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Shiro Ono, Kiyomi Yoshimoto, Masaki Matsubara, Nobushiro Nishimura, Hiromasa Kawashima, Ryo Yoneima, Noritaka Yada, Kenji Nishio Department of General Medicine, Nara Medical University, Kashihara, Nara, JapanCorrespondence: Shiro Ono, Department of General Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, 634-8522, Japan, Tel +81-744-29-8905, Fax +81-744-24-5739, Email shiroono0207@naramed-u.ac.jpPurpose: This study aims to investigate the characteristics of Epstein–Barr virus associated-hemophagocytic lymphohistiocytosis (EBV-HLH) and HLH caused by a severe form of infectious mononucleosis (IM-HLH) compared to IM by EBV, and thus also to assist in early diagnosis and providing appropriate treatment.Methods: Data for this analysis were collected from patients at the Department of General Medicine, Nara Medical University, between April 1, 2012, and August 1, 2020. EBV infection was diagnosed using clinical presentation and laboratory tests. HLH diagnosis followed the HLH-2004 protocol, supplemented by plasma EBV DNA detection. A range of clinical and laboratory parameters were collected, including age, sex, clinical outcomes, blood cell counts, hemoglobin, platelets, and various serum values. Plasma EBV DNA levels and flow cytometric analysis (FCM) of bone marrow were performed for HLH cases.Results: Among 1850 hospitalized patients, 14 cases were identified, including 2 HLH cases and 12 IM cases. Comparative analysis revealed distinctive features of HLH, including lower lymphocyte and platelet counts and higher levels of ferritin, soluble interleukin 2 receptor (sIL-2R), and D dimer compared to IM. Notably, one HLH case responded well to corticosteroid monotherapy, while the other case did not, resulting in a fatal outcome. Detection of a cluster of CD5-CD7 lymphocytes in bone marrow is a hallmark of EBV-HLH and useful to distinguish from IM-HLH.Conclusion: This study underscores the importance of early differentiation among EBV-HLH, IM-HLH, and IM in adults to guide appropriate treatment strategies. While specific laboratory markers help distinguish HLH from IM, a more detailed analysis of FCM is crucial for precise diagnosis of HLH cases and tailored therapeutic interventions.Keywords: hemophagocytic lymphohistiocytosis, Epstein–Barr virus infection, Epstein–Barr virus–associated hemophagocytic lymphohistiocytosis, diagnosis of EBV-HLH, flow cytometric analysis of bone marrow

Shiro Ono,1 Chikara Kurata,2 Nobushiro Nishimura,1 Hiromasa Kawashima,1 Ryo Yoneima,1 Yoshiaki Tai,1 Emiko Tatsumi,1 Makiko Miyamoto,1 Noritaka Yada,1 Kiyomi Yoshimoto,1 Kenji Nishio1 1Department of General Medicine, Nara Medical University, Nara, Japan; 2Department of Clinical Laboratory, Nara Medical University, Nara, JapanCorrespondence: Kenji NishioDepartment of General Medicine, Nara Medical University, 840 Shijo-Cho, Kashihara City, Nara 634-8522, JapanTel +81-744-29-8905Fax +81-744-24-5739Email knishio@naramed-u.ac.jpAbstract: The macroenzyme form of aspartate aminotransferase (macro-AST) is formed by the binding of AST with immunoglobulins. Macro-AST excretion from serum is prolonged because of its high molecular weight, leading to increased AST activities. Because of the difficulty in detecting macro-AST through routine laboratory tests, affected patients often undergo repeated examinations, with associated anxiety. We report a case in which macro-AST was detected by assaying the patient’s serum after refrigeration at 4ºC for 3 days. The sample showed progressive loss of AST activity compared with that frozen in the refrigerator, indicating the presence of macro-AST, which was confirmed as a complex with IgG-κ. The cold storage method was validated using many samples obtained from several patients. Use of this simple method to detect macro-AST may avoid unnecessary examinations and patient anxiety even at primary care facilities.Keywords: isolated elevation of aspartate aminotransferase, macroenzyme, macro-AST, cold storage method

Abstract The preferences of consumers for different flavours and aromas in wine are varied and may be explained by inherent factors such as cultural background, wine education and personal taste of the wine consumer. Wine flavour, as perceived in the mouth, includes aroma compounds released through the retronasal pathway, which are shaped by interactions with saliva. Saliva and wine interactions could provide an explanation as to why wine tasters express different preferences for wine. To test this hypothesis, 13 Western and 13 Chinese experienced wine tasters were recruited. Sensory evaluation was performed in formal surroundings to acquire free description-based and perceived sensory intensity data using the Pivot® Profile and continuous scale assessment, respectively. Participants’ saliva samples were collected before the sensory evaluation and spiked into a wine sample to investigate the impact on the wine’s volatile release using comprehensive two-dimensional gas chromatography–mass spectrometry (GC × GC–MS). Saliva samples were subjected to enzyme activity assays and protein composition profiling by Tandem Mass Tag (TMT) quantitative proteomics. The wine tasters showed differences in wine flavour perception, which was supported by the difference in wine volatile release resulting from the addition of saliva. The two groups of participants did not have significant differences in total salivary protein concentrations or the amounts of esterase and α-amylase. However, statistically significant variations in the concentrations of specific proteins (proline-rich proteins (PRPs) and lipocalin-1 (LCN-1); p

