Virtual Populations for Quantitative Systems Pharmacology Models.

Quantitative systems pharmacology (QSP) places an emphasis on dynamic systems modeling, incorporating considerations from systems biology modeling and pharmacodynamics. The goal of QSP is often to quantitatively predict the effects of clinical therapeutics, their combinations, and their doses on clinical biomarkers and endpoints. In order to achieve this goal, strategies for incorporating clinical data into model calibration are critical. Virtual population (VPop) approaches facilitate model calibration while faced with challenges encountered in QSP model application, including modeling a breadth of clinical therapies, biomarkers, endpoints, utilizing data of varying structure and source, capturing observed clinical variability, and simulating with models that may require more substantial computational time and resources than often found in pharmacometrics applications. VPops are frequently developed in a process that may involve parameterization of isolated pathway models, integration into a larger QSP model, incorporation of clinical data, calibration, and quantitative validation that the model with the accompanying, calibrated VPop is suitable to address the intended question or help with the intended decision. Here, we introduce previous strategies for developing VPops in the context of a variety of therapeutic and safety areas: metabolic disorders, drug-induced liver injury, autoimmune diseases, and cancer. We introduce methodological considerations, prior work for sensitivity analysis and VPop algorithm design, and potential areas for future advancement. Finally, we give a more detailed application example of a VPop calibration algorithm that illustrates recent progress and many of the methodological considerations. In conclusion, although methodologies have varied, VPop strategies have been successfully applied to give valid clinical insights and predictions with the assistance of carefully defined and designed calibration and validation strategies. While a uniform VPop approach for all potential QSP applications may be challenging given the heterogeneity in use considerations, we anticipate continued innovation will help to drive VPop application for more challenging cases of greater scale while developing new rigorous methodologies and metrics.

Epigenetic modification in 4T1 mouse breast cancer model by artificial light at night and melatonin - the role of DNA-methyltransferase.

Currently, one of the most disputed hypotheses regarding breast cancer (BC) development is exposure to short wavelength artificial light at night (ALAN) as multiple studies suggest a possible link between them. This link is suggested to be mediated by nocturnal melatonin suppression that plays an integral role in circadian regulations including cell division. The objective of the research was to evaluate effects of 1 × 30 min/midnight ALAN (134 µ Wcm-2, 460 nm) with or without nocturnal melatonin supplement on tumor development and epigenetic responses in 4T1 tumor-bearing BALB/c mice. Mice were monitored for body mass (Wb) and tumor volume for 3 weeks and thereafter urine samples were collected at regular intervals for determining daily rhythms of 6-sulfatoxymelatonin (6-SMT). Finally, mice were sacrificed and the tumor, lungs, liver, and spleen were excised for analyzing the total activity of DNA methyltransferases (DNMT) and global DNA methylation (GDM) levels. Mice exposed to ALAN significantly reduced 6-SMT levels and increased Wb, tumor volume, and lung metastasis compared with controls. These effects were diminished by melatonin. The DNMT activity and GDM levels showed tissue-specific response. The enzymatic activity and GDM levels were lower in tumor and liver and higher in spleen and lungs under ALAN compared with controls. Our results suggest that ALAN disrupts the melatonin rhythm and potentially leading to increased BC burden by affecting DNMT activity and GDM levels. These data may also be applicable to early detection and management of BC by monitoring melatonin and GDM levels as early biomarker of ALAN circadian disruption.

The loss of ecosystem-services emerging from artificial light at night.

