Site-specific changes in cytokine response to septic dose of lipopolysaccharide in ovariectomized female rats.

PURPOSE: The immune response is altered according to hormonal and metabolic status. Obesity increases the inflammatory and fever response, whereas loss of gonadal steroid decreases behavioral response to immune stress. However, the immune systems of ovariectomized animals exhibiting obesity and gonadal steroid deficiency, particularly under septic conditions, have not been fully examined. In the present study, we evaluated the ovariectomy-induced changes of central and peripheral immune responses to life-threatening septic stimulus. METHODS AND RESULTS: Ovariectomized rats showed heavier body weight and lighter uterine weight when compared with gonadally intact rats. Fever response to septic dose of lipopolysaccharide (LPS) in ovariectomized rats was less evident when compared with that in gonadally intact rats. In addition, under LPS-injected septic conditions, hypothalamic gene levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and serum protein levels of IL-1ß and TNF-α in ovariectomized rats were lower than those in gonadally intact rats. On the other hand, IL-6 levels in visceral fat under septic conditions were higher in ovariectomized rats than in gonadally intact rats. CONCLUSIONS: These findings indicate that ovariectomy-induced site-specific changes in cytokine response under septic conditions. As hypothalamic, but not peripheral, pro-inflammatory cytokines are directly involved in the fever response, the attenuation of fever response observed in ovariectomized rats may be caused by a reduction in central cytokine responses.

Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats.

Recent studies have suggested that intrauterine undernutrition is closely associated with the pathogenesis of diseases after birth. Perinatal undernutrition is known to disturb the development of reproductive function and delay the onset of puberty in some species. Using a rat model, we determined the effects of prenatal undernutrition on the development of the hypothalamic kisspeptin system and evaluated whether the alteration of the kisspeptin system contributes to the delayed onset of puberty induced by prenatal undernutrition. We also evaluated the effects of prenatal undernutrition on the developmental changes in serum leptin levels because leptin was a putative positive regulator of the hypothalamic kisspeptin system. We compared the timing of vaginal opening (VO) and the developmental changes in body weight, hypothalamic Kiss1 mRNA levels, and serum leptin concentrations between offspring with prenatal undernutrition (UN offspring) and normal nutrition (NN offspring). After birth, the UN offspring showed rapid growth and had caught up to body weight of the NN offspring by postnatal day 12. After postnatal day 16, the UN offspring showed significantly lower Kiss1 mRNA levels than the NN offspring, despite their significantly higher serum leptin levels (at days 20 and 28). The timing of VO in the UN offspring was delayed compared with that in the NN offspring, and chronic central injection of kisspeptin normalized the timing of VO in the UN offspring. These results suggest that decreased hypothalamic kisspeptin action contributes to the delayed onset of puberty in prenatally undernourished female rats. Increased leptin resistance in the kisspeptin system might be involved in these alterations.

Decreased expression of kisspeptin mediates acute immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat.

Kisspeptin and its corresponding receptor, the G protein-coupled receptor 54, play an important role in reproductive systems. It has been suggested that reproductive disorders in metabolically disrupted animals are caused by the alteration of hypothalamic KiSS-1 systems. Immune/inflammatory challenge is also known to disrupt reproductive function. However, the effects of immune/inflammatory challenge on KiSS-1 systems have not been investigated. In this study, we showed that lipopolysaccharide (LPS) injection decreased hypothalamic KiSS-1 mRNA expression as well as plasma LH levels in ovariectomized rats. Indomethacin completely blocked the suppressive effects of LPS on LH secretion and KiSS-1 mRNA level. Furthermore, we showed that i.v. injection of kisspeptin increased plasma LH levels in LPS-administrated rats to the same degree as in saline-injected rats. These results suggest that KiSS-1 systems are sensitive to immune/inflammatory challenge conditions and transmit these signals into the central reproductive system.

The type 2 corticotrophin-releasing hormone receptor mediates orexin A-induced luteinising hormone suppression in ovariectomised rats.

