Publications by authors named "Yumiko Higuchi"

22 Publications

  • Page 1 of 1

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

Int J Hematol 2021 Jun 12. Epub 2021 Jun 12.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.

Introduction: We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder.

Materials And Methods: Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting.

Results: Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells.

Conclusion: We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.
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http://dx.doi.org/10.1007/s12185-021-03174-yDOI Listing
June 2021

A Novel Amino Acid Substitution, Fibrinogen Bβp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

Int J Mol Sci 2020 Dec 10;21(24). Epub 2020 Dec 10.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto 390-8621, Japan.

We identified a novel heterozygous variant, Bβp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of fibrinogen.
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http://dx.doi.org/10.3390/ijms21249422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764081PMC
December 2020

Changes in serum citrullinated fibrinogen concentration associated with the phase of bacteremia patients.

Clin Chim Acta 2021 Jan 4;512:127-134. Epub 2020 Nov 4.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan.

Background: Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase.

Methods: We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers.

Result: Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p < 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p < 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01).

Conclusion: Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.
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http://dx.doi.org/10.1016/j.cca.2020.10.038DOI Listing
January 2021

Congenital fibrinogen disorder with a compound heterozygote possessing two novel FGB mutations, one qualitative and the other quantitative.

Thromb Res 2020 12 20;196:152-158. Epub 2020 Aug 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.

Introduction: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia.

Materials And Methods: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP).

Results And Conclusions: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bβp.N170K substitution) on different alleles. We did not detect shortened Bβ-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bβp.N170K-fibrinogen-CHO was almost same as normal Bβ-fibrinogen-CHO. TCFP of plasma Bβp.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bβp.N170K fibrinogen in patient's plasma. Bβp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.
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http://dx.doi.org/10.1016/j.thromres.2020.08.031DOI Listing
December 2020

Comparison of molecular structure and fibrin polymerization between two Bβ-chain N-terminal region fibrinogen variants, Bβp.G45C and Bβp.R74C.

Int J Hematol 2020 Sep 19;112(3):331-340. Epub 2020 Jun 19.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

We identified two heterozygous dysfibrinogenemias, Bβp.Gly45Cys (Kyoto VII; K-VII) and Bβp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bβp.G45C has been well analyzed; however, that of Bβp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bβp.G45C and Bβp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bβp.G45C was impaired more than Bβp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bβp.G45C and thrombosis for patients with Bβp.R74C.
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http://dx.doi.org/10.1007/s12185-020-02919-5DOI Listing
September 2020

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

Int J Lab Hematol 2020 Apr 20;42(2):190-197. Epub 2020 Jan 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

Introduction: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens.

Methods: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking.

Results: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results.

Conclusion: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.
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http://dx.doi.org/10.1111/ijlh.13152DOI Listing
April 2020

In Vivo Administration of Recombinant Human Granulocyte Colony-Stimulating Factor Increases the Immune Effectiveness of Dendritic Cell-Based Cancer Vaccination.

Vaccines (Basel) 2019 Sep 19;7(3). Epub 2019 Sep 19.

Department of Cancer Immunology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.

Significant recent advances in cancer immunotherapeutics include the vaccination of cancer patients with tumor antigen-associated peptide-pulsed dendritic cells (DCs). DC vaccines with homogeneous, mature, and functional activities are required to achieve effective acquired immunity; however, the yield of autologous monocyte-derived DCs varies in each patient. Priming with a low dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16-18 h prior to apheresis resulted in 50% more harvested monocytes, with a significant increase in the ratio of CD11cCD80 DCs/apheresed monocytes. The detection of antigen-specific cytotoxic T lymphocytes after Wilms' tumor 1-pulsed DC vaccination was higher in patients treated with rhG-CSF than those who were not, based on immune monitoring using tetramer analysis. Our study is the first to report that DC vaccines for cancer immunotherapy primed with low-dose rhG-CSF are expected to achieve higher acquired immunogenicity.
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http://dx.doi.org/10.3390/vaccines7030120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789603PMC
September 2019

γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal.

Thromb Res 2019 Oct 20;182:150-158. Epub 2019 Aug 20.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address:

Background: The fibrinogen γ-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including γD318Y and γΔN319D320, have been reported at the high affinity Ca-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, γD318Y and γK321E.

