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Aims: Myocardial calcification is an unusual condition in which excess calcium is deposited in the myocardium. Herein, we report two cases of myocardial calcification from our clinical experience. Furthermore, we conduct a systematic review to examine the clinical course and associated pathologies of myocardial calcification., Methods and Results: This systematic review was registered in PROSPERO (CRD42023463285). PubMed and Scopus were searched according to the following inclusion criteria: (i) case reports or case series describing patients with myocardial calcification; (ii) diagnosis of myocardial calcification by computed tomography (CT); (iii) adequate description of patients, including their chief complaint, medical history, evaluations, and treatments; and (iv) publication in English. Among the 75 patients, 24 had sepsis, 14 had myocarditis, and 37 had other pathologies. The mortality rate was 33% for patients with sepsis, 14% for patients with myocarditis, and 11% for patients with other pathologies. Follow-up CT findings beyond 2 years were reported in six patients, showing that the CT findings of myocardial calcification persisted but subsided over time. Autopsy was performed in seven patients, and extensive interstitial fibrosis and collection of inflammatory cells were observed in patients with myocarditis, sepsis, and ischaemic heart disease., Conclusion: While various medical conditions can cause myocardial calcification, accompanying conditions commonly reported with myocardial calcification were sepsis and myocarditis. The CT findings of myocardial calcification tend to regress over time if the underlying disease can be treated., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Ventricular-assist device therapy for small patients with congenital heart disease is challenging due to its complex anatomy and hemodynamics. We describe a 3-year-old patient with heart failure with truncus arteriosus in the palliative stage. The patient underwent palliative right ventricular outflow tract reconstruction following bilateral pulmonary artery banding. At 6 months of age, the patient developed severe truncal valve regurgitation and left ventricular dysfunction. Emergent truncal valve replacement with a mechanical valve was performed, but left ventricular dysfunction persisted. At 3 years of age, the patient developed acute progression of heart failure triggered by influenza infection. The patient was intubated and transferred to our center to determine the indication for heart transplantation. On the second day after admission, signs of multiorgan failure appeared. Emergent ventricular-assist device implantation for both ventricles was performed with truncal valve closure, ventricular septal defect closure, atrial septal defect closure, and re-right ventricular outflow tract reconstruction. The right ventricular-assist device was successfully removed on the seventh postoperative day. Due to the small pulmonary arteries, severe pulmonary stenosis persisted after ventricular-assist device implantation, but it gradually improved with multiple pulmonary angioplasties. The patient was registered in the Japanese organ transplant network and is awaiting a donor organ in a stable condition., (© 2024. The Author(s).)

This open-label, extension study assessed long-term safety, tolerability, and efficacy of ambrisentan in a pediatric population (age 8- < 18 years) with pulmonary arterial hypertension (PAH). Following completion of a 6-month, randomized study, participants entered the long-term extension at individualized ambrisentan dosages (2.5/5/7.5 or 10 mg/day). Safety assessments included adverse events (AEs), AEs of special interest, and serious AEs (SAEs); efficacy outcomes included 6-min walking distance (6MWD) and World Health Organization functional class (WHO FC). Thirty-eight of 41 (93%) randomized study participants entered the extension; 21 (55%) completed (reaching age 18 years). Most participants received concomitant phosphodiesterase-5 inhibitors (n = 25/38, 66%). Median ambrisentan exposure was 3.5 years. Most participants experienced ≥ 1 AE (n = 34/38, 89%), and 21 (55%) experienced SAEs, most commonly worsening PAH (n = 3/38, 8%), acute cardiac failure, pneumonia, or anemia (n = 2/38; 5% each); none considered ambrisentan-related. Seven participants (18%) died, with recorded reasons (MedDRA preferred term): cardiac failure (n = 2), PAH (n = 2), COVID-19 (n = 1), acute right ventricular failure (n = 1), and failure to thrive (n = 1); median time to death: 5.2 years. Anemia and hepatotoxicity AEs were generally mild to moderate and did not require ambrisentan dose adjustment. Assessed at study end in 29 participants (76%), mean 6MWD improved by 17% (standard deviation: 34.3%), and all (29/29, 100%) had improved or unchanged WHO FC.    Conclusion: Long-term weight-based ambrisentan dosing, alone or combined with other PAH therapies in children with PAH aged 8- < 18 years, exhibited tolerability and clinical improvements consistent with prior randomized study results.    Trial registration: NCT01342952, April 27, 2011. What is Known: • The endothelin receptor antagonist, ambrisentan, is indicated for treatment of pulmonary arterial hypertension (PAH). Previous studies have shown similar efficacy and tolerability in pediatric patients as in adults. What is New: • This open-label extension study assessed the long-term use of ambrisentan in pediatric patients (8-<18 years) with PAH, most of whom were also receiving recommended background PAH treatment. • Weight-based dosing of ambrisentan, given alone or in combination with other PAH therapies, was well tolerated with clinical improvements consistent with prior randomized study results., (© 2024. The Author(s).)

