Yutaka Kimura

Hgs (Hrs), a FYVE Domain Protein, Is Involved in Smad Signaling through Cooperation with SARA

ABSTRACT Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor β (TGF-β) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-β. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

Loss of Hippocampal CA3 Pyramidal Neurons in Mice Lacking STAM1

ABSTRACT STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1−/− mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1−/− mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1−/− mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1−/− mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.

An in vitro Test to Screen Skin Sensitizers Using a Stable THP-1-derived IL-8 Reporter Cell Line, THP-G8

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Optimization of the IL-8 Luc assay as an in vitro test for skin sensitization

We previously reported a dataset of the IL-8 Luc assay covering reference chemicals published by ECVAM, in which the effects of chemicals on IL-8 promoter activity were evaluated by an IL-8 reporter cell line, THP-G8 cells. To clarify its performance, we created another dataset of 88 sensitizers and 34 non-sensitizers. Simultaneously, to improve its performance, we changed the incubation time from 5 h to 16 h, deleted the criterion regarding the effects of N-acetylcysteine, and set an exclusion criterion for detergents. These modifications significantly improved its performance. In addition, we examined the following three criteria to judge chemicals as sensitizers: Criterion 1: Fold induction of SLO luciferase activity (FlnSLO-LA)⩾1.4, Criterion 2: the lower limit of the 95% confidence interval of FInSLO-LA⩾1.0, Criterion 3: the intersection of criteria 1 and 2. Among them, Criterion 1 produced the best performance, demonstrating that the accuracy, sensitivity and specificity were 81%, 79%, and 90%, respectively. In addition, we found that the IL-8 Luc assay solubilizing chemicals with X-VIVO substantially improved its performance. Finally, the IL-8 Luc assay combined with DPRA and DEREK could improve substantially its performance. These data suggest that the IL-8 Luc assay is a promising test method to screen skin sensitizers.

Delayed development of foreign body granuloma from an implanted permanent cardiac pacemaker.

We report a case of a late‐developing cutaneous mass on the chest wall that proved to be non‐specific mixed cell granuloma adjacent to the lead‐electrode parts of a permanent cardiac‐pacemaker that had been implanted in the left chest of an 81‐year‐old man 6 years previously. The lesion was treated by cardiac surgeons, whose management consisted of cutting only the intradermal part of the lead‐electrodes followed by tissue curettage, leaving the other portion embedded in the subendocardium because its removal could cause serious complications. Dermatologists should be alert to such late complications of embedded permanent pacemakers. Their removal requires close cooperation with cardiac surgeons to avoid unexpected complications.

Decreased Pulmonary Function in School Children in Western Japan after Exposures to Asian Desert Dusts and Its Association with Interleukin-8

The objective of the study was to investigate the influence of Asian dust storms (ADS) on pulmonary function of school children and the relationship of this effect with interleukin-8. Morning peak expiratory flow (PEF) was measured daily in 399 children from April to May 2012 and in 384 of these children from March to May 2013. The data were analyzed for an association between ADS events and PEF by linear mixed models. Interleukin-8 transcriptional activity was assessed in THP-G8 cells stimulated by airborne particles collected on ADS days. Seven ADS days were identified: April 23 and 24, 2012; March 8 to 10, 2013; and March 19 and 20, 2013. Changes in PEF after ADS exposure were −8.17 L/min (95% confidence interval, −11.40 to −4.93) in 2012 and −1.17 L/min (−4.07 to 1.74) in 2013, and there was a significant difference between 2012 and 2013. Interleukin-8 transcriptional activity was significantly higher in 2012 at 10.6 ± 2.9-fold compared to 3.7 ± 0.4 in March 8 to 10, 2013, and 2.3 ± 0.2 in March 19 and 20, 2013. The influence of ADS events on pulmonary function of children differs with each ADS event and may be related to interleukin-8 production.

