About-Weekly Pattern in the Dynamic Complexity of a Healthy Subject's Cellular Immune Activity: A Biopsychosocial Analysis.

In a previous integrative single-case study, we collected biological, psychological and social time-series data on a 25-year-old healthy woman over the course of 126 12-h intervals (63 days) and used urinary neopterin as an indicator of cellular immune activity [Schubert et al. 2012 (1)]. The present re-evaluation introduced Dynamic Complexity (DC) as an additional non-linear and non-stationary measure to further investigate the subject's biopsychosocial dynamics during the study. The new time series dealing with urinary neopterin complexity revealed a cyclic, circaseptan (about-weekly) repeating pattern (6.59 days). The only weekly reoccurring events over the course of the study that were associated with this immunological pattern were the in-depth interviews with the subject (mean distance between interviews: 6.5 days). Superposed epoch analysis (SEA) revealed a U-shaped relation between neopterin complexity and interviews, with a decrease in neopterin complexity before and during interviews and an increase after interviews. Furthermore, the complexity scores for irritation, anxiousness/depressiveness and mental activity were positively correlated with neopterin complexity. The results suggest that the interviews, which had been found to be related to the subject's need for educational and/or social accomplishment, were marked by stress (decrease in psycho-immunological flexibility and adaptability), which was then relieved after the interviews (increase in psycho-immunological flexibility and adaptability). It appears that the subject's cellular immune activity, as indicated by neopterin complexity, functionally mirrored the emotional meaning she ascribed to the in-depth interviews. This re-evaluation is in line with the view that biopsychosocial research requires multimodal analysis of single cases based on qualitative (e.g., in-depth interviews) and quantitative (e.g., time series analysis) data under conditions of "life as it is lived".

Metabolomics Pilot Study Identifies Desynchronization of 24-H Rhythms and Distinct Intra-patient Variability Patterns in Critical Illness: A Preliminary Report.

Background: Synchronized circadian rhythms play a key role in coordinating physiologic health. Desynchronized circadian rhythms may predispose individuals to disease or be indicative of underlying disease. Intensive care unit (ICU) patients likely experience desynchronized circadian rhythms due to disruptive environmental conditions in the ICU and underlying pathophysiology. This observational pilot study was undertaken to determine if 24-h rhythms are altered in ICU patients relative to healthy controls by profiling 24-h rhythms in vital signs and plasma metabolites. Methods: We monitored daily rhythms in 5 healthy controls and 5 ICU patients for 24 h. Heart rate and blood pressure were measured every 30 min, temperature was measured every hour, and blood was sampled for mass spectrometry-based plasma metabolomics every 4 h. Bedside sound levels were measured every minute. Twenty-four hours rhythms were evaluated in vitals and putatively identified plasma metabolites individually and in each group using the cosinor method. Results: ICU patient rooms were significantly louder than healthy controls' rooms and average noise levels were above EPA recommendations. Healthy controls generally had significant 24-h rhythms individually and as a group. While a few ICU patients had significant 24-h rhythms in isolated variables, no significant rhythms were identified in ICU patients as a group, except in cortisol. This indicates a lack of coherence in phases and amplitudes among ICU patients. Finally, principal component analysis of metabolic profiles showed surprising patterns in plasma sample clustering. Each ICU patient's samples were clearly discernable in individual clusters, separate from a single cluster of healthy controls. Conclusions: In this pilot study, ICU patients' 24-h rhythms show significant desynchronization compared to healthy controls. Clustering of plasma metabolic profiles suggests that metabolomics could be used to track individual patients' clinical courses longitudinally. Our results show global disordering of metabolism and the circadian system in ICU patients which should be characterized further in order to determine implications for patient care.

Some Near- and Far-Environmental Effects on Human Health and Disease with a Focus on the Cardiovascular System.

Environmental effects on human physiopathology are revisited herein from a chronobiologic viewpoint, with a focus on the cardiovascular system. Physiological variables undergo recurring changes that are predictable in a statistical, albeit not deterministic way. Biological rhythms cover a broad range of frequencies, which are usually shared by the environment as "co-periodisms". Some of these photic and non-photic periodicities shared by the environment and physiopathology are reviewed herein, together with their possible underlying mechanisms. A plausible cascade of events from the long-period cycles found in the cosmic environment to those affecting the Earth's atmosphere and weather conditions is presented, which may shed light on how they may shape the cycles characterizing human health. Maps of important cycles shared between the environment and physiopathology are being catalogued in an atlas of chronomes with the goal of distinguishing between strong and weak associations and providing an estimate of the lag that can be anticipated before observing physiological changes.

Circadian rhythm of autophagy proteins in hippocampus is blunted by sleep fragmentation.

