Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Identification of viral protein R of human immunodeficiency virus-1 (HIV) and interleukin-6 as risk factors for malignancies in HIV-infected individuals: A cohort study.

BACKGROUND: Despite effective antiretroviral therapy, patients with human immunodeficiency virus type-1 (HIV) suffer from a high frequency of malignancies, but related risk factors remain elusive. Here, we focused on blood-circulating viral protein R (Vpr) of HIV, which induces proinflammatory cytokine production and genotoxicity by exogenous functions. METHODS AND FINDINGS: A total 404 blood samples of HIV patients comprising of 126 patients with malignancies (tumor group) and 278 patients without malignancies (non-tumor group), each of 96 samples was first selected by one-to-one propensity score matching. By a detergent-free enzyme-linked immunosorbent assays (detection limit, 3.9 ng/mL), we detected Vpr at a higher frequency in the matched tumor group (56.3%) than in the matched non-tumor group (39.6%) (P = 0.030), although there was no different distribution of Vpr levels (P = 0.372). We also detected anti-Vpr immunoglobulin (IgG), less frequently in the tumor group compared with the tumor group (22.9% for tumor group vs. 44.8% for non-tumor group, P = 0.002), and the proportion of patients positive for Vpr but negative of anti-Vpr IgG was significantly higher in the tumor group than in the non-tumor group (38.6% vs. 15.6%, respectively, P < 0.001). Additionally, Interleukin-6 (IL-6), the levels of which were high in HIV-1 infected patients (P < 0.001) compared to non-HIV-infected individuals, was significantly higher in advanced cases of tumors (P < 0.001), and IL-6 level was correlated with Vpr in the non-tumor group (P = 0.010). Finally, multivariate logistic regression analysis suggested a positive link of Vpr with tumor occurrence in HIV patients (P = 0.002). CONCLUSION: Vpr and IL-6 could be risk factors of HIV-1 associated malignancies, and it would be importance to monitor these molecules for well managing people living with HIV-1.

Efficacy and Safety of Treatment with Plasma from COVID-19-Recovered Individuals.

Convalescent plasma therapy, which involves administering plasma from recovered coronavirus disease 2019 (COVID-19) patients to infected individuals, is being explored as a potential treatment for severe cases of COVID-19. This study aims to evaluate the efficacy and safety of convalescent plasma therapy in COVID-19 patients with moderate to severe illness. An open-label, single-arm intervention study was conducted without a control group. Plasma collected from recovered COVID-19 patients was administered to eligible participants. The primary endpoint was the proportion of patients who were placed on artificial ventilation or died within 14 days of transfusion. Secondary endpoints included clinical improvement, viral load measurements, and adverse event monitoring. A total of 59 cases were included in the study. The primary endpoint was evaluated by comparing the rate obtained in the study to an existing rate of 25%. The study also assessed clinical improvement, viral load changes, and safety endpoints through adverse event monitoring. Convalescent plasma therapy shows potential as a treatment option for COVID-19. This study aimed to provide evidence for the efficacy and safety of this therapy and may contribute to its future use in treating severe cases of COVID-19.

Trends of participants in convalescent plasma donation for COVID-19 in Japan as the pandemic evolved.

Background: We aimed to investigate chronological changes in the characteristics of participants in a coronavirus disease 2019 convalescent plasma donation study that may benefit optimal collection methods in the future. Methods: Data from a convalescent plasma donation study from April 30, 2020 to November 5, 2021 were collected and analyzed. After August 23, 2021, an interim analysis of factors linked to higher antibody titers led us to restrict our participant recruitment criteria to participants who were within 4 months of disease onset and to patients who were otherwise most likely to have sufficiently high antibody titers. Overall, 1299 samples from 1179 patients were analyzed. Results: Over the duration of the study, 35.9% of the samples were deemed eligible for convalescent plasma collection. The overall eligibility rate initially declined, dipping to <20% after one year. During this period, the proportion of enrolled samples from patients who had severe illness also declined, and the proportion of samples from participants who were >120 days post disease onset increased. After the addition of days from onset and vaccination status to our participant recruitment criteria, the eligibility rate improved significantly. Conclusions: As outbreaks of emerging infectious disease occur, it is desirable to construct and implement a scheme for convalescent plasma donation promptly and to monitor the eligibility rate over time. If it declines, promptly analyze and resolve the associated factors. Additionally, vaccine development and infection prevalence are likely to influence the effective recruitment of participants with high antibody titers.

