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Abstract In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti-inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, β-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by β-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity. Graphical abstract Image 1 Highlights • This assay could monitor expression of IL-6 and IL-10 reporters in real-time. • The assay continuously determines expression of reporters in living single cells. • Lipopolysaccharide strongly induces expression of IL-6 and IL-10 reporters. [ABSTRACT FROM AUTHOR]

Sleep fragmentation due to intermittent nocturnal arousal resulting in a reduction of total sleep time and sleep efficiency is a common symptom among people with Alzheimer’s disease (AD) and elderly people with normal cognitive function. Although epidemiological studies have indicated an association between sleep fragmentation and elevated risk of AD, a relevant disease model to elucidate the underlying mechanisms was lacking owing to technical limitations. Here we successfully induced chronic sleep fragmentation in AD model mice using a recently developed running-wheel-based device and demonstrate that chronic sleep fragmentation increases amyloid β deposition. Notably, the severity of amyloid β deposition exhibited a significant positive correlation with the extent of sleep fragmentation. These findings provide a useful contribution to the development of novel treatments that decelerate the disease course of AD in the patients, or decrease the risk of developing AD in healthy elderly people through the improvement of sleep quality. [ABSTRACT FROM AUTHOR]

We previously reported that heat-killed Lactobacillus brevis SBC8803 enhances appetite via changes in autonomic neurotransmission. Here we assessed whether a diet supplemented with heat-killed SBC8803 affects circadian locomotor rhythmicity and sleep architecture. Main methods and key findings Daily total activity gradually increased in mice over 4weeks and supplementation with heat-killed SBC8803 significantly intensified the increase, which reached saturation at 25days. Electroencephalography revealed that SBC8803 supplementation significantly reduced the total amount of time spent in non-rapid eye movement (NREM) sleep and increased the amount of time spent being awake during the latter half of the nighttime, but tended to increase the total amount of time spent in NREM sleep during the daytime. Dietary supplementation with SBC8803 can extend the duration of activity during the nighttime and of sleep during the daytime. Daily voluntary wheel-running and sleep rhythmicity become intensified when heat-killed SBC8803 is added to the diet. Significance Dietary heat-killed SBC8803 can modulate circadian locomotion and sleep rhythms, which might benefit individuals with circadian rhythms that have been disrupted by stress or ageing. [ABSTRACT FROM AUTHOR]

Psychological stressors prominently affect diurnal rhythms, including locomotor activity, sleep, blood pressure, and body temperature, in humans. Here, we found that a novel continuous stress imposed by the perpetual avoidance of water on a wheel (PAWW) affected several physiological diurnal rhythms in mice. One week of PAWW stress decayed robust circadian locomotor rhythmicity, while locomotor activity was evident even during the light period when the mice are normally asleep. Daytime activity was significantly upregulated, whereas nighttime activity was downregulated, resulting in a low amplitude of activity. Total daily activity gradually decreased with increasing exposure to PAWW stress. The mice could be exposed to PAWW stress for over 3 weeks without adaptation. Furthermore, continuous PAWW stress enhanced food intake, but decreased body weight and plasma leptin levels, indicating that sleep loss and PAWW stress altered the energy balance in these mice. The diurnal rhythm of corticosterone levels was not severely affected. The body temperature rhythm was diurnal in the stressed mice, but significantly dysregulated during the dark period. Plasma catecholamines were elevated in the stressed mice. Continuous PAWW stress reduced the duration of daytime sleep, especially during the first half of the light period, and increased nighttime sleepiness. Continuous PAWW stress also simultaneously obscured sleep/wake and locomotor activity rhythms compared with control mice. These sleep architecture phenotypes under stress are similar to those of patients with insomnia. The stressed mice could be entrained to the light/dark cycle, and when they were transferred to constant darkness, they exhibited a free-running circadian rhythm with a timing of activity onset predicted by the phase of their entrained rhythms. Circadian gene expression in the liver and muscle was unaltered, indicating that the peripheral clocks in these tissues remained intact. [ABSTRACT FROM AUTHOR]

Some reports have indicated that the core clock gene, Per2 regulates the cell cycle, immune system and neural functions. To understand the effects of PER2 on tumor growth in vivo, stable transformants of murine sarcoma 180 (S-180) cell lines expressing different levels of PER2 were established. The growth of stable PER2 transformants in vivo was significantly and dose-dependently suppressed according to the amount of PER2 expressed, indicating that PER2 plays a role in the growth suppression of sarcoma cells. The anchorage-dependent and -independent growth in vitro and expression of the clock controlled cell-cycle related genes, wee1, myc, and VEGF were not altered in stable PER2 transformants. In contrast, susceptibility to murine natural killer (NK) cell cytolytic activity was enhanced in PER2 transformants. Furthermore, PER2 transformants suppressed cell motility and reduced fibronectin expression, but the expression of integrin receptors was not affected. These results suggest that sarcoma cells overexpressing PER2 suppress tumors in vivo by changing the nature of tumor cell adhesion. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the amino acid sequences of rat PERIOD2 (rPER2) that are required for interaction with CRYPTOCHROME1 (CRY1) to understand the molecular mechanism of the circadian clock. Co-immunoprecipitation assays using various C-terminal fragments of rPER2 with internal deletions revealed that amino acid residues 1179–1198 are necessary for interaction with CRY1. To identify precisely which amino acid residues are responsible for the interaction, we substituted alanine for residues conserved among PER isoforms and species. We found that more than three mutations of conserved PER2 residues impaired not only binding to CRY1 but also subsequent nuclear translocation, although mutations of non-conserved residues did not affect interaction with CRY1. Thus, the conserved amino acid residues of 1179–1198 in PER2 are apparently responsible for binding to CRY1. [Copyright &y& Elsevier]

Abstract: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. To determine whether the circadian clock component PERIOD2 (PER2) is involved in the regulation of PAI-1 gene expression, we performed transient transfection assays in vitro, and generated transgenic (Tg) mice overexpressing PER2. We then compared PAI-1 expression in Tg and wild-type (WT) mice with or without obesity induced by a high-fat/high-sucrose diet. PER2 suppressed CLOCK:BMAL1- and CLOCK:BMAL2-dependent transactivation of the PAI-1 promoter in vitro. Furthermore, nuclear translocation is dispensable for PER2 to suppress CLOCK:BMAL1-dependent transactivation of the PAI-1 promoter, because functional loss of the nuclear localization domain did not affect either the interaction with BMAL1 or the suppressive role of PER2. The diurnal expression of clock and clock-controlled genes was disrupted in a gene-specific manner, whereas that of PAI-1 mRNA was significantly damped in the hearts of PER2 Tg mice fed with a normal diet. Obesity-induced plasma PAI-1 increase was significantly suppressed in Tg mice in accordance with cardiac PAI-1 mRNA levels, whereas body weight gain and changes in metabolic parameters were identical between WT and Tg mice. Endogenous PAI-1 gene expression induced by transforming growth factor-β1 was significantly attenuated in embryonic fibroblasts derived from Tg mice compared with those from WT mice. Our results demonstrated that PER2 represses PAI-1 gene transcription in a BMAL1/2-dependent manner. The present findings also suggest that PER2 attenuates obesity-induced hypofibrinolysis by downregulating PAI-1 expression independently of metabolic disorders. [Copyright &y& Elsevier]

Molecular circadian clock regulation engages a negative feedback loop comprising components of the negative limb, PERs and CRYs. In addition to the rhythmic transcriptional regulation of clock genes, controlled subcellular localization might contribute to the molecular mechanism of the mammalian circadian clock. To address this issue, we generated transgenic (TG) mice lines harboring either rat PER2 (rPER2) with a deleted nuclear localizing domain [NLD(–)] or intact PER2. In comparison with wild-type (WT) control, the period of the circadian locomotor rhythm in TG mice over-expressing NLD(–) PER2 was longer, while that in TG mice over-expressing intact PER2 was shorter. The nuclear entry of endogenous PER2, CRY1 and CRY2 was delayed in the suprachiasmatic nucleus (SCN) of NLD(–) PER2 TG mice under constant darkness, whereas that of mouse PER2 (mPER2) is accelerated in the SCN of intact PER2 TG mice. Under constant light, the locomotor activity of NLD(–) PER2 TG mice became arrhythmic, whereas WT animals remained rhythmic. These data indicate that PER2 controls circadian periods through nuclear localization in the SCN. In addition, sleep architecture was also affected in intact PER2 TG mice, suggesting PER2 can modulate a sleep molecular mechanism. [ABSTRACT FROM AUTHOR]