Anaphylaxis due to burdock

A 53‐year‐old Japanese man, with a history of developing urticaria (once after consuming mackerel and 10 times after consuming boiled burdock, carrot, curry, and rice), presented with redness over his entire body and dyspnea 1 h after eating boiled burdock. Physical examination revealed a low blood pressure of 64/29 mmHg and stridor, together with striking redness of the whole body; he was diagnosed to be in anaphylactic shock.

CCR4 Expression by Atypical T Cells in Systemic Pilotropic Lymphoma: Its Behavior under Treatment with Interferon Gamma, Topical PUVA and Systemic Retinoid

We describe an 88-year-old Japanese patient with pilotropic T cell lymphoma involving the peripheral blood as well as lymph nodes. This patient presented with multiple red follicular papules, confluent, infiltrated erythematous plaques and nodules. Moreover, he was conspicuous for the presence of total alopecia of the scalp and eyebrows. Histopathologically, the lesional skin showed dense follicular and perifollicular infiltrates of atypical lymphocytes. The flow cytometry disclosed the presence of weakly CD4+ CCR4+ cell populations that would not be detected in the peripheral blood from healthy controls. The patient responded well to topical PUVA and systemic etretinate (retinoid-PUVA) and intravenous IFN-γ. Parallel with the decrease in atypical cells in the peripheral blood, the percentage of weakly CD4+ CCR4+ T cells declined. However, about 1 week after we discontinued this treatment because of the side effects, the lymph node swelling became prominent, and, 4 weeks later, the patient died before restarting any specific chemotherapy.

Variation in the Effect of Particulate Matter on Pulmonary Function in Schoolchildren in Western Japan and Its Relation with Interleukin-8

This study aimed to investigate the effects of particulate matter (PM) on pulmonary function in schoolchildren, as well as the relationships of these effects with interleukin-8. Morning peak expiratory flow (PEF) was measured daily in 399 children during April–May 2012, and in 384 of these children during March–May 2013. PEF’s association with the daily levels of suspended particulate matter (SPM) and PM < 2.5 μm (PM2.5) was estimated using a linear mixed model. Interleukin-8 promoter activity was assessed in THP-G8 cells stimulated by fallen PM collected at Tottori University Hospital during four periods (two in 2012 and two in 2013). An increase of 14.0 μg/m3 in SPM led to PEF changes of −2.16 L/min in 2012 and −0.81 L/min in 2013, respectively. An increment of 10.7 μg/m3 in PM2.5 was associated with PEF changes of −2.58 L/min in 2012 and −0.55 L/min in 2013, respectively. These associations were only significant in 2012. Interleukin-8 promoter activity was significantly higher in both periods of 2012 than in 2013. There was a significant association between pulmonary function in schoolchildren and daily levels of SPM and PM2.5, but this association may differ depending on the PM’s ability to elicit interleukin-8 production.

Evaluation of the Multi-ImmunoTox Assay composed of 3 human cytokine reporter cells by examining immunological effects of drugs

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), to evaluate the effects on key predictivein vitro components of the human immune system. The system is composed of 3 stable reporter cell lines transfected with 3 luciferase genes, SLG, SLO, and SLR, under the control of 4 cytokine promoters, IL-2, IFN-γ, IL-1β, and IL-8, and the G3PDH promoter. We first compared the effects of dexamethasone, cyclosporine, and tacrolimus on these cell lines stimulated with phorbol 12-myristate 13-acetate and ionomycin, or lipopolysaccharides, with those on mRNA expression by the mother cell lines and human whole blood cells after stimulation. The results demonstrated that MITA correctly reflected the change of mRNA of the mother cell lines and whole blood cells. Next, we evaluated other immunosuppressive drugs, off-label immunosuppressive drugs, and non-immunomodulatory drugs. Although MITA did not detect immunosuppressive effects of either alkylating agents or antimetabolites, it could demonstrate those of the off-label immunosuppressive drugs, sulfasalazine, chloroquine, minocycline, and nicotinamide. Compared with the published immunological effects of the drugs, these data suggest that MITA can present a novel high-throughput approach to detect immunological effects of chemicals other than those that induce immunosuppressive effects through their inhibitory action on cell division.