Background: Restrictive cardiomyopathy (RCM) is characterized by impaired diastolic function with preserved ventricular contraction. Several pathogenic variants in sarcomere genes, including TNNI3 , are reported to cause Ca 2+ hypersensitivity in cardiomyocytes in overexpression models; however, the pathophysiology of induced pluripotent stem cell (iPSC)-derived cardiomyocytes specific to a patient with RCM remains unknown., Methods and Results: We established an iPSC line from a pediatric patient with RCM and a heterozygous TNNI3 missense variant, c.508C>T (p.Arg170Trp; R170W). We conducted genome editing via CRISPR/Cas9 technology to establish an isogenic correction line harboring wild type TNNI3 as well as a homozygous TNNI3 -R170W. iPSCs were then differentiated to cardiomyocytes to compare their cellular physiological, structural, and transcriptomic features. Cardiomyocytes differentiated from heterozygous and homozygous TNNI3 -R170W iPSC lines demonstrated impaired diastolic function in cell motion analyses as compared with that in cardiomyocytes derived from isogenic-corrected iPSCs and 3 independent healthy iPSC lines. The intracellular Ca 2+ oscillation and immunocytochemistry of troponin I were not significantly affected in RCM-cardiomyocytes with either heterozygous or homozygous TNNI3 -R170W. Electron microscopy showed that the myofibril and mitochondrial structures appeared to be unaffected. RNA sequencing revealed that pathways associated with cardiac muscle development and contraction, extracellular matrix-receptor interaction, and transforming growth factor-β were altered in RCM-iPSC-derived cardiomyocytes., Conclusions: Patient-specific iPSC-derived cardiomyocytes could effectively represent the diastolic dysfunction of RCM. Myofibril structures including troponin I remained unaffected in the monolayer culture system, although gene expression profiles associated with cardiac muscle functions were altered.

Background: Anti-α6β4 integrin autoantibodies can be observed in some patients with mucous membrane pemphigoid. We have previously identified anti-α6β4 integrin extracellular domain autoantibodies together with anti-BP180 NC16A antibodies in a patient with DPP-4 inhibitor-induced bullous pemphigoid. However, the significance and impact of anti-α6β4 integrin antibodies are unknown., Objectives: To characterize anti-α6β4 integrin extracellular domain autoantibodies in pemphigoid patients, to determine whether these antibodies inhibit laminin-α6β4 integrin binding and to observe their systemic effects., Methods: Anti-α6β4 integrin autoantibodies were analysed by staining cells expressing the extracellular region of α6β4 integrin with sera from 20 patients with pemphigoid. The anti-α6β4 integrin autoantibodies were characterized using different transfectants. The binding of laminins to α6β4 integrin was studied using cells expressing the activated conformation of α6β4 integrin and the inhibitory effect of the autoantibodies on the binding of laminins to α6β4 integrin was tested. Trends in antibody titres and clinical symptoms were quantified and analysed., Results: IgG autoantibodies against the extracellular domain of anti-α6β4 integrin were found in some patients with pemphigoid. Laminin binding to α6β4 integrin was observed in the active conformation of α6β4 integrin, and serum from a patient with a high titre of anti-α6β4 integrin antibodies inhibited the binding of both laminin-511 and laminin-332 to α6β4 integrin. α6β4 integrin is expressed on the basement membrane of both skin and small intestine, and exfoliation was observed in the patient's epidermis and small intestinal epithelium. A reduction in the titre of the anti-α6β4 integrin antibody was associated with improvement in both skin and gastrointestinal symptoms., Conclusions: This study demonstrated the presence of anti-α6β4 integrin extracellular domain-specific autoantibodies in some patients with pemphigoid. In addition, these autoantibodies showed inhibitory activity on α6β4 integrin-laminin binding. Anti-α6β4 integrin antibodies can affect the gastrointestinal tract as well as the skin and oral mucosa., (© 2023 European Academy of Dermatology and Venereology.)