Light pollution is increasing worldwide, affecting human health and ecosystem quality. The adverse effect of this novel pollution, mediated in mammals by suppression of the pineal neuro-hormone melatonin production and secretion, particularly by short wavelength (SWL) illumination. Currently, this problem is not challenged sufficiently, even ignored by decision-makers at local and national levels, as well as other related organizations. Therefore, we assume that the correct way to deal with it will be by treating the dark night as an ecosystem-service for temporal organization of humans as other organisms. Therefore, chasing darkness away and mainly by SWL illumination is as giving up the natural light/dark cycles offered as an ecosystem-service. So far, we have no environmental economic tools for assessing the real coast of the health damages or reduction in pollination caused by light pollution. Using Artificial Light at Night (ALAN) as a loss of ecosystem-services will enable us to give it a realistic economic value thus an opportunity to re-evaluate the environmental cost of SWL efficient illumination. This will also help decision-makers to move to the next stage of illumination preferring sustainable illumination.

Artificial light-at-night - a novel lifestyle risk factor for metabolic disorder and cancer morbidity.

Both obesity and breast cancer are already recognized worldwide as the most common syndromes in our modern society. Currently, there is accumulating evidence from epidemiological and experimental studies suggesting that these syndromes are closely associated with circadian disruption. It has been suggested that melatonin (MLT) and the circadian clock genes both play an important role in the development of these syndromes. However, we still poorly understand the molecular mechanism underlying the association between circadian disruption and the modern health syndromes. One promising candidate is epigenetic modifications of various genes, including clock genes, circadian-related genes, oncogenes, and metabolic genes. DNA methylation is the most prominent epigenetic signaling tool for gene expression regulation induced by environmental exposures, such as artificial light-at-night (ALAN). In this review, we first provide an overview on the molecular feedback loops that generate the circadian regulation and how circadian disruption by ALAN can impose adverse impacts on public health, particularly metabolic disorders and breast cancer development. We then focus on the relation between ALAN-induced circadian disruption and both global DNA methylation and specific loci methylation in relation to obesity and breast cancer morbidities. DNA hypo-methylation and DNA hyper-methylation, are suggested as the most studied epigenetic tools for the activation and silencing of genes that regulate metabolic and monostatic responses. Finally, we discuss the potential clinical and therapeutic roles of MLT suppression and DNA methylation patterns as novel biomarkers for the early detection of metabolic disorders and breast cancer development.

Artificial light at night: melatonin as a mediator between the environment and epigenome.

The adverse effects of excessive use of artificial light at night (ALAN) are becoming increasingly evident and associated with several health problems including cancer. Results of epidemiological studies revealed that the increase in breast cancer incidents co-distribute with ALAN worldwide. There is compiling evidence that suggests that melatonin suppression is linked to ALAN-induced cancer risks, but the specific genetic mechanism linking environmental exposure and the development of disease is not well known. Here we propose a possible genetic link between environmental exposure and tumorigenesis processes. We discuss evidence related to the relationship between epigenetic remodelling and oncogene expression. In breast cancer, enhanced global hypomethylation is expected in oncogenes, whereas in tumour suppressor genes local hypermethylation is recognized in the promoter CpG chains. A putative mechanism of action involving epigenetic modifications mediated by pineal melatonin is discussed in relation to cancer prevalence. Taking into account that ALAN-induced epigenetic modifications are reversible, early detection of cancer development is of great significance in the treatment of the disease. Therefore, new biomarkers for circadian disruption need to be developed to prevent ALAN damage.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity.

