Evaluation of the prognostic impact of pathologic tumor regression grade on patients with colorectal cancer after preoperative chemoradiotherapy.

BACKGROUND: The prognostic value of the pathological response to preoperative chemoradiotherapy (CRT) in rectal cancer (RC) remains unknown. OBJECTIVES: We aimed to assess the predictive value of the response to CRT that was derived from an evaluation of the histological findings (whole-section vs. representative-section sampling) and attempted to determine an objective cut-off value for the tumor regression grade (TRG). METHODS: We examined the association of the TRG with the outcomes (recurrence-free survival [RFS] and overall survival [OS]) of 78 patients with RC. Patients with RC treated with preoperative CRT were divided into development (30 cases) and validation (48 cases) cohorts. The TRG was classified as grades I (Ia, Ib), II, and III. The cut-off value was determined by receiver operating characteristic (ROC) curve analysis. RESULTS: The TRG determined from whole-section sampling versus representative-section sampling was more strongly correlated with patient survival. We found that in both cohorts, patients with a cut-off value of <73% had a poor prognosis. Finally, the cut-off value was found to be an independent predictive factor in both univariate and multivariate analysis. CONCLUSIONS: The TRG that was used to evaluate patients with RC who underwent preoperative CRT was an independent prognostic factor for outcome.

Mitochondrial-derived microprotein MOTS-c attenuates immobilization-induced skeletal muscle atrophy by suppressing lipid infiltration.

Mitochondrial open reading frame of the 12S ribosomal RNA type-c (MOTS-c), a mitochondrial microprotein, has been described as a novel regulator of glucose and lipid metabolism. In addition to its role as a metabolic regulator, MOTS-c prevents skeletal muscle atrophy in high fat-fed mice. Here, we examined the preventive effect of MOTS-c on skeletal muscle mass, using an immobilization-induced muscle atrophy model, and explored its underlying mechanisms. Male C57BL/6J mice (10 wk old) were randomly assigned to one of the three experimental groups: nonimmobilization control group (sterilized water injection), immobilization control group (sterilized water injection), and immobilization and MOTS-c-treated group (15 mg/kg/day MOTS-c injection). We used casting tape for the immobilization experiment. After 8 days of the experimental period, skeletal muscle samples were collected and used for Western blotting, RNA sequencing, and lipid and collagen assays. Immobilization reduced ∼15% of muscle mass, whereas MOTS-c treatment attenuated muscle loss, with only a 5% reduction. MOTS-c treatment also normalized phospho-AKT, phospho-FOXO1, and phospho-FOXO3a expression levels and reduced circulating inflammatory cytokines, such as interleukin-1b (IL-1ß), interleukin-6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1), in immobilized mice. Unbiased RNA sequencing and its downstream analyses demonstrated that MOTS-c modified adipogenesis-modulating gene expression within the peroxisome proliferator-activated receptor (PPAR) pathway. Supporting this observation, muscle fatty acid levels were lower in the MOTS-c-treated group than in the casted control mice. These results suggest that MOTS-c treatment inhibits skeletal muscle lipid infiltration by regulating adipogenesis-related genes and prevents immobilization-induced muscle atrophy.NEW & NOTEWORTHY MOTS-c, a mitochondrial microprotein, attenuates immobilization-induced skeletal muscle atrophy. MOTS-c treatment improves systemic inflammation and skeletal muscle AKT/FOXOs signaling pathways. Furthermore, unbiased RNA sequencing and subsequent assays revealed that MOTS-c prevents lipid infiltration in skeletal muscle. Since lipid accumulation is one of the common pathologies among other skeletal muscle atrophies induced by aging, obesity, cancer cachexia, and denervation, MOTS-c treatment could be effective in other muscle atrophy models as well.

Autoantibodies to nuclear valosin-containing protein-like protein: systemic sclerosis-specific antibodies revealed by in vitro human proteome.

OBJECTIVES: To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. METHODS: We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases, and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The Presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electric medical records. RESULTS: PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. CONCLUSION: Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement.

Luteolin Protects Against 6-Hydoroxydopamine-Induced Cell Death via an Upregulation of HRD1 and SEL1L.