Autophagy is essential for normal cellular survival and activity. Circadian rhythms of autophagy have been studied in several peripheral organs but not yet reported in the brain. Here, we measured the circadian rhythm of autophagy-related proteins in mouse hippocampus and tested the effect of sleep fragmentation (SF). Expressions of the autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and beclin were determined by western blotting and immunohistochemistry. Both the hippocampal LC3 signal and the ratio of its lipid-conjugated form LC3-II to its cytosolic form LC3-I showed a 24 h rhythm. The peak was seen at ZT6 (1 pm) and the nadir at ZT16 (1 am). The LC3 immunoreactivity in hippocampal CA1 pyramidal neurons also distributed differently, with more diffuse cytoplasmic appearance at ZT16. Chronic SF had a mild effect to disrupt the 24 h rhythm of LC3 and beclin expression. Interestingly, a greater effect of SF was seen after 24 h of recovery sleep when LC3-II expression was attenuated at both the peak and trough of circadian activities. Overall, the results show for the first time that the hippocampus has a distinct rhythm of autophagy that can be altered by SF.

Diminished leptin signaling can alter circadian rhythm of metabolic activity and feeding.

Leptin, a hormone mainly produced by fat cells, shows cell-specific effects to regulate feeding and metabolic activities. We propose that an important feature of metabolic dysregulation resulting in obesity is the loss of the circadian rhythm of biopotentials. This was tested in the pan-leptin receptor knockout (POKO) mice newly generated in our laboratory. In the POKO mice, leptin no longer induced pSTAT-3 signaling after intracerebroventricular injection. Three basic phenotypes were observed: the heterozygotes had similar weight and adiposity as the wild-type (WT) mice (>60% of the mice); the homozygotes were either fatter (∼30%), or rarely leaner (<5%) than the WT mice. By early adulthood, the POKO mice had higher average body weight and adiposity than their respective same-sex WT littermate controls, and this was consistent among different batches. The homozygote fat POKO showed significant reduction of midline estimating statistic of rhythm of circadian parameters, and shifts of ultradian rhythms. The blunted circadian rhythm of these extremely obese POKO mice was also seen in their physical inactivity, longer feeding bouts, and higher food intake. The extent of obesity correlated with the blunted circadian amplitude, accumulative metabolic and locomotor activities, and the severity of hyperphagia. This contrasts with the heterozygote POKO mice which showed little obesity and metabolic disturbance, and only subtle changes of the circadian rhythm of metabolic activity without alterations in feeding behavior. The results provide a novel aspect of leptin resistance, almost manifesting as an "all or none" phenomenon.

Astrocytic leptin-receptor knockout mice show partial rescue of leptin resistance in diet-induced obesity.

To determine how astrocytic leptin signaling regulates the physiological response of mice to diet-induced obesity (DIO), we performed metabolic analyses and hypothalamic leptin signaling assays on astrocytic leptin-receptor knockout (ALKO) mice in which astrocytes lack functional leptin receptor (ObR) signaling. ALKO mice and wild-type (WT) littermate controls were studied at different stages of DIO with measurement of body wt, percent fat, metabolic activity, and biochemical parameters. When fed regular chow, the ALKO mice had similar body wt, percent fat, food intake, heat dissipation, respiratory exchange ratio, and activity as their WT littermates. There was no change in blood concentrations of triglyceride, soluble leptin receptor (sObR), mRNA for leptin and uncoupling protein 1 (UCP1) in adipose tissue, and insulin sensitivity. Unexpectedly, in response to a high-fat diet the ALKO mice had attenuated hyperleptinemia and sObR, a lower level of leptin mRNA in subcutaneous fat, and a paradoxical increase in UCP1 mRNA. Thus, ALKO mice did not show the worsening of obesity that occurs with normal WT mice and the neuronal ObR mutation that results in morbid obesity. The findings are consistent with a competing, counterregulatory model between neuronal and astrocytic leptin signaling.

Free-running circadian rhythms of muscle strength, reaction time, and body temperature in totally blind people.

Light is the major synchronizer of circadian rhythms. In the absence of light, as for totally blind people, some variables, such as body temperature, have an endogenous period that is longer than 24 h and tend to be free running. However, the circadian rhythm of muscle strength and reaction time in totally blind people has not been defined in the literature. The objective of this study was to determine the period of the endogenous circadian rhythm of the isometric and isokinetic contraction strength and simple reaction time of totally blind people. The study included six totally blind people with free-running circadian rhythms and four sighted people (control group). Although the control group required only a single session to determine the circadian rhythm, the blind people required three sessions to determine the endogenous period. In each session, isometric strength, isokinetic strength, reaction time, and body temperature were collected six different times a day with an interval of at least 8 h. The control group had better performance for strength and reaction time in the afternoon. For the blind, this performance became delayed throughout the day. Therefore, we conclude that the circadian rhythms of strength and simple reaction time of totally blind people are within their free-running periods. For some professionals, like the blind paralympic athletes, activities that require large physiological capacities in which the maximum stimulus should match the ideal time of competition may result in the blind athletes falling short of their expected performance under this free-running condition.

Endothelial leptin receptor mutation provides partial resistance to diet-induced obesity.