Down syndrome (DS) is the most prevalent chromosomal disorder associated with a higher incidence of pulmonary arterial hypertension (PAH). The dysfunction of vascular endothelial cells (ECs) is known to cause pulmonary arterial remodeling in PAH, although the physiological characteristics of ECs harboring trisomy 21 (T21) are still unknown. In this study, we analyzed the human vascular ECs by utilizing the isogenic pairs of T21-induced pluripotent stem cells (iPSCs) and corrected disomy 21 (cDi21)-iPSCs. In T21-iPSC-derived ECs, apoptosis and mitochondrial reactive oxygen species (mROS) were significantly increased, and angiogenesis and oxygen consumption rate (OCR) were significantly impaired as compared with cDi21-iPSC-derived ECs. The RNA-sequencing identified that EGR1 on chromosome 5 was significantly upregulated in T21-ECs. Both EGR1 suppression by siRNA and pharmacological inhibitor could recover the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Alternately, the study also revealed that DYRK1A was responsible to increase EGR1 expression via PPARG suppression, and that chemical inhibition of DYRK1A could restore the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Finally, we demonstrated that EGR1 was significantly upregulated in the pulmonary arterial ECs from lung specimens of a patient with DS and PAH. In conclusion, DYRK1A/PPARG/EGR1 pathway could play a central role for the pulmonary EC functions and thus be associated with the pathogenesis of PAH in DS., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Impella is a device effective for the treatment of cardiogenic shock. However, among small children, its application has limitations due to left ventricle size and vasculature and the turning diameter of the aortic arch. Herein, we report an 11-year-old girl with fulminant myocarditis who was successfully managed with Impella CP implantation via the right subclavian artery using a polyethylene terephthalate chimney graft. Compared with insertion via the femoral artery, this method has several advantages. That is, it can address limitations in aortic arch diameter and facilitate equable fixation of the Impella device in small pediatric patients., (© 2022. The Japanese Society for Artificial Organs.)

Microbubbles have potential applications as drug and gene carriers, and drug release can be triggered by externally applied ultrasound irradiation while inside blood vessels. Desorption of molecules forming the microbubble shell can be observed under ultrasound irradiation of a single isolated microbubble, and the volume of desorbed molecules can be quantitatively estimated from the contact angle between the bubble and a glass plate. Microbubbles composed of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) shell and a poorly-soluble gas are created. When the microbubbles are exposed to a pulsed ultrasound, the contact angles increase dramatically; the percentage of DMPC molecules desorbed from the bubble surface reaches 70%. Vibration of a single bubble in the radial direction is measured using a laser Doppler vibrometer. The relationship between the vibrational characteristics and the amount of molecular desorption reveals that a larger vibrational amplitude of the bubble around the resonance size induces a larger amount of molecular desorption. These results support the possibility of controlling molecular desorption with pulsed ultrasound., (© 2023. Springer Nature Limited.)

Background: Restrictive cardiomyopathy in children is rare and outcomes are very poor. However, little information is available concerning genotype-outcome correlations., Methods: We analyzed the clinical characteristics and genetic testing, including whole exome sequencing, of 28 pediatric restrictive cardiomyopathy patients who were diagnosed from 1998 to 2021 at Osaka University Hospital in Japan., Results: The median age at diagnosis (interquartile range) was 6 (2.25-8.5) years. Eighteen patients received heart transplantations and 5 patients were on the waiting list. One patient died while waiting for transplantation. Pathologic or likely-pathogenic variants were identified in 14 of the 28 (50%) patients, including heterozygous TNNI3 missense variants in 8 patients. TNNT2 , MYL2 , and FLNC missense variants were also identified. No significant differences in clinical manifestations and hemodynamic parameters between positive and negative pathogenic variants were detected. However, 2- and 5-year survival rates were significantly lower in patients with pathogenic variants (50% and 22%) compared with survival in patients without pathogenic variants (62% and 54%; P =0.0496, log-rank test). No significant differences were detected in the ratio of patients diagnosed at nationwide school heart disease screening program between positive and negative pathogenic variants. Patients diagnosed by school screening showed better transplant-free survival compared with patients diagnosed by heart failure symptoms ( P =0.0027 in log-rank test)., Conclusions: In this study, 50% of pediatric restrictive cardiomyopathy patients had pathogenic or likely-pathogenic gene variants, and TNNI3 missense variants were the most frequent. Patients with pathogenic variants showed significantly lower transplant-free survival compared with patients without pathogenic variants., Competing Interests: Disclosures None.

Background: In adult patients, after a Fontan procedure, high central venous pressure (CVP) is a hemodynamic risk factor associated with poor prognosis. High liver stiffness (LS) on transient elastography (TE) is associated with high CVP in patients with heart failure without liver disease. Here, we investigated whether LS assessment using TE is a reliable method to noninvasively evaluate CVP in adult patients after a Fontan procedure, who can present varying degrees of liver fibrosis as a complication., Methods: We measured LS using TE and CVP by cardiac catheterization in 24 adult patients who had undergone a Fontan procedure. The estimated CVP was calculated using the previously reported formula: -5.8 + 6.7 × ln[LS]. We examined the correlation between LS and CVP, and degree of agreement between the estimated and measured CVPs. Patients were divided into two groups, with or without suspected liver cirrhosis, based on abdominal imaging studies., Results: The median patient age was 35 years (interquartile range 25, 39). Overall, there was a strong correlation between LS and CVP (ρ = 0.83, p < 0.001). The estimated CVP based on LS and the CVP measured using cardiac catheterization were positively correlated; however, the estimated CVP tended to be higher than the measured CVP (mean difference 0.9 mmHg [95% limits of agreement: -2.8 to 4.6 mmHg]). These results were consistent across all groups., Conclusions: In adult patients after a Fontan procedure, LS measured by TE showed a positive correlation with CVP by cardiac catheterization. TE can be useful as a noninvasive estimation of CVP., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (© 2023 The Authors.)