Solid phase microextraction (SPME) is a convenient, effective and green sample preparation technique, offering excellent compatibility with different chromatography methods. This review presents an overview of the history of the SPME technique and its early adaptation based on reported patents, followed by some current SPME designs, and intended usage, especially for gas chromatography (GC) applications. Particular focus on quantification approaches in SPME is included, with emphases on the methodology chosen for implementation, correct performance of quantification and validation, and the calibration method. The article concludes by highlighting some innovative SPME-related techniques, some of which are chosen for their quantitative coverage of analytes and their reported applications.

The volatile profile of raw beef contains vital information related to meat quality and freshness. This qualitative study examines the effect of packaging system on the formation and release of volatile organic compounds (VOCs) from raw beef over time, relative to the packaging best before date (BBD). The three packaging systems investigated were modified atmospheric packaging, vacuum packaging, and cling-wrapped packaging. Porterhouse steak samples with the same BBD were analysed from 3 days before to 3 days after the BBD. VOCs were detected via preconcentration using solid-phase microextraction combined with gas chromatography–accurate mass quadrupole time-of-flight mass spectrometry. In total, 35 different VOCs were tentatively identified. Interestingly, there was no clear relationship of the VOCs detected between the three packaging systems, with only carbon disulphide and acetoin, both known volatiles of beef, detected in all three. This is the first study to investigate the effects of commercial packaging systems on VOC formation; it provides an understanding of the relationship of VOCs to the BBD that is essential for the development of on-pack freshness and quality sensors.

Molecular spectroscopic detection techniques, such as Fourier transform infrared spectroscopy (FTIR), provides additional specificity for isomers where often mass spectrometry (MS) fails, due to similar fragmentation patterns. A hyphenated system of gas chromatography (GC) with FTIR via a light-pipe interface is reported in this study to explore a number of GC–FTIR analytical capabilities. Various compound classes were analyzed—aromatics, essential oils and oximes. Variation in chromatographic peak parameters due to the light-pipe was observed via sequentially-located flame ionization detection data. Unique FTIR spectra were observed for separated mixtures of essential oil isomers having similar mass spectra. Presentation of GC×FTIR allows a ‘comprehensive’-style experiment to be developed. This was used to obtain spectroscopic/separation profiles for interconverting oxime species with their individual spectra in the overlap region being displayed on a color contour plot. Partial least square regression provides multivariate quantitative analysis of co-eluting cresol isomers derived from GC–FTIR data. The model resulted in an R2 of 0.99. Prediction was obtained with R2 prediction value of 0.88 and RMSEP of 0.57, confirming the method’s suitability. This study explores the potential of GC–FTIR hyphenation and re-iterates its value to derive unambiguous and detailed molecular information which is complementary to MS.

Biofiltration of air contaminated with VOCs is inexpensive compared with the conventional techniques and very effective for treating large volumes of moist air streams with low concentrations of VOCs. In this study, biofiltration for the purification of polluted air from methanol, a hydrophilic VOC, and toluene, a hydrophobic VOC, was investigated. The experiments were operated using three separated stainless steel biofilters, for methanol, toluene, and a mixture of methanol and toluene, respectively. Biofilter consisted of a mixture of palm shells and activated sludge as a filter-bed material. Only the indigenous microorganisms of the bed medium without any addition of extra inoculum were used throughout the whole process. The polluted air inlet concentration was varied from 0.3-4.7 g/m3 with flow rates ranging from 0.06-0.45 m3/h, equivalent to the empty bed residence times of 9-71 sec. Polluted air was successfully treated by biofiltration, 100% removal efficiencies would be obtained when the air flow rate was lower than 0.45 m3/h. The presence of toluene did not affect the removal rate of methanol while the removal rate of toluene was decreased with the presence of methanol in air stream according to the competition phenomenon.

The article presents a case study of a 30-year-old woman who was taken to the hospital for foetal cardiac abnormality and echogenic foci. Topics discussed include her reproductive history suggesting previous pregnancies and abortions, administration of drugs such as clomiphene citrate and cyclofenil, no history of family genetic disorder, and her foetal echocardiography showing cardiac abnormality.

