Publications by authors named "Yuki Moritoki"

65 Publications

Eosinophil-mediated inflammation in the absence of eosinophilia.

Asia Pac Allergy 2021 Jul 13;11(3):e30. Epub 2021 Jul 13.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

The increase of eosinophil levels is a hallmark of type-2 inflammation. Blood eosinophil counts act as a convenient biomarker for asthma phenotyping and the selection of biologics, and they are even used as a prognostic factor for severe coronavirus disease 2019. However, the circulating eosinophil count does not always reflect tissue eosinophilia and vice versa. The mismatch of blood and tissue eosinophilia can be seen in various clinical settings. For example, blood eosinophil levels in patients with acute eosinophilic pneumonia are often within normal range despite the marked symptoms and increased number of eosinophils in bronchoalveolar lavage fluid. Histological studies using immunostaining for eosinophil granule proteins have revealed the extracellular deposition of granule proteins coincident with pathological conditions, even in the absence of a significant eosinophil infiltrate. The marked deposition of eosinophil granule proteins in tissue is often associated with cytolytic degranulation. Recent studies have indicated that extracellular trap cell death (ETosis) is a major mechanism of cytolysis. Cytolytic ETosis is a total cell degranulation in which cytoplasmic and nuclear contents, including DNA and histones that act as alarmins, are also released. In the present review, eosinophil-mediated inflammation in such mismatch conditions is discussed.
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http://dx.doi.org/10.5415/apallergy.2021.11.e30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8331253PMC
July 2021

The Curcumin Analog GO-Y030 Controls the Generation and Stability of Regulatory T Cells.

Front Immunol 2021 23;12:687669. Epub 2021 Jun 23.

Department of Clinical Oncology, Graduate School of Medicine, Akita University, Akita, Japan.

Regulatory T cells (Tregs) play a crucial role in preventing antitumor immune responses in cancer tissues. Cancer tissues produce large amounts of transforming growth factor beta (TGF-β), which promotes the generation of Foxp3 Tregs from naïve CD4 T cells in the local tumor microenvironment. TGF-β activates nuclear factor kappa B (NF-κB)/p300 and SMAD signaling, which increases the number of acetylated histones at the locus and induces gene expression. TGF-β also helps stabilize Foxp3 expression. The curcumin analog and antitumor agent, GO-Y030, prevented the TGF-β-induced generation of Tregs by preventing p300 from accelerating NF-κB-induced Foxp3 expression. Moreover, the addition of GO-Y030 resulted in a significant reduction in the number of acetylated histones at the Foxp3 promoter and at the conserved noncoding sequence 1 regions that are generated in response to TGF-β. tumor models demonstrated that GO-Y030-treatment prevented tumor growth and reduced the Foxp3 Tregs population in tumor-infiltrating lymphocytes. Therefore, GO-Y030 exerts a potent anticancer effect by controlling Treg generation and stability.
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http://dx.doi.org/10.3389/fimmu.2021.687669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261301PMC
June 2021

Clonal hematopoiesis in adult pure red cell aplasia.

Sci Rep 2021 01 26;11(1):2253. Epub 2021 Jan 26.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.
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http://dx.doi.org/10.1038/s41598-021-81890-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838416PMC
January 2021

How to detect eosinophil ETosis (EETosis) and extracellular traps.

Allergol Int 2021 Jan 12;70(1):19-29. Epub 2020 Nov 12.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan. Electronic address:

Eosinophils are short-lived and comprise only a small population of circulating leukocytes; however, they play surprisingly multifunctional roles in homeostasis and various diseases including allergy and infection. Recent research has shed light on active cytolytic eosinophil cell death that releases eosinophil extracellular traps (EETs) and total cellular contents, namely eosinophil extracellular trap cell death (EETosis). The pathological contribution of EETosis was made more cogent by recent findings that a classical pathological finding of eosinophilic inflammation, that of Charcot-Leyden crystals, is closely associated with EETosis. Currently no gold standard methods to identify EETosis exist, but "an active eosinophil lysis that releases cell-free granules and net-like chromatin structure" appears to be a common feature of EETosis. In this review, we describe several approaches that visualize EETs/EETosis in clinical samples and in vitro studies using isolated human eosinophils. EETs/EETosis can be observed using simple chemical or fluorescence staining, immunostaining, and electron microscopy, although it is noteworthy that visualization of EETs is greatly changed by sample preparation including the extracellular space of EETotic cells and shear flow. Considering the multiple aspects of biological significance, further study into EETs/EETosis is warranted to give a detailed understanding of the roles played in homeostasis and disease pathogenesis.
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http://dx.doi.org/10.1016/j.alit.2020.10.002DOI Listing
January 2021

Distinguishing coagulase-negative Staphylococcus bacteremia from contamination using blood-culture positive bottle detection pattern and time to positivity.

