PER2 regulation of mammary gland development.

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.

Efficacy of pharmacological therapies in patients with IBS with diarrhoea or mixed stool pattern: systematic review and network meta-analysis.

OBJECTIVE: Over half of patients with IBS have either diarrhoea (IBS-D) or a mixed stool pattern (IBS-M). The relative efficacy of licenced pharmacological therapies is unclear in the absence of head-to-head trials. We conducted a network meta-analysis to resolve this uncertainty. DESIGN: We searched MEDLINE, Embase, Embase Classic, the Cochrane central register of controlled trials, and Clinicaltrials.gov through January 2019 to identify randomised controlled trials (RCTs) assessing the efficacy of licenced pharmacological therapies (alosetron, eluxadoline, ramosetron and rifaximin) in adults with IBS-D or IBS-M. Trials included in the analysis reported a dichotomous assessment of overall response to therapy, and data were pooled using a random effects model. Efficacy and safety of all pharmacological therapies were reported as a pooled relative risk with 95% CIs to summarise the effect of each comparison tested. Treatments were ranked according to their p score. RESULTS: We identified 18 eligible RCTs (seven alosetron, five ramosetron, two rifaximin and four eluxadoline), containing 9844 patients. All were superior to placebo for the treatment of IBS-D or IBS-M at 12 weeks, according to the Food and Drug Administration (FDA)-recommended endpoint for trials in IBS. Alosetron 1 mg twice daily was ranked first for efficacy, based on the FDA-recommended composite endpoint of improvement in both abdominal pain and stool consistency, effect on global symptoms of IBS and effect on stool consistency. Ramosetron 2.5µg once daily was ranked first for effect on abdominal pain. Total numbers of adverse events were significantly greater with alosetron 1 mg twice daily and ramosetron 2.5µg once daily, compared with placebo. Rifaximin 550 mg three times daily ranked first for safety. Constipation was significantly more common with all drugs, except rifaximin 550 mg three times daily. CONCLUSION: In a network meta-analysis of RCTs of pharmacological therapies for IBS-D and IBS-M, we found all drugs to be superior to placebo, but alosetron and ramosetron appeared to be the most effective.

Correction to: inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness.

Following publication of the original article [1], we have been notified that the tagging of one of the author names was done incorrectly in the XML version of the paper. The online and pdf versions of this paper are not affected by the change. Original and corrected tagging can be seen below. The original article has been corrected.

Shift work cycle-induced alterations of circadian rhythms potentiate the effects of high-fat diet on inflammation and metabolism.

Based on genetic models with mutation or deletion of core clock genes, circadian disruption has been implicated in the pathophysiology of metabolic disorders. Thus, we examined whether circadian desynchronization in response to shift work-type schedules is sufficient to compromise metabolic homeostasis and whether inflammatory mediators provide a key link in the mechanism by which alterations of circadian timekeeping contribute to diet-induced metabolic dysregulation. In high-fat diet (HFD)-fed mice, exposure to chronic shifts of the light-dark cycle (12 h advance every 5 d): 1) disrupts photoentrainment of circadian behavior and modulates the period of spleen and macrophage clock gene rhythms; 2) potentiates HFD-induced adipose tissue infiltration and activation of proinflammatory M1 macrophages; 3) amplifies macrophage proinflammatory cytokine expression in adipose tissue and bone marrow-derived macrophages; and 4) exacerbates diet-induced increases in body weight, insulin resistance, and glucose intolerance in the absence of changes in total daily food intake. Thus, complete disruption of circadian rhythmicity or clock gene function as transcription factors is not requisite to the link between circadian and metabolic phenotypes. These findings suggest that macrophage proinflammatory activation and inflammatory signaling are key processes in the physiologic cascade by which dysregulation of circadian rhythmicity exacerbates diet-induced systemic insulin resistance and glucose intolerance.-Kim, S.-M., Neuendorff, N., Alaniz, R. C., Sun, Y., Chapkin, R. S., Earnest, D. J. Shift work cycle-induced alterations of circadian rhythms potentiate the effects of high-fat diet on inflammation and metabolism.

Inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness.

BACKGROUND: The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro. METHODS: The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg-Marquardt algorithm. Nonlinear regression and Akaike's information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests. RESULTS: We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness. CONCLUSIONS: Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.