Orexins are thought to be regulatory factors of the arousal and sleep patterns. They also affect immune, feeding, autonomic and neuroendocrine systems. We have previously shown that intracerebroventricular (i.c.v.) injection of orexin decreases pulsatile luteinising hormone (LH) secretion in ovariectomised (OVX) rats. However, the details of this mechanism have not been fully examined. Intracerebroventricular injection of orexin A also stimulates corticotrophin-releasing hormone (CRH) systems, which have been implicated in the stress-induced suppression of reproductive function. In the present study, we investigated the role of CRH systems in orexin-induced LH suppression. OVX rats were implanted with i.c.v. and intravenous (i.v.) cannulae. After i.c.v. injection of orexin and/or CRH receptor antagonists, blood samples were collected through the i.v. cannula at 6-min intervals for 120 min for LH measurement. Intracerebroventricular injection of orexin A or B (3 nmol/2.5 microl) suppressed pulsatile LH secretion. Coadministration of orexin A and alpha-helical corticotrophic-releasing factor (CRF), a nonselective CRH receptor antagonist (13 nmol/2.5 microl), or astressin(2)B, a selective type2 (CRH-R2) CRH receptor antagonist (28 nmol/2.5 microl), partly restored pulsatile LH secretion. Orexin B-induced LH suppression was not restored by alpha-helical CRF. In addition, i.c.v. injection of orexin A increased CRH and urocortin II (UcnII), but not Ucn mRNA levels, in the hypothalamus. These findings suggest that CRH-R2 mediates orexin A-induced LH suppression and it is possible that CRH and UcnII in the hypothalamus are involved in this pathway.

Mirthful laughter differentially affects serum pro- and anti-inflammatory cytokine levels depending on the level of disease activity in patients with rheumatoid arthritis.

OBJECTIVES: To examine the effect of mirthful laughter in rheumatoid arthritis (RA), we evaluated the levels of serum cytokines before and after patients experienced mirthful laughter. METHODS: Forty-one patients with RA and 23 healthy subjects were enrolled. They listened to 'Rakugo', a traditional Japanese comic story, to induce mirthful laughter. We measured serum IL-6, IL-1beta, TNF-alpha, IL-4 and IL-1 receptor antagonist (IL-1Ra) concentrations before and after patients listened to the story. The RA subjects were divided into two groups. One was designated the 'difficult-to-control RA' group (CRP > or =1.0 mg/dl); The other group was regarded as the 'easily controlled RA' group (CRP <1.0 mg/dl). RESULTS: The basal levels of serum IL-6 and TNF-alpha in the RA patients were significantly higher than those in the healthy group. After experiencing mirthful laughter, the levels of serum IL-6 decreased significantly in the RA group but not in the healthy subjects. Interestingly, the level of serum TNF-alpha decreased only in the easily controlled RA group. Serum IL-4 concentration in the RA group was significantly higher than that in healthy subjects before the story. After the story, the level of serum IL-4 significantly decreased in the RA group, especially in the difficult-to-control RA group. In contrast, serum IL-1Ra concentration was statistically higher in the RA group than that in healthy subjects before the story, and a further increase was observed after the story, especially in the easily controlled RA group. CONCLUSIONS: Our findings suggest that mirthful laughter affects the levels of serum pro- and anti-inflammatory cytokines differentially, depending on the RA disease activity.

Serum osteoprotegerin/osteoclastogenesis-inhibitory factor during pregnancy and lactation and the relationship with calcium-regulating hormones and bone turnover markers.

Pregnancy and lactation induce dynamic changes in maternal bone and calcium metabolism. A novel cytokine termed osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF) was recently isolated; this cytokine inhibits osteoclast maturation. To define the effects of pregnancy and lactation on circulating OPG/OCIF in mothers, we studied the changes in the levels of OPG/ OCIF as well as those of calcium-regulating hormones and biochemical markers of bone turnover in the maternal circulation during pregnancy (at 8-11 weeks, at 22-30 weeks, at 35-36 weeks and immediately before delivery) and lactation (at 4 days and at 1 month postpartum). Serum intact parathyroid hormone levels did not change and were almost within the normal range in this period. In contrast, serum 1,25-dihydroxyvitamin D levels increased with gestational age and were above the normal range during pregnancy. After delivery, they fell rapidly and significantly (P<0.01) to the normal range. The levels of serum bone-specific alkaline phosphatase, one of the markers of bone formation, increased with gestational age. After delivery, these levels were further increased at 1 month postpartum. The levels at 1 month postpartum were significantly higher than those at 8-11 and 22-30 weeks of pregnancy (P<0.01 and P<0.05 respectively). The levels of serum C-terminal telopeptides of type I collagen, one of the markers of bone resorption, did not change during pregnancy. After delivery, they rapidly and significantly (P<0.01) rose at 4 days postpartum, and had then fallen by 1 month postpartum. Circulating OPG/OCIF levels gradually increased with gestational age and significantly (P<0.01) increased immediately before delivery to 1.40+/-0.53 ng/ml (means+/-S.D.) compared with those in the non-pregnant, non-lactating controls (0.58+/-0.11 ng/ml). After delivery, they fell rapidly to 0.87+/-0.27 ng/ml at 4 days postpartum and had fallen further by 1 month postpartum. These results suggest that the fall in OPG/OCIF levels may be partially connected with the marked acceleration of bone resorption after delivery.