J Infect Chemother 2020 Jul 2;26(7):672-675. Epub 2020 Mar 2.

Central Laboratory, Teikyo University Mizonokuchi Hospital, Kanagawa, Japan; Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, Kanagawa, Japan.

Aim: Detection of coagulase-negative Staphylococcus in blood culture may be a result of either bacteremia or contamination. This often leads to diagnostic uncertainly. Our objective was to develop a method for differentiating whether a coagulase-negative Staphylococcus sp. positive blood culture represents bacteremia or contamination based on positive bottle detection pattern and time to positivity (TTP).

Methods: This study included 155 and 51 adults with positive blood cultures for Staphylococcus epidermidis and Staphylococcus hominis, respectively, over a three-year period from 2016 to 2018. Positive blood culture cases were categorized as either bacteremia or contamination based on the clinically available information, and the detection pattern and TTP in each category were investigated.

Results: A total of 57, 92, and 6 S. epidermidis positive blood cultures were categorized as bacteremia, contamination, and undetermined, respectively, whereas 15 and 36 S. hominis positive blood cultures were categorized as bacteremia and contamination, respectively. For positive blood cultures categorized as bacteremia, all four bottles in two sets of blood cultures were positive in 47/47 S. epidermidis and 14/14 S. hominis, respectively, whereas either one bottle in each of two sets or three bottles in two sets were positive in 10/19 S. epidermidis and 1/4 S. hominis, respectively; most of those TTPs were <48 h. Among them, the TTP in catheter-related blood stream infection was <24 h.

Conclusion: Although clinical assessment is crucial to differentiate between bacteremia and contamination, a combination of positive bottle detection pattern and TTP is a valuable diagnostic auxiliary tool.
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http://dx.doi.org/10.1016/j.jiac.2020.02.004DOI Listing
July 2020

Fructo-oligosaccharides ameliorate steatohepatitis, visceral adiposity, and associated chronic inflammation via increased production of short-chain fatty acids in a mouse model of non-alcoholic steatohepatitis.

BMC Gastroenterol 2020 Feb 27;20(1):46. Epub 2020 Feb 27.

Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, 5-1-1 Futako, Takatsu-ku, Kawasaki-shi, Kanagawa, 213-8507, Japan.

Background: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Within the spectrum of NAFLD, non-alcoholic steatohepatitis (NASH) in combination with hepatic inflammation and fibrosis can lead to liver cirrhosis and hepatocellular carcinoma. Dysbiosis was reported to contribute to NASH pathogenesis. This study aimed to determine the effects of fructo-oligosaccharides (FOS) on steatohepatitis and visceral adiposity in an obese mouse model of NASH.

Methods: Twelve newborn C57BL/6 J male mice were subcutaneously injected with monosodium glutamate (MSG) to induce obesity on a conventional diet. Six mice were also administered 5% FOS via drinking water from 10 weeks of age. At 18 weeks, histological characteristics of the liver and epididymal fat were compared between the groups. Hepatic mRNA expression of lipid metabolism enzymes and SCFA in feces and sera were measured.

Results: Hepatic steatosis, inflammatory cell infiltration, and hepatocyte ballooning in the liver and increased hepatic mRNA expression of fatty acid synthase and glycerol-3-phosphate acyltransferase were observed in the MSG-treated mice. FOS treatment improved the liver pathology and blunted the increases in the mRNA expression levels of lipid metabolism enzymes. In addition, FOS inhibited adipocyte enlargement and formation of crown-like structures and reduced the M1 macrophage frequency in the epididymal fat of the MSG mice (39.4% ± 3.0% vs. 22.8% ± 0.7%; P = 0.001). FOS increased not only the fecal concentrations of n-butyric acid (0.04 ± 0.01 vs. 0.38 ± 0.14 mg/g, P = 0.02), propionic acid (0.09 ± 0.03 vs. 0.42 ± 0.16 mg/g, P = 0.02), and acetic acid (0.65 ± 0.16 vs. 1.48 ± 0.29 mg/g, P = 0.03) but also the serum concentration of propionic acid (3.9 ± 0.5 vs. 8.2 ± 0.5 μmol/L, P = 0.001).

