Publications by authors named "Masashi Iwasaki"

17 Publications

  • Page 1 of 1

Negative-pressure wound therapy in combination with bronchial occlusion to treat bronchopleural fistula: a case report.

Surg Case Rep 2021 Mar 2;7(1):61. Epub 2021 Mar 2.

Department of General Thoracic Surgery, Ayabe City Hospital, 20-1 Otsuka, Aono-cho, Ayabe, Kyoto, 623-0011, Japan.

Background: Bronchopleural fistula, which usually accompanies bronchial fistula and empyema, is a severe complication of lung cancer surgery. Negative-pressure wound therapy can enhance drainage and reduce the empyema cavity, potentially leading to early recovery. This therapy is not currently indicated for bronchopleural fistulas because of the risk of insufficient respiration due to air loss from the fistula.

Case Presentation: A 73-year-old man, who was malnourished because of peritoneal dialysis, was referred to our hospital for the treatment of lung cancer. Right lower lobectomy with mediastinal lymph node dissection was performed via posterolateral thoracotomy, and the bronchial stump was covered with the intercostal muscle flap. His postoperative course was uneventful and he was discharged. However, he was readmitted to our hospital because of respiratory failure and diagnosed as having bronchopleural fistula on the basis of the bronchoscopic finding of a 10-mm hole at the membranous portion of the inlet of the remnant lower lobe bronchus. Thus, thoracotomy debridement and open window thoracostomy were immediately performed. After achieving infection control, bronchial occlusion was performed using fibrin glue and a polyglycolic acid sheet was inserted through a fenestrated wound. Bronchial fistula closure was observed on bronchoscopy; therefore, a negative-pressure wound therapy system was applied to close the fenestrated wound. The collapsed lung was re-expanded and the granulation tissue around the wound increased; therefore, thoracic cavity size decreased and thoracoplasty using the latissimus dorsi was performed.

Conclusions: This bronchopleural fistula was treated successfully after a right lower lobectomy using an extra-pleural bronchial occlusion and negative-pressure wound therapy.
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http://dx.doi.org/10.1186/s40792-021-01144-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922724PMC
March 2021

Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells.

Front Immunol 2020 21;11:1405. Epub 2020 Jul 21.

Department of Respiratory Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.

Increasing attention has been paid to human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) in the field of cancer immunotherapy. We have previously demonstrated that a novel bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA), efficiently expands peripheral blood Vγ2Vδ2 T cells to purities up to 95-99% in 10-11 days. In the present study, we first examined the effect of PTA on farnesyl diphosphate synthase (FDPS) using liquid chromatography mass spectrometry (LC-MS) to analyze the mechanism underlying the PTA-mediated expansion of Vγ2Vδ2 T cells. We find that the prodrug induced the accumulation of both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), direct upstream metabolites of FDPS. This indicates that not only IPP but also DMAPP plays an important role in PTA-mediated stimulation of Vγ2Vδ2 T cells. We next analyzed TCR-independent cytotoxicity of Vγ2Vδ2 T cells. When human lung cancer cell lines were challenged by Vγ2Vδ2 T cells, no detectable cytotoxicity was observed in 40 min. The lung cancer cell lines were, however, significantly killed by Vγ2Vδ2 T cells after 4-16 h in an effector-to-target ratio-dependent manner, demonstrating that Vγ2Vδ2 T cell-based cell therapy required a large number of cells and longer time when tumor cells were not sensitized. By contrast, pulsing tumor cell lines with 10-30 nM of PTA induced significant lysis of tumor cells by Vγ2Vδ2 T cells even in 40 min. Similar levels of cytotoxicity were elicited by ZOL at concentrations of 100-300 μM, which were much higher than blood levels of ZOL after infusion (1-2 μM), suggesting that standard 4 mg infusion of ZOL was not enough to sensitize lung cancer cells in clinical settings. In addition, Vγ2Vδ2 T cells secreted interferon-γ (IFN-γ) when challenged by lung cancer cell lines pulsed with PTA in a dose-dependent manner. Taken together, PTA could be utilized for both expansion of Vγ2Vδ2 T cells and sensitization of tumor cells in Vγ2Vδ2 T cell-based cancer immunotherapy. For use in patients, further studies on drug delivery are essential because of the hydrophobic nature of the prodrug.
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http://dx.doi.org/10.3389/fimmu.2020.01405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385076PMC
April 2021

Successful thoracoscopic evacuation of an extrapleural hematoma with delayed symptomatic pleural effusion: a case report.