Materials And Methods: γD318Y and γK321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking.

Results: γD318Y did not show any polymerization by thrombin or batroxobin, similar to γΔN319D320, whereas γK321E had slightly impaired polymerization. The functions of Ca binding, hole 'a', and the "D-D" interaction were markedly reduced in γD318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the γ-module of these variants were inconsistent with polymerization results. The degree of structural changes in γD318Y was moderate relative to those in γD318A and γD320A, which had markedly impaired polymerization, and γK321E, which showed slightly impaired polymerization.

Conclusion: Our results suggest that no polymerization of γD318Y or γΔN319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring γD318A and γD320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the γD318Y γ-module influenced the location and/or orientation of the adjacent β-module, which led to impaired "B-b" interactions.
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http://dx.doi.org/10.1016/j.thromres.2019.08.017DOI Listing
October 2019

Leaf shape deters plant processing by an herbivorous weevil.

Nat Plants 2019 09 2;5(9):959-964. Epub 2019 Sep 2.

The Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

The shapes of plant leaves are remarkably diverse, but their ecological functions are largely unknown. Reports on the effects of leaf shape on biotic interactions such as herbivory are especially scarce, partly because herbivorous insects rarely rely on leaf shape for host selection. Here, we show that leaf shape acts as a physical deterrent against a leaf-processing herbivore. Plants in the genus Isodon (Lamiaceae) host a specialized leaf-rolling weevil (Apoderus praecellens) whose ovipositing females process an entire leaf into a leaf roll to serve as larval food and shelter. Among the species of Isodon, I. umbrosus var. hakusanensis is exceptional in that it has deeply lobed leaves. Because leaf processing follows a consistent sequence of complex behaviours, the unusual shape of I. umbrosus leaves may disrupt this process. Under both natural and laboratory conditions, female weevils preferred I. trichocarpus, a close relative with non-lobed leaves, over I. umbrosus. Nutritional properties of the leaves do not explain this preference because weevil larvae developed equally well on both hosts. Modifying the non-lobed I. trichocarpus leaves to mimic the shape of I. umbrosus leaves also discouraged leaf processing. Leaf processing often terminated because weevils failed to complete the inspection routine on I. umbrosus leaves. Leaf shape may be an important but overlooked factor that affects the interactions between plants and leaf-processing herbivores.
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http://dx.doi.org/10.1038/s41477-019-0505-xDOI Listing
September 2019

Fibrin monomers derived from thrombogenic dysfibrinogenemia, Naples-type variant (BβAla68Thr), showed almost entirely normal polymerization.

Thromb Res 2018 12 4;172:1-3. Epub 2018 Oct 4.

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.

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http://dx.doi.org/10.1016/j.thromres.2018.10.004DOI Listing
December 2018

Feasibility and Immune Response of WT1 Peptide Vaccination in Combination with OK-432 for Paediatric Solid Tumors.

Anticancer Res 2018 04;38(4):2227-2234

Department of Regenerative Medicine, Kanazawa Medical University, Ishikawa, Japan.

Background/aim: Wilms' tumor 1 (WT1) peptide-based vaccination has been reported for its potential usefulness in targeting several cancers. The adjuvant drug OK-432 is known to have potent immunomodulation and therapeutic properties when applied in cancer treatment and may, thus, be important to trigger the appropriate immunological response in paediatric patients with a solid tumor that are vaccinated with a WT1 peptide.

Patients And Methods: Paediatric patients with a solid tumor were vaccinated with a WT1 peptide and OK-432 once every 2 weeks, for a total of seven times.

Results: Of the 24 patients, 18 completed the scheduled vaccinations. Sixteen patients had local skin symptoms and/or fever. In 1 patient, anaphylactic symptoms emerged at the time of the final injection, but these quickly subsided after the treatment. WT1-specific immunological responses were observed in 4 patients (22.2%). WT1 and HLA class I expression were confirmed in 100% and 85% of primary tumors, respectively.

Conclusion: WT1 peptide vaccine therapy combined with OK-432 appears to be relatively safe for children. However further studies in a larger number of patients are necessary to confirm its safety and efficacy.
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http://dx.doi.org/10.21873/anticanres.12465DOI Listing
April 2018

Interferon-α-inducible Dendritic Cells Matured with OK-432 Exhibit TRAIL and Fas Ligand Pathway-mediated Killer Activity.

Sci Rep 2017 02 13;7:42145. Epub 2017 Feb 13.

Centre for Advanced Cell Therapy, Shinshu University Hospital, Matsumoto, Nagano, Japan.

Active human dendritic cells (DCs), which efficiently induce immune responses through their functions as antigen-presenting cells, exhibit direct anti-tumour killing activity in response to some pathogens and cytokines. These antigen-presenting and tumour killing abilities may provide a breakthrough in cancer immunotherapy. However, the mechanisms underlying this killer DC activity have not been fully proven, despite the establishment of interferon-α (IFN-α)-generated killer DCs (IFN-DCs). Here mature IFN-DCs (mIFN-DCs), generated from IFN-DCs primed with OK-432 (streptococcal preparation), exhibited elevated expression of CD86 and human leukocyte antigen-DR (minimum criteria for DC vaccine clinical trials) as well as antigen-presenting abilities comparable with those of mature IL-4-DCs (mIL-4-DCs). Interestingly, the killing activity of mIFN-DCs, which correlated with the expression of CD56 (natural killer cell marker) and was activated via the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand pathway, was stronger than that of IFN-DCs and remarkably stronger than that of mIL-4-DCs. Therefore, mIFN-DCs exhibit great potential as an anti-cancer vaccine that would promote both acquired immunity and direct tumour killing.
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http://dx.doi.org/10.1038/srep42145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304184PMC
February 2017

Dendritic Cell-Based Adjuvant Vaccination Targeting Wilms' Tumor 1 in Patients with Advanced Colorectal Cancer.

Vaccines (Basel) 2015 Dec 11;3(4):1004-18. Epub 2015 Dec 11.

Shinshu Cancer Center, Shinshu University Hospital, Matsumoto 8621, Japan.

Despite significant recent advances in the development of immune checkpoint inhibitors, the treatment of advanced colorectal cancer involving metastasis to distant organs remains challenging. We conducted a phase I study to investigate the safety and immunogenicity of Wilms' tumor (WT1) class I/II peptides-pulsed dendritic cell DC vaccination for patients with advanced colorectal cancer. Standard treatment comprising surgical resection and chemotherapy was followed by one course of seven biweekly administrations of 1-2 × 10⁷ DCs with 1-2 KE of OK-432 (streptococcal preparation) in three patients. Clinical efficacy was confirmed based on WT1 expression using immunohistochemistry on paraffin-embedded tissues and immune monitoring using tetramer analysis and enzyme-linked immunosorbent spot (ELISPOT) assays. WT1 expression with human leukocyte antigen (HLA)-class I molecules was detected in surgical resected tissues. Adverse reactions to DC vaccinations were tolerable under an adjuvant setting. WT1-specific cytotoxic T cells were detected by both modified WT1-peptide/HLA-A*24:02 tetramer analysis and/or interferon-γ-producing cells through the use of ELISPOT assays after the first DC vaccination. Immunity acquired from DC vaccination persisted for two years with prolonged disease-free and overall survival. The present study indicated that DC vaccination targeting WT1 demonstrated the safety and immunogenicity as an adjuvant therapy in patients with resectable advanced colorectal cancer.
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http://dx.doi.org/10.3390/vaccines3041004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4693229PMC
December 2015

Enzyme-Linked Immunosorbent Spot Assay for the Detection of Wilms' Tumor 1-Specific T Cells Induced by Dendritic Cell Vaccination.

Biomedicines 2015 Dec 4;3(4):304-315. Epub 2015 Dec 4.

Center for Advanced Cell Therapy, Shinshu University Hospital, Matsumoto 390-8621, Japan.

Background: Despite recent advances in cancer immunotherapy and the development of various assays for T cell assessment, a lack of universal standards within immune monitoring remains. The objective of this study was to evaluate the enzyme-linked immunosorbent spot (ELISpot) assay in comparison with major histocompatibility complex-tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy.

Methods: The ELISpot assay was performed on peripheral blood mononuclear cells to assess reproducibility, daily precision, and linearity using HLA-A*24:02-restricted Cytomegalovirus peptide. Wilms' tumor 1 (WT1) antigen-specific cytotoxic T cells were then evaluated by both the ELISpot assay and WT1 tetramer analysis in peripheral blood from 46 cancer patients who received DC vaccinations pulsed with human leukocyte antigen (HLA)-A*24:02-restricted modified WT1 peptides.