Conclusions: FOS ameliorates steatohepatitis, visceral adiposity, and chronic inflammation by increasing SCFA production.
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http://dx.doi.org/10.1186/s12876-020-01194-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045471PMC
February 2020

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia.

Sci Rep 2019 06 14;9(1):8645. Epub 2019 Jun 14.

Repertoire Genesis Incorporation, Ibaraki, Japan.

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.
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http://dx.doi.org/10.1038/s41598-019-45264-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570656PMC
June 2019

Umbilical catheterization training: Tissue hybrid versus synthetic trainer.

Pediatr Int 2019 Jul;61(7):664-671

SimTiki Simulation Center, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA.

Background: This study of umbilical catheterization deliberate practice training compared skill and knowledge outcomes of umbilical catheterization using a tissue-hybrid simulator (REAL) versus a synthetic simulated umbilical cord task trainer (ART).

Methods: This was a prospective randomized control study. Pediatric residents were randomized to REAL or ART umbilical catheterization deliberate practice training. Pre-post-training changes in skill performance and knowledge scores for REAL and ART groups were compared. Fidelity of REAL and ART were compared by neonatologists.

Results: Twenty-seven pediatric residents completed training. Post-training mean skill scores were improved compared to pre-test scores (REAL, P < 0.001; ART, P < 0.0001). Post-training skill, knowledge, and self-efficacy scores were not different between the REAL and ART groups. Fidelity of REAL was higher than ART for neonatologists (P < 0.01).

Conclusions: The face validity of REAL was superior to ART, but resident umbilical cord deliberate practice training demonstrated no difference in skill, knowledge, and self-efficacy improvements between REAL and ART. Further studies on real patients are needed to evaluate the impact of using real or simulated umbilical cords for umbilical venous catheter/umbilical arterial catheter training.
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http://dx.doi.org/10.1111/ped.13904DOI Listing
July 2019

Comparison of CD16-negative selection vs. MACSxpress system for isolation of blood eosinophils.

Allergol Int 2019 Sep 6;68S:S11-S13. Epub 2019 May 6.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

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http://dx.doi.org/10.1016/j.alit.2019.04.005DOI Listing
September 2019

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

Front Immunol 2018 2;9:2534. Epub 2018 Nov 2.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4-6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8 T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells ( = 0.7426, = 0.0006) and between numbers of liver memory CD8 T cells and B cells ( = 0.6423, = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.
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http://dx.doi.org/10.3389/fimmu.2018.02534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224429PMC
September 2019

ICAM-1 upregulation is not required for retinoic acid-induced human eosinophil survival.

Immunol Lett 2018 04 1;196:68-73. Epub 2018 Feb 1.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.

Active metabolites of vitamin A, retinoic acids (RAs), are known to play critical roles in mucosal immune responses and dramatically inhibit human eosinophil apoptosis, but the detailed mechanisms have not been elucidated. We previously screened for ICAM-1 (CD54) upregulation in RA-stimulated human eosinophils by gene microarray analysis. As ICAM-1 induction and activation were observed to have a role in maintenance of eosinophil survival, we tested the hypothesis that RAs prolong eosinophil survival through ICAM-1 outside-in signaling. Blood-derived isolated eosinophils cultured with 9-cis RA and all-trans RA showed significant upregulation of ICAM-1 mRNA and cell surface expression. TTNPB, a retinoic acid receptor agonist, also induced ICAM-1 expression, while HX630, a retinoid X receptor agonist, did not. Furthermore, an RAR antagonist, HX531, completely inhibited the effect of RAs. Upregulated ICAM-1 was associated with altered kinetics of Akt, ERK, and p38 MAP kinase phosphorylation through ICAM-1 cross-linking, but an ICAM-1-blocking antibody did not affect RA-mediated cell survival. These findings indicate that RAs induce functional ICAM-1 expression through RARs, but the induced ICAM-1 does not contribute to prolongation of eosinophil survival.
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http://dx.doi.org/10.1016/j.imlet.2018.01.013DOI Listing
April 2018

Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis.

