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The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5'-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 10 5 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird's-eye view of the antibody repertoire, and our computational methods. Video LinkThe video component of this article can be found at https://www.jove.com/video/58804/ 8 . In addition to V-gene usage profile, the observed frequency of VDJ-profile in naive peripheral B cells is highly similar between individuals 8 . The analysis of the amino acid sequences of the VDJ-region also showed the occurrence of the same junctional

Background: Polymerase chain reaction (PCR) is highly sensitive and is thus the standard method for diagnosing pertussis. Real-time PCR is widely used because of its accuracy and the simplicity of the simultaneous cycle threshold (Ct) value, which represents the copy numbers of the target gene. Little is known of the association of Ct value with pertussis severity in neonates and infants. Methods: This study determined Ct values in neonates and infants diagnosed with pertussis by real-time PCR using nasopharyngeal samples at Vietnam National Children's Hospital in Hanoi in 2017 and 2019. The association of disease severity and clinical parameters were analyzed using univariate and multivariate analyses. Results: We evaluated 108 patients with pertussis [median age: 63 days, interquartile range (IQR): 41-92 days]. Only 6/108 (6%) received at least 1 dose of a pertussis-containing vaccine. Among them, 24 (22.2%) had severe disease requiring care in a pediatric intensive care unit, 16 (13.8%) required mechanical ventilation, and 3 (2.6%) died. The median Ct value was lower in patients with severe disease (19.0, IQR: 16.5-22.0, n = 24) than in those without severe disease (25.5, IQR: 20.0-30.0, n = 84) (P = 0.002). Logistic regression analyses demonstrated that PCR Ct value [odds ratio (OR): 1.783, 95% confidence interval (CI): 1.013-3.138, P = 0.045], age (OR: 3.118, 95% CI: 1.643-5.920, P = 0.001), and white blood cell counts (OR: 0.446, 95% CI: 0.261-0.763, P = 0.003) remained significantly associated with severe disease. Conclusions: Real-time PCR Ct values for pertussis might be useful as a predictor of severe disease in neonates and infants.

Obesity is one of the most significant risk factors for the deterioration and mortality associated with COVID-19 [1]. A certain proportion of COVID-19 patients experience marked elevations of inflammatory mediators, termed “cytokine storm”, resulting in the deterioration of the respiratory condition [2,3]. In the present study, we elucidate that the high visceral adipose tissue (VAT) burden was more closely related to accelerated inflammatory responses and the mortality of Japanese COVID-19 patients than other obesity-associated markers, including body mass index (BMI). To explore a novel stratification of COVID-19 patients, we infected mouse-adapted SARS-CoV-2 in several obese mice, revealing that VAT-dominant ob/ob mice and diet-induced obesity obese mice died after infection with low-titer mouse-adapted SARS-CoV-2 virus due to the subsequent cytokine storm, whereas none of the subcutaneous adipose tissue (SAT) dominant db/db mice or control lean wild-type mice died. SARS-CoV-2 genome and proteins were more abundant in the lungs of ob/ob mice, engulfed in macrophages, resulting in increased production of inflammatory cytokine represented by IL-6. As well as the anti-IL-6 treatment, the prevention of obesity by leptin administration improved the survival of SARS-CoV-2 infected ob/ob mice by reducing the viral protein burden and excessive immune responses.

Background Recombinant human soluble thrombomodulin (rTM) has been used to treat disseminated intravascular coagulation (DIC). Recent studies have shown the efficacy of rTM through its anti-inflammatory effects for treatment of adults with acute respiratory distress syndrome (ARDS). However, the safety and efficacy of rTM in children with severe ARDS complicated by DIC have not been reported. In this study, we investigated the feasibility of using rTM for the treatment of pneumonia-induced severe ARDS complicated by DIC in children. Methods Six children (age: median 10 month-old) with pneumonia-induced severe ARDS complicated by DIC were enrolled in this feasibility study. rTM (380 U/kg) was administered for a maximum of 6 days, in addition to conventional therapies including cardiopulmonary support, antibiotics and/or antivirus drugs administration, steroid administration and intravenous immunoglobulin after diagnosis of severe ARDS complicated by DIC. After administration of rTM, we measured changes in the plasma TM concentration and evaluated the clinical course, status of DIC and ARDS, and other laboratory findings, including levels of cytokines, chemokines, and biomarkers. Results In all six children, the plasma concentration of TM increased and DIC scores decreased after administration of rTM. Four of the six children recovered from the severe ARDS complicated by DIC after treatment in the pediatric intensive care unit, and were discharged from the hospital with no complications. In surviving children, levels of soluble receptors for advanced glycation end products, interleukin-6, interleukin-8 and monocyte chemotactic protein-1 decreased after administration of rTM. Conclusions The rTM administration is feasible as a therapeutic strategy for children over 2 months with pneumonia-induced severe ARDS complicated by DIC.