wake cycle. The authors examined subjects who had a high (cervical) or a low (thoracic) lesion. Intestinal Tcore (telemetry system) and physical activity (ambulatory activity monitor) levels were measured continuously and simultaneously in 8 tetraplegics, 7 paraplegics, and 8 able-bodied controls during one 24-h period of “normal” living. The regression slope between activity and Tcore was also calculated for each 2-h bin. Circadian rhythm parameters were estimated with partial Fourier time-series analysis, and groups were compared with general linear models, adjusted for the influence of individual wake-time. The (mean?±?SD) dominant period length for controls, paraplegics, and tetraplegics were 24.4?±?5.4?h, 22.5?±?5.0?h, and 16.5?±?5.1?h, respectively (p?=?.02). A significantly more pronounced 8-h harmonic was found for the variation in Tcore of SCI individuals (p = .05). Tetraplegics showed the highest nocturnal mean Tcore (p = .005), a 5-h phase-advanced circadian trough time (p = .04), and more variable relationships between physical activity and Tcore (p = .03). Taken together, tetraplegics demonstrate a pronounced disturbance of the circadian variation of Tcore, whereas the variation of Tcore in paraplegics was comparable to able-bodied controls. (Author correspondence: D.Thijssen@fysiol.umcn.nl)     

DIURNAL VARIATION IN ENDOTOXIN-INDUCED MORTALITY IN MICE: CORRELATION WITH PROINFLAMMATORY FACTORS

Many immune parameters exhibit daily and circadian oscillations, including the number of circulating cells and levels of cytokines in the blood. Mice also have a differential susceptibility to lipopolysaccharide (LPS or endotoxin)-induced endotoxic shock, depending on the administration time in the 24?h light-dark (LD) cycle. We replicated these results in LD, but we did not find temporal differences in LPS-induced mortality in constant darkness (DD). Animals challenged with LPS showed only transient effects on their wheel locomotor activity rhythm without modification of circadian period and phase. Levels of several key factors involved in the pathology of sepsis and septic shock were tested in LD. We found that LPS-induced levels of interleukin (IL)-1β, IL-6, JE (MCP-1), and MIP1α were significantly higher at zeitgeber time (ZT) 11 (time of increased mortality) than at ZT19 (ZT12?=?time of lights-off in the animal quarters for the 12L:12D condition). Our results indicate that the differences found in mortality that are dependent on the time of LPS-challenge are not directly related to an endogenous circadian clock, and that some relevant immune factors in the development of sepsis are highly induced at ZT11, the time of higher LPS-induced mortality, compared to ZT19. (Author correspondence: dgolombek@unq.edu.ar)     

Ring the Bell for Matins: Circadian Adaptation to Split Sleep by Cloistered Monks and Nuns

Cloistered monks and nuns adhere to a 10-century-old strict schedule with a common zeitgeber of a night split by a 2- to 3-h-long Office (Matins). The authors evaluated how the circadian core body temperature rhythm and sleep adapt in cloistered monks and nuns in two monasteries. Five monks and five nuns following the split-sleep night schedule for 5 to 46 yrs without interruption and 10 controls underwent interviews, sleep scales, and physical examination and produced a week-long sleep diary and actigraphy, plus 48-h recordings of core body temperature. The circadian rhythm of temperature was described by partial Fourier time-series analysis (with 12- and 24-h harmonics). The temperature peak and trough values and clock times did not differ between groups. However, the temperature rhythm was biphasic in monks and nuns, with an early decrease at 19:39?±?4:30?h (median?±?95% interval), plateau or rise of temperature at 22:35?±?00:23?h (while asleep) lasting 296?±?39?min, followed by a second decrease after the Matins Office, and a classical morning rise. Although they required alarm clocks to wake-up for Matins at midnight, the body temperature rise anticipated the nocturnal awakening by 85?±?15?min. Compared to the controls, the monks and nuns had an earlier sleep onset (20:05?±?00:59?h vs. 00:00?±?00:54?h, median?±?95% confidence interval, p?=?.0001) and offset (06:27?±?0:22?h, vs. 07:37?±?0:33?h, p?=?.0001), as well as a shorter sleep time (6.5?±?0.6 vs. 7.6?±?0.7?h, p?=?.05). They reported difficulties with sleep latency, sleep duration, and daytime function, and more frequent hypnagogic hallucinations. In contrast to their daytime silence, they experienced conversations (and occasionally prayers) in dreams. The biphasic temperature profile in monks and nuns suggests the human clock adapts to and even anticipates nocturnal awakenings. It resembles the biphasic sleep and rhythm of healthy volunteers transferred to a short (10-h) photoperiod and provides a living glance into the sleep pattern of medieval time. (Author correspondence: isabelle.arnulf@psl.aphp.fr)     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