Background Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. Methods and Results Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, apoptosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several humoral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor-β signaling pathways were significantly affected in DCM CFs. In contrast, single-cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. Conclusions Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell-cell contact.

Objectives: The goal of this study was to identify the clinical significance of the deoxyribonucleic acid (DNA) damage response marker, phosphorylated H2A histone variant X, on the bridge to recovery in low-weight paediatric patients with dilated cardiomyopathy (DCM) after having a Berlin Heart EXCOR implanted., Methods: Consecutive paediatric patients with DCM who had an EXCOR implanted for DCM at our hospital between 2013 and 2021 were reviewed. Patients were classified into 2 groups according to the degree of DNA damage in the left ventricular cardiomyocytes-the low DNA damage group and the high DNA damage group-using the median value as the threshold. We examined and compared the preoperative factors and histologic findings associated with cardiac functional recovery following the explant procedure in the 2 groups., Results: Competing outcome analysis of 18 patients (median body weight, 6.1 kg) showed that the incidence of an EXCOR explant was 40% at 1 year after the implant procedure. Serial echocardiography revealed significant left ventricular functional recovery in the low DNA damage group 3 months after the implant. The univariable Cox proportional hazards model revealed that the percentage of phosphorylated H2A histone variant X-positive cardiomyocytes was the significant factor associated with cardiac recovery and the EXCOR explant (hazard ratio, 0.16; 95% confidence interval, 0.027-0.51; P = 0.0096)., Conclusions: The degree of DNA damage response to the EXCOR implant may aid in predicting the bridge to recovery with EXCOR among low-weight paediatric patients with DCM., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Cardiac allograft vasculopathy (CAV) sometimes leads to restenosis, even after percutaneous transcatheter intervention. Recently, drug-coated balloons (DCBs) have been successfully used to treat coronary artery disease, especially CAVs, in adults. However, no studies have used DCBs in pediatric CAVs. We encountered a patient with CAV who underwent cardiac transplantation for restrictive cardiomyopathy at the age of 2 years. Nine years after the transplantation, severe stenosis of the proximal left anterior descending branch was observed. Considering the patient's young age and the possibility of restenosis, we performed an intervention with DCB. Follow-up conducted 7 months after the intervention showed no restenosis. Cardiac coronary artery lesions following transplantation are more likely to result in restenosis earlier than arteriosclerotic lesions. In pediatric patients, restenosis might require multiple stents and prolonged antiplatelet therapy. Our findings provide evidence supporting the possibility of an effective treatment of CAV in children., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Annals of Pediatric Cardiology.)

Restrictive cardiomyopathy (RCM) is a rare disease characterized by increased ventricular stiffness and preserved ventricular contraction. Various sarcomere gene variants are known to cause RCM; however, more than a half of patients do not harbor such pathogenic variants. We recently demonstrated that cardiac fibroblasts (CFs) play important roles in inhibiting the diastolic function of cardiomyocytes via humoral factors and direct cell-cell contact regardless of sarcomere gene mutations. However, the mechanical properties of CFs that are crucial for intercellular communication and the cardiomyocyte microenvironment remain less understood. In this study, we evaluated the rheological properties of CFs derived from pediatric patients with RCM and healthy control CFs via atomic force microscopy. Then, we estimated the cellular modulus scale factor related to the cell stiffness, fluidity, and Newtonian viscosity of single cells based on the single power-law rheology model and analyzed the comprehensive gene expression profiles via RNA-sequencing. RCM-derived CFs showed significantly higher stiffness and viscosity and lower fluidity compared to healthy control CFs. Furthermore, RNA-sequencing revealed that the signaling pathways associated with cytoskeleton elements were affected in RCM CFs; specifically, cytoskeletal actin-associated genes (ACTN1, ACTA2, and PALLD) were highly expressed in RCM CFs, whereas several tubulin genes (TUBB3, TUBB, TUBA1C, and TUBA1B) were down-regulated. These results implies that the signaling pathways associated with cytoskeletal elements alter the rheological properties of RCM CFs, particularly those related to CF-cardiomyocyte interactions, thereby leading to diastolic cardiac dysfunction in RCM., Competing Interests: The authors have declared that no competing interests exist.