Cadexomer iodine provides superior efficacy against bacterial wound biofilms in vitro and in vivo.

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds.

Myeloid cell-specific disruption of Period1 and Period2 exacerbates diet-induced inflammation and insulin resistance.

The circadian clockworks gate macrophage inflammatory responses. Given the association between clock dysregulation and metabolic disorders, we conducted experiments to determine the extent to which over-nutrition modulates macrophage clock function and whether macrophage circadian dysregulation is a key factor linking over-nutrition to macrophage proinflammatory activation, adipose tissue inflammation, and systemic insulin resistance. Our results demonstrate that 1) macrophages from high fat diet-fed mice are marked by dysregulation of the molecular clockworks in conjunction with increased proinflammatory activation, 2) global disruption of the clock genes Period1 (Per1) and Per2 recapitulates this amplified macrophage proinflammatory activation, 3) adoptive transfer of Per1/2-disrupted bone marrow cells into wild-type mice potentiates high fat diet-induced adipose and liver tissue inflammation and systemic insulin resistance, and 4) Per1/2-disrupted macrophages similarly exacerbate inflammatory responses and decrease insulin sensitivity in co-cultured adipocytes in vitro. Furthermore, PPARγ levels are decreased in Per1/2-disrupted macrophages and PPARγ2 overexpression ameliorates Per1/2 disruption-associated macrophage proinflammatory activation, suggesting that this transcription factor may link the molecular clockworks to signaling pathways regulating macrophage polarization. Thus, macrophage circadian clock dysregulation is a key process in the physiological cascade by which diet-induced obesity triggers macrophage proinflammatory activation, adipose tissue inflammation, and insulin resistance.

Toward Precision Medicine: Circadian Rhythm of Blood Pressure and Chronotherapy for Hypertension - 2021 NHLBI Workshop Report.

Healthy individuals exhibit blood pressure variation over a 24-hour period with higher blood pressure during wakefulness and lower blood pressure during sleep. Loss or disruption of the blood pressure circadian rhythm has been linked to adverse health outcomes, for example, cardiovascular disease, dementia, and chronic kidney disease. However, the current diagnostic and therapeutic approaches lack sufficient attention to the circadian rhythmicity of blood pressure. Sleep patterns, hormone release, eating habits, digestion, body temperature, renal and cardiovascular function, and other important host functions as well as gut microbiota exhibit circadian rhythms, and influence circadian rhythms of blood pressure. Potential benefits of nonpharmacologic interventions such as meal timing, and pharmacologic chronotherapeutic interventions, such as the bedtime administration of antihypertensive medications, have recently been suggested in some studies. However, the mechanisms underlying circadian rhythm-mediated blood pressure regulation and the efficacy of chronotherapy in hypertension remain unclear. This review summarizes the results of the National Heart, Lung, and Blood Institute workshop convened on October 27 to 29, 2021 to assess knowledge gaps and research opportunities in the study of circadian rhythm of blood pressure and chronotherapy for hypertension.

Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes.

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca(2+) and ATP accumulation and have demonstrated that intracellular Ca(2+) levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca(2+) levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca(2+). In contrast, our finding that peak mitochondrial Ca(2+) occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca(2+) uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca(2+) mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

The Zelnorm epidemiologic study (ZEST): a cohort study evaluating incidence of abdominal and pelvic surgery related to tegaserod treatment.

BACKGROUND: Pre-marketing clinical studies of tegaserod suggested an increased risk of abdominal surgery, particularly cholecystectomy. We sought to quantify the association between tegaserod use and the occurrence of abdominal or pelvic surgery, including cholecystectomy. METHODS: This cohort study was conducted within an insured population. Tegaserod initiators and similar persons who did not initiate tegaserod were followed for up to six months for the occurrence of abdominal or pelvic surgery. Surgical procedures were identified from health insurance claims validated by review of medical records. The incidence of confirmed outcomes was compared using both as-matched and as-treated analyses. RESULTS: Among 2,762 tegaserod initiators, there were 94 abdominal or pelvic surgeries (36 gallbladder): among 2,762 comparators there were 134 abdominal or pelvic surgeries (37 gallbladder) (hazard ratio HR] = 0.70, 95% confidence interval [C.I.] = 0.54-0.91 overall, HR = 0.98, 95% C.I. = 0.62-1.55 for gallbladder). Current tegaserod exposure compared to nonexposure was associated with a rate ratio [RR] of 0.68 (95% C.I. = 0.48-0.95) overall, while the RR was 0.99 (95% C.I. = 0.56-1.77) for gallbladder surgery. CONCLUSIONS: In this study, tegaserod use was not found to increase the risk of abdominal or pelvic surgery nor the specific subset of gallbladder surgery.