PLoS One 2017 20;12(6):e0175406. Epub 2017 Jun 20.

The Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, Takatsu-ku, Kawasaki-city, Kanagawa, Japan.

Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175406PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478096PMC
September 2017

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

Cytokine 2016 12 20;88:45-50. Epub 2016 Aug 20.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-β and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-β transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-β contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation.
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http://dx.doi.org/10.1016/j.cyto.2016.08.013DOI Listing
December 2016

Accumulated myeloid-derived suppressor cells demonstrate distinct phenotypes and functions in two non-alcoholic steatohepatitis mouse models.

Hepatobiliary Surg Nutr 2015 Oct;4(5):313-9

1 Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, Kawasaki-shi, Kanagawa 213-8507, Japan ; 2 Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Science, University of Toyama, Toyama-shi, Toyama 930-0194, Japan ; 3 Department of General Medical Practice and Laboratory Diagnostic Medicine, Akita University Graduate School of Medicine, Akita-shi, Akita 010-8543, Japan.

Background: To examine the steady state of hepatic myeloid-derived suppressor cells (MDSCs) and the lipid accumulation and inflammation-related changes in these cells, we analyzed the presence and functions of hepatic MDSCs in the following two non-alcoholic steatohepatitis (NASH) mouse models.

Methods: Monosodium glutamate (MSG) model; MSG was subcutaneously injected into neonatal male C57BL/6J mice that were fed with normal diet up to 18 weeks of age. Methionine/choline-deficient diet (MCD) model; 16-week-old male C57BL/6J mice were fed with an MCD for 2 weeks. Those hepatic MDSCs were evaluated by flow cytometry and immunohistochemically.

Results: Both MSG and MCD mice exhibited greater numbers of hepatic lipid droplets than 18-week-old male control mice. Hepatocellular ballooning was obvious in MSG, whereas inflammatory cell infiltration were apparent in MCD mice. CD11b, CD115, and Gr-1-positive hepatic MDSCs were increased in both models but higher in MCD mice, and demonstrated higher expression of an M2 macrophage marker CD206 mean fluorescence intensity (MFI) in MSG compared to MCD mice. Degree of reactive oxygen species production was evaluated using the DCFDA MFI values, which were significantly elevated in hepatic MDSCs from MCD mice. MSG mouse livers demonstrated Gr-1 positive cell accumulation around lipid droplets, mimicking crown-like structures in adipose tissues. In contrast, hepatic Gr-1 positive cells were primarily located in inflammatory cell aggregates in MCD mice.

Conclusions: These results suggest that hepatic fatty changes promote MDSC accumulation, and inflammatory changes induce phenotypic and functional alteration in hepatic MDSCs in NASH mouse models.
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http://dx.doi.org/10.3978/j.issn.2304-3881.2015.04.08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607837PMC
October 2015

Eosinophil extracellular trap cell death-derived DNA traps: Their presence in secretions and functional attributes.

J Allergy Clin Immunol 2016 Jan 9;137(1):258-267. Epub 2015 Jun 9.

Divisions of Allergy and Inflammation and Infectious Diseases, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.

Background: Activated human eosinophils, as well as neutrophils, can release extracellular chromatin to form DNA traps through cytolytic extracellular trap cell death (ETosis). Although formations of neutrophil DNA traps are recognized in patients with various inflammatory conditions, neither the presence of ETosis-derived eosinophil DNA traps in human allergic diseases nor the characteristics of these DNA traps have been studied.

Objective: We investigated the presence of ETosis-derived DNA traps in eosinophil-rich sinus and ear secretions and the functional attributes of ETosis DNA traps.

Methods: Eosinophil-rich secretions obtained from patients with eosinophilic chronic rhinosinusitis and eosinophilic otitis media were studied microscopically. In vitro studies of ETosis and DNA trap formation used blood-derived eosinophils and neutrophils, and studies of the binding capacities of DNA traps used labeled bacteria and fluorescent microbeads. Stabilities of DNA traps were evaluated by using fluorescence microscopy.

Results: Abundant nuclear histone H1-bearing DNA traps formed in vivo in the eosinophilic secretions and contributed to their increased viscosity. In vitro, after brief shear flow, eosinophil ETosis-elicited DNA traps assembled to form stable aggregates. Eosinophil DNA traps entrapped bacteria and fungi and, through hydrophobic interactions, microbeads. In comparison with neutrophil-derived DNA traps, eosinophil DNA traps ultrastructurally exhibited thicker fibers with globular structures and were less susceptible to leukocyte-derived proteolytic degradation, likely because of the lesser protease activities of eosinophils.