Pulmonary atresia and ventricular septal defect (PA/VSD) demonstrate a wide variety of pulmonary and coronary artery abnormalities; additionally, coronary-to-pulmonary artery fistula (CPAF) is a rare manifestation of PA/VSD and is seldom detected during pregnancy. In this report, we present a case of prenatal diagnosis of CPAF in PA/VSD and impactful images in a neonate, which were obtained using fetal echocardiography and postnatal electrocardiography-gated 320-row CT. Prenatal diagnosis of CPAF can facilitate the provision of better therapeutic strategies after birth., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

A 92-year-old woman diagnosed with dementia and end-stage gastric cancer received end-of-life care on the island where she lived. Informed consent concerning remote death certification based on the Japanese government's guidelines was obtained from a family member in case a physician was unavailable. A physical examination after cardiopulmonary arrest was conducted, supported by telemedicine and a well-trained registered nurse under remote supervision of the physician who last saw the deceased directly. Death certification was provided accordingly. To our knowledge, this was the first case of remote death certification using telemedicine in Japan.

Flowering plant species and their nectar-feeding vertebrates exemplify some of the most remarkable biotic interactions in the Neotropics. In the Brazilian Atlantic Forest, several species of birds (especially hummingbirds), bats, and non-flying mammals, as well as one lizard feed on nectar, often act as pollinators and contribute to seed output of flowering plants. We present a dataset containing information on flowering plants visited by nectar-feeding vertebrates and sampled at 166 localities in the Brazilian Atlantic Forest. This dataset provides information on 1902 unique interactions among 515 species of flowering plants and 129 species of potential vertebrate pollinators and the patterns of species diversity across latitudes. All plant-vertebrate interactions compiled were recorded through direct observations of visits, and no inferences of pollinators based on floral syndromes were included. We also provide information on the most common plant traits used to understand the interactions between flowers and nectar-feeding vertebrates: plant growth form, corolla length, rate of nectar production per hour in bagged flowers, nectar concentration, flower color and shape, time of anthesis, presence or absence of perceptible fragrance by human, and flowering phenology as well as the plant's threat status by International Union for Conservation of Nature (IUCN) classification. For the vertebrates, status of threat by IUCN classification, body mass, bill or rostrum size are provided. Information on the frequency of visits and pollen deposition on the vertebrate's body is provided from the original source when available. The highest number of unique interactions is recorded for birds (1771) followed by bats (110). For plants, Bromeliaceae contains the highest number of unique interactions (606), followed by Fabaceae (242) and Gesneriaceae (104). It is evident that there was geographical bias of the studies throughout the southeast of the Brazilian Atlantic Forest and that most effort was directed to flower-hummingbird interactions. However, it reflects a worldwide tendency of more plants interacting with birds compared with other vertebrate species. The lack of similar protocols among studies to collect basic data limits the comparisons among areas and generalizations. Nevertheless, this dataset represents a notable effort to organize and highlight the importance of vertebrate pollinators in this hotspot of biodiversity on Earth and represents the data currently available. No copyright or proprietary restrictions are associated with the use of this data set. Please cite this data paper when the data are used in publications or scientific events., (© 2022 The Ecological Society of America.)

Background: Although widely reported to affect older adults more, coronavirus disease 2019 (COVID-19) also affects adolescents, especially those with co-morbidities, including heart diseases. The safety and efficacy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines has been established in healthy adolescents, yet there are few data for humoral and cellular immunogenicity in adolescents with cardiac diseases., Methods: We evaluated anti-spike antibodies, neutralizing activities, and interferon-gamma production prior to and after SARS-CoV-2 vaccination in adolescents with cardiac diseases and healthy controls., Results: Five healthy adolescents and 26 patients with cardiac diseases, including congenital heart disease (CHD, n = 10), dilated cardiomyopathy (DCM, n = 4), idiopathic pulmonary arterial hypertension (IPAH, n = 4), and those post-heart transplantation (post-HTx, n = 8) were enrolled. No severe adverse events, including myocarditis and pericarditis, were noted, even in patients with severe heart failure. Febrile events were noted after 21 of 62 injections (34%). All the healthy adolescents and 21 of the 26 patients (81%) showed sufficient elevation of neutralizing antibodies after the second dose of vaccination. Neutralizing antibodies and cellular immunity were absent in four of the eight post-HTx patients and one with single ventricle CHD. There was no correlation between the anti-spike and neutralizing antibody titers and interferon-gamma levels. When comparing the clinical characteristics of the patients post-HTx who did or did not acquire antibodies, there was no significant difference in the immunosuppressant types and trough levels., Conclusions: SARS-CoV-2 mRNA vaccine has efficient immunogenicity for adolescents with CHD, IPAH, and DCM. Half of post-HTx patients could not acquire sufficient humoral immunity., (© 2022 Japan Pediatric Society.)