Leptin, a polypeptide hormone produced mainly by adipocytes, has diverse effects in both the brain and peripheral organs, including suppression of feeding. Other than mediating leptin transport across the blood-brain barrier, the role of the endothelial leptin receptor remains unclear. We recently generated a mutant mouse strain lacking endothelial leptin receptor signaling, and showed that there is an increased uptake of leptin by brain parenchyma after its delivery by in situ brain perfusion. Here, we tested the hypothesis that endothelial leptin receptor mutation confers partial resistance to diet-induced obesity. These ELKO mice had similar body weight and percent fat as their wild-type littermates when fed with rodent chow, but blood concentrations of leptin were significantly elevated. In response to a high-fat diet, wild-type mice had a greater gain of body weight and fat than ELKO mice. As shown by metabolic chamber measurement, the ELKO mice had higher oxygen consumption, carbon dioxide production, and heat dissipation, although food intake was similar to that of the wild-type mice and locomotor activity was even reduced. This indicates that the partial resistance to diet-induced obesity was mediated by higher metabolic activity in the ELKO mice. Since neuronal leptin receptor knockout mice show obesity and diabetes, the results suggest that endothelial leptin signaling shows opposite effects from that of neuronal leptin signaling, with a facilitatory role in diet-induced obesity.

Potential protective role of IL15Rα during inflammation.

We have shown that TNFα specifically activates the interleukin-15 (IL15) system in cerebral endothelial cells composing the blood-brain barrier. To determine the functions of cerebral IL15 signaling in inflammation, we first treated mice with lipopolysaccharide (LPS) and determined the expression of the three receptor subtypes of IL15. Robust time-dependent upregulation occurred in all subunits. We then tested whether IL15Rα knockout (KO) affected the maintenance of body temperature and activity level after a single dose of LPS. Circadian telemetry data were analyzed by the cosinor method. Both wild-type and KO mice had clear 24-h rhythms of basal temperature and activity. KO mice had a significantly higher midline estimating statistic of rhythm (MESOR; approximating 24 h mean) of temperature and delayed 24-h acrophase (peak) of activity than the wild-type mice. LPS disrupted the circadian rhythm of activity more severely in the KO group. Besides a decrease in MESOR and 24-h amplitude of activity after LPS, the KO mice showed a significant reduction of MESOR, amplitude, and changed acrophase for temperature on the second of 2 days. The disrupted circadian rhythm of temperature and activity in the KO mice after LPS suggests that upregulated IL15 receptors may serve a beneficial role to counteract the consequences of neuroinflammation.

IL-15 receptor deletion results in circadian changes of locomotor and metabolic activity.

Interleukin-15 (IL-15) is a cytokine produced in the normal brain that acts on its specific receptor IL-15Ralpha and co-receptors IL-2Rbeta and IL-2Rgamma in neuronal cells. The functions of the cerebral IL-15 system, however, are not yet clear. To test the hypothesis that IL-15Ralpha regulates metabolic activity and body temperature, we quantified the specific metabolic phenotype of IL-15Ralpha knockout mice. These normal-appearing mice were leaner with lower fat composition. During the entire circadian cycle, the knockout mice had a significantly higher acrophase in locomotor activity and heat dissipation. During the light phase, there was significantly greater food intake, oxygen consumption, and carbon dioxide production. The difference in the dark and light phases suggests that IL-15Ralpha participates in circadian rhythm regulation. The higher oxygen consumption in the light phase indicates adaptive thermogenesis in the knockout mice. The body temperature of the receptor knockout mice was significantly higher than the control in the light phase, and this was mainly caused by a large difference occurring between 0600 and 0900 h. In addition to the metabolic chamber studies and circadian rhythm analyses, qPCR of hypothalamic homogenates indicated higher mRNA expression of orexin and transient receptor potential vanilloid 4 cation channels. Consistent with a direct role of IL-15Ralpha in the hypothalamus, IL-15 treatment of the wild-type mice induced c-Fos expression in the preoptic area. We conclude that activation of hypothalamic neurons by IL-15 in mice contributes to thermoregulation and modifies the metabolic phenotype.

Circadian gene mPer2 overexpression induces cancer cell apoptosis.

The Period2 gene, an indispensable component of the circadian clock, not only modulates circadian oscillations, but also regulates organic function. We examined whether overexpression of the mouse Period2 gene (mPer2) in tumor cells influences cell growth and induces apoptosis. Overexpression of PERIOD2 in the mouse Lewis lung carcinoma cell line (LLC) and mammary carcinoma cell line (EMT6) results in reduced cellular proliferation and rapid apoptosis, but not in NIH 3T3 cells. Overexpressed mPER2 also altered the expression of apoptosis-related genes. The mRNA and protein levels of c-Myc, Bcl-X(L) and Bcl-2 were downregulated, whereas the expression of p53 and bax was upregulated in mPER2-overexpressing LLC cells compared with control cells transferred with empty plasmid. Our results suggest that the circadian gene mPeriod2 may play an important role in tumor suppression by inducing apoptotic cell death, which is attributable to enhanced pro-apoptotis signaling and attenuated anti-apoptosis processes.