Background:  Neutrophil NETosis and neutrophil extracellular traps (NETs) play a critical role in pathogenesis of coronavirus disease 2019 (COVID-19)-associated thrombosis. However, the extents and reserve of NETosis, and potential of thrombus formation under shear in whole blood of patients with COVID-19 are not fully elucidated. Neither has the role of recombinant ADAMTS13 or caplacizumab on the accumulation of NETs and thrombus in COVID-19 patients' whole blood under shear been investigated., Methods:  Flow cytometry and microfluidic assay, as well as immunoassays, were employed for the study., Results:  We demonstrated that the percentage of H3Cit + MPO+ neutrophils, indicative of NETosis, was dramatically increased in patients with severe but not critical COVID-19 compared with that in asymptomatic or mild disease controls. Upon stimulation with poly [I:C], a double strain DNA mimicking viral infection, or bacterial shigatoxin-2, the percentage of H3Cit + MPO+ neutrophils was not significantly increased in the whole blood of severe and critical COVID-19 patients compared with that of asymptomatic controls, suggesting the reduction in NETosis reserve in these patients. Microfluidic assay demonstrated that the accumulation of NETs and thrombus was significantly enhanced in the whole blood of severe/critical COVID-19 patients compared with that of asymptomatic controls. Like DNase I, recombinant ADAMTS13 or caplacizumab dramatically reduced the NETs accumulation and thrombus formation under arterial shear., Conclusion:  Significantly increased neutrophil NETosis, reduced NETosis reserve, and enhanced thrombus formation under arterial shear may play a crucial role in the pathogenesis of COVID-19-associated coagulopathy. Recombinant ADAMTS13 or caplacizumab may be explored for the treatment of COVID-19-associated thrombosis., Competing Interests: X.L.Z. is a consultant for Alexion, Sanofi, Takeda, Apollo, GC Biopharma, and Stago. X.L.Z. is also the co-founder of Clotsolution. All other authors have declared no relevant conflict., (Thieme. All rights reserved.)

Necrotising sialometaplasia (NSM) is a non-neoplastic lesion mainly arising in the minor salivary glands of the oral cavity. In the clinical features, NSM shows swelling with or without ulceration, and can mimic a malignant disease such as squamous cell carcinoma. Histopathologically, NSM usually shows the lobular architecture that is observed in the salivary glands. Additionally, acinar infarction and squamous metaplasia of salivary ducts and acini are observable. The aetiology of this lesion remains unknown, although it has a characteristic feature that sometimes requires clinical and histopathological differentiation from malignancy. In this study, we investigated upregulated genes in NSM compared with normal salivary glands, and focused on the TGF-β3 (TGFB3) gene. The results of the histopathological studies clarified that fibroblasts surrounding the lesion express TGF-β3. Moreover, in vitro studies using mouse salivary gland organoids revealed that TGF-β3 suppressed salivary gland cell proliferation and induced squamous metaplasia. We demonstrated a possible aetiology of NSM by concluding that increased TGF-β3 expression during wound healing or tissue regeneration played a critical role in cell proliferation and metaplasia. © 2024 The Pathological Society of Great Britain and Ireland., (© 2024 The Pathological Society of Great Britain and Ireland.)

Background: The response of Canada's research community to the COVID-19 pandemic provides a unique opportunity to examine the country's clinical health research ecosystem. We sought to describe patterns of enrolment across Canadian Institutes of Health Research (CIHR)-funded studies on COVID-19., Methods: We identified COVID-19 studies funded by the CIHR and that enrolled participants from Canadian acute care hospitals between January 2020 and April 2023. We collected information on study-and site-level variables from study leads, site investigators, and public domain sources. We described and evaluated factors associated with cumulative enrolment., Results: We obtained information for 23 out of 26 (88%) eligible CIHR-funded studies (16 randomized controlled trials [RCTs] and 7 cohort studies). The 23 studies were managed by 12 Canadian and 3 international coordinating centres. Of 419 Canadian hospitals, 97 (23%) enrolled a total of 28 973 participants - 3876 in RCTs across 78 hospitals (median cumulative enrolment per hospital 30, interquartile range [IQR] 10-61), and 25 097 in cohort studies across 62 hospitals (median cumulative enrolment per hospital 158, IQR 6-348). Of 78 hospitals recruiting participants in RCTs, 13 (17%) enrolled 50% of all RCT participants, whereas 6 of 62 hospitals (9.7%) recruited 54% of participants in cohort studies., Interpretation: A minority of Canadian hospitals enrolled the majority of participants in CIHR-funded studies on COVID-19. This analysis sheds light on the Canadian health research ecosystem and provides information for multiple key partners to consider ways to realize the full research potential of Canada's health systems., Competing Interests: Competing interests:: Christopher Yarnell reports receiving a Vanier Scholarship from the Canadian Institutes of Health Research (CIHR), outside the submitted work. Kathryn Rowan reports holding the role of programme director, Health & Social Care Delivery Research Programme, UK National Institute for Health and Care Research (NIHR), a part-time, paid secondment from the Intensive Care National Audit and Research Centre. Kusum Menon reports receiving salary support from a CHEO Foundation Research Chair in Pediatric Intensive Care Medicine. Dr. Menon has also received a CIHR grant for researching stress hydrocortisone in pediatric septic shock. Dr. Menon is the chair of the Canadian Critical Care Trials Group. Robert Fowler reports receiving a grant from CIHR in support of the COVID-19 Network of Clinical Trials Networks. Alison Fox-Robichaud is the nominated principal applicant and holder of the CIHR Sepsis Canada network grant. As scientific director of Sepsis Canada, Dr. Fox-Robichaud also reports receiving additional support from McMaster University and Hamilton Health Sciences. John Marshall reports receiving support from CIHR in support of the current manuscript. Dr. Marshall has also participated on a data safety monitoring or advisory board for the AM Pharma REVIVAL Trial and the SHIPSS Trial, and is the chair of the International Forum for Acute Care Trialists. Michelle Kho reports receiving support as the Canada Research Chair in Critical Care Rehabilitation and Knowledge Translation and from Sepsis Canada and the COVID-19 Network of Clinical Trials Networks, in support of the current manuscript. No other competing interests were declared., (© 2024 CMA Impact Inc. or its licensors.)

Purpose: Whole-body silicon photomultiplier positron emission tomography (WB SiPM PET) could be used to diagnose breast cancer spread before lumpectomy. We aimed to investigate the method of measuring the tumor size by WB SiPM PET as a basis for diagnosing breast cancer spread in the breast., Materials and Methods: We retrospectively reviewed 35 breast cancer lesions in 32 patients who underwent WB SiPM PET/CT in the prone position as preoperative breast cancer examinations from September 2020 to March 2022. In all cases, a 20-mm spherical VOI was placed in the normal mammary gland to measure the mean standardised uptake value (SUVmean) and the standard deviation (SD) of 18 F-fluorodeoxyglucose (FDG) uptake. We prepared four types of candidates (SUVmean + 2 SD, SUVmean + 3 SD, 1.5 SUVmean + 2 SD, 1.5 SUVmean + 3 SD) for thresholds for delineating tumor contours on PET images. On the semiautomatic viewer soft, the maximum tumor sizes were measured at each of the four thresholds and compared with the pathological tumor sizes, including the extensive intraductal component (EIC)., Results: The lesion detection sensitivity was 97% for WB SiPM PET. PET detected 34 lesions, excluding 4-mm ductal carcinomas in situ (DCIS). PET measurements at the '1.5 SUVmean + 2 SD' threshold demonstrated values closest to the pathological tumor sizes, including EIC. Moreover, '1.5 SUVmean + 2 SD' had the highest concordance (63%)., Conclusions: The study demonstrated that among various PET thresholds, the '1.5 SUVmean + 2 SD' threshold exhibited the best performance. However, even with this threshold, the concordance rate was limited to only 63%., (© 2024. The Author(s).)

Abstract: Neutrophil NETosis is a unique form of cell death, characterized by the release of decondensed chromatin and antimicrobial contents to the extracellular space, which is involved in inflammation and thrombosis. However, the role of NETosis in the pathogenesis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) and how a targeted therapy affects the accumulation of neutrophil extracellular traps (NETs) under flow remain unknown. Flow cytometry demonstrated that the percentage of neutrophils undergoing NETosis in whole blood from patients with iTTP on admission was significantly increased, with a concurrent decrease in the capacity of inducible NETosis by shigatoxin. After therapy, the percentage of H3Cit+MPO+ neutrophils was significantly reduced, with an improvement in inducible NETosis in these patients. Additionally, little to no NET and thrombus formation was detected underflow in the whole blood from patients with iTTP when platelet counts were very low, but the NET and thrombus formation was dramatically increased following therapy when platelet counts rose to ≥50 × 109/L or were restored to normal with donor platelets. Similarly, there was no thrombus or NET accumulation under flow in the whole blood from vwf-/- mice, but NET accumulation was significantly higher in Adamts13-/- mice than in wild-type mice. Finally, recombinant ADAMTS13 or caplacizumab (or anfibatide) prevented NET and thrombus formation under flow in whole blood from patients with iTTP or from Adamts13-/- mice. These results indicate that neutrophil NETosis and NET formation depend on platelets and von Willebrand factor (VWF) in iTTP, and a targeted therapy such as recombinant ADAMTS13 or caplacizumab may prevent NET and thrombus formation under flow in iTTP., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In this paper, we report a low-cost printing process of carbon nanotube (CNT)-based, all-organic microelectrode arrays (MEAs) suitable for in vitro neural stimulation and recording. Conventional MEAs have been mainly composed of expensive metals and manufactured through high-cost and complex lithographic processes, which have limited their accessibility for neuroscience experiments and their application in various studies. Here, we demonstrate a printing-based fabrication method for microelectrodes using organic CNT/paraffin ink, coupled with the deposition of an insulating layer featuring single-cell-sized sensing apertures. The simple microfabrication processes utilizing the economic and readily available ink offer potential for cost reduction and improved accessibility of MEAs. Biocompatibility of the fabricated microelectrode was suggested through a live/dead assay of cultured neural cells, and its large electric double layer capacitance was revealed by cyclic voltammetry that was crucial for preventing cytotoxic electrolysis during electric neural stimulation. Furthermore, the electrode exhibited sufficiently low electric impedance of 2.49 Ω·cm 2 for high signal-to-noise ratio neural recording, and successfully captured model electric waves in physiological saline solution. These results suggest the easily producible and low-cost printed all-organic microelectrodes are available for neural stimulation and recording, and we believe that they can expand the application of MEA in various neuroscience research.

The aim of this study was to estimate the administered dosage of 81mKr noble gas as calculated by the radioactivity of 81Rb-rubidium hydroxide (81RbOH). The administered dosage was regarded as the total amount of 81mKr noble gas. The radioactivity of 81mKr was calculated using the radioactivity of 81RbOH at the examination, the beginning of inhalation, the inhalation duration and the attenuation volume from the generator to the patient for 81mKr noble gas. In addition, we created an Internet survey and asked National University Hospital in Japan to respond to questions regarding the parameters of concern. Survey responses were provided by 38 hospitals (response rate was 90.5%). Twenty-seven hospitals (64.3%) examined lung ventilation scintigraphy using 81mKr noble gas. The mean administered dosage and the effective dose of lung ventilation scintigraphy using 81mKr noble gas were 35.8 ± 22.1 GBq and 0.97 ± 0.60 mSv, respectively., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII's half-life in circulation and modulates FVIII's immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood., Objectives: To determine the effect of FVIII in VWF proteolysis and homeostasis in vivo., Methods: Mouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question., Results: An intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13 +/- and Adamts13 -/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13 -/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy., Conclusion: Our results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions., Competing Interests: Declaration of competing interests X.L.Z. is a consultant and a member of the advisory boards for Alexion, Apollo, GC Biopharma, Sanofi, Stago, and Takeda. X.L.Z. is also the cofounder of Clotsolution. W.C. holds equity in Ivygen. All other authors have declared no relevant conflict., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Plasma levels of von Willebrand factor (VWF) are significantly elevated in patients with coronavirus disease 2019 (COVID-19). However, dynamic changes and prognostic value of this biomarker in hospitalized patients with COVID-19 have not been determined., Methods: A total of 124 patients infected with SARS-CoV-2 were prospectively recruited for the study. Serial blood samples were obtained at the time of admission (D1), 3-4 days following standard-care treatments (D2), and 1-2 days prior to discharge or any time collected prior to death (D3). Plasma VWF antigen, ADAMTS13 antigen, and ADAMTS13 proteolytic activity, as well as the ratio of VWF/ADAMTS13 were determined, followed by various statistical analyses., Results: On admission, plasma levels of VWF in COVID-19 patients were significantly elevated compared with those in the healthy controls, but no statistical significance was detected among patients with different disease severity. Plasma ADAMTS13 activity but not its antigen levels were significantly lower in patients with severe or critical COVID-19 compared with that in other patient groups. Interestingly, the ratios of plasma VWF antigen to ADAMTS13 antigen were significantly higher in patients with severe or critical COVID-19 than in those with mild to moderate disease. More importantly, plasma levels of VWF and the ratios of VWF/ADAMTS13 were persistently elevated in patients with COVID-19 throughout hospitalization. Kaplan-Meier and Cox proportional hazard regression analyses demonstrated that an increased plasma level of VWF or ratio of VWF/ADAMTS13 at D2 and D3 was associated with an increased mortality rate., Conclusions: Persistent endotheliopathy, marked by the elevated levels of plasma VWF or VWF/ADAMTS13 ratio, is present in all hospitalized patients following SARS-CoV-2 infection, which is strongly associated with mortality.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers ( Cd44 , Oct4 , Nanog , and Sox2 ) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