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature neonates. Classical BPD is caused by hyperoxia and high-pressure mechanical ventilation, whereas BPD in recent era is caused by impaired pulmonary angiogenesis and alveolarization in extreme prematurity. Although sildenafil was reported to be effective in a hyperoxia-induced rat BPD model, several clinical trials could not demonstrate any significant improvement in the respiratory statuses of BPD infants. Riociguat is a soluble guanylate cyclase stimulator that increases cyclic guanosine monophosphate activity in a nitric oxide independent manner. However, a beneficial effect in BPD has not been established yet., Methods and Results: We established BPD model in rats by injection of SU5416 on day 1 followed by maintenance under normoxia, which resulted in oversimplified alveoli, sparse pulmonary capillary vessels, severe pulmonary hypertension, and growth retardation, which mimicked the features observed in recent clinical management of BPD. We administered riociguat from day 10, when BPD rats exhibited growth retardation. Histological analyses demonstrated that riociguat treatment significantly but partially ameliorated lung alveolarization, vascularization, and pulmonary hypertension. However, the survival rate was not significantly improved by riociguat treatment., Conclusions: Riociguat could ameliorate pulmonary alveolarization, vascularization, and hypertension in the SU5416 induced BPD rat model, but could not improve the overall survival.

Liver X receptors (LXR) α and β are a family of nuclear receptors that regulate lipogenesis by controlling the expression of the genes involved in the synthesis of fatty acids. MID1IP1, which encodes MIG12, is a target gene of LXR. MIG12 induces fatty acid synthesis by stimulating the polymerization-mediated activation of acetyl-CoA carboxylase (ACC). Here, we show that LXR's activation stimulates ACC polymerization in HepG2 cells by increasing the expression of MIG12. A knockdown of MID1IP1 abrogated the stimulation completely. The mutations of MIG12's leucine-zipper domain reduced the interaction between MIG12 and ACC, thus decreasing the MIG12's capacity to stimulate ACC polymerization. These results indicate that LXR's activation stimulates lipogenesis not only through the induction of the genes encoding lipogenic enzymes but also through MIG12's stimulation of ACC polymerization., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease caused by vascular remodeling of the pulmonary arteries with elevated pulmonary vascular resistance. Recently, various pulmonary vasodilator drugs have become available in the clinical field, and have dramatically ameliorated the prognosis of IPAH. However, little is known about how the mechanical properties of pulmonary arterial smooth muscle cells (PASMCs) are altered under drug supplementation., Methods: Atomic force microscopy (AFM) was used to investigate the mechanical properties of PASMCs derived from a patient with IPAH (PAH-PASMCs) and a healthy control (N-PASMCs) which received the supplementation of clinically used drugs for IPAH: sildenafil, macitentan, and riociguat., Results: PASMCs derived from PAH-PASMCs were stiffer than those derived from N-PASMCs. With sildenafil treatment, the apparent Young's modulus ( E 0 ) of cells significantly decreased in PAH-PASMCs but remained unchanged in N-PASMCs. The decrease in E 0 of PAH-PASMCs was also observed in macitentan and riociguat treatment. The stress relaxation AFM revealed that the decrease in E 0 of PAH-PASMCs resulted from a decrease in the cell elastic modulus and/or increase in cell fluidity. The combination treatment of macitentan and riociguat showed an additive effect on cell mechanical properties, implying that this clinically accepted combination therapy for IPAH influences the intracellular mechanical components., Conclusions: Pulmonary vasodilator drugs affect the mechanical properties of PAH-PASMCs, and there exists a mechanical effect of combination treatment on PAH-PASMCs., Competing Interests: None to declare., (Copyright 2021, Katsuragi et al.)

Pure restrictive cardiomyopathy is a strong risk factor for poor outcomes in children with cardiomyopathy on ventricular assist devices. Owing to concomitant right heart failure, children with end-staged restrictive cardiomyopathy who are supported with a ventricular assist device often require a biventricular assist device, which is another risk factor for waitlist mortality in heart transplantation candidates. Herein, we report the case of a 3-year-old boy with pure restrictive cardiomyopathy who successfully underwent heart transplantation after 12 months of support with staged biventricular assist devices. Owing to the progression of diastolic dysfunction, the left ventricular assist device could not provide adequate circulation support. Despite the provision of biventricular assist device support, the patient required a complex management strategy that involved balancing the left and right ventricular assist device supports. We were able to stabilize the patient by careful synchronization of the supports and proceeded to heart transplantation. TRIAL REGISTRATION: Clinical Registration No.: Institutional Review Board of Osaka University Hospital, approval no. 16105.