Parkinson's Disease (PD) is caused by many factors and endoplasmic reticulum (ER) stress is considered as one of the responsible factors for it. ER stress induces the activation of the ubiquitin-proteasome system to degrade unfolded proteins and suppress cell death. The ubiquitin ligase 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation 1 (HRD1) and its stabilizing molecule, the suppressor/enhancer lin-12-like (SEL1L), can suppress the ER stress via the ubiquitin-proteasome system, and that HRD1 can also suppress cell death in familial and nonfamilial PD models. These findings indicate that HRD1 and SEL1L might be key proteins for the treatment of PD. Our study aimed to identify the compounds with the effects of upregulating the HRD1 expression and suppressing neuronal cell death in a 6-hydroxydopamine (6-OHDA)-induced cellular PD model. Our screening by the Drug Gene Budger, a drug repositioning tool, identified luteolin as a candidate compound for the desired modulation of the HRD1 expression. Subsequently, we confirmed that low concentrations of luteolin did not show cytotoxicity in SH-SY5Y cells, and used these low concentrations in the subsequent experiments. Next, we demonsrated that luteolin increased HRD1 and SEL1L mRNA levels and protein expressions. Furthermore, luteolin inhibited 6-OHDA-induced cell death and suppressed ER stress response caused by exposure to 6-OHDA. Finally, luteolin did not reppress 6-OHDA-induced cell death when expression of HRD1 or SEL1L was suppressed by RNA interference. These findings suggest that luteolin might be a novel therapeutic agent for PD due to its ability to suppress ER stress through the activation of HRD1 and SEL1L.

The clinicopathological impact of tumor-associated macrophages in patients with cutaneous malignant melanoma.

BACKGROUND: Tumor-associated macrophages (TAMs) are an immune component of the cutaneous malignant melanoma (CMM) microenvironment and affect tumor growth. TAMs can polarize into different phenotypes, that is, proinflammatory M1 and anti-inflammatory M2 macrophages. However, the role of the macrophage phenotype in CMM remains unclear. METHODS: We examined 88 patients with CMM. Tissue microarrays were constructed, and the density of M1 and M2 macrophages was analyzed by immunohistochemistry. Immune cells coexpressing CD68 and phosphorylated signal transducer and activator of transcription 1 (pSTAT1) were considered M1 macrophages, whereas those coexpressing CD68 and c-macrophage activating factor (c-Maf) were defined as M2 macrophages. These TAMs were counted, and the relationships between the density of M1 and M2 macrophages and clinicopathological factors including prognosis were investigated. RESULTS: The CD68/c-Maf score ranged from 0 to 34 (median: 5.5). The patients were divided based on the median score into the CD68/c-Maf high (≥5.5) and low (<5.5) expression groups. Univariate and multivariate analyses revealed that CD68/c-Maf expression was an independent predictive factor for progression-free survival and an independent prognostic factor for overall survival. CD68/pSTAT1 expression was found in only two patients. CONCLUSION: We suggest that CD68/pSTAT1 coexpression is rarely observed in patients with CMM, and high CD68/c-Maf expression is a predictor of worse prognosis in these patients.

A Case of Autoimmune Hepatitis Complicated by Undiagnosed Factor VII Deficiency: A Pitfall of Coagulopathy.

Prothrombin time (PT) is a key parameter for assessing the severity of liver disease. We present the case of a 37-year-old woman with severe acute liver injury due to autoimmune hepatitis. Although prednisolone drastically improved her hepatocyte function, her PT did not recover to the reference range. A review of her medical records revealed that the patient had normal transaminase levels and prolonged PT 2 years previously. Further examinations of her coagulopathy revealed that she had low factor VII activity, suggesting a diagnosis of factor VII deficiency. Our experience suggests that altered coagulopathy should be considered in cases of liver injury with an extraordinary PT.

A novel reagent for the screening of haptoglobin-deficient blood donors.

BACKGROUND AND OBJECTIVES: In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization, leading to serious transfusion reactions. Therefore, implementation of a consistent supply of haptoglobin-deficient fresh frozen plasma is crucial. We developed a novel reagent to facilitate large-scale identification of haptoglobin-deficient individuals as potential donors of plasma products. MATERIALS AND METHODS: We established mouse monoclonal anti-haptoglobin-producing cell lines (three clones) using the hybridoma method by immunizing mice with the haptoglobin protein. Purified antibodies were conjugated with carboxylate-modified polystyrene latex beads and used for haptoglobin measurements by the latex agglutination method using an automatic analyser (LABOSPECT008). Samples with low protein concentrations were re-examined by enzyme-linked immunosorbent assay to confirm the results. Additionally, the haptoglobin gene was amplified by polymerase chain reaction to confirm the haptoglobin deletion allele (Hpdel ). RESULTS: From February to October 2022, 7476 blood donor samples were screened. Two haptoglobin-deficient and 21 low-haptoglobin-expressing individuals were identified. Two haptoglobin-deficient donors were found homozygous for Hpdel , and 19 (90%) of the 21 low-haptoglobin-expressing individuals were heterozygous for Hpdel , which includes the first reported case of heterozygous Hpdel /HpJohnson . CONCLUSION: We developed a new reagent for the detection of haptoglobin deficiency, which is automatable and inexpensive and appears useful for large-scale screening of blood donors.

Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc-scOX40L, Fc-sc4-1BBL, Fc-scCD27L (CD70) and Fc-scGITRL. Without intentional cross-linking, these soluble Fc-scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc-scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc-scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

A multifunction flow channel switching module for multi-column capillary gas chromatography using silicon pneumatic microvalves.

Decades ago, multi-column GC was developed for separating analytes which cannot be separated with a single column by switching the flow channels and introducing them into multiple columns. Formerly, a channel switching system consisted of stainless-tube-type packed columns and multi-port switching valves which were connected by stainless-steel pipes. These systems utilizing packed columns are still widely used today, mainly for gas analysis, especially for refinery gas analysis and natural gas analysis related to oil refining. However, there have been no new development updates in terms of hardware for decades. Since multi-port switching valves are used, there are problems such as the high frequency of valve replacements due to wear of seals, high running costs as well as long downtimes. In recent years, a heart-cutting multidimensional capillary GC using pressure switching devices such as the Deans switch has been put into practical use. Compared to the switching valves, the pressure switching devices have no wearing parts and are maintenance-free. Multi-dimensional capillary GC using the pressure-switching method cannot be applied to the analysis of low-boiling-point gasses because the retention capacity of the capillary column is low and the column inlet pressure is far from the optimum value for switching. Because of this another related factors, it has not been able to completely replace the traditional methods. In this study, an ON/OFF type silicon pneumatic microvalve was designed and fabricated by semiconductor manufacturing technology, and a flow channel switching module was developed by mounting microvalves on a metallic channel plate which is made by diffusion bonding. The flow channel switching module using silicon pneumatic microvalves has a heat resistance of up to 310 °C, can withstand pressures up to 1.5 MPA or more, and a durability that can withstand over 2 million opening and closing operations. In addition, the reproducibility of the gas sample analysis showed good reproducibility values of RSD 0.1% or less for peak areas and RSD 0.01 to 0.04% for retention time. Flow path switching without the use of pressure switching simplifies method development in several ways. For instance, by incorporating the use of valves that can be opened and closed independently, it was possible to integrate several methods such as heart cut, precut, column switching, and backflushing flow path layouts.

Integrated Evaluation of Inflammatory, Nutritional, and Sarcopenia Markers to Predict Survival in Metastatic Breast Cancer Patients.

BACKGROUND/AIM: The prognosis of a cancer patient is influenced by the tumor-related factors, as well as by various patient-related factors. We evaluated the association between inflammatory and nutritional factors and their outcomes, including the prognosis and therapeutic course, in patients with metastatic breast cancer. PATIENTS AND METHODS: In this observational retrospective study, we evaluated 35 patients. The inflammatory and nutritional markers before systemic therapy included the lymphocyte count, neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), systemic immune-inflammatory index (SII), systemic inflammatory response index (SIRI), pan-immuno-inflammatory values (PIV), prognostic nutritional index (PNI), Glasgow prognostic score (GPS), and psoas muscle index (PMI). RESULTS: Triple-negative, low PNI, and GPS 2 were correlated with worse overall survival in the univariable analysis. The GPS was the only independent predictor of overall survival [hazard ratio=5.85, 95% confidence interval=1.15-29.68, p<0.01]. The time to treatment failure of first-line therapy in patients with GPS 2 was significantly shorter than that in patients with GPS 0/1 (p<0.01). CONCLUSION: The GPS was an independent predictive marker for overall survival in patients with metastatic breast cancer.

The Tokyo 2020 Olympic and Paralympic pharmacy services during the COVID-19 pandemic.