Background: We compared the prognostic value of the Japanese Society on Thrombosis and Hemostasis (JSTH) disseminated intravascular coagulation (DIC) diagnostic criteria with that of the International Society on Thrombosis and Haemostasis (ISTH) DIC diagnostic criteria for 28-day in-hospital mortality., Methods: We conducted a multicenter prospective cohort study involving two hematology departments, four emergency departments, and one general medicine department in Japan between August 2017 and July 2021. We assessed three ISTH DIC diagnostic criteria categories using low cutoff levels of D-dimer (low D-dimer), high cutoff levels of D-dimer (high D-dimer), and fibrinogen/fibrin degradation products (FDP) as fibrin-related markers. The main outcome was diagnosis-based category additive net reclassification index (NRI)., Results: A total of 222 patients were included: 82 with hematopoietic disorders, 86 with infections, and 54 with other diseases. The 28-day in-hospital mortality rate was 14% (n = 31). The DIC rates diagnosed by the JSTH, ISTH-low D-dimer, high D-dimer, and FDP DIC diagnosis were 52.7%, 47.3%, 42.8%, and 27.0%, respectively. The overall category additive NRI by JSTH DIC diagnosis vs. ISTH-low D-dimer, high D-dimer, and FDP DIC diagnosis were - 10 (95% confidence interval [CI]: -28 to 8, p = 0.282), - 7.8 (95% CI: -26 to 10, p = 0.401), and - 11 (95% CI: -26 to 3, p = 0.131), respectively., Conclusions: JSTH criterion showed the highest sensitivity for DIC diagnosis that did not improve but reflected the same prognostic value for mortality evaluated using ISTH DIC diagnosis criteria. This finding may help clinicians to use JSTH DIC criterion as an early intervention strategy in patients with coagulopathy., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Background: Convalescent plasma infusion (CPI) was given to patients with COVID-19 during the early pandemic with mixed therapeutic efficacy. However, the impacts of CPI on the ADAMTS13-von Willebrand factor (VWF) axis and vascular endothelial functions are not known., Objectives: To determine the impacts of CPI on the ADAMTS13-VWF axis and vascular endothelial functions., Methods: Sixty hospitalized patients with COVID-19 were enrolled in the study; 46 received CPI and 14 received no CPI. Plasma ADAMTS13 activity, VWF antigen, endothelial syndecan-1, and soluble thrombomodulin (sTM) were assessed before and 24 hours after treatment., Results: Patients with severe and critical COVID-19 exhibited significantly lower plasma ADAMTS13 activity than the healthy controls. Conversely, these patients showed a significantly increased VWF antigen. This resulted in markedly reduced ratios of ADAMTS13 to VWF in these patients. The levels of plasma ADAMTS13 activity in each patient remained relatively constant throughout hospitalization. Twenty-four hours following CPI, plasma ADAMTS13 activity increased by ∼12% from the baseline in all patients and ∼21% in those who survived. In contrast, plasma levels of VWF antigen varied significantly over time. Patients who died exhibited a significant reduction of plasma VWF antigen from the baseline 24 hours following CPI, whereas those who survived did not. Furthermore, patients with severe and critical COVID-19 showed significantly elevated plasma levels of syndecan-1 and sTM, similar to those found in patients with immune thrombotic thrombocytopenic purpura. Both syndecan-1 and sTM levels were significantly reduced 24 hours following CPI., Conclusion: Our results demonstrate the relative deficiency of plasma ADAMTS13 activity and endothelial damage in patients with severe and critical COVID-19, which could be modestly improved following CPI therapy., (© 2022 The Author(s).)

In some cases, differentiating thrombotic thrombocytopenic purpura (TTP) from septic disseminated intravascular coagulation (DIC) without measuring ADAMTS13 activity is critical for urgent lifesaving plasma exchange. To investigate whether PLASMIC score without identifying the presence of schistocytes, D-dimer, fibrin/fibrinogen degradation products (FDP), FDP/D-dimer ratio, prothrombin time-international normalized ratio (PT-INR), lactate dehydrogenase (LD), hemoglobin (Hb), and LD/Hb ratio are useful in differentiating patients with TTP from those with septic DIC. Retrospective analysis was conducted on the medical records of the patients with septic DIC (32 patients) or TTP (16 patients). The PLASMIC score and other laboratory measurements all were helpful in differentiating TTP from septic DIC. When dichotomized between high risk (scores 6-7) and intermediate-low risk (scores 0-5), the PLASMIC score predicted TTP with a sensitivity of 75.0% and a specificity of 100%. However, 4 of 16 patients with TTP and 19 of 32 patients with septic DIC showed comparable PLASMIC scores of 4 or 5, making it difficult to distinguish between the two by PLASMIC score alone. Among the measurements examined, the LDH/Hb ratio was the most useful for differentiation. Receiver operating characteristic analysis of the LD/Hb ratio for predicting TTP revealed a cutoff of 53.7 (IU/10 g) (sensitivity 0.94, specificity 0.91). If the LD/Hb ratio was less than 53.7, it was unlikely that the patient had TTP. A combination of the LD/Hb ratio and the PLASMIC score may be useful for distinguishing between TTP and DIC and identifying patients who need rapid plasma exchange or caplacizumab administration., Competing Interests: Declaration of Conflicting InterestsMM, the inventor of the ELISA used to assess ADAMTS13 activity, received funding from Chugai Pharmaceutical. The remaining authors declare no conflicts of interest..

