Hidetsugu Fujigaki

ObjectivesReports have shown that the kynurenine pathway, one of the pathways by which tryptophan is metabolized, is activated in patients with diffuse large B-cell lymphoma (DLBCL). Activation of the kynurenine pathway triggers the production of various metabolites, such as kynurenine (Kyn), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), kynurenic acid (KA), and anthranilic acid (AA), which contribute to immune tolerance. The current study aimed to investigate the changes in metabolites of kynurenine pathway in DLBCL patients and evaluate their performance predicting DLBCL.MethodsChanges in metabolites of kynurenine pathway were examined using high-performance liquid chromatography in 35 DLBCL patients (age 61.2 ± 13.5 years) and 44 healthy controls (age 58.5 ± 12.5 years).ResultsDLBCL patients had significantly higher levels of 3-HK, AA, and 3-HAA but lower levels of tryptophan (Trp) and KA compared to healthy controls. Given that the ratio of each metabolite represents the change in the Kyn pathway, the 3-HK/KA ratio was examined. Notably, DLBCL patients had a significantly higher 3-HK/KA ratio compared to healthy controls. In DLBCL, the area under the receiver operative characteristic (ROC) curve for 3-HK/KA (0.999) was higher than that for lactate dehydrogenase (0.885) and comparable to that for soluble interleukin-2 receptor (sIL-2R) (0.997). Based on ROC curve analysis, the 3-HK/KA ratio was found to be useful biomarker for the diagnosis of DLBCL.ConclusionOur results suggest that the 3-HK/KA ratio is a clinically useful biomarker of DLBCL. Moreover, its combination with existing markers, such as sIL-2R, can improve its effectiveness of diagnosing DLBCL.

Background and Purpose Alterations in tryptophan‐kynurenine (TRP‐KYN) pathway are implicated in major depressive disorder (MDD). α7 nicotinic acetylcholine (α7nACh) receptor regulates the hypothalamic–pituitary–adrenal (HPA) axis. We have shown that deficiency of kynurenine 3‐monooxygenase (KMO) induces depression‐like behaviour via kynurenic acid (KYNA; α7nACh antagonist). In this study, we investigated the involvement of the TRP‐KYN pathway in stress‐induced behavioural changes and the regulation of the HPA axis. Experimental Approach Mice were exposed to chronic unpredictable mild stress (CUMS) and subjected to behavioural tests. We measured TRP‐KYN metabolites and the expression of their enzymes in the hippocampus. KMO heterozygous mice were used to investigate stress vulnerability. We also evaluated the effect of nicotine (s.c.) on CUMS‐induced behavioural changes and an increase in serum corticosterone (CORT) concentration. Key Results CUMS decreased social interaction time but increased immobility time under tail suspension associated with increased serum corticosterone concentration. CUMS increased KYNA levels via KMO suppression with microglial decline in the hippocampus. Kmo^+/− mice were vulnerable to stress: they exhibited social impairment and increased serum corticosterone concentration even after short‐term CUMS. Nicotine attenuated CUMS‐induced behavioural changes and increased serum corticosterone concentration by inhibiting the increase in corticotropin‐releasing hormone. Methyllycaconitine (α7nACh antagonist) inhibited the attenuating effect of nicotine. Conclusions and Implications CUMS‐induced behavioural changes and the HPA axis dysregulation could be induced by the increased levels of KYNA via KMO suppression. KYNA plays an important role in the pathophysiology of MDD as an α7nACh antagonist. Therefore, α7nACh receptor is an attractive therapeutic target for MDD.