Background: Restrictive cardiomyopathy (RCM) is characterized by impaired ventricular relaxation. Although several mutations were reported in some patients, no mutations were identified in cardiomyocyte expressing genes of other patients, indicating that pathological mechanisms underlying RCM could not be determined by cardiomyocytes only. Cardiac fibroblasts (CFs) are a major cell population in the heart; however, the pathological roles of CFs in cardiomyopathy are not fully understood., Methods and results: This study established 4 primary culture lines of CFs from RCM patients and analyzed their cellular physiology, the effects on the contraction and relaxation ability of healthy cardiomyocytes under co-culture with CFs, and RNA sequencing. Three of four patients hadTNNI3mutations. There were no significant alterations in cell proliferation, apoptosis, migration, activation, and attachment. However, when CFs from RCM patients were co-cultured with healthy cardiomyocytes, the relaxation velocity of cardiomyocytes was significantly impaired both under direct and indirect co-culture conditions. RNA sequencing revealed that gene expression profiles of CFs in RCM were clearly distinct from healthy CFs. The differential expression gene analysis identified that several extracellular matrix components and cytokine expressions were dysregulated in CFs from RCM patients., Conclusions: The comprehensive gene expression patterns were altered in RCM-derived CFs, which deteriorated the relaxation ability of cardiomyocytes. The specific changes in extracellular matrix composition and cytokine secretion from CFs might affect pathological behavior of cardiomyocytes in RCM.

Clinical outcomes of pulmonary arterial hypertension (PAH) may be improved using targeted delivery system. We investigated the efficacy of ONO1301 (prostacyclin agonist) nanospheres (ONONS) in Sugen5416/hypoxia rat models of PAH. The rats were injected with saline (control) or ONONS (n = 10, each) on days 21 and 28, respectively. Hepatocyte growth factor (HGF)-expressing fibroblasts and inflammatory cytokines were measured. Cardiac performance was assessed and targeted delivery was monitored in vivo, using Texas red-labeled nanoparticles. Compared with control, HGF-expressing fibroblasts and HGF expression levels were significantly higher in the ONONS group, while the levels of interleukin-6, interleukin-1β, transforming growth factor-β, and platelet-derived growth factor were lower. Histological assessment revealed significant amelioration of the percent medial wall thickness in pulmonary vasculature of rats in the ONONS group. Rats in the ONONS group showed decreased proliferating cell nuclear antigen-positive smooth muscle cells and improved right ventricle pressure/left ventricle pressure. No difference was seen in the accumulation of Texas red-labeled nanoparticles in the brain, heart, liver, and spleen between PAH and normal rats. However, a significant area of nanoparticles was detected in the lungs of PAH rats. ONONS effectively ameliorated PAH, with selective delivery to the damaged lung.

Objective: To assess the safety and efficacy of the endothelin receptor antagonist ambrisentan in pediatric pulmonary arterial hypertension (PAH)., Study Design: In this open-label, phase IIb study, patients with PAH aged 8 to <18 years were randomized to low- or high-dose ambrisentan for 24 weeks. Most patients were receiving other PAH medication(s) that could not be changed during the trial. The primary outcome was safety (treatment-emergent adverse events [TEAEs]); secondary outcome was efficacy (including change from baseline to week 24 in 6-minute walking distance and World Health Organization functional class). Study staff were blinded to treatment. No statistical testing was performed., Results: Most of the 41 patients randomized (80%) experienced ≥1 TEAE; most were mild (22%) or moderate (49%) in severity (no difference between dose groups). Most common TEAEs were headache (24%), nausea (17%), abdominal pain (12%), and nasopharyngitis (12%). Eight patients had serious TEAEs; 2 were fatal (unrelated to study treatment). Improved 6-minute walking distance was observed from baseline to week 24: total mean (SD) change, +40.69 (84.58) meters; World Health Organization functional class was maintained or improved in 70% and 27% patients, respectively., Conclusions: Ambrisentan was well tolerated; TEAEs were consistent with the adult safety profile. Efficacy was similar to previous findings in adult PAH; however, interpretation is limited by small sample size. Findings support a potentially similar benefit:risk profile in pediatric (8 to <18 years) and adult patients with PAH., Trial Registration: ClinicalTrials.gov: NCT01332331., (© 2020 GlaxoSmithKline.)

