サイトカインを指標としたマウスアレルギー性接触皮膚炎の解析と化学物質スクリーニングへの応用に関する研究

皮膚は環境からの種々の物質に曝され第一線の反応をする組織である。皮膚炎や微生物感染、皮膚腫瘍など、炎症や免疫に関与する免疫担当細胞だけでなく、皮膚を構成する細胞自体も種々のサイトカインを産生分泌することが確かめられている。サイトカインは生体の細胞により産生、分泌され細胞間の情報伝達を司りつつ、免疫応答、炎症、細胞増殖、分化、造血など、さまざまな生体の機構を制御している。　一方、皮膚科領域の温疹・皮膚炎のうち、アレルギー性接触皮膚炎(以下、ACD)は、単純化学物質により惹起される最も一般的な遅延型アレルギーの一つである。また、ACDは動物を用いて比較的容易に誘導することが可能なことより、免疫学的あるいは分子生物学的解析手法等を用い、その発症機構の解析が行われている。しかし、ACD解析の多くは惹起誘発時における(皮膚所属)リンパ節内あるいは脾細胞内における解析が主体であり、皮膚反応局所についての解析は少ない。　この研究の目的は化学物質による遅延型アレルギーの発症機構解明に向けて、マウスACDの皮膚反応局所におけるサイトカインプロフィールを解析し、その特徴を明らかにするとともに、サイトカインを指標とした化学物質による感作性等に対する評価系として有用性について検討することである。　はじめに、マウスでの実験的ACDの惹起誘発期の皮膚反応局所におけるT細胞の関与と皮膚反応局所に発現するサイトカイン産生パターンについてRT-PCR法にて解析した。DNFB(2,4-dinitro-fluorobenzene)を用いたACDにおいて惹起誘発期の耳介腫脹反応は惹起誘発24時間後に最大となった。病理学的所見では、皮膚病変形成に伴ってリンパ球の浸潤が顕著となり、T細胞群のうちCD4＾+ T細胞及びCD8＾+ T細胞の関与がACDの反応成立に必要であることが示唆された。また、これらの細胞群より産生されるサイトカイン類については、IL-4を中心としたTh2型サイトカインに比較してIFN-γを中心としたTh1型サイトカインのmRNAの発現が顕著であった。さらにIFN-γ促進因子であるIL-12の発現や抗IFN-γ抗体処理マウスを用いた解析結果から、ACD反応成立には、Th1型サイトカインのうち特にIFN-γの皮膚組織内におけるmRNA発現の重要性が示唆された。　第2として、ACDの惹起誘発期と同様に感作誘導期におけるT細胞とサイトカイン産生パターンについて解析した。すなわち、感作誘導の有無による反応について比較解析した。その結果、CD4＾+ T細胞並びにCD8＾+ T細胞のmRNA発現が、感作誘導期及び惹起誘発期のいずれにおいても認められた。また、この結果は免疫組織学的解析データとも一致しており、両細胞がACDの感作誘導期及び惹起誘発期の両過程に深く関与していることが示された。次に、これらの細胞から産生されるサイトカインについて解析した。その結果、ACDの反応成立過程(惹起誘発後)の皮膚反応局所において、主に活性化CD4＾+ T細胞から分泌されると考えられるサイトカイン(特にIFN-γ、IL-2等のTh1型サイトカイン)が、ACDの感作誘導期の時点ですでに皮膚反応局所に発現することを認めた。　第3として、ACDと刺激性接触皮膚炎と皮膚反応局所に発現するサイトカイン産生パターンにより比較解析した。各炎症反応局所では、感作性物質では特に、IL-2及びIFN-γのmRNAの顕著な発現を認め、Th1リンパ球を主体とした免疫応答を誘導するのに対し、刺激性物質ではIL-2及びIFN-γなどのmRNA発現抑制と、TNF-αなどの産生により好中球を主体とした刺激性反応が示された。本解析結果から、感作性物質と刺激性物質とを識別する際に、皮膚反応局所におけるTh1型サイトカイン(IL-2やIFN-γ等)mRNA発現の有無の重要性が示唆された。　第4として、フェノール系化合物を被験材料として、サイトカインmRNA発現を指標としたACDの感作性評価への可能性について検討した。その結果、本解析により、ACDによる耳介腫脹率が強度な被験物質に限らず、軽度な被験物質においても効率良く、Th1型サイトカイン(Th1細胞)のmRNAが検出されており感作性評価法としての可能性が示唆された。　第5として、光感作誘発性物質と紫外線(特にUVA)とに起因する光アレルギー性接触皮膚炎(PACD)の皮膚反応局所におけるサイトカインmRNAの発現パターンについて検討した。PACDの惹起誘発過程においては、早期よりIL-4、IL-6及びIL-10のTh2型サイトカインmRNAの顕著な発現が認められるのに対し、IL-2及びIFN-γのTh1型サイトカインmRNAはACDと比較してその発現は極めて軽度であった。Th1/Th2細胞のバランスから病態を把握をすると、ACDは促進回路を担うTh1細胞が誘導され易い反応系(Thl易誘導性)であるのに対し、PACDは、抑制回路を担うTh2細胞が誘導され易い反応系(Th2易誘導性)であることが示唆された。　本研究では示されたサイトカインを指標としたマウスACDの反応局所における解析はACDの免疫応答また、皮膚と免疫担当細胞(リンパ節、脾細胞等)との関連性等を追求する際に有効な解析法になり得ると考える。また、今後、未知の化学物質の刺激性、感作性、光感作性等の検出に向けて、簡便にして高感度なスクリーニング法(化学物質によるアレルギー原性の評価系)として発展することも期待される。Recent advance in researches on cytokines enabled us to understand the mechanisms of inflammatory skin diseases. Keratinocytes can synthesize and release cytokines in their microenviroment in response to external stimuli such as ultraviolet light, chemical substances, and bacterial toxins. Cytokines released from keratinocytes affect functions of other types of cells in the skin such as mast cells, endothelial cells, and fibroblasts which can also release cytokines to contribute to various kinds of inflammatory reactions in the skin. Today, cytokine action has been extensively studied to elucidate the precise mechanism of skin disease. Contact dermatitis is one of the most common skin disease. Recently, many analyses on mechanism of contact dermatitis have been carried out on cytokines. But analysis of