Preexisting cardiovascular disease (CVD) is a pivotal risk factor for severe coronavirus disease 2019 (COVID-19). We investigated the longitudinal (over 1 year and 9 months) humoral and cellular responses to primary series and booster doses of mRNA COVID-19 vaccines in patients with CVD. Twenty-six patients with CVD who received monovalent mRNA COVID-19 vaccines were enrolled in this study. Peripheral blood samples were serially drawn nine times from each patient. IgG against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) was measured using an enzyme-linked immunosorbent assay. The numbers of interferon-γ-releasing cells in response to SARS-CoV-2 peptides were measured using an enzyme-linked immunospot assay. The RBD-IgG titers increased 2 weeks after the primary series and booster vaccination and waned 6 months after vaccination. The S1-specific T cell responses in patients aged < 75 years were favorable before and after booster doses; however, the Omicron BA.1-specific T cell responses were poor. These results suggest that regular vaccination is useful to maintain long-term antibody levels and has implications for booster dose strategies in patients with CVD. Additional booster doses, including Omicron variant-adapted mRNA vaccines, may be recommended for patients with CVD, regardless of age.

Indoleamine 2,3-dioxygenase 2 (IDO2) is an enzyme of the tryptophan-kynurenine pathway that is constitutively expressed in the brain. To provide insight into the physiological role of IDO2 in the brain, behavioral and neurochemical analyses in IDO2 knockout (KO) mice were performed. IDO2 KO mice showed stereotyped behavior, restricted interest and social deficits, traits that are associated with behavioral endophenotypes of autism spectrum disorder (ASD). IDO2 was colocalized immunohistochemically with tyrosine-hydroxylase-positive cells in dopaminergic neurons. In the striatum and amygdala of IDO2 KO mice, decreased dopamine turnover was associated with increased α-synuclein level. Correspondingly, levels of downstream dopamine D1 receptor signaling molecules such as brain-derived neurotrophic factor and c-Fos positive proteins were decreased. Furthermore, decreased abundance of ramified-type microglia resulted in increased dendritic spine density in the striatum of IDO2 KO mice. Both chemogenetic activation of dopaminergic neurons and treatment with methylphenidate, a dopamine reuptake inhibitor, ameliorated the ASD-like behavior of IDO2 KO mice. Sequencing analysis of exon regions in IDO2 from 309 ASD samples identified a rare canonical splice site variant in one ASD case. These results suggest that the IDO2 gene is, at least in part, a factor closely related to the development of psychiatric disorders.

Background: mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became common. We investigated the optimal timing for inoculation against SARS-COV-2 in the candidates for cardiac surgery under cardiopulmonary bypass (CPB). Methods: In 100 patients with preoperative vaccination, who underwent CPB surgery between July 2021 and February 2022, the IgG against the receptor binding domain (RBD-IgG), with a threshold of >100 binding antibody unit (BAU)/mL for adequate immunity, was measured. Results: The vaccines, including 87 BNT162b2 (Pfizer/BioNTech) and 13 mRNA-1273 (Moderna), were inoculated at 98.8 ± 59.4 days before surgery. The median RBD-IgG titers before surgery, 1 day after surgery, and 1 month after surgery were 166.8, 100.0, and 84.0 BAU/mL, respectively. The standby interval (SBI) from the vaccination to the surgery showed a significantly negative correlations with the RBD-IgG titer before the surgery ( p < 0.001). A cut-off SBI for RBD-IgG >100 BAU/mL before surgery was <81 days with a sensitivity of 76%, specificity of 62%, and area under ROC value of 0.73 ( p = 0.03). The patients with SBI <81 days ( n = 48) had significantly higher RBD-IgG (>100 BAU/mL) than those with SBI ⩾81 days ( n = 52) at all perioperative periods. Conclusions: Although 40% of the RBD-IgG titers reduce 1 day after CPB surgery, the patients who received the SARS-COV-2 vaccination within an 81-day window prior to the surgery maintained a desirable RBD-IgG level, even up to 1 month after surgery. It may be important to schedule the surgery no later than 81 days after the vaccination.

Niacin is a cofactor in many biological reactions related to energy metabolism, redox reactions, DNA repair and longevity. Although it has been considered that increasing energy expenditure increases NAD consumption, little study has directly demonstrated the effect of exercise on niacin nutritional status. We have recently established the niacin insufficient model mice using kynurenine 3-monooxygenase knock out (KMO-/-) mice with niacin-limited diet, which lack the de novo NAD synthesis pathway from tryptophan. To evaluate the effects of chronic endurance exercise on niacin nutritional status, 4 wk old KMO-/- mice were fed 4 or 30 mg/kg nicotinic acid containing diets, and forced to swim in a running water pool every other day for 35 d. The swim-exercised mice fed 4 mg/kg nicotinic acid diet showed lower body weight gain and niacin nutritional markers such as liver and blood NAD, and urine nicotinamide metabolites than the sedentary mice. These animals did not show any difference in the NAD synthesis, NAD salvage and nicotinamide catabolic pathways. Chronic endurance exercise failed to affect any indices in the mice fed the 30 mg/kg nicotinic acid diet. When the diet was exchanged the 4 mg/kg for 30 mg/kg nicotinic acid diet to the mice showed chronic endurance exercise-induced growth retardation, their body weight rapidly increased. These results show that chronic endurance exercise impairs niacin nutritional status in the niacin insufficient mice, and enough niacin intake can prevent this impairment. Our findings also suggest that chronic endurance exercise increases niacin requirement by increase of NAD consumption.

mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have played a key role in reducing morbidity and mortality from coronavirus disease 2019 (COVID-19). We conducted a double-blind, placebo-controlled phase I/II trial to evaluate the safety, tolerability, and immunogenicity of EXG-5003, a two-dose, controllable self-replicating RNA vaccine against SARS-CoV-2. EXG-5003 encodes the receptor binding domain (RBD) of SARS-CoV-2 and was administered intradermally without lipid nanoparticles (LNPs). The participants were followed for 12 months. Forty healthy participants were enrolled in Cohort 1 (5 µg per dose, n = 16; placebo, n = 4) and Cohort 2 (25 µg per dose, n = 16; placebo, n = 4). No safety concerns were observed with EXG-5003 administration. SARS-CoV-2 RBD antibody titers and neutralizing antibody titers were not elevated in either cohort. Elicitation of antigen-specific cellular immunity was observed in the EXG-5003 recipients in Cohort 2. At the 12-month follow-up, participants who had received an approved mRNA vaccine (BNT162b2 or mRNA-1273) >1 month after receiving the second dose of EXG-5003 showed higher cellular responses compared with equivalently vaccinated participants in the placebo group. The findings suggest a priming effect of EXG-5003 on the long-term cellular immunity of approved SARS-CoV-2 mRNA vaccines.

Objective The objective of this study was to estimate the humoral immune response evaluated by immunoglobulin G (IgG) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD-IgG) following the third mRNA COVID-19 vaccination in patients with kidney disease who received immunosuppressive treatment. Methods The primary outcome was RBD-IgG levels after the third SARS-CoV-2 vaccination. The primary comparison was the RBD-IgG levels between patients with kidney disease who received immunosuppressive treatment (n=124) and those who did not (n=33). Results The RBD-IgG levels were significantly lower in the patients with kidney disease who received immunosuppressive treatment than in those who did not receive immunosuppressive treatment. The RBD-IgG levels were lower in patients treated with glucocorticoid monotherapy than in those who did not receive immunosuppressive treatment. Even in patients who received ≤ 5 mg prednisolone, the RBD-IgG levels were significantly lower. Nine of the 10 patients who received rituximab within one year before the first vaccination did not experience seroconversion after the third vaccination. Meanwhile, all nine patients who received rituximab only after the second vaccination experienced seroconversion, even if B cell recovery was insufficient. Patients treated with mycophenolate mofetil plus glucocorticoid plus belimumab had significantly lower RBD-IgG levels than those treated with mycophenolate mofetil plus glucocorticoid. Conclusions The RBD-IgG levels were lower in patients with kidney disease who received immunosuppressive treatment than in those who did not receive immunosuppressive treatment. Low-dose glucocorticoid monotherapy affected the humoral immune response following the third mRNA COVID-19 vaccination.

Cisplatin is a chemotherapeutic agent used to treat many types of malignant tumors. However, irrespective of its potent anticancer properties and efficacy, nephrotoxicity is the dose-limiting factor of cisplatin treatment. Cisplatin infiltrates renal tubular cells in the kidneys and is metabolized by cysteine conjugate-beta lyase 1 (CCBL1) to form highly reactive thiol-cisplatin; this may mediate cisplatin's nephrotoxicity. Therefore, CCBL1 inhibition may prevent cisplatin-induced nephrotoxicity. Using a high-throughput screening assay, we identified 2',4',6'-trihydroxyacetophenone (THA) as an inhibitor of CCBL1. THA inhibited human CCBL1 β-elimination activity in a concentration-dependent manner. We further investigated the preventive effect of THA on cisplatin-induced nephrotoxicity. THA attenuated the effect of cisplatin on the viability of confluent renal tubular cells (LLC-PK1 cells) but had no effect on cisplatin-induced reduction of proliferation in the tumor cell lines (LLC and MDA-MB-231). THA pretreatment significantly attenuated cisplatin-induced increases in blood urea nitrogen, creatinine, cell damage score, and apoptosis of renal tubular cells in mice in a dose-dependent manner. Furthermore, THA pretreatment attenuated cisplatin-induced nephrotoxicity without compromising its antitumor activities in mice bearing subcutaneous syngeneic LLC tumors. THA could help prevent cisplatin-induced nephrotoxicity and may provide a new strategy for cisplatin-inclusive cancer treatments.

OBJECTIVES: We evaluated the applicability of a machine learning based Low-density lipoprotein-cholesterol (LDL-C) estimation method and the influence of the characteristics of the training datasets. METHODS: Three training datasets were chosen from training datasets: health check-up participants at the Resource Center for Health Science (N = 2664), clinical patients at Gifu University Hospital (N = 7409), and clinical patients at Fujita Health University Hospital (N = 14842). Nine different machine learning models were constructed through hyperparameter tuning and 10-fold cross-validation. Another test dataset of another 3711 clinical patients at Fujita Health University Hospital was selected as the test set used for comparing and validating the model against the Friedewald formula and the Martin method. RESULTS: The coefficients of determination of the models trained on the health check-up dataset produced coefficients of determination that were equal to or inferior to those of the Martin method. In contrast, the coefficients of determination of several models trained on clinical patients exceeded those of the Martin method. The means of the differences and the convergences to the direct method were higher for the models trained on the clinical patients' dataset than for those trained on the health check-up participants' dataset. The models trained on the latter dataset tended to overestimate the 2019 ESC/EAS Guideline for LDL-cholesterol classification. CONCLUSION: Although machine learning models provide valuable method for LDL-C estimates, they should be trained on datasets with matched characteristics. The versatility of machine learning methods is another important consideration.

BACKGROUND/AIM: Cancer cells with high anchorage independence can survive and proliferate in the absence of adhesion to the extracellular matrix. Under anchorage-independent conditions, cancer cells adhere to each other and form aggregates to overcome various stresses. In this study, we investigated the cytomorphology and gene expression signatures of oral cancer cell aggregates. MATERIALS AND METHODS: Two oral cancer-derived cell lines, SAS and HSC-3 cells, were cultured in a low-attachment plate and their cytomorphologies were observed. The transcriptome between attached and detached SAS cells was examined using gene expression microarrays. Subsequently, gene enrichment analysis and Ingenuity Pathway Analysis were performed. Gene expression changes under attached, detached, and re-attached conditions were measured via RT-qPCR. RESULTS: While SAS cells formed multiple round-shaped aggregates, HSC-3 cells, which had lower anchorage independence, did not form aggregates efficiently. Each SAS cell in the aggregate was linked by desmosomes and tight junctions. Comparative transcriptomic analysis revealed 1,698 differentially expressed genes (DEGs) between attached and detached SAS cells. The DEGs were associated with various functions and processes, including cell adhesion. Moreover, under the detached condition, the expression of some epithelial genes (DSC3, DSP, CLDN1 and OCLN) were up-regulated. The changes in both cytomorphology and epithelial gene expression under the detached condition overall returned to their original ones when cells re-attached. CONCLUSION: The results suggest specific cytomorphological and gene expression changes in oral cancer cell aggregates. Our findings provide insights into the mechanisms underlying anchorage-independent oral cancer cell aggregation and reveal previously unknown potential diagnostic and therapeutic molecules.

Phencyclidine (PCP) causes mental symptoms that closely resemble schizophrenia through the inhibition of the glutamatergic system. The kynurenine (KYN) pathway (KP) generates metabolites that modulate glutamatergic systems such as kynurenic acid (KA), quinolinic acid (QA), and xanthurenic acid (XA). Kynurenine 3-monooxygenase (KMO) metabolizes KYN to 3-hydroxykynurenine (3-HK), an upstream metabolite of QA and XA. Clinical studies have reported lower KMO mRNA and higher KA levels in the postmortem brains of patients with schizophrenia and exacerbation of symptoms in schizophrenia by PCP. However, the association between KMO deficiency and PCP remains elusive. Here, we demonstrated that a non-effective dose of PCP induced impairment of prepulse inhibition (PPI) in KMO KO mice. KA levels were increased in the prefrontal cortex (PFC) and hippocampus (HIP) of KMO KO mice, but 3-HK levels were decreased. In wild-type C57BL/6 N mice, the PPI impairment induced by PCP is exacerbated by KA, while attenuated by 3-HK, QA and XA. Taken together, KMO KO mice were vulnerable to the PPI impairment induced by PCP through an increase in KA and a decrease in 3-HK, suggesting that an increase in the ratio of KA to 3-HK (QA and XA) may play an important role in the pathophysiology of schizophrenia.

The COVID-19 vaccine will be safe and effective in solid organ transplant recipients (SOTs). However, the blunted antibody responses were also of concern. Few studies have reported prolonged serologic follow-up after 2 doses of BNT162b2 vaccine in SOTs. We performed a single-center, prospective observational study of 78 SOTs who received 2 doses of BNT162b2 vaccine. We identified the trajectory of antibody titers after vaccination among SOTs with or without mycophenolate mofetil (MMF) or withdrawn from MMF. We found low seroconversion rates (29/42: 69%) and low antibody titers in SOTs treated with MMF. An inverse linear relationship between neutralizing antibody titers and MMF concentration was confirmed in restricted cubic spline plots (P for effect < .01, P for nonlinearity = .08). For the trajectory of antibody responses, seroconversion and improved antibody titers were observed after withdrawal from MMF in SOTs who showed seronegative or low antibody titers at the first visit after 2 doses of vaccine (P for effect < .01, P for nonlinearity < .05, and P for interaction < .01). We identified increased B-cell counts after withdrawal from MMF (P < .01). The recovery of antibody responses was seen in SOTs withdrawn from MMF. The trajectories of antibody responses were modified by MMF administration.