Surg Case Rep 2019 Aug 14;5(1):133. Epub 2019 Aug 14.

Division of Thoracic Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, 602-8566, Japan.

Background: Traumatic extrapleural hematoma is a rare condition and is usually managed conservatively until spontaneous resolution unless active bleeding or expansion is found.

Case Presentation: An 80-year-old man taking an anticoagulant medication was referred to our hospital after accidentally falling in a street ditch while riding a bike. Chest X-ray and computed tomography (CT) scan showed multiple fractures on ribs 7-9, hemothorax, and extrapleural hematoma in the posterior chest wall. Though the patient's hemothorax was improved by chest tube drainage, the extrapleural hematoma still remained. He was transferred to another hospital for rehabilitation, but he was readmitted to our hospital because of dyspnea with accumulation of left pleural effusion, including a subpopulation of neutrophils, but without bacterial infection. We performed thoracoscopic evacuation of the hematoma on day 57 after the initial blunt chest trauma. The patient has had no recurrence of pleuritis for 6 months after surgery.

Conclusion: Since posttraumatic extrapleural hematoma may result in delayed secondary intractable pleural effusion causing dyspnea, careful observation is necessary when considering indications of surgical intervention.
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http://dx.doi.org/10.1186/s40792-019-0691-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694359PMC
August 2019

Volume-Based Consolidation-to-Tumor Ratio Is a Useful Predictor for Postoperative Upstaging in Stage I and II Lung Adenocarcinomas.

Thorac Cardiovasc Surg 2019 Aug 8. Epub 2019 Aug 8.

Division of Thoracic Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Background:  We investigated the postoperative upstaging of stage I and II lung adenocarcinoma patients to identify useful predictors for accurate staging.

Methods:  We retrospectively reviewed data from 80 consecutive patients undergoing lobectomy and mediastinal lymph node dissection for clinical stage I and II lung adenocarcinomas. We evaluated clinical variables, including the preoperative serum carcinoembryonic antigen (CEA), tumor diameter, consolidation-to-tumor ratio (CTR), maximum standardized uptake value (SUVmax) on FDG- PET, expression of epithelial growth factor receptor mutations, and pathological invasion to the pleura (pl), lymph duct (ly), and vein (v).

Results:  Eleven patients (13.8%) showed postoperative upstaging. Three cases had pN1 migrating from cN0, four cases had pN2 from cN0, and four cases showed malignant pleural effusion. The CEA level and CTR were significantly higher in the upstaging group. The tumors in the upstaging group showed more frequent pathological invasion to the visceral pleura and vein. In patients with 3 cm or smaller consolidation, two-dimensional (2D)-CTR and volume-based CTR were independent predictors of upstaging.

Conclusions:  Volume-based CTR could be a useful predictor for accurate clinical staging in stage I and II adenocarcinoma patients in addition to consolidation size, serum CEA level, and 2D-CTR. Both volume-based and 2D-CTRs might be especially useful in T1 diseases.
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http://dx.doi.org/10.1055/s-0039-1694061DOI Listing
August 2019

Bioluminescence Microplate Assay of Cyanide with Escherichia coli Harboring a Plasmid Responsible for Cyanide-dependent Light Emission in Alginate Microenvironment.

Anal Sci 2019 Jul 17;35(7):821-825. Epub 2019 May 17.

Center of Environmental Science, Kyoto Institute of Technology.