Development of an age-dependent cognitive index: relationship between impaired learning and disturbances in circadian timekeeping.

Preclinical quantitative models of cognitive performance are necessary for translation from basic research to clinical studies. In rodents, non-cognitive factors are a potential influence on testing outcome and high variability in behavior requires multiple time point testing for better assessment of performance in more sophisticated tests. Thus, these models have limited translational value as most human cognitive tests characterize cognition using single digit scales to distinguish between impaired and unimpaired function. To address these limitations, we developed a cognitive index for learning based on previously described scores for strategies used by mice to escape the Barnes maze. We compared the cognitive index and circadian patterns of light-dark entrainment in young (4-6 months), middle-aged (13-14 months), and aged (18-24 months) mice as cognitive changes during aging are often accompanied by pronounced changes in sleep-wake cycle. Following continuous analysis of circadian wheel-running activity (30-40 days), the same cohorts of mice were tested in the Barnes maze. Aged mice showed significant deficits in the learning and memory portions of the Barnes maze relative to young and middle-aged animals, and the cognitive index was positively correlated to the memory portion of the task (probe) in all groups. Significant age-related alterations in circadian entrainment of the activity rhythm were observed in the middle-aged and aged cohorts. In middle-aged mice, the delayed phase angle of entrainment and increased variability in the daily onsets of activity preceded learning and memory deficits observed in aged animals. Interestingly, learning-impaired mice were distinguished by a positive relationship between the extent of Barnes-related cognitive impairment and variability in daily onsets of circadian activity. While it is unclear whether changes in the sleep-wake cycle or other circadian rhythms play a role in cognitive impairment during aging, our results suggest that circadian rhythm perturbations or misalignment may nevertheless provide an early predictor of age-related cognitive decline.

Sex differences in the diathetic effects of shift work schedules on circulating cytokine levels and pathological outcomes of ischemic stroke during middle age.

Shift work is associated with increased risk for vascular disease, including stroke- and cardiovascular-related mortality. However, evidence from these studies is inadequate to distinguish the effect of altered circadian rhythms in isolation from other risk factors for stroke associated with shift work (e.g., smoking, poor diet, lower socioeconomic status). Thus, the present study examined the diathetic effects of exposure to shifted LD cycles during early adulthood on circadian rhythmicity, inflammatory signaling and ischemic stroke pathology during middle age, when stroke risk is high and outcomes are more severe. Entrainment of circadian activity was stable in all animals maintained on a fixed light:dark 12:12 cycle but was severely disrupted during exposure to shifted LD cycles (12hr advance/5d). Following treatment, circadian entrainment in the shifted LD group was distinguished by increased daytime activity and decreased rhythm amplitude that persisted into middle-age. Circadian rhythm desynchronization in shifted LD males and females was accompanied by significant elevations in circulating levels of the inflammatory cytokine IL-17A and gut-derived inflammatory mediator lipopolysaccharide (LPS) during the post-treatment period. Middle-cerebral artery occlusion, 3 months after exposure to shifted LD cycles, resulted in greater post-stroke mortality in shifted LD females. In surviving subjects, sensorimotor performance, assessed 2- and 5-days post-stroke, was impaired in males of both treatment groups, whereas in females, recovery of function was observed in fixed but not shifted LD rats. Overall, these results indicate that early exposure to shifted LD cycles promotes an inflammatory phenotype that amplifies stroke impairments, specifically in females, later in life.

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties.

In the mammalian circadian system, cell-autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 (Luc) knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 (Luc) SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2 (Luc) fibroblasts. SCN mPer2 (Luc) cells generated self-sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co-cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 (Luc) fibroblasts decayed after 3-4 cycles in serum-shocked cultures but robustly persisted for 6-7 cycles in the presence of SCN2.2 cells. In the co-culture model, the circadian behavior of mPer2 (Luc) fibroblasts was dependent on the integrity of the molecular clockworks in co-cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1(ldc) /Per2 (ldc) ). Because immortalized mPer2 (Luc) SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real-time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.