Inhaled ciclesonide does not affect production of antibodies or elimination of virus in patients with COVID-19: Subanalysis of a multicenter, open-label randomized trial.

During an earlier multicenter, open-label, randomized controlled trial designed to evaluate the effectiveness of high-dose inhaled ciclesonide in patients with asymptomatic or mild coronavirus disease 2019 (COVID-19), we observed that worsening of shadows on CT without worsening of clinical symptoms was more common with ciclesonide. The present study sought to determine if an association exists between worsening CT shadows and impaired antibody production in patients treated with inhaled ciclesonide. Eighty-nine of the 90 patients in the original study were prospectively enrolled. After exclusions, there were 36 patients each in the ciclesonide and control groups. We analyzed antibody titers against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein at various time points. Changes in viral load during treatment were compared. There was no significant difference in age, sex, body mass index, background clinical characteristics, or symptoms between the two groups. Although evaluation on day 8 suggested a greater tendency for worsening shadows on CT in the ciclesonide group (p = 0.072), there was no significant difference between them in the ability to produce antibodies (p = 0.379) or the maximum antibody titer during the clinical course. In both groups, worsening CT shadows and higher viral loads were observed on days 1-8, suggesting ciclesonide does not affect clearance of the virus (p = 0.134). High-dose inhaled ciclesonide did not impair production of antibodies against SARS-CoV-2 or affect elimination of the virus, suggesting that this treatment can be used safely in patients with COVID-19 patients who use inhaled steroids for asthma and other diseases.

Association between SARS-CoV-2 anti-spike antibody titers and the development of post-COVID conditions: A retrospective observational study.

The symptoms that persist after an acute coronavirus disease 2019 (COVID-19) are referred to as post- COVID conditions. Although the cause of post-COVID conditions remains unclear, the host immune response to SARS-CoV-2 may be involved. Hence, we aimed to investigate the effect of serum antibody titers against SARS-CoV-2 on the development of post-COVID conditions. We conducted a retrospective observational study of COVID-19-recovered individuals who attended the clinic at the National Center for Global Health and Medicine between January 2020 and April 2021. Serum SARS-CoV-2 anti-spike antibody titers were measured and a questionnaire survey was used to collect information on the presence of post-COVID conditions and demographic characteristics of the participants. Participants were then divided into two groups: high peak antibody titer group [≥ 0.759 OD450 value], and low peak antibody titer group [< 0.759 OD450 value] and compared their frequency of post-COVID conditions. Of 526 individuals attending the clinic, 457 (86.9%) responded to the questionnaire. We analyzed the data of 227 (49.7%) participants with measurements of serum antibody titers during the peak period. The incidence of depressed mood was significantly higher in the group with higher antibody titers (odds ratio: 2.34, 95% CI: 1.17-4.67, p = 0.016). There was no significant difference in the frequency of the remaining symptoms between the two groups. Among post-COVID conditions, the depressed mood was more frequent in the group with high serum antibody titers which suggests a difference in pathogenesis between depressive mood and other post-COVID conditions that requires further investigation.

Efficacy of convalescent plasma therapy for COVID-19 in Japan: An open-label, randomized, controlled trial.

BACKGROUND: Convalescent plasma is a potential therapeutic option for patients with coronavirus disease 2019 (COVID-19). Despite its use for treating several viral infections, we lack comprehensive data on its efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We conducted a multicenter, open-label, randomized controlled trial of convalescent plasma therapy with high neutralizing activity against SARS-CoV-2 in high-risk patients within five days after the onset of COVID-19 symptoms. The primary endpoint was the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs from days 0-5. RESULTS: Between February 24, 2021, and November 30, 2021, 25 patients were randomly assigned to either convalescent plasma (n = 14) or standard of care (n = 11) groups. Four patients discontinued their allocated convalescent plasma, and 21 were included in the modified intention-to-treat analysis. The median interval between the symptom onset and plasma administration was 4.5 days (interquartile range, 3-5 days). The primary outcome of the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs did not significantly differ between days 0-5 (1.2 log10 copies/mL in the convalescent plasma vs. 1.2 log10 copies/mL in the standard of care (effect estimate, 0.0 [95% confidence interval, -0.8-0.7]; P = 0.94)). No deaths were observed in either group. CONCLUSIONS: The early administration of convalescent plasma with high neutralizing activity did not contribute to a decrease in the viral load within five days compared with the standard of care alone.

Robust protein-based engineering of hepatocyte-like cells from human mesenchymal stem cells.