PURPOSE: Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts is involved in the progression of osteoarthritis (OA). Human osteoblasts isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin pathway, and a reduced mineralization in vitro as well as in vivo. These alterations were linked with an abnormal response to BMP-2. OA osteoblasts release factors such as the hepatocyte growth factor (HGF) that contribute to cartilage loss whereas chondrocytes do not express HGF. HGF can stimulate BMP-2 expression in human osteoblasts, however, the role of HGF and its effect in OA osteoblasts remains unknown. Here we investigated whether elevated endogenous HGF levels in OA osteoblasts are responsible for their altered response to BMP-2. METHODS: We prepared primary human subchondral osteoblasts using the sclerotic medial portion of the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals obtained at autopsy. The expression of HGF was evaluated by qRT-PCR and the protein production by western blot analysis. HGF expression was reduced with siRNA technique whereas its activity was inhibited using the selective inhibitor PHA665752. Alkaline phosphatase activity (ALPase) and osteocalcin release were measured by substrate hydrolysis and EIA respectively. Canonical Wnt/ß-catenin signaling (cWnt) was evaluated both by target gene expression using the TOPflash TCF/lef luciferase reporter assay and western blot analysis of ß-catenin levels in response to Wnt3a stimulation. Mineralization in response to BMP-2 was evaluated by alizarin red staining. RESULTS: The expression of HGF was increased in OA osteoblasts compared to normal osteoblasts and was maintained during their in vitro differentiation. OA osteoblasts released more HGF than normal osteoblasts as assessed by western blot analysis. HGF stimulated the expression of TGF-ß1. BMP-2 dose-dependently (1 to 100 ng/ml) stimulated both ALPase and osteocalcin in normal osteoblasts whereas, it inhibited them in OA osteoblasts. HGF-siRNA treatments reversed this response in OA osteoblasts and restored the BMP-2 response. cWnt is reduced in OA osteoblasts compared to normal, and HGF-siRNA treatments increased cWnt in OA osteoblasts almost to normal. Smad1/5/8 phosphorylation in response to BMP-2, which is reduced in OA osteoblasts, was corrected when these cells were treated with PHA665752. The BMP-2-dependent mineralization of OA osteoblasts, which is also reduced compared to normal, was only partially restored by PHA665752 treatment whereas 28 days treatment with HGF reduced the mineralization of normal osteoblasts. CONCLUSION: OA osteoblasts expressed more HGF than normal osteoblasts. Increased endogenous HGF production in OA osteoblasts stimulated the expression of TGF-ß1 and reduced their response to BMP-2. Inhibiting HGF expression or HGF signaling restored the response to BMP-2 and Smad1/5/8 signaling. In addition, decreased HGF signaling partly corrects the abnormal mineralization of OA osteoblasts while increased HGF prevents the normal mineralization of normal osteoblasts. In summary, we hypothesize that sustained elevated HGF levels in OA osteoblasts drive their abnormal phenotype and is implicated in OA pathophysiology.

Long-day photoperiod interacts with vasopressin and food restriction to modulate reproductive status and vasopressin receptor expression of male golden spiny mice.

We tested the effects of photoperiod, water and food availability on body mass, reproductive status and arginine vasopressin receptor 1A (Avpr1a) mRNA expression in males of desert-adapted golden spiny mice, Acomys russatus. In Experiment 1, males were acclimated to short-day (SD; 8 h:16 h light:dark) or long-day (LD; 16 h:8 h light:dark) photoperiods with either saline (control) or vasopressin treatment for 3 weeks. The results of this experiment revealed that under control conditions, SD mice increased body mass by ~5% while LD mice decreased it by ~4%. SD photoperiod had no effect on reproductive status and leptin levels, whereas LD males increased testes mass and serum testosterone, but the photoperiod had no effect on leptin levels. Vasopressin administration decreased LD-induced reproductive enhancement. Because no consistent effect of SD treatment was found on reproductive status, Experiment 2 was carried out only on LD-acclimated males kept under 75% food restriction (decrease from ad libitum) with saline or leptin treatment. Body mass, testes mass, serum testosterone, leptin concentrations and Avpr1a mRNA expression were measured. Food restriction remarkably decreased body mass, with a more potent effect in leptin-treated males, showing enhanced reproductive status and a significant increase in serum leptin compared with controls. Avpr1a expression was significantly upregulated in LD, vasopressin-treated and food-restricted males, with higher levels in the hypothalamus compared with the testes. We conclude that in A. russatus, LD photoperiod interacts with water and food availability to advance reproductive responses. Avpr1a is suggested to integrate nutritional and osmotic signals to optimize reproduction by modulating reproductive and energetic neuroendocrine axes at the central level. The interaction between photoperiod and other environmental cues is of an adaptive value to desert-adapted small rodents for timing reproduction in unpredictable ecosystems such as extreme deserts.