Amitriptyline Decreases GABAergic Transmission in Basal Forebrain Neurons Using an Optogenetic Model of Aging.

The antidepressant drug amitriptyline is used in the treatment of clinical depression and a variety of neurological conditions such as anxiety, neuropathic pain disorders and migraine. Antidepressants are associated with both therapeutic and untoward effects, and their use in the elderly has tripled since the mid-1990s. Because of this widespread use, we are interested in testing the acute effects of amitriptyline on synaptic transmission at therapeutic concentrations well below those that block voltage-gated calcium channels. We found that 3 µM amitriptyline reduced the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) and reduced quantal content in mice at ages of 7-10 mo. and 23-25 mo., suggesting a presynaptic mechanism of action that does not diminish with age. We employed a reduced synaptic preparation of the basal forebrain (BF) and a new optogenetic aging model utilizing a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R specific for GABAergic neurons [VGAT-ChR2(H134R)-EYFP]. This model enables optogenetic light stimulation of specific GABAergic synaptic terminals across aging. Age-related impairment of circadian behavior was used to confirm predictable age-related changes associated with this model. Our results suggest that low concentrations of amitriptyline act presynaptically to reduce neurotransmitter release and that this action is maintained during aging.

Long-term tegaserod treatment for dysmotility-like functional dyspepsia: results of two identical 1-year cohort studies.

BACKGROUND: Functional dyspepsia (FD) is a chronic disorder that adversely affects health-related quality of life (HRQoL). Published information on its long-term management is minimal and treatment options are limited. AIM: The aim of this study was to evaluate safety, efficacy and HRQoL with tegaserod 6 mg twice daily over 1 year in women with FD who completed one of two 6-week, randomized, placebo-controlled, double-blind studies. METHODS: About 780 patients received tegaserod 6 mg twice daily in two identical 1-year extension studies. Scheduled assessments included adverse events, the Short-Form Nepean Dyspepsia Index (SF-NDI), Work Productivity and Activity Impairment-Dyspepsia (WPAI-Dyspepsia) questionnaire, and patient perceptions of treatment efficacy. RESULTS: Mean tegaserod treatment duration in the two studies was 236 and 222 days. Most adverse events occurred in the first 6 months, were similar to previous reports (commonly diarrhea), and were transient and well tolerated. SF-NDI, WPAI-Dyspepsia scores and perceived symptom relief improved from baseline over the 1-year evaluation. CONCLUSIONS: The long-term safety profile of tegaserod in women with FD was consistent with that of short-term treatment and accompanied by improvements in HRQoL, work productivity and symptom relief. These long-term results add to the clinical experience with FD and support the potential value of a 5-HT(4) agonist in the management of FD.

Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes.

The master circadian pacemaker located within the suprachiasmatic nucleus (SCN) of the mammalian brain controls system-level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. ATP is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferin-luciferase chemiluminescence assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (tau) of 23.7 h in untreated as well as vehicle-treated and forskolin-treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to 12 h of light and 12 h of dark or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23 h. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.

Disruption of period gene expression alters the inductive effects of dioxin on the AhR signaling pathway in the mouse liver.

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 (Per1(ldc) and Per1(ldc)/Per2(ldc)). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.

Role of Proinflammatory Cytokines in Feedback Modulation of Circadian Clock Gene Rhythms by Saturated Fatty Acids.

Proinflammatory signaling cascades have been implicated in the mechanism by which high fat diet (HFD) and saturated fatty acids (SFA) modulate fundamental circadian properties of peripheral clocks. Because the cytokines TNFα and IL-6 are key signals in HFD- and SFA-induced proinflammatory responses that ultimately lead to systemic insulin resistance, the present study examined the roles of these cytokines in the feedback modulation of peripheral circadian clocks by the proinflammatory SFA, palmitate. IL-6 and TNFα secretion in Bmal1-dLuc fibroblast cultures was increased during palmitate treatment although the time course and amplitude of the inductive response differed between these cytokines. Similar to the time-dependent phase shifts observed in response to palmitate, treatment with IL-6 or with the low dose (0.1 ng/ml) of TNFα at hour 12 (i.e., after forskolin synchronization) induced phase advances of fibroblast Bmal1-dLuc rhythms. In complementary experiments, treatment with neutralizing antibodies against these proinflammatory cytokines or their receptors to inhibit of IL-6- or TNFα-mediated signaling repressed palmitate-induced phase shifts of the fibroblast clock. These studies suggest that TNFα, IL-6 and other proinflammatory cytokines may mediate the feedback modulation of peripheral circadian clocks by SFA-induced inflammatory signaling.