The Drosophila shaggy gene product is a mammalian glycogen synthase kinase-3β (GSK-3β) homologue that contributes to the circadian clock of the Drosophila through TIMELESS phosphorylation, and it regulates nuclear translocation of the PERIOD/TIMELESS heterodimer. We found that mammalian GSK-3β is expressed in the suprachiasmatic nucleus and liver of mice and that GSK-3β phosphorylation exhibits robust circadian oscillation. Rhythmic GSK-3β phosphorylation is also observed in serum-shocked NIH3T3 cells. Exposing serum-shocked NIH3T3 cells to lithium chloride, a specific inhibitor of GSK-3β, increases GSK-3β phosphorylation and delays the phase of rhythmic clock gene expression. On the other hand, GSK-3β overexpression advances the phase of clock gene expression. We also found that GSK-3β interacts with PERIOD2 (PER2) in vitro and in vivo. Recombinant GSK-3β can phosphorylate PER2 in vitro. GSK-3β promotes the nuclear translocation of PER2 in COS1 cells. The present data suggest that GSK-3β plays important roles in mammalian circadian clock. [ABSTRACT FROM AUTHOR]

CLOCK is a positive component of a transcription/ translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5'-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues. [ABSTRACT FROM AUTHOR]

The mammalian circadian timing system consists of a central pacemaker in brain hypothalamus and damping oscillators in most peripheral tissues. To investigate the mechanism that controls circadian rhythms in the mammalian peripheral tissues, we examined the expression rhythm of mPer2, BMAL1, albumin D-site binding protein (DBP), and Rev-erbα mRNAs in the heart of homozygous Clock mutant mice on Jcl:ICR background under the temporal feeding restriction. Unexpectedly, the restricted feeding (RF) shifted the circadian phase of both mPer2 and BMAL1 mRNA expressions in the heart not only of wild-type mice but also of Clock mutant mice. Furthermore, in the Clock mutant mice, the amplitude of the circadian expression of mPer2 and BMAL1 mRNAs was dramatically increased by the RF. These data indicate that functional CLOCK is not required for an entrainment of peripheral clocks to RF. On the other hand, the expression levels of DBP and Rev-erbα mRNAs were blunted in Clock mutant mice not only under ad libitum but also under RF conditions. Thus, it seems that the rhythmic expression of Rev-erbα is not involved in the RF-induced circadian expression of BMAL1 mRNA, although REV-ERBα has been identified as a major regulator of BMAL1 transcription. Thus, the entraining mechanism of peripheral tissues to the RF seems to be different from that to the central clock in the suprachiasmatic nucleus. [Copyright &y& Elsevier]

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL‐8) and ubiquitin‐C (UBC) promoters, respectively. Subsequently, we generated a three‐dimensional (3D) skin‐like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non‐woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real‐time bioluminescence monitoring, allowing the assessment of time‐ and dose‐dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin. [ABSTRACT FROM AUTHOR]

Sleep disturbances can contribute to cognitive decline and neuropsychiatric disorders. However, the underlying mechanisms of these processes are poorly understood. The present study evaluated the effects of a chronic sleep disorder (CSD) on long-term memory formation and anxiety-like behavior in our originally established mouse model of psychophysiological stress-induced CSD characterized by disrupted circadian rhythms of wheel-running activity and sleep-wake cycles. Model mice are continuously exposed to mild stress imposed by perpetually staying on a running-wheel to avoid water. The findings of novel object recognition (NORT) and open field (OFT) tests showed that CSD impaired recognition memory and elicited anxiety-like behavior, respectively. These results suggested that the CSD impaired cognitive function and emotional status. Thus, this CSD model could be useful for studying the underlying mechanisms of neurobehavioral difficulties caused by sleep disorders. We then examined the hippocampal mRNA expression of genes associated with learning and memory, and anxiety and depression. The CSD increased the mRNA expression of Crhr1 , Ngf and Phlpp1 , and suppressed that of Ace , Egr2 and Slc6a4. Based on the functions of these genes, we inferred that the increase in Crhr1 mRNA was associated with the pathogenesis of psychiatric conditions, whereas mRNA levels of the other five genes were directed towards symptom relief. Upregulating hippocampal Crhr1 expression might contribute in part to the activation of corticotropin-releasing hormone (CRH)-CRH receptor1 signaling that mediates CSD-evoked mental disorders. • We created a mouse model of a stress-induced chronic sleep disorder (CSD). • CSD impaired long-term memory and elicited anxiety-like behavior. • Increased expression of hippocampal Crhr1 might contribute to the mental disorders. [ABSTRACT FROM AUTHOR]