BACKGROUND: Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs). METHODS: MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4). Then, engineered MSCs (4F-Heps) were subjected to epigenetic analysis, biochemical analysis and flow cytometry analysis with antibodies to marker proteins of mature hepatocytes and hepatic progenitors such as delta-like homolog 1 (DLK1) and trophoblast cell surface antigen 2 (TROP2). Functional properties of the cells were also examined by injecting them to mice with lethal hepatic failure. RESULTS: Epigenetic analysis revealed that a 5-day treatment of 4F upregulated the expression of genes involved in hepatic differentiation, and repressed genes related to pluripotency of MSCs. Flow cytometry analysis detected that 4F-Heps were composed of small numbers of mature hepatocytes (at most 1%), bile duct cells (~19%) and hepatic progenitors (~50%). Interestingly, ~20% of 4F-Heps were positive for cytochrome P450 3A4, 80% of which were DLK1-positive. Injection of 4F-Heps significantly increased survival of mice with lethal hepatic failure, and transplanted 4F-Heps expanded to more than 50-fold of human albumin-positive cells in the mouse livers, well consistent with the observation that 4F-Heps contained DLK1-positive and/or TROP2-positive cells. CONCLUSION: Taken together with observations that 4F-Heps were not tumorigenic in immunocompromised mice for at least 2 years, we propose that this artificial transcription system is a versatile tool for cell therapy for hepatic failures.

Resveratrol blocks retrotransposition of LINE-1 through PPAR α and sirtuin-6.

The retroelement long interspersed element-1 (LINE-1 or L1) comprises about 17% of the human genome. L1 retrotransposition is known to cause genomic instability and related disorders, and resveratrol suppresses this retrotransposition; however, the underlying mechanism is still not elucidated. Recent observations showed that low-molecular-weight compounds might induce L1 retrotransposition through unknown mechanisms. This study aimed to determine polyphenol resveratrol (RV)'s effect on L1-RTP (retrotransposition) in somatic cells. Surprisingly, RV completely blocked L1-RTP. Experiments using the PPARα inhibitor GW6471 or siRNA-mediated PPARα depletion showed that RV-mediated L1-RTP's inhibition depended on peroxisome proliferator-activated receptor α (PPARα). We demonstrated that RV inhibits p38 and cAMP response element binding protein phosphorylation, which are involved in MAPK signaling, and the L1-ORF1 protein's chromatin recruitment. Furthermore, RV increased the expression of sirtuin-6 (SIRT6), which inhibited the activation of L1. The sirtuins family, SIRT1, SIRT6, and SIRT7, but not SIRT3, are involved in RV-mediated inhibition of L1-RTP. Overall, our findings suggest that RV directly modulates PPARα-mediated L1-RTP in somatic cells and that MAPK signaling interacts with SIRT6 closely and may play a role in preventing human diseases such as cancer.

Long COVID: current status in Japan and knowledge about its molecular background.

Even after recovering from coronavirus disease 2019 (COVID-19), patients can experience prolonged complaints, referred to as "long COVID". Similar to reports in Caucasians, a follow-up study in Japan revealed that fatigue, dyspnea, cough, anosmia/dysgeusia, and dyssomnia are common symptoms. Although the precise mode of long COVID remains elusive, multiple etiologies such as direct organ damage by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), autoimmunity, prolonged inflammatory reactions, and psychiatric impairment seem to be involved. Notably, SARS-CoV-2 is neurotropic, and viral RNA and proteins are continuously detectable in multiple organs, including the brain. Viral proteins exert a number of different toxic effects on cells, suggesting that persistent infection is a key element for understanding long COVID. Here, we first reviewed the current status of long COVID in Japan, and then summarized literature that help us understand the molecular background of the symptoms. Finally, we discuss the feasibility of vaccination as a treatment for patients with long COVID.

A case report of breakthrough infection with the SARS-CoV-2 delta variant and household transmission: Role of vaccination, anti-spike IgG and neutralizing activity.