Spectral and duration sensitivity to light-at-night in 'blind' and sighted rodent species.

Light-at-night (LAN) has become a defining feature of human and animal ecosystems and may possibly compromise human and animal physiology and health. Spectral and acclimation duration (AD) sensitivity were compared between social voles (Microtus socialis) and 'blind' mole rats (Spalax ehrenbergi) in four increasing ADs (0, 1, 7 and 21 days) to LAN (1×30 min, 293 µW cm(-2)) of three different monochromatic lights [blue (479 nm), yellow (586 nm) and red (697 nm)]. Animals were sampled for urine and oxygen consumption (V(O(2))) promptly after each LAN-AD. Urine samples were analyzed for production rate, urinary 6-sulfatoxymelatonin and urinary metabolites of adrenalin and cortisol. Overall, the blue light elicited the greatest effects on the biological markers of M. socialis, whereas similar effects were detected for S. ehrenbergi in response to red light. The increasing LAN-AD resulted in a dose-dependent decrement of all markers tested, except of stress hormones, which showed a direct positive correlation with LAN-AD. Our results suggest that: (1) photoperiod is an important cue for entraining physiological functions in the 'blind' S. ehrenbergi, which is essentially characterized by red-shifted sensitivity compared with the blue-shifted sensitivity detected for the sighted counterpart species, and (2) there is a strong association between LAN of the appropriate wavelength and adrenal endocrine responses, suggesting that LAN is a potential environmental stressor.

Photoentrainment in blind and sighted rodent species: responses to photophase light with different wavelengths.

Our study examined the impact of daylight (photophase) wavelength on the photoentrainment sensitivity of two species with vastly different visual systems. Social voles (Microtus socialis) and 'blind' mole rats (Spalax ehrenbergi) were exposed to short-wavelength (479 nm) or long-wavelength (697 nm) light at an intensity of 293 µW cm(-2). Rhythms of urine production, urinary 6-sulfatoxymelatonin (6-SMT), urinary metabolites of adrenaline and cortisol, and oxygen consumption (VO(2)) were used as markers for the sensitivity of the photoentrainment system. Significant 24-h rhythms were detected in all variables for both species under short-wavelength light, whereas ultradian rhythms of 12- or 8-h were detected under long-wavelength light. Wavelength inversely affected 6-SMT levels in M. socialis (negative correlation) and S. ehrenbergi (positive correlation). Increased levels of stress hormone metabolites were detected in M. socialis under the long-wavelength light whereas, in S. ehrenbergi elevated levels were secreted under short-wavelength light. Long-wavelength light increased VO(2) in M. socialis and decreased it in S. ehrenbergi; short-wavelength light elicited the opposite effects. Our results indicate that photophase wavelength is an integral light property for modulating photoperiodic responses in mammals, including visually challenged species. Finally, the spectral-induced differential responses between the two species potentially represent adaptive physiological flexibility in species with contrasting visual and habitat challenges.

Differential effects of photophase irradiance on metabolic and urinary stress hormone concentrations in blind and sighted rodents.