Abstract: Circadian dysfunction perturbs the female reproductive cycle. In particular, mice lacking the clock gene Bmal1 show severe infertility, implying that BMAL1 plays roles in ovulation and luteinization. Here, we examined temporal changes in clock gene expression in the ovary and oviduct before and during gonadotropin‐induced follicular growth, ovulation, and luteinization in sexually immature mice. While the oviduct did not show a drastic change in clock gene expression, Bmal1 expression in the ovary was higher than that in control mice during the period from 4 to 16 hr after human chorionic gonadotropin (hCG) administration. Bmal1 expression reached a maximum at 16 hr after hCG administration, when follicle luteinization occurred. In an interesting manner, administration of hCG to ex vivo‐cultured oviduct triggered a shorter circadian period and inevitably resulted in phase advance. Together, our present data suggest that LH surge induces continuous expression of BMAL1 in the mouse ovary and modulates circadian phase in the mouse oviduct. [ABSTRACT FROM AUTHOR]

Abstract: Lactobacilli provide several health benefits to mammals, including humans. We previously observed that in rats, intraduodenal injection of Lactobacillus johnsonii La1 elevated efferent gastric vagal nerve activity (efferent-GVNA), while Lactobacillus paracasei ST11 suppressed efferent-GVNA, and thereby increased or decreased food intake. To determine the function of Lactobacillus brevis (SBC8803), its effect on food intake was examined by providing food containing heat-killed SBC8803 to mice. We observed that administration of SBC8803 elevated food intake. Because the afferent intestinal vagal nerve (IVN) is hypothesized to be involved in efferent-GVNA changes, we examined the effect of intraduodenal administration of heat-killed SBC8803 on efferent-GVNA and afferent-IVN activity (IVNA) in rats. In this study, we found that intraduodenal administration of heat-killed SBC8803 increased both efferent-GVNA and afferent-IVNA in rats. Moreover, IV administration of the serotonin 3 receptor antagonist granisetron eliminated the effects of SBC8803 on efferent-GVNA and afferent-IVNA. These findings suggest that heat-killed SBC8803 enhances appetite by elevating digestion and absorption abilities via changes in autonomic neurotransmission that might be mediated by the serotonin 3 receptor. [Copyright &y& Elsevier]

Abstract: Circadian clock genes are involved in mammalian reproductive physiology. We examined the expression of circadian clock genes such as Per2, Bmal1, Clock and Cry in granulosa cells from follicles at different developmental stages and in follicle-stimulating hormone (FSH)-treated granulosa cells. The expression of Clock mRNA was significantly increased in granulosa cells from pre-selection follicles as compared to that in the granulosa cells from other follicles. In contrast, the expression of Per2 mRNA was significantly increased in the granulosa cells from preovulatory follicles as compared to that in the granulosa cells from other follicles. Bmal1 and Cry mRNA expressions did not change across different follicular stages. FSH stimulated the expression of Clock mRNA in the cultured granulosa cells. The expression of Bmal1, Cry and Per2 mRNAs was not affected by FSH. Our results suggested that Clock may be associated with follicular development in the bovine ovary. In summary, our results suggested that circadian clock genes play an important role during follicular development in the bovine ovary. [Copyright &y& Elsevier]

Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions. [Copyright &y& Elsevier]