Results: The ELISpot assay was proven to have reproducibility (coefficient of variation (CV) ranged from 7.4% to 16.3%), daily precision (CV ranged from 5.0% to 17.3%), and linearity ( = 0.96-0.98). WT1-specific immune responses were detected by the ELISpot assay in 34 out of 46 patients (73.9%) post-vaccination. A Spearman's rank-correlation coefficient of 0.82 between the ELISpot assay and WT1 tetramer analysis was obtained.

Conclusion: This is the first report of a comparison of an ELISpot assay and tetramer analysis in the context of dendritic cell (DC)-based cancer immunotherapy. The ELISpot assay has reproducibility, linearity, and excellent correlation with the WT1 tetramer analysis. These findings suggest that the validated ELISpot assay is useful to monitor the acquired immunity by DC vaccination targeting WT1.
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http://dx.doi.org/10.3390/biomedicines3040304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5344226PMC
December 2015

Dendritic cell-based immunotherapy targeting Wilms' tumor 1 in patients with recurrent malignant glioma.

J Neurosurg 2015 Oct 7;123(4):989-97. Epub 2015 Aug 7.

Department of Neurosurgery, Shinshu University School of Medicine;

Object: Dendritic cell (DC)-based vaccination is considered a potentially effective therapy against advanced cancer. The authors conducted a Phase I study to investigate the safety and immunomonitoring of Wilms' tumor 1 (WT1)-pulsed DC vaccination therapy for patients with relapsed malignant glioma.

Methods: WT1-pulsed and/or autologous tumor lysate-pulsed DC vaccination therapy was performed in patients with relapsed malignant gliomas. Approximately 1 × 10(7) to 2 × 10(7) pulsed DCs loaded with WT1 peptide antigen and/or tumor lysate were intradermally injected into the axillary areas with OK-432, a streptococcal preparation, at 2-week intervals for at least 5-7 sessions (1 course) during an individual chemotherapy regimen.

Results: Ten patients (3 men, 7 women; age range 24-64 years [median 39 years]) with the following tumors were enrolled: glioblastoma (6), anaplastic astrocytoma (2), anaplastic oligoastrocytoma (1), and anaplastic oligodendroglioma (1). Modified WT1 peptide-pulsed DC vaccine was administered to 7 patients, tumor lysate-pulsed DC vaccine to 2 patients, and both tumor lysate-pulsed and WT1-pulsed DC vaccine to 1 patient. The clinical response was stable disease in 5 patients with WT1-pulsed DC vaccination. In 2 of 5 patients with stable disease, neurological findings improved, and MR images showed tumor shrinkage. No serious adverse events occurred except Grade 1-2 erythema at the injection sites. WT1 tetramer analysis detected WT1-reactive cytotoxic T cells after vaccination in patients treated with WT1-pulsed therapy. Positivity for skin reaction at the injection sites was 80% (8 of 10 patients) after the first session, and positivity remained for these 8 patients after the final session.

Conclusions: This study of WT1-pulsed DC vaccination therapy demonstrated safety, immunogenicity, and feasibility in the management of relapsed malignant gliomas.
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http://dx.doi.org/10.3171/2015.1.JNS141554DOI Listing
October 2015

Safety and tolerability of allogeneic dendritic cell vaccination with induction of Wilms tumor 1-specific T cells in a pediatric donor and pediatric patient with relapsed leukemia: a case report and review of the literature.

Cytotherapy 2015 Mar 4;17(3):330-5. Epub 2014 Dec 4.

Center for Advanced Cell Therapy, Shinshu University Hospital, Matsumoto, Japan. Electronic address:

A 15-year-old girl with acute lymphoblastic leukemia received allogeneic dendritic cell vaccination, pulsed with Wilms tumor 1 (WT1) peptide, after her third hematopoietic stem cell transplantation (HSCT). The vaccines were generated from the third HSCT donor, who was her younger sister, age 12 years. The patient received 14 vaccines and had no graft-versus-host disease or systemic adverse effect, aside from grade 2 skin reaction at the injection site. WT1-specific immune responses were detected after vaccination by both WT1-tetramer analysis and enzyme-linked immunosorbent spot assay. This strategy may be safe, tolerable and even feasible for patients with a relapse after HSCT.
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http://dx.doi.org/10.1016/j.jcyt.2014.10.003DOI Listing
March 2015

CD20-negative CD138-positive leukemic large cell lymphoma with plasmablastic differentiation with an IgH/MYC translocation in an HIV-positive patient.