We describe the bioluminescence of a genetically engineered Escherichia coli harboring a recombined plasmid with a catalase gene promoter fused lux gene cluster, responsible for the generation of photons closely associated with respiratory inhibition, with the aim of applying it for cyanide sensing. This E. coli construct was favorably utilized for the microplate assay of cyanide by leveraging the microenvironment of the biocompatible alginate. The brightness of the bioluminescence, induced by cyanide stimulation of the respiration causative of the production of hydrogen peroxide, positively correlates with its concentration. Moreover, visualization of cyanide with a consumer digital camera, ranging in concentration from about 0.01 mg CN·L in the alginate sol to around 100 mg CN·L in its gel, was attained.
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http://dx.doi.org/10.2116/analsci.19N014DOI Listing
July 2019

Determination of human γδ T cell-mediated cytotoxicity using a non-radioactive assay system.

J Immunol Methods 2019 03 14;466:32-40. Epub 2019 Jan 14.

Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Program for Nurturing Global Leaders in Tropical and Emerging Infectious Diseases, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshidakonoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan; Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan. Electronic address:

The adoptive transfer of immune effector cells, such as CD8 killer αβ T cells, γδ T cells, NK (natural killer) cells, and genetically-modified T cells, has been receiving increasing attention. It is essential to determine cellular cytotoxicity so as to monitor the function and quality of ex vivo-expanded immune effector cells before infusion. The most common method is the [Cr]-sodium chromate release assay. It is, however, preferable to avoid the use of radioactive materials in clinical laboratories. In order to establish a non-radioactive alternative to the standard radioactive assay, we previously synthesized a chelate-forming prodrug (BM-HT) and demonstrated that a combination of BM-HT and europium (Eu) was useful to determine NK cell-mediated cytotoxicity. In the present study, we examined whether or not this improved assay system could be used to determine the cellular cytotoxicity exhibited by Vγ2Vδ2 γδ T cells. In addition, we compared Eu and terbium (Tb) in the measurement of cellular cytotoxicity. Our assay system using BM-HT could be used successfully for the analysis of both γδ T cell receptor (TCR)- and CD16-mediated cytotoxicity. When the intensity of fluorescence was compared between Eu and Tb, Tb chelate was more sensitive than Eu chelate, suggesting that the detection system using Tb is superior to Eu when tumor cells are not efficiently labeled with BM-HT. The method established herein is expected to promote the development of novel adoptive cell therapies for cancer.
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http://dx.doi.org/10.1016/j.jim.2019.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817948PMC
March 2019

Synthesis and Immunomodulatory Activity of Fluorine-Containing Bisphosphonates.

ChemMedChem 2019 02 25;14(4):462-468. Epub 2019 Jan 25.

Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan.

Immune checkpoint blockade using anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibodies (mAbs) has revolutionized cancer treatment. However, many types of cancer do not respond and for those that do, only a minority of patients achieve durable remissions. Therefore, oncoimmunologists are working to develop adoptive cell therapies for non-hematopoietic tumors by harnessing immune effector cells such as αβ T cells and γδ T cells. In contrast to conventional αβ T cells that recognize peptides in the context of MHC class I or II molecules, γδ T cells expressing Vγ2Vδ2 T cell receptors (also termed Vγ9Vδ2) are stimulated by isoprenoid metabolites (phosphoantigens) such as isopentenyl diphosphate in a butyrophilin-3A1-dependent manner. Vγ2Vδ2 T cells kill almost all types of tumor cells that have been treated with bisphosphonates. In this study, we synthesized a series of fluorine-containing bisphosphonates based on current drugs and found that they stimulated Vγ2Vδ2 T cell killing of tumor cells. A fluorine-containing prodrug analogue of zoledronate where phosphonate moieties were masked with pivaloyloxymethyl groups markedly enhanced Vγ2Vδ2 T-cell-mediated cytotoxicity, and also promoted the expansion of peripheral blood Vγ2Vδ2 T cells. These results demonstrate that a prodrug of a fluorine-containing zoledronate analogue can sensitize tumor cells for killing as well as expand Vγ2Vδ2 T cells for adoptive cell therapy.
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http://dx.doi.org/10.1002/cmdc.201800764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818088PMC
February 2019

Intractable pneumothorax due to rupture of subpleural rheumatoid nodules: a case report.

Surg Case Rep 2018 Aug 8;4(1):89. Epub 2018 Aug 8.

Department of General Thoracic Surgery, Ayabe City Hospital, 20-1 Otsuka, Aono-cho, Ayabe, Kyoto, 623-0011, Japan.

Background: In rare cases, rheumatoid pleural nodules can rupture into the pleural cavity to cause pneumothorax or empyema. We report successful surgical treatment of a patient with an intractable secondary pneumothorax due to rupture of a subpleural rheumatoid nodule into the pleural cavity.

Case Presentation: A 75-year-old man with a medical history of rheumatoid arthritis, acute coronary syndrome, and diabetes was admitted to our hospital because of left chest pain and dyspnea. A chest X-ray and chest computed tomography (CT) scan showed a left pneumothorax and several small subpleural nodules with cavitation. Repeated pleurodesis via a chest tube failed to improve the pneumothorax, so we decided to perform thoracoscopic surgery. Air leakage was detected in the left upper lobe where the subpleural nodule was visible on chest CT. Resection of the lesion successfully resulted in resolution of the air leakage. The final pathological diagnosis of the subpleural nodule was a pulmonary rheumatoid nodule. The patient has had no evidence of recurrence of pneumothorax after surgery.

Conclusions: We obtained a final pathological diagnosis of a rheumatoid nodule that caused an intractable pneumothorax. Pneumothorax associated with rupture of rheumatoid nodules in the subpleural cavitary is difficult to treat with thoracoscopic surgery as a second-line treatment.
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http://dx.doi.org/10.1186/s40792-018-0502-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082747PMC
August 2018

Expansion of human γδ T cells for adoptive immunotherapy using a bisphosphonate prodrug.

Cancer Sci 2018 Mar 4;109(3):587-599. Epub 2018 Feb 4.

Department of Internal Medicine and the Interdisciplinary Graduate Program in Immunology, University of Iowa Carver College of Medicine, Iowa City Veterans Affairs Health Care System, Iowa City, IA, USA.

Cancer immunotherapy with human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) has shown promise because of their ability to recognize and kill most types of tumors in a major histocombatibility complex (MHC) -unrestricted fashion that is independent of the number of tumor mutations. In clinical trials, adoptive transfer of Vγ2Vδ2 T cells has been shown to be safe and does not require preconditioning. In this report, we describe a method for preparing highly enriched human Vγ2Vδ2 T cells using the bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA). PTA stimulated the expansion of Vγ2Vδ2 cells to purities up to 99%. These levels were consistently higher than those observed after expansion with zoledronic acid, the most commonly used stimulator for clinical trials. Cell numbers also averaged more than those obtained with zoledronic acid and the expanded Vγ2Vδ2 cells exhibited high cytotoxicity against tumor cells. The high purity of Vγ2Vδ2 cells expanded by PTA increased engraftment success in immunodeficient NOG mice. Even low levels of contaminating αβ T cells resulted in some mice with circulating human αβ T cells rather than Vγ2Vδ2 cells. Vγ2Vδ2 cells from engrafted NOG mice upregulated CD25 and secreted tumor necrosis factor-α and interferon-γ in response to PTA-treated tumor cells. Thus, PTA expands Vγ2Vδ2 T cells to higher purity than zoledronic acid. The high purities allow the successful engraftment of immunodeficient mice without further purification and may speed up the development of allogeneic Vγ2Vδ2 T cell therapies derived from HLA-matched normal donors for patients with poor autologous Vγ2Vδ2 T cell responses.
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http://dx.doi.org/10.1111/cas.13491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834800PMC
March 2018

Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug.

Sci Rep 2017 07 20;7(1):5987. Epub 2017 Jul 20.

Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

Bisphosphonates have benefits in breast cancer and multiple myeloma patients and have been used with adoptive immunotherapy with γδ T cells expressing Vγ2 Vδ2 TCRs. Although treatment with γδ T cells is safe, it has shown limited efficacy. Present bisphosphonates stimulate γδ T cells but were designed to inhibit bone resorption rather than treating cancer and have limited oral absorption, tumor cell entry, and cause bone side effects. The development of phosphate and phosphonate nucleotide prodrugs has led to important drugs for hepatitis C and HIV. Using a similar approach, we synthesized bisphosphonate prodrugs and found that they efficiently limit tumor cell growth. Pivoxil bisphosphonate esters enter cells where esterases convert them to their active acids. The bisphosphonate esters stimulated γδ T cells to secrete TNF-α in response to a variety of tumor cells more efficiently than their corresponding acids. The most active compound, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1- bisphosphonate (7), specifically expanded γδ T cells and stimulated them to secrete interferon-γ and kill tumor cells. In preclinical studies, combination therapy with compound 7 and γδ T cells prolonged survival of mice inoculated with either human bladder cancer or fibrosarcoma cells. Therefore, bisphosphonate prodrugs could enhance the effectiveness of adoptive cancer immunotherapy with γδ T cells.
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http://dx.doi.org/10.1038/s41598-017-05553-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519590PMC
July 2017

Targeting Cancer Cells with a Bisphosphonate Prodrug.

ChemMedChem 2016 12 27;11(24):2656-2663. Epub 2016 Oct 27.

Center for Innovation in Immunoregulative Technology and Therapeutics, Department of Immunology and Cell Biology, Graduate School ofMedicine, Kyoto University, Kyoto, 606-8501, Japan.

Nitrogen-containing bisphosphonates have antitumor activity in certain breast cancer and myeloma patients. However, these drugs have limited oral absorption, tumor cell entry and activity, and cause bone side effects. The potencies of phosphorylated antiviral drugs have been increased by administering them as prodrugs, in which the negative charges on the phosphate moieties are masked to make them lipophilic. We synthesized heterocyclic bisphosphonate (BP) prodrugs in which the phosphonate moieties are derivatized with pivaloyloxymethyl (pivoxil) groups and that lack the hydroxy "bone hook" on the geminal carbon. When the lipophilic BP prodrugs enter tumor cells, they are converted into their active forms by intracellular esterases. The most active BP prodrug, tetrakispivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (7), was found to potently inhibit the in vitro growth of a variety of tumor cell lines, especially hematopoietic cells, at nanomolar concentrations. Consistent with this fact, compound 7 inhibited the prenylation of the RAP1A small GTPase signaling protein at concentrations as low as 1-10 nm. In preclinical studies, 7 slowed the growth of human bladder cancer cells in an immunodeficient mouse model. Thus, 7 is significantly more active than zoledronic acid, the most active FDA-approved BP, and a potential anticancer therapeutic.
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http://dx.doi.org/10.1002/cmdc.201600465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605902PMC
December 2016

Zoledronic acid-induced expansion of γδ T cells from early-stage breast cancer patients: effect of IL-18 on helper NK cells.

Cancer Immunol Immunother 2013 Apr 15;62(4):677-87. Epub 2012 Nov 15.

Department of Surgery, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Human γδ T cells display potent cytotoxicity against various tumor cells pretreated with zoledronic acid (Zol). Zol has shown benefits when added to adjuvant endocrine therapy for patients with early-stage breast cancer or to standard chemotherapy for patients with multiple myeloma. Although γδ T cells may contribute to this additive effect, the responsiveness of γδ T cells from early-stage breast cancer patients has not been fully investigated. In this study, we determined the number, frequency, and responsiveness of Vγ2Vδ2 T cells from early- and late-stage breast cancer patients and examined the effect of IL-18 on their ex vivo expansion. The responsiveness of Vγ2Vδ2 T cells from patients with low frequencies of Vγ2Vδ2 T cells was significantly diminished. IL-18, however, enhanced ex vivo proliferative responses of Vγ2Vδ2 T cells and helper NK cells from patients with either low or high frequencies of Vγ2Vδ2 T cells. Treatment of breast cancer patients with Zol alone decreased the number of Vγ2Vδ2 T cells and reduced their ex vivo responsiveness. These results demonstrate that Zol can elicit immunological responses by γδ T cells from early-stage breast cancer patients, but that frequent in vivo treatment reduces Vγ2Vδ2 T cell numbers and their responsiveness to stimulation.
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http://dx.doi.org/10.1007/s00262-012-1368-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639312PMC
April 2013

Expression and function of PD-1 in human γδ T cells that recognize phosphoantigens.