The effects of different photophase irradiance levels on the daily rhythms of energy expenditure (DEE, calculated from oxygen consumption, VO(2)) and urinary metabolites of stress hormones in sighted (Microtus socialis) and blind (Spalax ehrenbergi) rodents were compared. Five groups of each species were exposed to different irradiance levels (73, 147, 293, 366, and 498 microW/cm(2)) under short photoperiod (8L:16D) condition with constant ambient temperature 25 +/- 2 degrees C for 21 days before assessments. As light intensity increased from 73 microW/cm(2), both species reduced DEE, especially among M. socialis. Cosinor analysis revealed significant ultradian rhythms in VO(2) of M. socialis with period length being inversely related to irradiance level. Conversely, in S. ehrenbergi, robust 24 h VO(2) rhythms were detected at all irradiances. In M. socialis, significant 24 h rhythms in urinary output of adrenaline were detected only at 293 microW/cm(2), whereas for cortisol, unambiguous rhythms were detected at 73 and 147 microW/cm(2). Distinct adrenaline daily rhythms of S. ehrenbergi were observed at 73 and 293 microW/cm(2), whereas this species exhibited significant rhythms in cortisol at 147 and 293 microW/cm(2). Changes in photophase irradiance levels affected stress hormone concentrations in a dose-dependent manner. There were significant negative and positive correlations of M. socialis and S. ehrenbergi stress hormones, respectively, with increasing irradiance. Our results indicate photophase light intensity is another environmental factor that can significantly affect entrainment of mammalian daily rhythms. Both low and high irradiance conditions can trigger stress responses, depending on the species' natural habitat.

Characterization of oxidized low-density lipoprotein-induced hormesis-like effects in osteoblastic cells.

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Atherogenic determinants such as oxidized low-density lipoprotein (oxLDL) particles have been shown both to stimulate the proliferation and promote apoptosis of bone-forming osteoblasts. Given such opposite responses, we characterized the oxLDL-induced hormesis-like effects in osteoblasts. Biphasic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reductive activity responses were induced by oxLDL where low concentrations (10-50 microg/ml) increased and high concentrations (from 150 microg/ml) reduced the MTT activity. Cell proliferation stimulation by oxLDL partially accounted for the increased MTT activity. No alteration of mitochondria mass was noticed, whereas low concentrations of oxLDL induced mitochondria hyperpolarization and increased the cellular levels of reactive oxygen species (ROS). The oxLDL-induced MTT activity was not related to intracellular ROS levels. OxLDL increased NAD(P)H-associated cellular fluorescence and flavoenzyme inhibitor diphenyleneiodonium reduced basal and oxLDL-induced MTT activity, suggesting an enhancement of NAD(P)H-dependent cellular reduction potential. Low concentrations of oxLDL reduced cellular thiol content and increased metallothionein expression, suggesting the induction of compensatory mechanisms for the maintenance of cell redox state. These concentrations of oxLDL reduced osteoblast alkaline phosphatase activity and cell migration. Our results indicate that oxLDL particles cause hormesis-like response with the stimulation of both proliferation and cellular NAD(P)H-dependent reduction potential by low concentrations, whereas high concentrations lead to reduction of MTT activity associated with the cell death. Given the effects of low concentrations of oxLDL on osteoblast functions, oxLDL may contribute to the impairment of bone remodeling equilibrium.

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation.

UNLABELLED: Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. OBJECTIVES: Here, we have investigated the roles of melastatin-like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca2+) and magnesium (Mg2+) conducting channels, during proliferation of human osteoblasts. RESULTS: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast-like MG-63, SaOS and U2-OS cells, and reduction of extracellular Mg2+ or Ca2+ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg2+ and Ca2+ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre-incubation of cells in reduced extracellular Mg2+ conditions led to activation of Ca2+ and Mg2+ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. CONCLUSIONS: Our results indicate that extracellular Mg2+ and Ca2+ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg2+ and Ca2+ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.

The effect of alpha- and beta-adrenergic blockade on daily rhythms of body temperature, urine production, and urinary 6-sulfatoxymelatonin of social voles Microtus socialis.

To examine the effect of adrenergic blockade on daily rhythms of rectal body temperature (T(b)), urine production rate, and melatonin (MEL; measured as urinary 6-sulfatoxymelatonin; 6-SMT), social voles Microtus socialis received a single intra-peritoneal injection of either prazosin (PRAZ, 1 mg/kg) or propranolol (PROP, 4.5 mg/kg); alpha- and beta-adrenergic blocking agents respectively, 1 h prior to scotophase onset (light/dark, 12L:12D; lights on 07:00 h). Both blockers caused significant decrease in T(b) values mainly during scotophase. Nocturnal urine production rates were higher for M. socialis treated with the drugs compared with controls. Overall, urine production rates were systematically higher in PROP-voles over the 24 h period when compared with PRAZ-voles; however these differences were not statistically significant. Interestingly, PROP caused significant elevation in urinary 6-SMT at the second half of the dark phase, whereas PRAZ had no effects. These data suggest that the mechanisms regulating MEL synthesis and thermoregulatory responses in M. socialis are different from those described in other rodents' species. Importantly, the data also suggest that the beta-blockade-induced elevation in MEL levels may be directly associated with increased urination in M. socialis.

Recurrent strokes in a young patient with papillary fibroelastoma: a case report and literature review.

Since the introduction of echocardiography the diagnosis of papillary fibroelastomas (PFEs) in living patients has increased. They are second most common after myxomas representing 10% of cardiac tumors. The present case is of a patient with recurrent cerebrovascular accidents and documented protein S deficiency who continued to stroke despite adequate anticoagulation. Mitral valve PFE was suspected on echocardiography and confirmed at surgical excision. Two large studies published in the last decade describe the echocardiographic and clinical characteristics of PFEs which although are histologically benign, may present with a clinical course of devastating consequences owing to its strategic location within the cardiac structures. Echocardiography, particularly transesophageal echocardiography (TEE), provides the necessary anatomical resolution and detail to ascertain location, extent of involvement, and hemodynamic implications. Tissue diagnosis is based on characteristic histopathological features of avascular fronds lined by endothelial cells, containing varying amounts of elastin. The echocardiographic characteristics along with treatment options are reviewed.

Endogenous versus exogenous IL-10 in postoperative intraperitoneal adhesion formation in a murine model.

BACKGROUND: Modulation of the immune response to peritoneal injury may prevent postoperative adhesion formation. Exogenous interleukin-10 (IL-10) limits postoperative adhesion formation. The objective of the present study is to determine (I) the IL-10 dose most effective at adhesion prevention, (II) the presence of endogenous IL-10 in peritoneal fluid, and (III) the ability of an anti-IL-10 monoclonal antibody to modify postoperative intraperitoneal adhesion formation. MATERIALS AND METHODS: Within parts I, II, and III of the study 8-week-old Swiss Webster mice were randomized to have no surgery or to undergo a standardized adhesion-inducing peritoneal injury. Animals were further randomized to different dosing schedules of IL-10 (10 ng/kg to 100 micrograms/kg; part I), different times of euthanasia and peritoneal lavage after surgery (part II), or intraperitoneal (i.p.) treatment with phosphate-buffered saline vehicle (1 ml), IL-10 (1 microgram/kg), or anti-IL-10 (1 microgram/kg; part III). All i.p. injections were given immediately after surgery and then every 24 hr for a total of four injections. Animals were sacrificed 7 days after surgery and adhesion formation was assessed. RESULTS: Maximal adhesion-limiting effects of exogenons IL-10 were reached at doses of 1 microgram/kg. Mean detectable endogenous IL-10 levels in the peritoneal fluid varied from 145 to 220 pg/ml throughout the postoperative course. There were significant (P < 0.0005) differences in adhesion scores between mice treated with IL-10 (3.39; 95% CI 0.39-6.39) or PBS postoperatively (7.46; 95% CI 5.28-9.64), untreated surgical control animals (5.98; 95% CI 2.55-9.41), or anti-IL-10-treated animals (10.11; 95% CI 8.50-11.72). CONCLUSION: Interleukin 10 is effective at reducing postoperative intraperitoneal adhesion formation. Low levels of endogenous IL-10 are detectable in peritoneal fluid throughout the postoperative course, not correlating with adhesion scores. Anti-IL-10, when administered daily postoperatively in pharmacologic doses, does not appear to significantly increase postoperative adhesion formation. This puts into question if endogenous IL-10 production indeed plays a role in nonadhesiogenic peritoneal healing.