cytokine production patterns is performed on almost lymph node cells or spleen cells, but not on skin tissues. Therefore, there still remain several points to be clarified for understanding of the precise mechanism of contact dermatitis. So, in this study, the author analyzed cytokine mRNA expression in the local dermal tissues in chemicalinduced contact dermatitis in mice.1. Cytokines and T lymphocytes in the elicitation phase of allergic contact dermatitis The studies on T lymphocytes involvement and kinetics of cytokine mRNA by the revers transcriptase - polymerase chain reaction (RT-PCR) method in the elicitation phase of allergic contact dermatitis were performed using skin tissues collected at regular intervals from BALB/c mice sensitized with 2,4- dinitrofluorobenzene. The results obtained from these studies and inhibition of dermatitis by anti-CD4 and anti-CD8 antibodies showed that participation of T lymphocytes and infiltration of T lymphocytes at local dermal sites were found to play an important role in formation of allergic contact dermatitis. On the basis of these results, cytokines formed at the local site of allergic contact dermatitis were examined at the mRNA level. It was found that IFN-γ, IL-6 and IL-1β were expressed 24 h after the elicitation and then IL-2, IL-4 and TNF-α 48 h after the elicitation. The detection of expression of IL-12(p35) and inhibitory effect of anti-IFN-γ antibody on allergic contact dermatitis suggested that IFN-γ among several Th1 types of cytokines is especially important for the formation of allergic contact dermatitis. Therefore, the author concluded that Th1 type cytokines , especially of IFN-γ , are essential for allergic contact dermatitis and the evaluation of these cytokines mRNA expression in the local skin tissues will be convenient method for screening chemicals for the potencial as contact allergens.2. Cytokines and T lymphocytes in the induction phase of allergic contact dermatitis The author examined the mechanism of onset of allergic contact dermatitis induced by several kinds of chemicals in BALB/c mice by focusing on cytokine mRNA expression. The involvement of T lymphocytes in the formation of allergic contact dermatitis was studied by the RT-PCR method using primers for CD4 and CD8 surface antigen, suggesting that both CD4^+ and CD8^+ cells participate in the formation of allergic contact dermatitis. On the basis of these results we analyzed cytokines produced in the reaction site (skin tissues) at the mRNA level with or without the pre-sensitization (induction) of two strong contactants (2,4-dinitrofluorobenzene , picryl chloride). Consequently, there was tendency that the expression of mRNA of Th1 types of cytokines was commonly observed around 24 h after the challenging irrespective of the pre-sensitization (induction). These data suggested that the expression of Th1 type cytokines in the local skin tissue after challenging might be indicative of the potencial of the chemicals as contact allergens.3. Analysis of cytokine profiles in allergic and irritant contact dermatitis Both allergic contact dermatitis and irritant contact dermatitis that are caused by