Eur J Immunol 2011 Feb 11;41(2):345-55. Epub 2011 Jan 11.

Laboratory of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

Programmed cell death-1 (PD-1) is an inhibitory receptor and plays an important role in the regulation of αβ T cells. Little is known, however, about the role of PD-1 in γδ T cells. In this study, we investigated the expression and function of PD-1 in human γδ T cells. Expression of PD-1 was rapidly induced in primary γδ T cells following antigenic stimulation, and the PD-1(+) γδ T cells produced IL-2. When PD-1(+) γδ T cells were stimulated with Daudi cells with and without programmed cell death ligand-1 (PD-L1) expression, the levels of IFN-γ production and cytotoxicity in response to PD-L1(+) Daudi cells were diminished compared to the levels seen in response to PD-L1(-) Daudi cells. The attenuated effector functions were reversed by anti-PD-L1 mAb. When PD-1(+) γδ T cells were challenged by PD-L1(+) tumors pretreated with zoledronate (Zol), which induced γδ TCR-mediated signaling, the resulting reduction in cytokine production was only slight to moderate compared to the reduction seen when PD-1(+) γδ T cells were challenged by PD-L1(-) tumors. In addition, cytotoxic activity of PD-1(+) γδ T cells against Zol-treated PD-L1(+) tumors was comparable to that against Zol-treated PD-L1(-) tumors. These results suggest that TCR triggering may partially overcome the inhibitory effect of PD-1 in γδ T cells.
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http://dx.doi.org/10.1002/eji.201040959DOI Listing
February 2011

Orally administered soymorphins, soy-derived opioid peptides, suppress feeding and intestinal transit via gut mu(1)-receptor coupled to 5-HT(1A), D(2), and GABA(B) systems.

Am J Physiol Gastrointest Liver Physiol 2010 Sep 8;299(3):G799-805. Epub 2010 Jul 8.

Kyoto Univ., Gokasho Uji, Japan.

We previously reported that soymorphins, mu-opioid agonist peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic-like activity. The aim of this study was to investigate the effects of soymorphins on food intake and gut motility, along with their mechanism. We found that soymorphins decreases food intake after oral administration in fasted mice. Orally administered soymorphins suppressed small intestinal transit at lower dose than that of anorexigenic activity. Suppression of food intake and small intestinal transit after oral administration of soymorphins was inhibited by naloxone or naloxonazine, antagonists of mu- or mu(1)-opioid receptor, respectively, after oral but not intraperitoneal administration. The inhibitory activities of small intestinal transit by soymorphins were also inhibited by WAY100135, raclopride, or saclofen, antagonists for serotonin 5-HT(1A), dopamine D(2), or GABA(B) receptor, respectively. We then examined the order of activation of 5-HT(1A), D(2), and GABA(B) receptors, using their agonists and antagonists. The inhibitory effect of 8-hydroxy-2-dipropylaminotetralin hydrobromide, a 5-HT(1A) agonist, after oral administration on small intestinal transit was blocked by raclopride or saclofen. Bromocriptine, a D(2) agonist-induced small intestinal transit suppression, was inhibited by saclofen, but not by WAY100135. Baclofen, a GABA(B) agonist-induced small intestinal transit suppression, was not blocked by WAY100135 or raclopride. These results suggest that 5-HT(1A) activation elicits D(2) followed by GABA(B) activations in small intestinal motility. We conclude that orally administered soymorphins suppress food intake and small intestinal transit via mu(1)-opioid receptor coupled to 5-HT(1A), D(2), and GABA(B) systems.
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http://dx.doi.org/10.1152/ajpgi.00081.2010DOI Listing
September 2010

Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP receptor followed by CCK(1) receptor.