BACKGROUND: During the Tokyo 2020 Games, pharmacists were required to provide appropriate pharmacotherapeutic care to athletes and officials at the polyclinic. Owing to the worldwide COVID-19 pandemic that was prevalent at the Games, it was imperative to strengthen infection control measures in the setting of such a major sporting event and to prevent and minimize the spread of COVID-19. OBJECTIVE: This study reports on the COVID-19 infection control measures and services provided by the pharmacy at the Tokyo 2020 Games. By evaluating pharmacy operations that took place under the COVID-19 protocol, this study provides insights for the organization of future sporting events, specifically their medical facilities. METHODS: Infection control measures in the pharmacy were implemented in accordance with the manual for dealing with COVID-19 infections. The number and content of issued and dispensed prescriptions were obtained from the electronic medical records and pharmacy department systems. These data were compared with those of the London 2012 Games, which were used as a reference for the pharmacy operations at the Tokyo 2020 Games. RESULTS: The participating pharmacists were fully trained in infection control measures. The number of prescriptions issued during the Olympics and Paralympics were 1120 and 1022, respectively. Prescriptions issued at the fever clinic accounted for 4% of the total number (77/2142). No influenza antiviral medications were prescribed, though medications to alleviate cold-like symptoms were issued. Compared to the London 2012 Games, there was a decrease (-59%) in the number of prescriptions. CONCLUSION: The positive impact of COVID-19 infection control measures was evident. The volume of prescriptions at the Tokyo 2020 Games was lower than that at the London 2012 Games. It was inferred that this was due to thorough infection control measures as well as enhanced pre-entry medical checkups before entering Japan, which reduced the incidence of diseases.

Genetic hyperactivation of Nrf2 causes larval lethality in Keap1a and Keap1b-double-knockout zebrafish.

The Keap1-Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds.

Exploring Predictive Risk Factors of Infusion Reactions with First Pertuzumab Administration in HER2-positive Breast Cancer Patients: A Single Institution Experience.

Introduction: Pertuzumab and trastuzumab are monoclonal antibodies used for treating HER2-positive breast cancer. These anti-HER2 antibodies may induce infusion reactions (IR), mainly upon first administration. We investigated factors predicting IR in the initial pertuzumab treatment for HER2-positive breast cancer. Methods: We retrospectively reviewed the medical records of 57 patients who first received pertuzumab-containing treatment in our hospital from January 2014 to February 2021. The frequency of IR during or immediately after pertuzumab administration was examined. We also analyzed patient characteristics that may represent possible risk factors for IR. Results: The incidence rate of IR was 44% (25/57). Red blood cell count (P < 0.001), hemoglobin (Hb) concentration (P = 0.0011), and hematocrit (P < 0.001) immediately before pertuzumab administration were significantly lower in patients with IR than in those without. In patients with IR, erythrocyte levels immediately before pertuzumab treatment were significantly lower than baseline when having received anthracycline-containing chemotherapy within three months. Logistic regression analysis showed that a decrease in Hb levels was a significant risk factor for IR (log odds ratio = -17). According to the receiver-operating characteristic analysis, a 10% decrease in Hb after anthracycline-containing treatment was the best cut-off value for predicting IR (sensitivity: 88%; specificity: 77%; area under the curve: 0.87). Conclusions: Our study showed a higher incidence of IR after pertuzumab treatment than in clinical trials. There was a strong association between IR occurrence and erythrocyte levels lower than baseline in the group that received anthracycline-containing chemotherapy immediately before.

Influence of different tooth etchants on bur-cut and uncut enamel.

This study aimed to evaluate the effect of pre-etching for two-step self-etch adhesive bonding to bur-cut and uncut enamel. Bur-cut and uncut enamel surfaces were assigned to surface treatments of no etchant (CT), Enamel Conditioner (EC; Shofu, Kyoto, Japan), or K-etchant syringe (KE; Kuraray Noritake Dental, Tokyo, Japan). The bonded samples were thermal cycled and evaluated by microshear bond strength (µSBS). The adhesive interface after acid-base challenge and the conditioned enamel surfaces were morphologically analyzed using scanning electron microscopy (SEM). For bur-cut enamel, EC and KE pre-etching significantly improved µSBS. For uncut enamel, KE showed higher µSBS than EC. SEM observation revealed that only KE removed the prismless layer of the uncut enamel surface. EC could improve enamel bonding and appears to be a substitute for phosphoric acid, especially for bur-cut enamel. However, uncut enamel could not be effectively conditioned by EC with a lower bond strength than KE.

Characterization of the Geometrical and Electronic Structures of the Active Site and Its Effects on the Surrounding Environment in Reduced High-Potential Iron-Sulfur Proteins Investigated by the Density Functional Theory Approach.

The high-potential iron-sulfur protein (HiPIP) is an electron-transporting protein that functions in the photosynthetic electron-transfer system and possesses a cubane-type [4Fe-4S] cluster in the active center. Characterization of the geometrical and electronic structures of the [4Fe-4S] cluster leads to an understanding of the functions in HiPIP, which are expected to be influenced by the environment surrounding the [4Fe-4S] cluster. This work characterized the geometrical and electronic structures of the [4Fe-4S] cluster in the reduced HiPIP and evaluated their effects on the protein environment using the density functional theory (DFT) approach. DFT calculations showed that the structural asymmetry and spin delocalization between iron atoms allowed for the acquisition of a unique stable geometrical and electronic structure in the open-shell singlet. In addition, the formation of an Fe-Fe bond accompanying the spin delocalization was found to depend on the interatomic distance. A comparison of the calculated stable structures with and without consideration of the amino acids around the [4Fe-4S] cluster demonstrated that the surrounding amino acids stabilized the unique geometrical and electronic structure of the [4Fe-4S] cluster in HiPIP.

Differential Expression in the Tumor Microenvironment of mRNAs Closely Associated with Colorectal Cancer Metastasis.

BACKGROUND: Metastasis of colorectal cancer (CRC) is a major cause of CRC-related mortality. However, the detailed molecular mechanism of CRC metastasis remains unknown. A recent study showed that the tumor microenvironment, which includes cancer cells and the surrounding stromal cells, plays a major role in tumor invasion and metastasis. Identification of altered messenger RNA (mRNA) expression in the tumor microenvironment is essential to elucidation of the mechanisms responsible for tumor progression. This study investigated the mRNA expression of genes closely associated with metastatic CRC compared with non-metastatic CRC. METHODS: The samples examined were divided into cancer tissue and isolated cancer stromal tissue. The study examined altered mRNA expression in the cancer tissues using The Cancer Genome Atlas (TCGA) (377cases) and in 17 stromal tissues obtained from our laboratory via stromal isolation using an array-based analysis. In addition, 259 patients with CRC were enrolled to identify the association of the candidate markers identified with the prognosis of patients with stage 2 or 3 CRC. The study examined the enriched pathways identified by gene set enrichment analysis (GSEA) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) module in both the TCGA dataset and isolated stromal tissue. RESULTS: As a result, whereas tenascin-C, secreted phosphoprotein 1 and laminin were expressed in metastatic CRC cells, olfactory receptors (ORs) 11H1 and OR11H4 were expressed in stromal tissue cells isolated from metastatic CRC cases. Finally, upregulated expression of tenascin-C and OR11H4 was correlated with the outcome for CRC patients. CONCLUSION: The authors suggest that upregulated expression levels of tenascin-C and OR11H1 play an important role in CRC progression.

Pharmacy services at the Tokyo 2020 Olympic and Paralympic Games: perspectives of the pharmacy workforce.

OBJECTIVES: To evaluate the awareness of the volunteer pharmacy workforce of medication use and their satisfaction with the pharmacy services of the Tokyo 2020 Olympic and Paralympic Games from a pharmacist's perspective. METHODS: A questionnaire was developed from related articles in published peer-reviewed journals and modified prior to distribution to the whole population of pharmacists serving at the Tokyo 2020 Olympic and Paralympic Games. Validity tests were conducted based on expert opinions and Cronbach's alpha (0.79). The questionnaire consisted of demographics (11 questions), knowledge of medication use in sports (8 questions) and satisfaction on the provision of the service (5 questions). Responses using a 5-point-Likert scale, from strongly agree (5) to strongly disagree (1), and two free text questions were analysed with descriptive statistics. RESULTS: The response rate was 86% (n=32/37). Overall, the pharmacists reported a high awareness of medication use. Specifically, questions on the prohibited list of medications (mean 4.0±SD 0.7), COVID-19 policy (3.8±0.9), use of alternative non-prohibited medications (3.6±1.0) and therapeutic use exemptions (3.5±0.9). Moreover, they rated high satisfaction with the pharmacy service they provided. However, rates were ≤3 for knowledge of the International Olympic Committee Needle Policy (2.6±1.0), Medication Importation Declaration (2.9±1.0) and communication skills (3.0±1.0). CONCLUSION: Pharmacists were confident and satisfied with the pharmacy service at the games. The study confirms the importance of prior training and education. Game-specific policies and strategies to improve communication skills should be included in the pharmacy education for future Games.

OX40 Ligand-Mannose-Binding Lectin Fusion Protein Induces Potent OX40 Cosignaling in CD4+ T Cells.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4+ T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4+ T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.