many environmental factors are common skin diseases. But differentiation between allergic contact dermatitis and irritant contact dermatitis is very difficult in vivo. So, we compared the expression of cytokine mRNA in the dermal tissue in murine experimental models of allergic contact dermatitis (2,4-dinitrofluorobenzene, picryl chloride, oxazolone) and irritant contact dermatitis (sodium lauryl sulfate, benzalkonium chloride, isopropyl myristate). As the results, different expression of cytokine mRNA in the ear site of each dermatitis was observed at 24 h after priming. The expression of mRNA of IL-2 and IFN-γ was observed at the local site of allergic dermatitis, but not in irritant dermatitis, suggesting that marked expression of Th1 type cytokines as IL-2 or IFN-γ in the reaction sites is very important to differentiate allergic contact dermatitis from irritant contact dermatitis.4. Evaluation of sensitizing effects of phenolic compounds by analysis of cytokine mRNA expression In this chapter, the author evaluated sensitizing effect by mouse ear swelling test with phenolic compounds. Skin specimens were collected from auricles of BALB/c mice on 24 h after elicitation of allergic contact dermatitis with six phenolic compounds. The expression of cytokine mRNA in the skin specimens was examined by the RT-PCR method. Phenolic compounds which caused varied intensity of ear swelling response induced expression of Th1 type cytokines (IL-2 and IFN-γ) on 24 h after elicitation. It was also found that among several phenolic compounds, dimerization of these compounds from monomer decreased sensitizing effect. These data suggested that analysis of cytokine mRNA expression in local skin tissue is highly sensitive method for screening the chemicals for their potential as contact allergens.5. Cytokine mRNA expression in challenged skin of murine contact photosensitivity The dynamics of expression of cytokine mRNA was examined by the RT-PCR method in the elicitation phase of photoallergic contact dermatitis. Skin specimens were collected from auricles of BALB/c mice that had been ultraviolet A (UVA)-photo-challenged with 3,3\u27,4\u27,5-tetrachlorosalicylanilide during 0 to 48 h after elicitation, along with samples from mice ordinarily challenged with 2,4-dinitrofluorobenzene. The levels of mRNA for IL-4, IL-6 and IL-10 were markedly elevated in the skin sites challenged with 3,3\u27,4\u27,5-tetrachlorosalicylanilide plus UVA during 0 to 48 h after elicitation. On the other hand, IL-2 and IFN-γ mRNA expression was characteristically obseved in allergic contact dermatitis induced by 2,4-dinitrofluorobenzene with modest expression of Th2 type cytokines. These data suggested that Th2 type cytokines are more readily inducible in photoallergic contact dermatitis by 3,3\u27,4\u27,5-tetrachlorosalicylanilide as compared with allergic contact dermatitis by 2,4-dinitrofluorobenzene. Finally, this study suggested that analysis of cytokine mRNA expression in local skin tissue is a convenient and highly sensitive method for screening varied new chemicals for their potentials of irritation, sensitization, and photosensitization.博士(獣医学)麻布大

Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

Glucokinase-mediated glucose signaling induces insulin secretion, proliferation, and apoptosis in pancreatic β-cells. However, the precise molecular mechanisms underlying these processes are not clearly understood. Here, we demonstrated that glucokinase activation using a glucokinase activator (GKA) significantly upregulated the expression of Fibulin-5 (Fbln5), a matricellular protein involved in matrix-cell signaling, in isolated mouse islets. The islet Fbln5 expression was induced by ambient glucose in a time- and dose-dependent manner and further enhanced by high-fat diet or the deletion of insulin receptor substrate 2 (IRS-2), whereas the GKA-induced increase in Fbln5 expression was diminished in Irs-2-deficient islets. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase inhibitor, KATP channel opener, Ca2+ channel blocker and calcineurin inhibitor, while it was augmented by harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1 A inhibitor. Although deletion of Fbln5 in mice had no significant effects on the glucose tolerance or β-cell functions, adenovirus-mediated Fbln5 overexpression increased glucose-stimulated insulin secretion in INS-1 rat insulinoma cells. Since the islet Fbln5 expression is regulated through a glucokinase/KATP channel/calcineurin/nuclear factor of activated T cells (NFAT) pathway crucial for the maintenance of β-cell functions, further investigation of Fbln5 functions in the islets is warranted

DPP-4 inhibition improves early mortality, β cell function, and adipose tissue inflammation in db/db mice fed a diet containing sucrose and linoleic acid

Additional file 3: Figure S2. Liver and epididymal fat weights in db/+ mice and db/db mice. The experiments were performed in db/+ or db/db mice fed an SL diet, SO diet, SL containing DPP-4 inhibitor (0.4% des-fluoro-sitagliptin) diet, or SO containing DPP-4 inhibitor diet for 8 weeks. (left) Liver weights as a proportion of body weight (n = 5). (right) Epididymal fat weights as a proportion of body weight (n = 5)

Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non-chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology

Bispectral index-guided propofol sedation during endoscopic ultrasonography

Background/Aims Bispectral index (BIS) monitors process and display electroencephalographic data are used to assess the depth of anesthesia. This study retrospectively evaluated the usefulness of BIS monitoring during endoscopic ultrasonography (EUS). Methods This study included 725 consecutive patients who underwent EUS under sedation with propofol. BIS monitoring was used in 364 patients and was not used in 361. The following parameters were evaluated: (1) median dose of propofol; (2) respiratory and circulatory depression; (3) occurrence of body movements; (4) awakening score >8 at the time; and (5) awakening score 2 hours after leaving the endoscopy room. Results The BIS group received a significantly lower median dose of propofol than the non-BIS group (159.2 mg vs. 167.5 mg; p=0.015) in all age groups. For patients aged ≥75 years, the reduction in heart rate was significantly lower in the BIS group than in the non-BIS group (1.2% vs. 9.1%; p=0.023). Moreover, the occurrence of body movements was markedly lower in the BIS group than in the non-BIS group (8.5% vs. 39.4%; p<0.001). Conclusions During EUS examination, BIS monitoring is useful for maintaining a constant depth of anesthesia, especially in patients 75 years of age or older

Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells

The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it

Prevalence and Distribution of Ossified Lesions in the Whole Spine of Patients with Cervical Ossification of the Posterior Longitudinal Ligament A Multicenter Study (JOSL CT study)

Ossification of the posterior longitudinal ligament (OPLL) can cause severe and irreversible paralysis in not only the cervical spine but also the thoracolumbar spine. To date, however, the prevalence and distribution of OPLL in the whole spine has not been precisely evaluated in patients with cervical OPLL. Therefore, we conducted a multi-center study to comprehensively evaluate the prevalence and distribution of OPLL using multi-detector computed tomography (CT) images in the whole spine and to analyze what factors predict the presence of ossified lesions in the thoracolumbar spine in patients who were diagnosed with cervical OPLL by plain X-ray. Three hundred and twenty-two patients with a diagnosis of cervical OPLL underwent CT imaging of the whole spine. The sum of the levels in which OPLL was present in the whole spine was defined as the OP-index and used to evaluate the extent of ossification. The distribution of OPLL in the whole spine was compared between male and female subjects. In addition, a multiple regression model was used to ascertain related factors that affected the OP-index. Among patients with cervical OPLL, women tended to have more ossified lesions in the thoracolumbar spine than did men. A multiple regression model revealed that the OP-index was significantly correlated with the cervical OP-index, sex (female), and body mass index. Furthermore, the prevalence of thoracolumbar OPLL in patients with a cervical OP-index ≥ 10 was 7.8 times greater than that in patients with a cervical OP-index ≤ 5. The results of this study reveal that the extent of OPLL in the whole spine is significantly associated with the extent of cervical OPLL, female sex, and obesity

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements