藤垣 英嗣

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.

プログラニュリン(PGRN)の測定方法の確立を目的として新たに開発されたPGRN測定試薬の基礎的検討をおこなった。未治療の関節リウマチ(RA)患者50例と変形性ひざ関節症(OA)患者37例,健常者100例についてPGRN測定試薬を用いて測定した。さらにRA治療薬のTNF関連生物学的製剤Infliximab(IFX)投与患者5例について経時変を観察した。再現性は2濃度の併行精度4.4%,4.2%,5日間の室内再現精度8.7%,7.8%であった。健常者のPGRN(Mean±SDng/mL)は男性37.9±8.9ng/ml,女性40.5±8.7ng/mlであった。RA患者は62.9±12.4ng/ml,OA患者55.1±12.0ng/mlでありRA患者が有意に高値であった。またIFX治療患者では治療前から約1年後のPGRNは軽度(約20%)の上昇が認められた。本試薬の基礎的検討は良好であり,RA患者ではOA患者,健常者対照に比べて有意に高く従来の報告と一致していた。(著者抄録)

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.

To determine the efficacy of SARS-CoV-2 mRNA vaccination for allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients, we measured antibody titer serially in 92 allo-HSCT patients. Among the evaluable 87 patients, median age at vaccination was 53 years (range, 18-75). The average time between allo-HSCT and vaccination was 3.3 years (range, 0.5-15.7). One month after the second dose, 70 patients (80.5%) had a positive response, whereas 17 patients (19.5%) had a negative response (< 20 U/mL). Only patients older than 44 years had a negative response. Low IgM level was the only significant predictor of vaccine failure in elderly patients. When antibody response before and after the third vaccination was examined in 47 patients, antibodies increased significantly from a median of 18.3 U/mL to 312.6 U/mL (P < 0.01). The median antibody titer after the third vaccination of healthy individuals (n = 203) was 426.4 U/mL, which was comparable to that of patients (P = 0.2). The antibody titer after the third mRNA vaccination increased even in patients whose first two mRNA vaccinations failed. These findings suggest that allo-HSCT recipients should receive the mRNA vaccine regularly.

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.

＜文献概要＞藤田医科大学は新型コロナウイルス(SARS-CoV-2)感染拡大の社会情勢を鑑み,大量処理が可能なPCR検査システムの構築を行った.運用は医療科学部の教員および附属病院臨床検査部の臨床検査技師によって行い,現在では1日約1,500検体のPCR検査が可能な大規模システムを構築した.本システムはSARS-CoV-2感染の終息後には研究用途へ返還するが,再度のパンデミックの発生時には即座に再構築が可能となるPCR検査システムとなっている.

LPSは炎症の成立に重要な役割を果たしている。LPSはLPS結合蛋白(LBP)と結合し、さらにCD14が結合することで炎症シグナルを細胞内に誘導する。今回、我々は長期療養(5年以上)のRA患者を対象に、高感度法によるLBPの定量とACPA抗体の変動を調査した。また炎症の指標とされているCRPおよびIL-6の測定を同時に行いバイオマーカーとしてのLBPの臨床的意義について検討を行った。血中LBP値(Mean±SD)は健常者3.69±1.26μg/mL、OA群6.05±2.40μg/mL、RA群11.10±5.16μg/mLであり、RA群で最も有意に高値を示した(p<0.0001)。さらにstage、classの亢進に伴いLBPが増加した。また、ACPAとは相関(r=0.410)を認め、陽性群と陰性群での比較では陽性例が有意(p<0.002)に高値であった。このことから高感度法によるLBPの測定はRAの新たな病態解析の指標の一つになる可能性が有るものと考える。(著者抄録)

ADVIAシリーズ用に開発されたIL-6測定試薬をCentaur XPTを用いてその有用性について検討した。併行精度(%)2.2〜7.7,室内再現精度(%)2.9〜6.8で精度について問題ないと考えられる。また従来法のELISA法(r=0.966),CLEIA法(r=0.977)との相関は良好であった。健常者(1.35±0.70pg/mL),不明関節炎患者(3.49±6.29pg/mL)と関節リウマチ患者(17.56±31.28pg/mL)の比較において関節リウマチ患者が有意(p<0.001)に高値であった。さらに操作性については,既に検査室に設置されている装置を用いるため操作手順を大きく変更することはなく日常検査項目と同時測定が可能である。今後,炎症性疾患や感染症などの早期診断バイオマーカーとして臨床の場における新たな診断価値が提唱されるものと考えている。(著者抄録)

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.

LPS結合蛋白(LBP)はLPSを単球に提示するなど,前炎症段階における初期免疫に関与する。今回我々は化学発光免疫測定装置へ搭載可能な新規LBP測定試薬の基本性能評価を行った。再現性は4%未満であり,共存物質の影響を受けないこと,良好な希釈直線性を有することが確認された。健常対照群のLBP値はELISA法やLA法の既報よりも高値であった。また,RA患者において健常対照群よりも有意に高値であった。本試薬は良好な基本性能を有しており新たな炎症関連マーカーとしての活用が期待される。(著者抄録)

汎用生化学自動分析装置を用いたSARS-CoV-2抗体定量の有用性について基礎的検討を行った。陰性検体としてSARS-CoV-2流行以前に健康診断を受診した300名から得られた300検体の血清を用い、新型コロナワクチンを2回接種した本学教職員30名から経時的に得られた血清150検体を用いた。異なる2種類の精度管理血清(血清1:陰性域、血清2:陽性域)を20回連続測定した。その結果、血清1と血清2の測定値はそれぞれ1.69±0.58、31.70±0.89、変動係数はそれぞれ34.5%と2.8%であった。期待値を大きく外れるポイントは認められず良好な希釈直線性が得られ、理論値3000BAU/mLの検体を2n倍希釈し測定した。その結果、300BAU/mLから400BAU/mL付近においてフッキング現象が観察され、プロゾーンの存在が示された。本試薬は多くの検査室が所有する汎用自動分析装置で使用できると考えられた。

＜文献概要＞藤田医科大学は新型コロナウイルス(SARS-CoV-2)感染拡大の社会情勢を鑑み,大量処理が可能なPCR検査システムの構築を行った.運用は医療科学部の教員および附属病院臨床検査部の臨床検査技師によって行い,現在では1日約1,500検体のPCR検査が可能な大規模システムを構築した.本システムはSARS-CoV-2感染の終息後には研究用途へ返還するが,再度のパンデミックの発生時には即座に再構築が可能となるPCR検査システムとなっている.

Concern has been raised about the effectiveness of the coronavirus disease 2019 (COVID-19) vaccine in the population of patients with various comorbidities such as heart disease. We investigated the humoral response to the BNT162b2 mRNA COVID-19 vaccine in patients with cardiovascular disease (CVD). We measured IgG against severe acute respiratory syndrome coronavirus 2 spike receptor-binding domain (RBD−IgG) in 85 CVD patients and 179 healthcare workers (HCWs). Blood samples were collected from patients and HCWs three times: (1) before the first dose of vaccination, (2) two weeks after the first dose of vaccination, and (3) two weeks after the second dose of vaccination. Patients with CVD showed a significantly inferior serological response to the BNT162b2 mRNA COVID-19 vaccine at 14 days after the prime dose compared to HCWs (21% vs. 95%, p < 0.001). Median RBD−IgG titers of patients with CVD at 14 days after the second dose were significantly lower than those of HCWs (137.2 U/mL (80.6–200.4 U/mL) vs. 176.2 U/mL (123.9–260.0 U/mL), p < 0.001). In multivariable analyses, CVD is significantly associated with seropositivity after first vaccination and RBD−IgG titers after second vaccination. CVD patients may have a poor humoral response to the BNT162b2 mRNA COVID-19 vaccine, need to be closely monitored, and require earlier revaccination to ensure stronger immunity and protection against infection.

Major depressive disorder (MDD) is a common and serious psychiatric disease that involves brain inflammation. Bifidobacterium breve is commonly used as a probiotic and was shown to improve colitis and allergic diseases by suppressing the inflammatory response. Heat-sterilized B. breve has beneficial effects on inflammation. We hypothesize, therefore, that this probiotic might reduce depression symptoms. We tested this is a mouse model of social defeat stress. C57BL/6J mice exposed to chronic social defeat stress (CSDS) for five consecutive days developed a mild depression-like behavior characterized by a social interaction impairment. CSDS also altered the gut microbiota composition, such as increased abundance of Bacilli, Bacteroidia, Mollicutes, and Verrucomicrobiae classes and decreased Erysipelotrichi class. The prophylactic effect of heat-sterilized B. breve as a functional food ingredient was evaluated on the depression-like behavior in mice. The supplementation started two weeks before and lasted two weeks after the last exposure to CSDS. Two weeks after CSDS, the mice showed deficits in social interaction and increased levels of inflammatory cytokines, including interleukin-1β (IL-1β) in the prefrontal cortex (PFC) and hippocampus (HIP). Heat-sterilized B. breve supplementation significantly prevented social interaction impairment, suppressed IL-1β increase in the PFC and HIP, and modulated the alteration of the gut microbiota composition induced by CSDS. These findings suggest that heat-sterilized B. breve prevents depression-like behavior and IL-1β expression induced by CSDS through modulation of the gut microbiota composition in mice. Therefore, heat-sterilized B. breve used as an ingredient of functional food might prevent MDD.

Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabolizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-inflammatory cytokines and correlates with the severity of major depressive disorder (MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development of neuropsychiatric disorders including MDD. However, the role of miRNAs in targeting IDO1 in the pathophysiology of MDD is still unknown. In this study, we investigated the role of novel miRNAs in the regulation of IDO1 activity and its effect on lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS up-regulated miR-874-3p concomitantly with increase in IDO1 expression in the prefrontal cortex (PFC), increase in immobility in the forced swimming test as depression-like behavior and decrease in locomotor activity as sickness behavior without motor dysfunction. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1 inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase in immobility in the forced swimming test, but did not decrease in locomotor activity induced by LPS. These results suggest that miR-874-3p may play an important role in preventing the LPS-induced depression-like behavior through inhibition of IDO1 expression. This may also serve as a novel potential target molecule for the treatment of MDD. (Figure presented.).

Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Abs in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect Abs against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various Ag-specific Ab isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via PCR test. We developed IgG, IgM, and IgA measurement assays for each Ag, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full-length S protein, S trimer, and nucleocapsid (N) domain, based on ELISA. The assays of the S protein for all isotypes showed high specificity, whereas the assays for all isotypes against N protein showed lower specificity. The sensitivity of all Ag-specific Ab isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 d postsymptom onset. The best correlation with virus-neutralizing activity was found for IgG against RBD, and levels of IgG against RBD in sera from four patients with severe COVID-19 increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

Tryptophan (TRP) is metabolized via the kynurenine (KYN) pathway, which is related to the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the metabolism of KYN to 3-hydroxykynurenine. In rodents, KMO deficiency induces a depression-like behavior and increases the levels of kynurenic acid (KA), a KYN metabolite formed by kynurenine aminotransferases (KATs). KA antagonizes α7 nicotinic acetylcholine receptor (α7nAChR). Here, we investigated the involvement of KA in depression-like behavior in KMO knockout (KO) mice. KYN, KA, and anthranilic acid but not TRP or 3-hydroxyanthranilic acid were elevated in the prefrontal cortex of KMO KO mice. The mRNA levels of KAT1 and α7nAChR but not KAT2−4, α4nAChR, or β2nAChR were elevated in the prefrontal cortex of KMO KO mice. Nicotine blocked increase in locomotor activity, decrease in social interaction time, and prolonged immobility in a forced swimming test, but it did not decrease sucrose preference in the KMO KO mice. Methyllycaconitine (an α7nAChR antagonist) antagonized the effect of nicotine on decreased social interaction time and prolonged immobility in the forced swimming test, but not increased locomotor activity. Galantamine (an α7nAChR allosteric agonist) blocked the increased locomotor activity and prolonged immobility in the forced swimming test, but not the decreased social interaction time in the KMO KO mice. In conclusion, elevation of KA levels contributes to depression-like behaviors in KMO KO mice by α7nAChR antagonism. The ameliorating effects of nicotine and galantamine on depression-like behaviors in KMO KO mice are associated with the activation of α7nAChR.

我が国の精神疾患患者数は400万人を超えている(平成29年度厚生労働省患者調査より)。この数は医療機関を受診した患者数から推計されており、受診していない者を含めると患者数はさらに多いと考えられる。精神疾患の早期発見・予後改善・再発予防のためには、客観的に判断できる早期診断マーカーや病態把握のための検査が必要である。我々はトリプトファン-キヌレニン代謝経路に注目し、精神神経疾患の診断薬・治療薬の開発を目的とした研究を行っている。本稿では、血中トリプトファン代謝産物の測定が抑うつ状態の早期発見に有効であることを示した研究について紹介する。また、統合失調症新規治療薬の開発を目指したキヌレニン経路中酵素の阻害剤探索に関する研究について報告する。(著者抄録)

高感度化学発光法(CLEIA)を測定原理とするSARS-CoV-2に対する抗体検出試薬について、その特性と臨床的有用性をVITROS 5600IIを用いて基礎的検討を行った。PCRで陰性または陽性に測定された患者血清を用い日内変動での再現性を確認した。陰性血清で測定値0.004±0.0004、変動係数(CV)10.85%、カットオフ値1.0に対してその平均数値は0.004と大きく離れていた。陽性血清では6.47±0.28、CV 4.31%であった。日差再現性は2種類の管理血清を用いて12日間の変動について検討を行った。その結果、同一ロット内で良好な変動係数であった。共存物質の影響の調査においては、測定結果に影響を及ぼす変化は認められなかった。各検体の反応実態をグラフに示したところ、陰性検体のシグナルは0.75〜1.12強度の間に分布しているのに対して、陽性検体では約300〜3000強度の高値幅に集積していた。日常検査においてはCOI(1.0付近)のシグナルは300強度付近であることから、陰性反応集積域から陽性反応域には大きな反応幅を持っており容易に分別が可能である。イムノクロマト法との比較したところ、イムノクロマト法ではIgG、IgMのどちらかに反応ラインを認めた例は100例中5例で確認された。しかし、本法で測定した結果はすべて陰性の結果であり、イムノクロマト法とは異なる結果が観察された。ELISA法との相関を調べたところ、ELISA法で数値化された値と本法の相関は係数(r)=0.838と高い関係を示していた。しかしながら一部の検体で本法に比しELISA法での反応性が高値傾向となる検体も散見された。

SARS-CoV-2; COVID-19; Antibody; Immunochromatography; Infectious disease; Virology; Immunology; Laboratory medicine; Diagnostics.

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of kynurenine (KYN) to kynurenic acid (KYNA). Although the isozymes KAT1–4 have been identified, KYNA is mainly produced by KAT2 in brain tissues. KNYA is an antagonist of N-methyl-D-aspartate and α-7-nicotinic acetylcholine receptors, and accumulation of KYNA in the brain has been associated with the pathology of schizophrenia. Therefore, KAT2 could be exploited as a therapeutic target for the management of schizophrenia. Although currently available KAT2 inhibitors irreversibly bind to pyridoxal 5′-phosphate (PLP), inhibition via this mechanism may cause adverse side effects because of the presence of other PLP-dependent enzymes. Therefore, we identified novel selective KAT2 inhibitors by screening approximately 13,000 molecules. Among these, glycyrrhizic acid (GL) and its analogues, glycyrrhetinic acid (GA) and carbenoxolone (CBX), were identified as KAT2 inhibitors. These compounds were highly selective for KAT2 and competed with its substrate KYN, but had no effects on the other 3 KAT isozymes. Furthermore, we demonstrated that in complex structures that were predicted in docking calculations, GL, GA and CBX were located on the same surface as the aromatic ring of KYN. These results indicate that GL and its analogues are highly selective and competitive inhibitors of KAT2.

Tryptophan metabolism is important to induce immune tolerance in tumors. To date, 3 types of tryptophan-metabolizing enzymes have been identified: indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and IDO2) and tryptophan 2,3-dioxygenase 2. Numerous studies have focused on IDO1 as its expression is enhanced in various cancers. Recently, IDO2 has been identified as a tryptophan-metabolizing enzyme that is involved in several immune functions and expressed in cancers such as pancreatic cancer. However, the biological role of IDO2 in the induction of immune tolerance in tumors has not yet been reported. In the present study, we examined the effects of Ido2 depletion on tumor growth in a mouse model of Lewis lung carcinoma by using Ido2-knockout mice. Ido2-knockout mice had reduced tumor volumes compared to WT mice. Furthermore, Ido2 depletion altered the tumor microenvironment, such as tryptophan accumulation and kynurenine reduction, leading to enhancement of immune cell invasion. Finally, enzyme-linked immunospot assay revealed that Ido2 depletion enhanced γ-interferon secretion in the tumor. In conclusion, Ido2 is an important immune regulator in the tumor microenvironment. Our data indicate that IDO2 is a potential target for cancer treatment and drug development.

Phencyclidine (PCP) is a dissociative anesthetic that induces psychotic symptoms and neurocognitive deficits in rodents similar to those observed in schizophrenia patients. PCP administration in healthy human subjects induces schizophrenia-like symptoms such as positive and negative symptoms, and a range of cognitive deficits. It has been reported that PCP, ketamine, and related drugs such as N-methyl-D-aspartate-type (NMDA) glutamate receptor antagonists, induce behavioral effects by blocking neurotransmission at NMDA receptors. Further, NMDA receptor antagonists reproduce specific aspects of the symptoms of schizophrenia. Neurochemical models based on the actions of PCP are well established, with increased focus on glutamatergic dysfunction as a basis for both symptoms and cognitive dysfunction in schizophrenia. On the other hand, the endogenous NMDA receptor antagonist, kynurenic acid (KYNA), which is a product of tryptophan-kynurenine pathway (KP) metabolism, is involved in schizophrenia pathogenesis. KYNA concentrations are elevated in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia. KYNA elevation affects neurotransmitter release in a similar manner to that of psychotomimetic agents such as PCP, underscoring a molecular basis of its involvement in schizophrenia pathophysiology. This review will highlight the relationship between PCP and KP metabolites based on evidence that both exogenous and endogenous NMDA receptor antagonists are involved in the pathogenesis of schizophrenia, and discuss our current understanding of the mechanisms underlying dysfunctional glutamatergic signaling as potential therapeutic targets for schizophrenia.

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function, we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice, which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover, the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast, the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally, RAW cells overexpressing Ido2 did not alter nuclear factor κB (NF-κB) or stat1 expression, but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS, providing novel options for the treatment of immune disorders.

The kynurenine pathway (KP) is the major route for tryptophan (TRP) metabolism in most mammalian tissues. The KP metabolizes TRP into a number of neuroactive metabolites, such as kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN). Elevated metabolite concentrations in the central nervous system are associated with the pathophysiology of several inflammation-related neuropsychiatric diseases. During an inflammatory response, the initial KP metabolic step is primarily regulated by indoleamine 2,3-dioxygenase 1 (IDO1), which produces KYN from TRP. Following this initial step, the KP has 2 distinct branches; one branch is regulated by kynurenine 3-monooxygenase (KMO) and is primarily responsible for the 3-HK and QUIN production, and the other branch is regulated by kynurenine aminotransferase (KAT), which produces KYNA, an N-methyl-D-aspartate receptor and alpha-7-nicotinic acetylcholine receptor antagonist. Unbalanced KP metabolism has been demonstrated in distinct neuropsychiatric diseases; thus, understanding the mechanisms that regulate KP enzyme expression and activity is important. These enzymes are expressed by specific cell types, and the induction of enzyme expression by inflammatory stimuli also shows cell type specificity. This review provides an overview and discusses the current understanding of the influence of KP enzyme expression and activity in different cell types on the pathophysiological mechanisms of specific neuropsychiatric diseases. Moreover, the potential use of KP enzyme inhibition as a therapeutic strategy for treating neurological diseases is briefly discussed. This article is part of the Special Issue entitled ‘The Kynurenine Pathway in Health and Disease’.

広島国際大学保健医療学部臨床工学科(2013年度より医療技術学科に名称変更)の2011年度と2012年度の臨床検査学専攻入学生のカリキュラムは、卒業時に臨床検査技師と臨床工学技士の両方の国家試験受験資格が得られるカリキュラムとなっている。本稿ではダブルライセンス取得に対する本学科の取り組みとその成果について報告する。2015年3月に本専攻の第1期生(2011年度入学生)が、2016年3月に第2期生(2012年度入学生)が本カリキュラムを修了し、それぞれ卒業時に施行された臨床検査技師国家試験を受験した。このうち、臨床工学技士国家試験も受験し、両国家試験に合格したのは第1期生が39人中6人(15.4%)、第2期生が39人中11人(28.2%)であった。ダブルライセンスを取得した学生は少数ではあったが、本カリキュラムは学生の向上心の育成やチーム医療への意識付け、就職の選択肢を広げることなどに貢献できたと考えられた。(著者抄録)

Wip1 (protein phosphatase Mg2+/Mn2+-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d -/- mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O2, Ppm1d-/- MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d-/- MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d-/- MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d-/- MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication. © 2014 Landes Bioscience.

Protein posttranslational modifications (PTMs) perform essential roles in the biological regulation of a cell. PTMs are extremely important because they can change a protein's physical or chemical properties, conformation, activity, cellular location, or stability. In fact, most proteins are altered by the addition or removal of a chemical moiety on either an amino acid or the protein's N- or C-terminus. Some PTMs can be added and removed dynamically as a mechanism for reversibly controlling protein function. Thus, identifying the PTM sites is critical to fully understand the biological roles of any given protein. Mass spectrometry (MS) is a widely used analytical strategy to identify PTMs. We have used an automated two-dimensional liquid chromatography (LC) system coupled with electrospray ionization quadrupole ion-trap MS to identify PTMs for indoleamine 2,3-dioxygenase 1 (IDO1), one of the tryptophan catabolic enzymes. IDO1 promotes immune tolerance by suppressing local T-cell responses under various physiological and pathophysiological conditions, such as pregnancy in mammals, tumor resistance, autoimmunity, and chronic inflammation. Although many studies have demonstrated the biological importance of IDO activity, the PTMs of IDO enzymes remain largely unknown. Only a few important PTMs of IDO1 have been found, such as nitration, N-terminal acetylation, and phosphorylation. In this review, we analyze the PTMs of IDO1 using our twodimensional LC-MS/MS system, and provide an overview of our current understanding. © Springer-Verlag 2012.