There have been several reports of breakthrough infections, which are defined as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections among individuals who had received at least two doses of vaccine at least 14 days before the onset of infection, but data on the antibody titers, including SARS-CoV-2 neutralizing antibody activity, and the clinical course of individuals with breakthrough infections are limited. We encountered a case of breakthrough infection with the SARS-CoV-2 delta variant in a 31-year-old female healthcare worker (the index case, Case 1) and a secondary case (Case 2) in her unvaccinated 33-year-old husband. We studied the role of the anti-spike immunoglobulin G (IgG) and neutralizing antibody activity in the two case patients. Case 1 had high anti-spike IgG detected on day 3 of the illness, with low neutralizing antibody activity. The neutralizing antibody activity started to increase on day 5 of the illness. In Case 2 both the anti-spike IgG and the neutralizing antibody activity remained low from days 4-11 of illness, and the anti-spike IgG gradually increased from day 9. In Case 1, the fever broke within 4 days of onset, coinciding with the rise in neutralizing antibodies, whereas the fever took 7 days to resolve in Case 2. SARS-CoV-2 infection can occur even in vaccinated individuals, but vaccination may contribute to milder clinical symptoms because neutralizing antibodies are induced earlier in vaccinated individuals than in unvaccinated individuals.

Isolation of human monoclonal antibodies with neutralizing activity to a broad spectrum of SARS-CoV-2 viruses including the Omicron variants.

Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection.

A case of COVID-19 reinfection in a hemodialysis patient: the role of antibody in SARS-CoV-2 infection.

Hemodialysis patients are vulnerable to severe and lethal COVID-19, and their protective immunity against COVID-19 is not yet fully understood. Therefore, we report a case of COVID-19 reinfection in a hemodialysis patient 81 days after the first episode and discuss the role of antibodies in SARS-CoV-2 infection. A hemodialysis patient developed asymptomatic COVID-19 due to an outbreak in a hospital on October 29th, 2020. As he was hospitalized and did not develop any symptoms, he was discharged on November 9th. On January 18th, he presented with symptomatic COVID-19 due to close household contact. Then, he developed respiratory failure and was transferred to National Center for Global Health and Medicine if he would need intensive care. He recovered with oxygen inhalation, favipiravir, and steroid treatment, and was discharged on February 12th. To evaluate anti-SARS-CoV-2 antibodies during two hospital stays, we measured immunoglobulin (Ig) G specific for S1 subunit of Spike (S) protein of SARS-CoV-2 (IgG-S1) , IgG specific for the full-length S protein (anti-Spike IgG) and neutralizing antibodies. No seroconversion occurred 5 days after initial infection, the seroconversion of IgG-S1 was observed 10 days after the second infection. Similar to IgG-S1 antibody titer results, anti-Spike IgG and neutralizing antibodies increased from 12 days after the second infection. In conclusion, we experienced a case of COVID-19 reinfection in a hemodialysis patient 81 days after the first episode and showed the kinetics and role of antibodies in SARS-CoV-2 infection. Further studies are needed to understand SARS-CoV-2 reinfection risk in hemodialysis patients and its clinical significance.

Antibody responses after two doses of SARS-CoV-2 mRNA-1273 vaccine in an individual with history of COVID-19 re-infection.

We present a case of a 58-year-old Japanese man with a history of 2 previous COVID-19 infections, who received 2 doses of mRNA-1273 vaccine. We are not aware of any previous study regarding antibody tendency after 2 infections and 2 vaccinations. We evaluated his IgG titer of antispike protein and neutralizing activity from the first infection before and after 2 doses of vaccine. Both antispike IgG titer and neutralizing activity showed a tendency to decline almost 1 year after initial infection; they rapidly increased after the first vaccination, and they remained high after the second vaccination. Although this is a single case report, it seems to have generalizability because the findings are consistent with previous reports regarding single infections or 3 doses of vaccination. Our findings suggest that a single booster shot may provide sufficient protection and aid the understanding of immunologic responses of vaccination in patients with COVID-19 with history of re-infection.

Prolonged SARS-CoV-2 infection associated with long-term corticosteroid use in a patient with impaired B-cell immunity.

Corticosteroids are widely used to treat severe COVID-19, but in immunocompromised individuals, who are susceptible to persistent infection, long term corticosteroid use may delay viral clearance. We present a case of prolonged SARS-CoV-2 infection in a man with significantly impaired B-cell immunity due to non-Hodgkin lymphoma which had been treated with rituximab. SARS-CoV-2 shedding persisted, despite treatment with remdesivir. Viral sequencing confirmed the persistence of the same viral strain, ruling out the possibility of reinfection. Although SARS-CoV-2 IgG, IgA and IgM remained negative throughout the treatment period, after reduction of the corticosteroid dose, PCR became negative. Long-term corticosteroid treatment, especially in immunocompromised individuals, may result in suppression of cell-mediated immunity and prolonged SARS-CoV-2 infection.

Multisystem Inflammatory Syndrome in Adult after First Dose of mRNA Vaccine.