Atherosclerosis is considered the major cause of cardiovascular and cerebrovascular diseases, which are the leading causes of death worldwide. Excessive nitric oxide production and inflammation result in dysfunctional vascular endothelial cells, which are critically involved in the initiation and progression of atherosclerosis. The present study aimed to identify a bioactive compound from Jerusalem artichoke leaves with anti-inflammatory activity that might prevent atherosclerosis. We isolated bioactive heliangin that inhibited NO production in LPS-induced macrophage-like RAW 264.7 cells. Heliangin suppressed ICAM-1, VCAM-1, E-selectin, and MCP-1 expression, as well as NF-κB and IκBα phosphorylation, in vascular endothelial cells stimulated with TNF-α. These results suggested that heliangin suppresses inflammation by inhibiting excessive NO production in macrophages and the expression of the factors leading to the development of atherosclerosis via the NF-κB signaling pathway in vascular endothelial cells. Therefore, heliangin in Jerusalem artichoke leaves could function in the prevention of atherosclerosis that is associated with heart attacks and strokes. [ABSTRACT FROM AUTHOR]

The criptochrome genes (Cry1 and Cry2) are involved in the molecular mechanism that controls the circadian clock, and mice lacking these genes (Cry1-/-/Cry2-/-) are behaviorally arrhythmic. It has been speculated that the circadian clock modulates the characteristics of thermoregulation, resulting in body temperature (Tb) rhythm. However, there is no direct evidence proving this speculation. We show here that Tb and heat production in Cry1-/-/Cry2-/- mice are arrhythmic under constant darkness. In contrast, both rhythms occur under a light-dark cycle and/or periodical food restriction linked with spontaneous activity and/or eating, although they are not robust as those in wild-type mice. The relationship between heat production and Tb in Cry1-/-/Cry2-/- mice is linear and identical under any conditions, indicating that their Tb rhythm is determined by heat production rhythm associated with activity and eating. However, Tb in wild-type mice is maintained at a relatively higher level in the active phase than the inactive phase regardless of the heat production level. These results indicate that the thermoregulatory responses are modulated according to the circadian phase, and the Cry genes are involved in this mechanism. [ABSTRACT FROM AUTHOR]

To investigate the molecular mechanisms in photo-entrainment of circadian rhythms, expression patterns of mouse period1 (mPer1) and mPer2 mRNAs were examined in the suprachiasmatic nucleus (SCN) and skin under a complete light–dark (LD) cycle and a corresponding skeleton photoperiod (SPP). Under the SPP, the robust daily expressions of mPer1 and mPer2 were maintained in the SCN and in the skin. A slower rise time for mPer1 mRNA expression was observed in the SPP compared with that in the LD condition both in the SCN and in the skin, although the expression pattern of mPer2 was not significantly different between the two lighting conditions. The results suggest that the expressions of mPer1 and mPer2 mRNAs are differentially regulated not only in the SCN but also in peripheral tissues. Furthermore, we propose that the persistent expression of mPer2 mRNA is required for the entrainment to the SPP. [Copyright &y& Elsevier]

Our previous study suggested that the interleukin (IL)-6 and IL-10 could serve as good biomarkers for chronic inflammatory disease. We previously established an IL-6 and IL-10 reporters assay that could examine reporter activity along with the reference gene in LPS-induced RAW 264.7 cells. In this study, we described new and stable RAW 264.7 derived dual-color IL-6/gapdh and IL-10/gapdh reporters. This assay allowed us to easily determine relative IL-6 and IL-10 levels with 96-well plate within one step. We evaluated the relative IL-6 and IL-10 levels in the LPS-induced stable cells testing 52 natural products by real-time bioluminescence monitoring and time-point determination using a microplate luminometer. The relative IL-6 and IL-6/IL-10 values decreased by the crude ethanol extracts from nutmeg and by 1′S-1′-acetoxychavicol from greater galangal using real-time bioluminescence monitoring. At the same time, the relative IL-10 was induced. The relative IL-6 and IL-6/IL-10 decreased by crude ethanol extracts from nutmeg and 1′S-1′-acetoxychavicol acetate at 6 h. Only crude ethanol extract from nutmeg induced IL-10 at 6 h. We suggested that the use of these stable cells by real-time monitoring could serve as a screening assay for anti-inflammatory activity and may be used to discover new drugs against chronic inflammatory disease. [ABSTRACT FROM AUTHOR]