Peptides 2010 May 25;31(5):909-14. Epub 2010 Feb 25.

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.

The anti-hypertensive peptide Arg-Ile-Tyr, which was isolated based on its inhibitory activity (IC(50)=28microM) for angiotensin I-converting enzyme (ACE) from the subtilisin digest of rapeseed protein, exhibited vasorelaxing activity (EC(50)=5.1microM) in an endothelium-dependent manner in the mesenteric artery of spontaneously hypertensive rats (SHRs). We named the peptide rapakinin. ACE inhibitors are reported to induce nitric oxide (NO)-dependent vasorelaxation by elevating the endogenous bradykinin level; however, the vasorelaxation induced by 10microM of rapakinin was blocked only insignificantly by HOE140 or N(G)-nitro-l-arginine methyl ester (l-NAME), antagonists of bradykinin B(2) receptor and an inhibitor of NO synthase, respectively. On the other hand, the vasorelaxation induced by 10microM rapakinin was significantly blocked by indomethacin and CAY10441, a cyclooxygenase (COX) inhibitor and an antagonist of the IP receptor, respectively. The vasorelaxing activity of rapakinin was also blocked by lorglumide, an antagonist of the cholecystokinin (CCK) CCK(1) receptor, although rapakinin has no affinity for the IP and CCK(1) receptors. The vasorelaxation induced by 10microM iloprost, an IP receptor agonist, was also blocked by lorglumide, suggesting that CCK-CCK(1) receptor system is activated downstream of the PGI(2)-IP receptor system. The anti-hypertensive activity of rapakinin after oral administration in SHRs was also blocked by CAY10441 and lorglumide. These results suggest that the anti-hypertensive activity of rapakinin might be mediated mainly by the PGI(2)-IP receptor, followed by CCK-CCK(1) receptor-dependent vasorelaxation.
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http://dx.doi.org/10.1016/j.peptides.2010.02.013DOI Listing
May 2010

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.

Proc Natl Acad Sci U S A 2008 Feb 14;105(8):3011-6. Epub 2008 Feb 14.

Structural Biology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook 2, 12441 Parklawn Drive, Rockville, MD 20852, USA.

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives "exhausted" virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.
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http://dx.doi.org/10.1073/pnas.0712278105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268576PMC
February 2008

Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer.

Proc Natl Acad Sci U S A 2007 Feb 21;104(9):3360-5. Epub 2007 Feb 21.

Department of Gynecology and Obstetrics, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

The ligands for programmed cell death 1 (PD-1), an immunoinhibitory receptor belonging to CD28/cytotoxic T lymphocyte antigen 4 family, are PD-1 ligand 1 and 2 (PD-Ls). Recent reports suggest that the aberrant expression of PD-Ls on tumor cells impairs antitumor immunity, resulting in the immune evasion of the tumor cells. Although an inverse correlation between the expression level of PD-Ls and patients' prognosis has been reported for several malignant tumors, the follow-up period was limited because of the lack of the antibody (Ab) applicable to paraffin-embedded specimens. Here we generated a new Ab against PD-1 ligand 1 (PD-L1) and analyzed the expression level of PD-Ls in human ovarian cancer using paraffin-embedded specimens. Patients with higher expression of PD-L1 had a significantly poorer prognosis than patients with lower expression. Although patients with higher expression of PD-1 ligand 2 also had a poorer prognosis, the difference was not statistically significant. A significant inverse correlation was observed between PD-L1 expression and the intraepithelial CD8(+) T lymphocyte count, suggesting that PD-L1 on tumor cells directly suppresses antitumor CD8(+) T cells. Multivariate analysis showed the expression of PD-L1 on tumor cells and intraepithelial CD8(+) T lymphocyte count are independent prognostic factors. The PD-1/PD-L pathway can be a good target for restoring antitumor immunity in ovarian cancer.
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http://dx.doi.org/10.1073/pnas.0611533104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1805580PMC
February 2007