A 32-year-old man in Japan experienced respiratory failure after receiving the first dose of coronavirus disease (COVID-19) vaccine. He was treated with noninvasive ventilation and corticosteroids. Serologic test results suggested previous COVID-19; therefore, he received a diagnosis of multisystem inflammatory syndrome. COVID-19 vaccination could be a trigger for this condition.

Suspected Tuberculous Pleurisy and Coronavirus Disease 2019 Comorbidity.

A 33-year-old woman with a fever, cough, and pharyngitis was admitted after left-sided pleural effusion was detected. The fever and upper respiratory symptoms were confirmed, and she was diagnosed with coronavirus disease (COVID-19) after showing a positive polymerase chain reaction (PCR) test. After thoracentesis, pleural fluid revealed elevated adenosine deaminase values and a positive QuantiFeron test; tuberculous pleurisy was thus suspected. Subsequent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR and anti-SARS-CoV-2 Spike IgG tests were negative, suggesting that the initial PCR result had been erroneous. However, we were unable to confirm this. Data concerning COVID-19 diagnostics are insufficient at present. It is important to make comprehensive judgments regarding the diagnosis and treatment of patients as well as public health.

Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide.

Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit ß1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296-516 aa) or TNPO1 fragment (1-297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296-516-aa fragment of IMB1 and 1-297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

Case Report: Changes in Cytokine Kinetics During the Course of Disease in a Japanese Patient With Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe disease that is reportedly linked to coronavirus disease 2019. Affected patients present with gastrointestinal symptoms and cardiovascular dysfunction, in addition to Kawasaki disease-like features, suggesting the potential for overlapping disease mechanisms. Kawasaki disease has been reported among individuals of East Asian ethnicities, whereas there is minimal clinical literature regarding the occurrence of MIS-C among individuals of Asian ethnicities. A few reports thus far have described changes in cytokine kinetics during the course of disease in patients with MIS-C. We followed the temporal cytokine kinetics in a 9-year-old Japanese girl who exhibited a classical trajectory of MIS-C. The patient exhibited right cervical swelling and pain, abdominal pain, vomiting, and lip reddening, which developed 31 days after she was diagnosed with severe acute respiratory syndrome coronavirus-2 infection. The patient was diagnosed with Kawasaki disease on her fifth day of illness; because she fulfilled the criteria for MIS-C, she was also diagnosed with this disease on her fifth day of illness. Her fever rapidly resolved upon administration of intravenous immunoglobulin, aspirin, and prednisolone. On the patient's sixth day of illness, she developed acute myocarditis, which was treated with two diuretics and one vasodilator; the myocarditis ameliorated within a few days. Analyses of temporal kinetics for 71 serum cytokines revealed several patterns of cytokine changes that were consistent with the patient's clinical course of disease. Importantly, there was a clear distinction between cytokines that did and did not decrease rapidly following post-treatment fever resolution. These findings may be useful for the assessment of disease status and selection of therapy in patients with similar symptoms; they may also provide insights for basic and clinical research regarding MIS-C.

How we secured a COVID-19 convalescent plasma procurement scheme in Japan.

BACKGROUND: In order to tackle the COVID-19 pandemic, a COVID-19 convalescent plasma (CCP) procurement program was initiated in Japan in April 2020. The program was a collaboration between a government-managed national hospital, an infectious disease research institute, and a blood banking organization. Each party assumed different responsibilities: recruitment, SARS-CoV-2 antibody profiling, and plasmapheresis; conduction of screening tests; and SARS-CoV-2 blood testing, respectively. METHODS: We adopted a two-point screening approach before the collected CCP was labeled as a CCP product for investigational use, for which we mainly tested anti-SARS-CoV-2 antibody eligibility and blood product eligibility. Anti-SARS-CoV-2 spike protein titer was measured using enzyme-linked immunosorbent assay, and the IC50 value was denoted as the neutralizing activity. Blood donor eligibility was extended beyond the normal blood donation guidelines to include a broader range of participants. After both eligibility criteria were confirmed, participants were asked to revisit the hospital for blood donation, which is a unique aspect of the Japanese CCP program, as most donations are taking place in normal blood donation venues in other countries. Some donors were re-scheduled for repeat plasma donations. As public interest in anti-SARS-CoV-2 antibodies increased, test results were given to the participants. RESULTS: As of September 17, 2020, our collection of CCP products was sufficient to treat more than 100 patients. As a result, projects for administration and distribution are also being conducted. CONCLUSIONS: We successfully implemented a CCP procurement scheme with the goal to expand to other parts of the country to improve treatment options for COVID-19.