OBJECTIVES: Patients with cardiovascular disease are vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. Although SARS-CoV2 vaccination may be effective, its impact on surgical patients is not well studied. We investigated the effects of cardiovascular surgery, especially under cardiopulmonary bypass (CPB), on the antibody titres after SARS-CoV2 vaccination. METHODS: A prospective observational study was designed for patients undergoing surgery between July and November 2021. The immunoglobulin G against the receptor-binding domain was measured and antibody preserved rate (APR) was calculated from perioperative titres comparison. RESULTS: Enrolled 63 study patients were divided into 39 undergoing surgery with CPB (Group CPB) and 24 without CPB (Group None). Preoperative vaccines were BNT162b2 (Pfizer/BioNTech) (n = 58, 92%) and mRNA-1273 (Moderna) (n = 5, 8%). While immunoglobulin G against the receptor-binding domain titres did not significantly decrease after surgery in Group None, they decreased significantly in Group CPB from 21.80 [11.15, 37.85] to 11.95 [6.80, 18.18] U/ml (P < 0.001) a day after surgery, 11.40 [7.85, 22.65] U/ml (P < 0.001) 14 days after surgery and 7.60 [4.80, 17.60] U/ml (P < 0.001) a month after surgery. The APRs a day after the surgery were significantly lower in Group CPB (0.46 [0.41, 0.60]) than in Group None (0.80 [0.68, 0.87]) (P < 0.001). CONCLUSIONS: The SARS-CoV2 antibody titres significantly decreased with lower APRs immediately after surgery under CPB. Based on our informative results, careful considerations of vaccination schedule might be required for surgery under CPB.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is known to cause not only respiratory but also neuropsychiatric symptoms, which are assumed to be derived from a cytokine storm and its effects on the central nervous systems. Patients with COVID-19 who develop severe respiratory symptoms are known to show severe neuropsychiatric symptoms such as cerebrovascular disease and encephalopathy. However, the detailed clinical courses of patients with neuropsychiatric symptoms caused by mild or asymptomatic COVID-19 remain poorly understood. Here, we present a case of COVID-19 who presented with severe and prolonged neuropsychiatric symptoms subsequent to mild respiratory symptoms. CASE PRESENTATION: A 55-year-old female with COVID-19 accompanied by mild respiratory symptoms showed delusion, psychomotor excitement, and poor communication ability during quarantine outside the hospital. Considering her diminished respiratory symptoms, her neuropsychiatric symptoms were initially regarded as psychogenic reactions. However, as she showed progressive disturbance of consciousness accompanied by an abnormal electroencephalogram, she was diagnosed with post-COVID-19 encephalopathy. Although her impaired consciousness and elevated cytokine level improved after steroid pulse therapy, several neuropsychiatric symptoms, including a loss of concentration, unsteadiness while walking, and fatigue, remained. CONCLUSIONS: This case suggests the importance of both recognizing that even apparently mild COVID-19-related respiratory symptoms can lead to severe and persistent neuropsychiatric symptoms, and elucidating the mechanisms, treatment, and long-term course of COVID-19-related neuropsychiatric symptoms in the future.

To fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), mass vaccination has begun in many countries. To investigate the usefulness of a serological assay to predict vaccine efficacy, we analyzed the levels of IgG, IgM, and IgA against the receptor-binding domain (RBD) of SARS-CoV-2 in the sera from BNT162b2 vaccinated individuals in Japan. This study included 219 individuals who received two doses of BNT162b2. The levels of IgG, IgM, and IgA against RBD were measured by enzyme-linked immunosorbent assay before and after the first and second vaccination, respectively. The relationship between antibody levels and several factors, including age, gender, and hypertension were analyzed. Virus-neutralizing activity in sera was measured to determine the correlation with the levels of antibodies. A chemiluminescent enzyme immunoassay (CLEIA) method to measure IgG against RBD was developed and validated for the clinical setting. The levels of all antibody isotypes were increased after vaccination. Among them, RBD-IgG was dramatically increased after the second vaccination. The IgG levels in females were significantly higher than in males. There was a negative correlation between age and IgG levels in males. The IgG levels significantly correlated with the neutralizing activity. The CLEIA assay measuring IgG against RBD showed a reliable performance and a high correlation with neutralizing activity. Monitoring of IgG against RBD is a powerful tool to predict the efficacy of SARS-CoV-2 vaccination and provides useful information in considering a personalized vaccination strategy for COVID-19. IMPORTANCE Mass vaccination campaigns using mRNA vaccines against SARS-CoV-2 have begun in many countries. Serological assays to detect antibody production may be a useful tool to monitor the efficacy of SARS-CoV-2 vaccination in individuals. Here, we reported the induction of antibody isotype responses after the first and second dose of the BNT162b2 vaccine in a well-defined cohort of employees in Japan. We also reported that age, gender, and hypertension are associated with differences in antibody response after vaccination. This study not only provides valuable information with respect to antibody responses after BNT162b2 vaccination in the Japanese population but also the usefulness of serological assays for monitoring vaccine efficacy in clinical laboratories to determine a personalized vaccination strategy for COVID-19.