Conclusions: In human allergic diseases local cytolysis of eosinophils not only releases free eosinophil granules but also generates nuclear-derived DNA traps that are major extracellular structural components within eosinophil-rich secretions.
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http://dx.doi.org/10.1016/j.jaci.2015.04.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674385PMC
January 2016

G-protein-coupled estrogen receptor agonist suppresses airway inflammation in a mouse model of asthma through IL-10.

PLoS One 2015 31;10(3):e0123210. Epub 2015 Mar 31.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita, 010-8543, Japan.

Estrogen influences the disease severity and sexual dimorphism in asthma, which is caused by complex mechanisms. Besides classical nuclear estrogen receptors (ERαβ), G-protein-coupled estrogen receptor (GPER) was recently established as an estrogen receptor on the cell membrane. Although GPER is associated with immunoregulatory functions of estrogen, the pathophysiological role of GPER in allergic inflammatory lung disease has not been examined. We investigated the effect of GPER-specific agonist G-1 in asthmatic mice. GPER expression in asthmatic lung was confirmed by immunofluorescent staining. OVA-sensitized BALB/c and C57BL/6 mice were treated with G-1 by daily subcutaneous injections during an airway challenge phase, followed by histological and biochemical examination. Strikingly, administration of G-1 attenuated airway hyperresponsiveness, accumulation of inflammatory cells, and levels of Th2 cytokines (IL-5 and IL-13) in BAL fluid. G-1 treatment also decreased serum levels of anti-OVA IgE antibodies. The frequency of splenic Foxp3+CD4+ regulatory T cells and IL-10-producing GPER+CD4+ T cells was significantly increased in G-1-treated mice. Additionally, splenocytes isolated from G-1-treated mice showed greater IL-10 production. G-1-induced amelioration of airway inflammation and IgE production were abolished in IL-10-deficient mice. Taken together, these results indicate that extended GPER activation negatively regulates the acute asthmatic condition by altering the IL-10-producing lymphocyte population. The current results have potential importance for understanding the mechanistic aspects of function of estrogen in allergic inflammatory response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123210PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380451PMC
March 2016

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

PLoS One 2015 19;10(3):e0120386. Epub 2015 Mar 19.

Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120386PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366258PMC
February 2016

Eliciting eosinophil CCR3 expression by synthetic retinoids.

Allergol Int 2014 May;63 Suppl 1:67-8

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

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http://dx.doi.org/10.2332/allergolint.13-LE-0641DOI Listing
May 2014

Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils.

Immunol Lett 2014 Jul 6;160(1):72-78. Epub 2014 Apr 6.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Electronic address:

Sexual dimorphism in asthma links the estrogen and allergic immune responses. The function of estrogen was classically believed to be mediated through its nuclear receptors, i.e., estrogen receptors (ERs). However, recent studies established the important roles of G-protein-coupled estrogen receptor (GPER/GPR30) as a novel membrane receptor for estrogen. To date, the role of GPER in allergic inflammation is poorly understood. The purpose of this study was to examine whether GPER might affect the functions of eosinophils, which play an important role in the pathogenesis of asthma. Here, we demonstrated that GPER was expressed in purified human peripheral blood eosinophils both at the mRNA and protein levels. Although GPER agonist G-1 did not induce eosinophil chemotaxis or chemokinesis, preincubation with G-1 enhanced eotaxin (CCL11)-directed eosinophil chemotaxis. G-1 inhibited eosinophil spontaneous apoptosis and caspase-3 activities. The anti-apoptotic effect was not affected by the cAMP-phospodiesterase inhibitor rolipram or phosphoinositide 3-kinase inhibitors. In contrast to resting eosinophils, G-1 induced apoptosis and increased caspase-3 activities when eosinophils were co-stimulated with IL-5. No effect of G-1 was observed on eosinophil degranulation in terms of release of eosinophil-derived neurotoxin (EDN). The current study indicates the functional capacities of GPER on human eosinophils and also provides the previously unrecognized mechanisms of interaction between estrogen and allergic inflammation.
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http://dx.doi.org/10.1016/j.imlet.2014.03.012DOI Listing
July 2014

Serological and Histological Examination of a Nonalcoholic Steatohepatitis Mouse Model Created via the Administration of Monosodium Glutamate.

Int Sch Res Notices 2014 30;2014:725351. Epub 2014 Oct 30.

Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, 3-8-3 Mizonokuchi, Takatsu-ku, Kawasaki-shi, Kanagawa 213-8507, Japan.

The administration of monosodium glutamate (MSG) to mice induces hepatic steatosis and inflammation. In this study, we investigated the metabolic features of MSG-treated mice and the histological changes that occur in their livers and adipose tissue. MSG mice were prepared by subcutaneously injecting MSG into newborn C57BL/6J male mice. The control mice were subcutaneously injected with saline. Another group of mice was fed a methionine- and choline-deficient diet (MCD). Compared with the control mice, the MSG mice had higher serum levels of insulin and cholesterol than the control mice, whereas the opposite was true for the MCD mice. Microvesicular steatosis and inflammatory cell infiltration were detected in both the MSG and MCD mouse livers. Enlarged adipocytes and crown-like structures were observed in the epididymal fat of the MSG mice, whereas neither of these features was seen in the MCD mice. Flow cytometric analysis revealed increased frequencies of monocytes and M1 macrophages in the livers and epididymal fat tissue of the MSG mice, respectively. The MSG mice exhibited the characteristic liver histopathology of nonalcoholic steatohepatitis (NASH) as well as metabolic syndrome-like features, which suggested that MSG mice are a better model of human NASH than MCD mice.
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http://dx.doi.org/10.1155/2014/725351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4897218PMC
July 2016

The effect of pharmacological PI3Kγ inhibitor on eotaxin-induced human eosinophil functions.

Pulm Pharmacol Ther 2014 Apr 9;27(2):164-9. Epub 2013 Dec 9.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

Background: Asthma is characterized by chronic inflammation caused by activation of immune cells including Th2 lymphocytes and eosinophils. Phosphoinositide 3-kinase (PI3K) γ deficient asthmatic mice did not develop lung eosinophilia, although the detailed mechanisms are not well known. A CC chemokine eotaxin (CCL11) plays a prominent role in developing eosinophilic inflammation through CCR3. In this study, we tested the roles of PI3Kγ in eotaxin-induced eosinophil functions using a pharmacological inhibitor.

Method: Human peripheral blood eosinophils were isolated by CD16-negative selection method. The effect of AS605240, synthetic PI3Kγ inhibitor on eotaxin-induced adhesion, chemotaxis, and degranulation were studied using intracellular adhesion molecule-1 (ICAM-1)-coated plates, Boyden chamber system, ELISA for eosinophil-derived neurotoxin (EDN) levels in the culture supernatant, respectively. CCR3 expression levels and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were assessed by flowcytometry. Involvement of PI3Kγ in spontaneous apoptosis was studied using flowcytometry.

Results: Although AS605240 did not affect the eosinophil spontaneous apoptosis, eotaxin-induced chemotaxis, adhesion to ICAM-1 coated plate, and EDN release were inhibited by AS605240. AS605240 also inhibited the eotaxin-induced ERK1/2 phosphorylation without down-regulation of surface CCR3 expression.

Conclusion: These results indicate that PI3Kγ inhibitor attenuates eotaxin-induced eosinophil functions by suppressing the downstream signaling of CCR3 without significant cytotoxicity. PI3Kγ plays an important role in the development of eosinophilic inflammation and blockade of PI3Kγ might be a therapeutic strategy for treatment of eosinophil-related diseases including asthma.
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http://dx.doi.org/10.1016/j.pupt.2013.11.006DOI Listing
April 2014

A case of pulmonary hamartoma showing rapid growth.

Case Rep Med 2013 19;2013:231652. Epub 2013 Sep 19.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.

A 65-year-old man was admitted for detailed examination of a growing nodular shadow in the left lung. The nodular shadow was initially detected in a routine chest X-ray check-up in March 2012 that warranted regular chest X-ray follow-up. The nodular shadow increased in size from 12 × 15 mm to 15 × 20 mm within five months. The calculated tumor doubling time (TDT) in our case was approximately 132.2 days. A malignant tumor was strongly suspected based on the rapid growth, and tumorectomy was thus performed. Cartilaginous tissue accounted for most of the pathological specimen, but a small amount of an epithelial component was observed histologically, and we diagnosed a hamartoma. Hamartoma generally shows slow annual growth, but it is important to recognize that rapid enlargement occurs in some cases.
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http://dx.doi.org/10.1155/2013/231652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792529PMC
October 2013

Splenic lymph follicles generate immunoglobulin M-producing B cells in primary biliary cirrhosis.

Hepatol Res 2014 Oct 7;44(10):E253-E256. Epub 2014 Jan 7.

The Fourth Department of Internal Medicine, Teikyo University Mizonokuchi Hospital, Kawasaki, Japan.

Aim: To reveal the site of immunoglobulin (Ig)M production in primary biliary cirrhosis (PBC) we performed immunohistochemical analysis on spleens collected from patients with PBC.

Methods: Splenic tissue samples were collected at the time of the autopsy from patients with hepatic failure. Immunostaining for IgM, CD21 and CXCL13 were performed using the splenic tissue samples.

Results: The samples from five out of eight cases with PBC but not in eight cases of chronic hepatitis C virus infection showed accumulation of IgM positive cells in CD21 positive lymph follicles. The CXCL13 positive cells also accumulated in the center of the lymph follicles where the IgM positive cells accumulated.

Conclusion: The present results suggest that excess IgM is produced from the spleen of PBC. Furthermore, it was suggested that CXCL13 positive follicular dendritic cells possibly contribute to this process.
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http://dx.doi.org/10.1111/hepr.12231DOI Listing
October 2014

Adiponectin attenuates human eosinophil adhesion and chemotaxis: implications in allergic inflammation.

J Asthma 2013 Oct 17;50(8):828-35. Epub 2013 Jul 17.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine , Akita , Japan.

Objective: Growing evidence has shown an association between obesity and asthma. Adiponectin, an adipocyte-derived cytokine, is known to have anti-inflammatory effects with reduced concentrations in obese subjects. Recent findings raised the intriguing possibility that adiponectin might play a role in allergic inflammation, although the mechanistic basis for their relationship remains unclear. The purpose of this study was to examine whether adiponectin might affect functions of eosinophils, which play an important role in the pathogenesis of asthma.

Methods: Human peripheral blood eosinophils were purified to study expression of adiponectin receptors AdipoR1 and AdipoR2 using RT-PCR and flow cytometry. The effect of adiponectin on eosinophil survival was investigated using annexin V and propidium iodide staining. Eotaxin-induced cell adhesion was investigated using ICAM-1-coated plates. A Boyden chamber and real-time horizontal migration system were used for eotaxin-directed chemotaxis assay. Expression of eotaxin receptor CCR3 and intracellular calcium influx were assessed by flow cytometry.

Results: AdipoR1 and AdipoR2 were expressed in human eosinophils. Adiponectin did not affect eosinophil survival or CCR3 expression; however, eotaxin-enhanced adhesion was inhibited by pretreatment with adiponectin. Adiponectin also diminished eotaxin-directed chemotactic responses by disturbing both velocity and directionality. Calcium influx in response to eotaxin was attenuated by adiponectin.

Conclusions: These results indicate that adiponectin attenuates the eosinophil functions induced by eotaxin without affecting cell viability. The inhibitory effect was associated with diminished calcium signaling rather than altering of surface receptor expression. Increasing circulating adiponectin might be a novel therapeutic modality for treatment of asthma, especially in obese asthmatics.
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http://dx.doi.org/10.3109/02770903.2013.816725DOI Listing
October 2013

Lung metastasis from perineal leiomyosarcoma: a case report and a review of the Japanese literature.

Case Rep Med 2013 5;2013:496304. Epub 2013 Mar 5.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan.

Pulmonary metastasis from leiomyosarcoma is rare and its clinical management is challenging. A single lung metastasis from a perineal leiomyosarcoma occurred in a 79-year-old woman. Five months after resection of the lung metastasis, a new metastatic tumor developed in the contralateral lung. Since the patient did not desire to receive hospitalized treatment, TS-1 (an oral agent consisting of a combination of tegafur, gimeracil, and oteracil potassium) therapy was started on an outpatient basis. The lung metastasis has been successfully controlled for at least 17 months with excellent tolerability. The clinical features and the treatment of this case are discussed.
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http://dx.doi.org/10.1155/2013/496304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603195PMC
March 2013

Gender difference in allergic airway remodelling and immunoglobulin production in mouse model of asthma.

Respirology 2013 Jul;18(5):797-806

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, Akita.

Background And Objective: Epidemiological studies have shown that the prevalence of adult asthma and severe asthma is higher in women. It has also been reported that female mice are more susceptible than males to the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The influence of gender difference in the pathogenesis of severe asthma, especially airway remodelling in an animal model, has been studied rarely. We investigated gender difference in the development of airway remodelling using a long-term antigen-challenged mouse asthma model.

Methods: Following ovalbumin (OVA)/alum intraperitoneal injection, male or female mice (BALB/c) were challenged with aerosolized 1% OVA on 3 days/week for 5 weeks, and differences in AHR, airway inflammation and airway remodelling between the sexes were investigated.

Results: In OVA-sensitized and OVA-challenged (OVA/OVA) female mice, eosinophils, lymphocytes, T-helper type 2 cytokines and growth factors in bronchoalveolar lavage fluid were increased compared with OVA/OVA male mice. Histological features of airway remodelling were also increased in OVA/OVA female mice. Serum total and OVA-specific immunoglobulin E (IgE) and serum IgA were significantly elevated in OVA/OVA female mice.

Conclusions: These results indicate that female mice experience more airway remodelling compared with male mice. These results suggest the involvement of sex hormones and gender differences in cellular functions in airway remodelling.
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http://dx.doi.org/10.1111/resp.12078DOI Listing
July 2013

Obesity and eosinophilic inflammation: does leptin play a role.

Int Arch Allergy Immunol 2012 15;158 Suppl 1:87-91. Epub 2012 May 15.

Department of Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan.

It has been pointed out that obesity is a risk factor for, and is involved in the exacerbation of asthma. Mounting evidence about adipose tissue-derived proteins (adipokines) gave rise to the current understanding of obesity as a systemic inflammatory disorder. In this review, we summarized the involvement of leptin, focusing on eosinophil functions. Several studies have indicated that leptin can restrain eosinophil apoptosis, enhance migration, increase adhesion molecules and induce cytokine production. Since leptin also acts on a variety of immune cells related to allergic response, increased leptin in obese individuals potentially explains the mechanism by which obesity leads to an exacerbation of asthma. Further studies targeting adipokines will delineate the association between obesity and eosinophil-associated diseases.
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http://dx.doi.org/10.1159/000337799DOI Listing
July 2012

Comparative analysis of portal cell infiltrates in antimitochondrial autoantibody-positive versus antimitochondrial autoantibody-negative primary biliary cirrhosis.

Hepatology 2012 May 4;55(5):1495-506. Epub 2012 Apr 4.

First Hospital, University of Jilin, Changchun, China.

Unlabelled: Substantial evidence supports dysregulated B-cell immune responses in patients with primary biliary cirrhosis (PBC), including the presence of serum antimitochondrial antibodies (AMAs). However, recent reports from murine models of PBC suggest that B cells may also provide regulatory function, and indeed the absence of B cells in such models leads to exacerbation of disease. The vast majority of patients with PBC have readily detectable AMAs, but a minority (<5%) are AMA negative (AMA(-)), even with recombinant diagnostic technology. This issue prompted us to examine the nature of B-cell infiltrates surrounding the portal areas in AMA-positive (AMA(+)) and AMA(-) patients, because they display indistinguishable clinical features. Of importance was the finding that the degree of bile duct damage around the portal areas was significantly milder in AMA(+) PBC than those observed in AMA(-) PBC patients. The portal areas from AMA(-) patients had a significant increase of cluster of differentiation (CD)5(+) cells infiltrating the ductal regions, and the levels of B-cell infiltrates were worse in the early phase of bile duct damage. The frequency of positive portal areas and the magnitude of CD5(+) and CD20(+) cellular infiltrates within areas of ductal invasion is associated with the first evidence of damage of biliary duct epithelia, but becomes reduced in the ductopenia stage, with the exception of CD5(+) cells, which remain sustained and predominate over CD20(+) cells.

Conclusion: Our data suggest a putative role of B-cell autoimmunity in regulating the portal destruction characteristic of PBC.
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http://dx.doi.org/10.1002/hep.25511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299932PMC
May 2012
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