Intern Med 2009 1;48(7):559-62. Epub 2009 Apr 1.

Nagano Red Cross Hospital, Matsumoto.

A 49-year-old HIV-positive Japanese man was referred to our hospital for multiple skin nodules. Many plasmablastic atypical lymphocytes were observed in the peripheral blood. He was diagnosed with diffuse large B cell lymphoma (DLBCL) by a biopsy of the inguinal lymph node. IgH/MYC translocation was detected by in situ hybridization of the lymph node and chromosomal analysis of bone marrow cells showed 46, XY, t(8 ; 14)(q24 ; q32)add(14)(q32), der(21)t(1 ; 21)(q12 ; p11). He showed a transient response to multi-agent chemotherapy, and during the course of salvage chemotherapy, he died of urinary infection. This case has unique clinical features compared with previously reported DLBCLs with plasmablastic differentiation.
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http://dx.doi.org/10.2169/internalmedicine.48.1359DOI Listing
July 2009

Simple polymerase chain reaction for the detection of mutations and deletions in the epidermal growth factor receptor gene: applications of this method for the diagnosis of non-small-cell lung cancer.

Clin Chim Acta 2009 Mar 24;401(1-2):68-72. Epub 2008 Nov 24.

Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

Background: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the responses to the tyrosine kinase inhibitors gefitinib and erlotinib in patients with non-small-cell lung cancer (NSCLC). Although various methods for detecting EGFR gene mutations have been developed, they have several disadvantages. We attempted to establish a new method for the detection of EGFR gene mutations with the use of paraffin-embedded samples.

Methods: The detections of T790M mutations in exon 20 and L858R mutations in exon 21 are based on the principle of allele-specific oligonucleotide polymerase chain reaction (PCR). We also designed PCR primers that enable to detect all types of deletions in exon 19. We assessed the basic performance efficiency of this method, and to confirm its clinical applicability, we performed PCR using DNA extracted from 66 tissue sections that were obtained from patients with NSCLC and embedded in paraffin.

Results: The sensitivity of this method for the detection of deletions or mutations was as low as 0.5%. In the 66 subjects whose samples were analyzed, we detected the following deletions and mutations in the EGFR gene: 11 deletions in exon 19, 8 L858R mutations, and 1 double mutation of L858R and T790M.

Conclusion: The present method is sensitive and specific for the detection of deletions and mutations in the EGFR gene and is thus suitable for use in laboratory tests.
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http://dx.doi.org/10.1016/j.cca.2008.11.018DOI Listing
March 2009

[Additional chromosomal changes in impaired chimeric analysis of a patient with relapsed leukemia after bone marrow transplantation].

Rinsho Ketsueki 2008 Feb;49(2):109-14

Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

Chimerism analysis by polymerase chain reaction amplification of short tandem repeats (PCR-STR) has become a routine diagnostic procedure for evaluating grafts and assessing the likeliness of original disease recurrence after allogeneic stem cell transplantation. Following a sex-mismatched hematopoietic stem cell transplantation (HSCT), we monitored the clinical course of a 61-year old male AML M6 patient with trisomy 8 using PCR-STR with a TH01 locus on 11p15 and fluorescence in situ hybridization (FISH) analysis specific for alpha satellite DNA on chromosome 8. Ten months after HSCT, FISH analysis showed 24.8% recipient cells, but PCR-STR demonstrated 100% donor type chimerism. Further XY FISH analysis of May-Grünwald-Giemsa-stained bone marrow samples clearly demonstrated relapse of the original disease and G-banding analysis of bone marrow samples at relapse showed that an additional chromosomal abnormality, del(11) (p10), had deleted the PCR-STR detection site in all recipient type cells. As such, clinicians should consider the possibility that unexpected karyotype changes may invalidate PCR-STR analysis findings, especially when conflicting results appear among chimerism analyses.
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February 2008