Hirofumi Inagaki

Interleukin-1β, interleukin-6, epidermal growth factor and transforming growth factor-α are elevated in the brain from parkinsonian patients

Interleukin (IL)-1 beta, IL-6, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) were measured for the first time in the brain (caudate nucleus, putamen and cerebral cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme immunoassays. The concentrations of IL-1 beta, IL-6, EGF, and TGF-alpha in the dopaminergic, striatal regions were significantly higher in parkinsonian patients than those in controls, whereas those in the cerebral cortex did not show significant differences between parkinsonian and control subjects. Since these cytokines and growth factors may play important roles as neurotrophic factors in the brain, the present results suggest that they may be produced as compensatory responses in the nigrostriatal dopaminergic regions in Parkinsons disease, and may be related, at least in part, to the process of neurodegeneration in Parkinsons disease.

Interleukin (IL)-1β, IL-2, IL-4, IL-6 and transforming growth factor-α levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease ☆

Interleukin (IL)-1β, IL-2, IL-4, IL-6, epidermal growth factor (EGF), and transforming growth factor (TGF)-α were measured for the first time in ventricular cerebrospinal fluid (VCSF) from control non-parkinsonian patients, patients with juvenile parkinsonism (JP) and patients with Parkinsons disease (PD) by highly sensitive sandwich enzyme immunoassays. All cytokines were detectable in VCSF from control and parkinsonian patients, and the concentrations were much higher than those in lumbar CFS. The concentrations of IL-1β, IL-2, IL-4 and TGF-α in VCSF were higher in JP than those in controls (P < 0.05). In contrast, the concentrations of IL-2 and IL-6 in VCSF from patients with PD were higher than those from control patients (P < 0.05). These results agree with our previous reports, in which the cytokine levels were elevated in the striatal dopaminergic region of the brain from patients with PD. Since VCSF is produced in the ventricles, the alteration of cytokines in VCSF may reflect the changes of cytokines in the brain. Because cytokines play an important role as mitogens and neurotrophic factors in the brain, the increases in cytokines as a compensatory response may occur in the brain of patients of JP or PD during the progress of neurodegeneration. Increase in cytokines may contribute not only as a compensatory response but as a primary initiating trigger for the neurodegeneration.

Interleukin 1β, interleukin 6, β2-microglobulin, and transforming growth factor-α in gingival crevicular fluid from human periodontal disease

Inflammatory mediators are central to the pathogenesis of periodontal diseases and may be used as markers in diagnosis. The aim of this study was to identify and quantify the various growth factors, apoptosis-related modifiers [soluble form of Fas (sFas) and bcl-2] and cytokines in the gingival crevicular fluid (GCF) of patients with different severities of periodontitis as compared with those of controls. GCF samples were taken from patients with periodontal disease and from controls. The concentrations of epidermal growth factor, transforming growth factor (TGF)-alpha, interleukin (IL)-1 beta, IL-6, interferon-gamma, beta 2-microglobulin (beta 2-MG), and apoptosis-related modifiers sFas and bcl-2 in the samples were determined by enzyme-linked immunosorbent assay. TGF-alpha was significantly lower in patients with periodontal disease than in the controls. In contrast, the concentrations of IL-1 beta, IL-6; and beta 2-MG were significantly higher in the group with severe periodontal disease than in the controls. The amount of total protein in the GCF was considerably higher in the disease group than the controls (p < 0.05). TGF-alpha, IL-1 beta, and beta 2-MG concentrations were associated (Spearman rank correlation, r < 0.05 for all) with clinical measures of disease severity (pocket depth) and inflammation (bleeding when probed). Apoptosis-related modifiers (sFas and bcl-2) could not be detected in any samples. These results suggest that the growth factor TGF-alpha and certain cytokines are associated with the presence of periodontal disease.

Forest bathing enhances human natural killer activity and expression of anti-cancer proteins.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37–55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50% increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

Method for the analysis of the methylphosphonic acid metabolites of sarin and its ethanol-substituted analogue in urine as applied to the victims of the Tokyo sarin disaster

An analysis method for the methylphosphonic acid metabolites of sarin in urine using trimethylsilyl derivatization and flame photometric detection is described in this report. Authentic reference standards of isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) as well as methylphosphonic acid were employed to estimate the concentration in human urine. A sample pretreatment procedure was developed for urine using a column of cation-loaded ion-exchange resins (Ag+ -, Ba2+ - or H+ -Dowex) and adjusting the pH of the eluate from the column to 3.75-3.85 improved recovery of the target compounds. The eluate was evaporated to dryness under vacuum prior to trimethylsilylation, to remove water and any hydroxy- or amino-carrying volatile substances. The sarin metabolites, because of their low volatility, were concentrated and could be derivatized for analysis. The use of synthesized authentic sarin and ethylsarin metabolites, i.e., IMPA and EMPA, made it possible to establish the necessary sample pretreatment procedures for derivatization and gas chromatography-flame photometric detection (GC-FPD) analysis. The detection limits were 0.025 ppm both for EMPA and [MPA, and 0.625 microM for MPA, respectively. This method can be useful for estimating the exposure level to sarin by assaying the metabolites in urine and it is applicable to a large numbers of samples.

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

BackgroundFourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.MethodsSeven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.ResultsBleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β.ConclusionThese findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

Effect of phytoncide from trees on human natural killer cell function.

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37–60 years, who stayed at an urban hotel for 3 nights from 7.00p.m. to 8.00a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as α-pinene and β-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

Early intervention with fluoxetine reverses abnormalities in the serotonergic system and behavior of rats exposed prenatally to dexamethasone.

Many psychiatric disorders emerge after adolescence. Among a variety of predisposing factors, prenatal stress has been thought to cause the symptoms of anxiety disorders. We recently reported that prenatal dexamethasone (DEX) exposure, which mimics some aspects of prenatal stress, induced anxiety-related behaviors in male offspring when they reached adulthood. Before the emergence of behavioral changes, abnormalities occurred in the hypothalamic-pituitary-adrenal axis during postnatal development. In the present study, we found abnormalities in serotonin (5-HT) signaling, including decreased expression of 5-HT(1A) receptor (5-HT(1A)-R) mRNA in the medial prefrontal cortex (mPFC) and 5-HT content in the hippocampus at postnatal week (PW) 4. These results support using early therapeutic interventions with serotonergic drugs to prevent late-emerging anxiety symptoms. To test this hypothesis, we treated rat pups born to DEX-administered mothers with fluoxetine (FLX), a selective serotonin reuptake inhibitor commonly used as an anti-anxiety medication, via breast milk from postnatal day (PD) 2-21. Anxiety-related behaviors examined at PW11-13 were not observed in the prenatally DEX-exposed offspring that were treated with FLX. Likewise, FLX increased 5-HT concentrations in the mPFC and ventral hippocampus at PW3 and normalized 5-HT(1A)-R mRNA concentrations in the mPFC at PW4. The decrease in brain-derived neurotrophic factor (BDNF) protein in the mPFC and dorsal hippocampus was also restored at PW4. Furthermore, administration of the 5-HT(1A)-R full agonist (R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin from PD2 to 21 also prevented the emergence of behavioral abnormalities in the prenatally DEX-exposed offspring, implicating the involvement of 5-HT(1A)-Rs in the neonatal FLX effect. Collectively, an early pharmacological intervention to normalize serotonergic transmission effectively suppressed the emergence of symptoms induced by prenatal DEX exposure in rats.

Impact of quadruple regimen of clarithromycin added to metronidazole-containing triple therapy against Helicobacter pylori infection following clarithromycin-containing triple-therapy failure.

Background: The establishment of an optimal second‐line regimen for Helicobacter pylori infection is required. Although quadruple therapy should overcome resistance to either clarithromycin or metronidazole, the effects of a quadruple regimen in second‐line therapy are unknown. This study aims to evaluate the efficacy of triple therapy composed of proton pump inhibitor/amoxicillin plus metronidazole with the combined additive effects of clarithromycin as a second‐line quadruple therapy against H. pylori infection.

Acute and subchronic immunotoxicity of p-chloronitrobenzene in mice. I. Effect on natural killer, cytotoxic T-lymphocyte activities and mitogen-stimulated lymphocyte proliferation

We evaluated the immunotoxicity of p-chloronitrobenzene (p-CNB) after intraperitoneal (i.p.) injection of p-CNB in BDF1 mice; single i.p. injection of 300 mg/kg (acute experiments), or 30 mg/kg three times a week for 4 weeks (subchronic experiments). The following items were investigated: number of splenocytes, natural killer (NK) activity, cytotoxic T-lymphocyte (CTL) activity and LPS-stimulated lymphocyte proliferation using splenocytes, hemoglobin (Hb) concentration in peripheral blood and body weight. NK activity in exposed mice significantly decreased compared to control in both acute and subchronic experiments. CTL activity in acute exposed mice showed a significant decrease on the 3rd day only after injection, and significant decrease at 3 and 4 weeks in subchronic exposed mice compared to controls. Comparing the effect of p-CNB on NK activity with that of CTL for both the acute and subchronic exposures, NK activity was more inhibited by p-CNB than CTL activity in the acute stage, whereas both the NK and CTL activities were inhibited by p-CNB in the subchronic stage. There was an indication that p-CNB also inhibited LPS- stimulated B-lymphocyte proliferation. On the other hand, Hb concentration did not show significant difference between the exposed and control mice in both acute and subchronic experiments. Body weight in subchronically exposed mice was significantly lower than the control from day 19. The above evidence indicated that p-CNB has an inherent immunotoxic effect on mice.