Background: Polymorphous adenocarcinoma is a common intraoral minor salivary gland carcinoma in Western countries but is extremely rare in Japan. The current study aimed to characterize the clinicopathological features and status of molecular alterations of polymorphous adenocarcinoma-associated genes, such as PRKD1/2/3, ARID1A, and DDX3X, in a large cohort of Japanese patients with polymorphous adenocarcinoma., Methods: We examined the cases of 36 Japanese patients with salivary gland polymorphous adenocarcinoma and 26 cases involving histopathological mimics. To detect gene splits, fluorescence in situ hybridization was carried out for polymorphous adenocarcinoma-associated genes. Additionally, we applied a SNaPshot multiplex assay to identify PRKD1 hotspot mutations., Results: This study revealed the indolent clinical course of polymorphous adenocarcinoma with a high 10-year overall survival rate (92.9%), accompanied by occasional local recurrences and cervical lymph node metastasis (23.3%). Twenty cases (55.6%) of polymorphous adenocarcinoma (but none of the mimics) exhibited alterations in at least one polymorphous adenocarcinoma-associated gene. Rearrangement of polymorphous adenocarcinoma-associated genes and PRKD1 E710D were identified in 17 (47.2%) and 4 (11.1%) cases, respectively; one case showed coexisting PRKD3 split and PRKD1 E710D. In the multivariate analysis, high clinical stage (p = 0.0005), the presence of prominent nucleoli (p = 0.0003), and ARID1A split positivity (p = 0.004) were independent risk factors for disease-free survival., Conclusion: Japanese patients with polymorphous adenocarcinoma showed clinicopathological features similar to those reported in Western countries. This study disclosed that polymorphous adenocarcinoma-associated genetic alterations were common and specific findings in polymorphous adenocarcinomas. The diagnostic role and possible prognostic significance of polymorphous adenocarcinoma-associated genetic alterations in polymorphous adenocarcinomas were suggested., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: We compared the prognostic value of serum high mobility group box 1 protein (HMGB1) and histone H3 levels with the International Society on Thrombosis and Haemostasis (ISTH) disseminated intravascular coagulation (DIC) scores for 28-day in-hospital mortality in patients with DIC caused by various underlying diseases., Methods: We conducted a multicenter prospective cohort study including two hematology departments, four emergency departments, and one general medicine department in Japan, between August 2017 and July 2021. We included patients diagnosed with DIC by the ISTH DIC scoring system., Results: Overall, 104 patients were included: 50 with hematopoietic disorders, 41 with infections, and 13 with the other diseases. The 28-day in-hospital mortality rate was 21%. The receiver operator characteristic (ROC) curve showed that a DIC score of 6 points, serum HMGB1 level of 8 ng/mL, and serum histone H3 level of 2 ng/mL were the optimal cutoff points. The odds ratios of more than these optimal cutoff points of the DIC score, serum HMGB1, and histone H3 levels were 1.58 (95% confidence interval [CI]: 0.60 to 4.17, p = 0.36), 5.47 (95% CI: 1.70 to 17.6, p = 0.004), and 9.07 (95% CI: 2.00 to 41.3, p = 0.004), respectively. The area under the ROC curve of HMGB1 (0.74, 95% CI: 0.63 to 0.85) was better than that of the ISTH DIC scores (0.55, 95% CI: 0.43 to 0.67, p = 0.03), whereas that of histone H3 was not (0.71, 95% CI: 0.60 to 0.82, p = 0.07). Calibration and net reclassification plots of HMGB1 identified some high-risk patients, whereas the ISTH DIC scores and histone H3 did not. The category-free net reclassification improvement of HMGB1 was 0.45 (95% CI: 0.01 to 0.90, p = 0.04) and that of histone H3 was 0.37 (95% CI: - 0.05 to 0.78, p = 0.08)., Conclusions: Serum HMGB1 levels have a prognostic value for mortality in patients with DIC. This finding may help physicians develop treatment strategies., (© 2022. The Author(s).)

Despite improvements in diagnosis and treatment, the 5-year survival of oral squamous cell carcinoma (OSCC), no matter the location, remains low, averaging 50%. Telomerase is expressed in 85% of malignancies and may play an important role in human carcinogenesis. Its catalytic component is human telomerase reverse transcriptase (hTERT), which has been thought, but not proven, to be involved in survival with OSCC. We investigated whether hTERT protein was a prognostic factor in OSCC by evaluating its association with clinicopathologic findings and OSCC survival. We found that in comparison to patients with high hTERT expression, patients with low hTERT expression survived significantly longer, including a longer 5-year overall survival. In addition, overall survival was significantly correlated to hTERT expression and the histologic grade and N status of the tumor. Disease-free survival was significantly related to hTERT expression, the histologic grade and N status of the tumor, and mode of invasion. These results suggest that hTERT protein is involved in cervical lymph node metastasis, that its levels may be increased during carcinogenesis, and that it may influence tumor invasion. We believe that this study is the first to demonstrate that OSCC with high hTERT expression carries a worse prognosis than cases with low hTERT expression., Competing Interests: None., (IJCEP Copyright © 2022.)

TAFRO syndrome is a systemic inflammatory, lymphoproliferative disorder, but the pathophysiology of the disease is unknown. It is typically characterized by thrombocytopenia, anasarca, a fever, reticulin fibrosis, renal dysfunction, and organomegaly. However, other manifestations have been also reported. We encountered a 43-year-old man with TAFRO syndrome who showed mediastinal panniculitis, liver damage, and adrenal lesions in addition to the core signs. He achieved complete remission with combination therapy of corticosteroids, tocilizumab, and cyclosporin, and remission was maintained even after drug discontinuation at 15 months. Atypical manifestations and complete remission of TAFRO syndrome were remarkable features of our case.

Purpose: The aim of this study was to evaluate the classification accuracy of specific blood flow reduction patterns in clinical images by deep learning using simulation data., Methods: We obtained Z-score maps for 100 cases each of simulated Alzheimer's disease (AD), simulated dementia with Lewy bodies (DLB), and simulated normal cognition (NC) by performing statistical analysis of the simulation data that provided defects and healthy patient data. The clinical images were determined by reference to radiological reports, and Z-score maps of AD (n=33), DLB (n=20), and NC (n=28) were used. A network was constructed with reference to AlexNet, 4-fold cross-validation was performed using only simulation data, and classification accuracy was evaluated. We also trained the model using the simulation data and classified the clinical images., Results: The accuracy rate of classification between simulations was 96.2% and that of the clinical images was 84.2%., Conclusion: Through deep learning using simulation data, clinical images may be classified with an accuracy of 84.2%.

Background: Two phase II studies in Japan examined the efficacy and safety of nivolumab, a programmed cell death 1 receptor inhibitor, in patients with advanced squamous and non-squamous non-small cell lung cancer (ONO-4538-05 and ONO-4538-06). We examined the long-term efficacy and safety of nivolumab in these patients treated for up to 5 years., Methods: Patients with squamous (N = 35) or non-squamous (N = 76) non-small cell lung cancer received nivolumab (3 mg/kg every 2 weeks) until disease progression/death. Overall survival and progression-free survival were assessed at 5 years after starting treatment in separate and pooled analyses. Safety was evaluated in terms of treatment-related adverse events., Results: A total of 17 patients were alive at the database lock (26 July 2019). The median overall survival (95% confidence interval) and 5-year survival rate were 16.3 (12.4-25.2) months and 14.3% in squamous patients, 17.1 (13.3-23.0) months and 19.4% in non-squamous patients and 17.1 (14.2-20.6) months and 17.8% in the pooled analysis, respectively. Programmed death ligand-1 expression tended to be greater among 5-year survivors than in non-survivors (P = 0.0703). Overall survival prolonged with increasing programmed death ligand-1 expression, with 5-year survival rates of 11.8, 21.8 and 41.7% in patients with programmed death ligand-1 expression of <1, ≥1-<50 and ≥50%, respectively. Treatment-related adverse events in ≥10% of patients (pooled analysis) included rash (15.3%), malaise (14.4%), decreased appetite (14.4%), pyrexia (14.4%) and nausea (10.8%)., Conclusions: Long-term survival with nivolumab was observed in patients with squamous or non-squamous non-small cell lung cancer. No new safety signals were reported after ≥5 years of follow-up., (© The Author(s) 2020. Published by Oxford University Press.)

Aquaporin 3 (AQP3) serves as a water and glycerol transporter facilitating epithelial cell hydration. Recently, the involvement of AQP3 in cancers has been reported. However, the immunohistochemical expression of AQP3 in carcinomas remains controversial. We hypothesized that differences in aquaporin 3 antigen recognition (AQP3 AR) may influence their expressions. Thus, our study aimed to assess the immunostaining patterns of 3 AQP3 AR sites in oral squamous cell carcinoma (OSCC) and to compare the adjacent areas of high-grade epithelial dysplasia (HG-ED) and normal oral mucosa (NOM). The study group included formalin-fixed OSCC samples (n=51) with adjacent regions of HG-ED (n=12) and NOM (n=51). The tissues were stained with anti-AQP3 antibodies (AR sites at amino acid (AA) 250-C terminus, AA180-228, and N terminus AA1-80) by immunohistochemistry. Our results showed that strong membranous immunostaining was observed for AQP3 AR sites at the AA250-C terminus and AA180-228 in all the samples for NOM and weak AQP3 immunostaining for both the AR sites in all the 12 samples for HG-ED. The invasive front of OSCC samples showed that AQP3 AR at the AA250-C terminus decreased in 42/51 samples (82.4%) and AA180-228 in 47/51 samples (92.2%). Conversely, in the AQP3 AR site at N terminus AA1-80, all samples of the NOM showed negative or slightly positive staining in the cytoplasm of the lower layers. AQP3 expression was increased in 12/12 cases (100%) and 46/51 cases (90.2%) in the HG-ED and invasive front of OSCC, respectively. AQP3 may be used as a biomarker for detecting malignant transformations. AQP3 AR site differences influence their immunohistochemical expression in OSCC.