Circadian Modulation of Acute Alcohol Sensitivity But Not Acute Tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between naïve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism. (Author correspondence: lyons@bio.fsu.edu)     

Daily Rhythms in the Hypothalamus-Pituitary-Interrenal Axis and Acute Stress Responses in a Teleost Flatfish,Solea senegalensis

The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p?<?.05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT]?=?14.5 and 14.4?h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT?=?1.2, 2.4, and 3.4?h, respectively), and crhbp and lactate at the end of the dark phase (ZT?=?22.3 and 23.0?h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2?±?10.7?ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6?±?11.1?ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture. (Author correspondence: jflopez@um.es)     

SLEEP PATTERNS IN HIGH SCHOOL AND UNIVERSITY STUDENTS: A LONGITUDINAL STUDY

We performed a longitudinal study to investigate whether changes in social zeitgebers and age alter sleep patterns in students during the transition from high school to university. Actimetry was performed on 24 high-school students (mean age?±?SD: 18.4?±?0.9 yrs; 12 females) for two weeks. Recordings were repeated in the same subjects 5 yrs later when they were university students. The sleep period duration and its center, the mid-sleep time, and total sleep time were estimated by actimetry. Actigraphic total sleep time was similar when in high school and at the university on school days (6.31?±?0.47 vs. 6.45?±?0.80?h, p?=?ns) and longer on leisure days by 1.10?±?1.10?h (p?<?0.0001 vs. school days) when in high school, but not at the university. Compared to the high school situation, the mid-sleep time was delayed when at the university on school days (03∶11?±?0.6 vs. 03∶55?±?0.7?h, p?<?0.0001), but not on leisure days. Individual mid-sleep times on school and leisure days when in high school were significantly correlated with the corresponding values 5 yrs later when at the university (r?=?0.58 and r?=?0.55, p?<?0.05, respectively). The large differences in total sleep time between school and leisure days when students attended high school and the delayed mid-sleep time on school days when students attended university are consistent with a circadian phase shift due to changes in class schedules, other zeitgebers, and lifestyle preferences. Age-related changes may also have occurred, although some individuality of the sleep pattern was maintained during the 5 yr study span. These findings have important implications for optimizing school and work schedules in students of different age and level of education. (Author correspondence: pneubloc@usz.uzh.ch)     

Cyclic Presence and Absence of Conspecifics Alters Circadian Clock Phase But Does Not Entrain the Locomotor Activity Rhythm of the Fruit Fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms. (Author correspondence: vsharma@jncasr.ac.in, vksharmas@gmail.com)     

Association Study of a Variable-Number Tandem Repeat Polymorphism in the Clock Gene PERIOD3 and Chronotype in Norwegian University Students

Circadian rhythms are endogenously generated cycles involving physiological parameters, such as core body temperature, hormone levels, blood pressure, sleep, and metabolism, with a period length of around 24?h. The circadian clock in mammals is regulated by a set of clock genes that are functionally linked together, and polymorphisms in clock genes could be associated with differences in circadian rhythms. A variable-number tandem repeat (VNTR) in the human clock gene PERIOD3 (PER3) has been suggested to correlate with a morning (lark) versus evening (owl) chronotype as well as with the circadian rhythm sleep disorder “delayed sleep phase disorder” (DSPD). The authors examined 432 healthy Norwegian university students in search of further support for an association between the PER3 polymorphism and diurnal preference. The Horne-Östberg Morningness-Eveningness Questionnaire (MEQ) and Preferences Scale (PS) were used to evaluate subjective chronotype. DNA samples were genotyped with respect to the 4-repeat and 5-repeat alleles of the VNTR PER3 polymorphism, and the genotype distribution was 192 (4-4), 191 (4-5), and 49 (5-5). The authors estimated that the power to detect an association of the 4-allele with preference for morningness or eveningness was 75%. The authors found no association between the PER3 clock gene and chronotype, indicating that the proposed role of PER3 needs further clarification. (Author correspondence: teresa.osland@med.uib.no)