Diarylheptanoids suppress expression of leukocyte adhesion molecules on human vascular endothelial cells.

Diarylheptanoids possess potent anti-inflammatory properties. However, the mechanism of their action is not fully understood. In this study, we found that three diarylheptanoids, 1-(3, 5-dimethoxy-4-hydroxyphenyl)-7-phenylhept-1-en-3-one (YPE-01), yakuchinone B and demethyl-yakuchinone B, reduced the adhesion of both human monocytic cell line U937 and human eosinophilic cell line EoL-1 cells to tumor necrosis factor-alpha (TNF-alpha)-treated human umbilical vein endothelial cells. In addition, they suppressed interleukin-1beta- or TNF-alpha-induced expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on the surface of the endothelial cells. Since YPE-01 reduced both VCAM-1 and ICAM-1 mRNA induction in TNF-alpha-stimulated endothelial cells, diarylheptanoids appeared to suppress adhesion molecule expression at the transcriptional level. Furthermore, YPE-01 suppressed both VCAM-1 and ICAM-1 mRNA induction as well as edema in 12-O-tetradecanoylphorbol 13-acetate (TPA)-inflamed mice ears in vivo. These results suggest that the anti-inflammatory action of diarylheptanoids is, at least in part, due to their suppressive effect on the surface expression of inducible adhesion molecules in endothelial cells, and subsequent leukocyte adhesion.

Disaccharide esters screened for inhibition of tumor necrosis factor-alpha release are new anti-cancer agents.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine playing a part in various pathological states. Non-toxic inhibitors of TNF-alpha release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF-alpha release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF-alpha release with synthetic disaccharide esters, such as 6,6'-di-O-alkanoyl-alpha, alpha-trehaloses (6,6'-diester-trehaloses), 4,4'-di-O-alkanoyl-alpha, alpha-trehaloses (4,4'-diester-trehaloses) and 6,6'-diamino-6,6'-dideoxy-N,N'-dialkanoyl-alpha, alpha-trehaloses (6,6'-diamide-trehaloses) bearing fatty acids of various chain lengths, and n-dodecyl-beta-D-maltoside as a disaccharide monoester. 6,6'-Diester-trehaloses and 4,4'-diester-trehaloses of C8 to C12 fatty acids, 6,6'-diamide-trehaloses of C8 to C14 fatty acids, and n-dodecyl-beta-D-maltoside all inhibited TNF-alpha release in a dose-dependent manner. The IC50 values are 7.4-14.8 microM for 6,6'-diester-trehaloses (C8 to C12), 14.6-21.6 microM 4,4'-diester-trehaloses (C8 to C12), 2.9-15.0 microM for 6,6'-diamide-trehaloses (C8 to C14) and 23 microM for dodecyl-beta-D-maltoside. Both 6,6'-di-O-octanoyl-alpha, alpha-trehalose (C8, designated as SS555) and n-dodecyl-beta-D-maltoside (C12) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor-bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n-dodecyl-beta-D-maltoside. Moreover, SS555 inhibited TNF-alpha gene expression mediated through inhibition of AP-1 activation, but not NF-kappa B activation. This paper reports that diester-trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n-dodecyl-beta-D-maltoside appear to be potential cancer-preventive agents of a new type.

HLA and HPA typing in idiopathic thrombocytopenic purpura patients treated with Kami-kihi-to.

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.

Isolation and identification of two new diterpene glycosides from Nicotiana tabacum.