Alosetron versus traditional pharmacotherapy in clinical practice: effects on resource use, health-related quality of life, safety and symptom improvement in women with severe diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: Severe diarrhea-predominant irritable bowel syndrome (IBS-D) is associated with decreased health-related quality of life (HRQOL) and increased health care costs. Treatment recommendations for IBS-D often start with traditional pharmacotherapy (TP), with escalation to alosetron, rifaximin or eluxadoline if there is no success. There has been no previous head-to-head clinical trial comparing IBS-D treatment outcome for alosetron versus TP. This study, GSK protocol S3B30020, evaluated resource use, work productivity, health-related quality of life and global symptom response in women with IBS-D who were treated with alosetron or TP. METHODS: A total of 1956 patients who met criteria for severe IBS-D were randomized to treatment with alosetron 1 mg twice daily (BID) or only TP for up to 24 weeks. Work productivity and resource use were evaluated by standard questionnaires, HRQOL by the IBSQOL instrument and IBS symptoms by the Global Improvement Scale (GIS). RESULTS: Compared to only TP, alosetron-treated patients reported: (1) fewer clinic/office visits for any health problem (p = .0181) or for IBS-D (p = .0004); (2) reduced use of over-the-counter medications for IBS-D (p < .0001); (3) fewer days of lost work productivity (p < .0001); (4) decreased restriction of social and outdoor activities (p < .0001); and (5) greater global improvement in IBS-D symptoms (p < .0001). Alosetron treatment improved HRQOL scores for all domains (p < .0001). Incidence of adverse events during alosetron use was not remarkable and was similar to that previously reported. CONCLUSIONS: Alosetron 1 mg BID significantly reduced health care utilization and lost productivity, and significantly improved global IBS symptoms, HRQOL, and participation in outdoor and social activities compared with treatment response to TP.

Tegaserod treatment for dysmotility-like functional dyspepsia: results of two randomized, controlled trials.

OBJECTIVES: Therapies for dysmotility-like functional dyspepsia (FD) are limited. We studied tegaserod, a selective serotonin type 4 receptor agonist, in patients with FD. METHODS: Two identical multicenter, double-blind, randomized, placebo-controlled trials enrolled women >/=18 yr with recurring mid-upper abdominal discomfort characterized by postprandial fullness, early satiety, and/or bloating. Patients were randomized to tegaserod 6 mg b.i.d. or placebo. Two patient-reported primary variables were assessed: percentage of days with satisfactory symptom relief, and symptom severity using the composite average daily severity score (CADSS). RESULTS: In total, 2,667 women were randomized with no differences between trials in terms of recruitment method, Helicobacter pylori status, heartburn, or medication use. Mean percentage of days with satisfactory symptom relief for tegaserod versus placebo in Trial 1: 32.2%versus 26.6% (95% CI of treatment difference 2.82, 9.27; P < 0.01), Trial 2: 31.9%versus 29.4% (95% CI of treatment difference -0.21, 6.53; P= 0.066). Mean CADSS in Trial 1: 3.14 versus 3.35 (95% CI of treatment difference -0.29, -0.10; P < 0.0001), Trial 2: 3.15 versus 3.23 (95% CI of treatment difference -0.18, 0.01; P= 0.094). Meta-analysis showed significant benefit for both end points: increase in days with satisfactory relief 4.6% (95% CI 2.29, 6.96); decrease in CADSS 0.14 (95% CI 0.21, 0.07). Treatment effect was greater in patients with severe baseline symptoms. Diarrhea requiring study discontinuation was more common with tegaserod than placebo (4.1%vs 0.3%). CONCLUSIONS: Some improvement in dysmotility-like FD was observed with tegaserod treatment. The clinical implication of this improvement is uncertain.