Two new diterpene glycosides containing 20-hydroxygeranyllinalool were isolated and identified from Nicotiana tabacum. These compounds consisted of five molecules of glucose and/or rhamnose. The locations of the aglycone and glycosides in the molecules were determined by 2D-NMR with the HMBC technique. The structures were (6E,10E,14Z)-20-hydroxygeranyllinalyl-3-O- [alpha-L-rhamnopyranosyl (1 --> 4)]-beta-D-glucopyranoside-20-O- [beta-D-glucopyranosyl (1 --> 2)]-[alpha-L-rhamnopyranosyl (1 --> 6)]-beta-D- glucopyranoside and (6E,10E,14Z)-20-hydroxygeranyllinalyl-3-O- [alpha-L-rhamnopyranosyl (1 --> 4)]-beta-D-glycopyranoside-20-O- [alpha-L-rhamnopyranosyl (1 --> 4)]-[alpha-L-rhamnopyranosyl (1 --> 6)]-beta-D- glucopyranoside.

Effects of a new antiarrhythmic drug, SD-3212, on canine ventricular arrhythmia models.

The antiarrhythmic effects of a new antiarrhythmic agent, SD-3212, (-)-(S)-3,4-Dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylene dioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H-1, 4-benzothiazine hydrogen fumarate, were investigated using canine models of ventricular arrhythmias, i.e. spontaneously occurring digitalis-, two-stage coronary ligation- and adrenaline-induced arrhythmias. SD-3212 suppressed adrenaline-induced arrhythmia and showed some antiarrhythmic effect on digitalis- and 48 hr coronary ligation-arrhythmias. These results indicate that SD-3212 has antiarrhythmic effects common among class IV antiarrhythmic drugs and also has additional efficacy common among class I antiarrhythmic drugs, thus when considering the level of experimental arrhythmias it somewhat resembles propafenone. It may therefore become a clinically useful antiarrhythmic drug among typical class I or class IV antiarrhythmic drugs.

Detection and chemical identification of natural bio-antimutagens. A case of the green tea factor.

A bio-antimutagen, isolated from Japanese green tea (leaves of Camellia sinensis), reduced high spontaneous mutations due to altered DNA-polymerase III in a mutator strain of Bacillus subtilis. Chemical studies showed that the factor was epigallo-catechin-gallate (EGCg).

Experimental immunohistochemical studies on the amygdalofugal peptidergic (substance P and somatostatin) fibers in the stria terminalis of the rat.

The amygdalofugal substance P (SP) and somatostatin (SRIF) neuron systems in the stria terminalis (ST) were investigated by means of the indirect immunofluorescence technique of Coons. SP- and SRIF-positive cells were mainly located in the area (Amc) between the central (ac) and medial (am) amygdaloid nuclei. Some extended medially into the am and laterally into the ac. Destruction of the Amc resulted in a marked reduction of SP- and SRIF-positive fibers in the ST. Furthermore, a substantial decrease in SP-positive fibers was seen in the dorsal part of the bed nucleus of the ST (stb), there was a small decrease in the SP-positive fibers in the lateral hypothalamus (LH), a significant decrease in the SRIF-positive fibers in the lateroventral part of the anterior hypothalamic nucleus (lvAH), and a small decrease in the SRIF-positive fibers in the LH. These facts indicate that the origins of a number of SP- and SRIF-positive fibers are the Amc and that the amygdalofugal SP pathway in the ST innervates stb and LH and the amygdalofugal SRIF pathway in the ST projects to lvAH and LH.

Topographic organization of the projection from the forebrain subcortical areas to the hippocampal formation of the rat.

Using the technique of iontophoretic microinjection of horseradish peroxidase, the present study disclosed the complexity and high degree of the topographic organization in the forebrain subcortical afferents to the different regions of rat hippocampus, e.g. diagonal band, posterior (PH), dorsomedial and rostral lateral hypothalamic nuclei chiefly project to the rostrodorsal part (DRA) and caudal gyrus dentatus including CA3, the supramammillary area predominantly to the rostroventral area (VRA), the area lateral to PH to the DRA and VRA, substantia innominata and some thalamic nuclei (n. reuniens, n. lateralis thalami, n. anterior ventralis and n. lateralis thalami pars posterior) to the dorsal subiculum, respectively.