Objectives: This study aimed to identify the histological characteristics associated with bridge to recovery using Berlin Heart EXCOR® (BHE) in paediatric patients <10 kg with dilated cardiomyopathy., Methods: Of the 10 consecutive patients <10 kg with dilated cardiomyopathy who underwent BHE implantation between 2013 and 2018, 4 patients showed improvement in left ventricular (LV) function, resulting in successful BHE explantation (recovery group). The remaining 6 patients showed persistent LV dysfunction and underwent heart transplantation (non-recovery group). The following variables were compared between the 2 groups: (i) histological findings in LV myocardium obtained at BHE implantation and (ii) LV function after BHE implantation assessed with echocardiography and cardiac catheterization., Results: The degree of myocardial fibrosis was significantly lower, and the capillary vascular density was significantly higher in the recovery group than in the non-recovery group [16% (standard deviation 5.9%) vs 28% (5.9%), P = 0.021, and 65 (11) vs 43 (18) units/high-power field, P = 0.037, respectively]. The changes during 3 months after BHE implantation in LV diastolic dimension (z-score) and ejection fraction were significantly greater in the recovery group than in the non-recovery group [-9.6 (3.5) vs -3.6 (4.5), P = 0.045, and 36% (13%) vs 13% (13%), P = 0.032, respectively]., Conclusions: In paediatric patients <10 kg with dilated cardiomyopathy, bridge to recovery with BHE implantation was achieved in patients with less injured LV myocardial histology at BHE implantation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Objectives: Tricuspid valve dysplasia (TVD) and Ebstein's anomaly (EA) diagnosed by fetal echocardiography vary greatly in terms of clinical severity and prognosis. The Celermajer index and Simpson-Andrews-Sharland (SAS) score have been reported previously for the prediction of prognosis in cases of TVD/EA; however, they do not take into account the hemodynamic impact of left ventricular (LV) function, which has recently been implicated as being important in the pathophysiology of TVD/EA. The aim of this study was to develop a novel scoring system that includes LV function for the prediction of perinatal death in fetuses diagnosed with TVD/EA., Methods: The clinical records of 36 fetuses diagnosed prenatally with TVD/EA between 2000 and 2015 in our hospital were reviewed. Univariate analysis was used to assess the association between perinatal death (defined as death between 22 weeks' gestation and 4 weeks after delivery) and gestational age at diagnosis, cardiothoracic area ratio (CTAR), degree of pulmonary artery flow, direction of ductal flow, right-to-left ventricular diameter ratio, tricuspid regurgitation (TR) maximum velocity, Celermajer index, SAS score and LV-Tei index. A new prognostic score, the TRIPP score (TRIcuspid malformation Prognosis Prediction score), was developed using the parameters found to be associated significantly with perinatal death. The predictive value of this score was assessed in an additional nine fetuses diagnosed with TVD/EA., Results: Thirty-six fetuses were diagnosed prenatally with TVD/EA, two of which were terminated, one was lost to follow-up and two died before 22 weeks' gestation. Of the 31 included fetuses, 10 (32%) died in the perinatal period. Univariate analysis demonstrated that TR maximum velocity was significantly lower (2.22 ± 0.17 m/s vs 3.26 ± 0.12 m/s; P < 0.001) and SAS score was significantly higher (5.7 ± 0.6 points vs 2.8 ± 0.4 points; P = 0.0014) in cases of perinatal death than in surviving fetuses. The degree of pulmonary artery flow and the direction of ductal flow were also associated significantly with perinatal death (P < 0.01 for both). Notably, LV-Tei index was significantly higher in cases of perinatal death than in surviving fetuses (0.81 ± 0.08 vs 0.50 ± 0.05; P < 0.001). In contrast, there was no significant difference in Celermajer index, CTAR or right-to-left ventricular diameter ratio. Finally, we established a novel combinatorial scoring system, the TRIPP score, including the four significant factors: TR maximum velocity, pulmonary artery flow, direction of ductal flow and LV-Tei index. The TRIPP score was found to predict efficiently perinatal mortality in fetuses with TVD/EA., Conclusions: Our novel combinatorial score of echocardiographic parameters, the TRIPP score, including LV-Tei index, is easy to measure and provides a good tool for the prediction of perinatal mortality in fetuses diagnosed prenatally with TVD/EA. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.)

Supraspinal motor control systems of pectoral fins remain unclear in teleosts. Nucleus ruber of Goldstein (1905; NRg), which has been identified as the probable homologue of nucleus ruber of tetrapods, is a candidate structure serving for such functions. In the present study, we investigated possible involvement of the NRg in the control of pectoral fin movement by tract-tracing experiments in the Nile tilapia Oreochromis niloticus. Tracer injections into the NRg revealed the fiber course of rubrospinal tract. Rubrospinal fibers crossed the midline at the level of midbrain, descended through the tegmentum, and terminated in a region ventrally adjacent to the dorsal horn at the spinomedullary junction, without reaching the ventral horn where pectoral fin motor neurons are present. Tracer injection experiments into the dorsal horn region resulted in labeled terminals in proximities of presumed pectoral fin motor neurons in the ventral horn. Tracer injection experiments into the ventral horn resulted in retrogradely labeled neurons ventrally adjacent to the dorsal horn, where labeled terminals were detected following rubral injections. These anatomical analyses suggest that the NRg of actinopterygians is involved in the control of pectoral fin motor neurons through an indirect pathway via interneurons in the dorsal horn., (© 2018 Wiley Periodicals, Inc.)

The clarification of public concerns regarding heart transplantation is important for improving low organ donation rates in Japan. In the present study, we used the Twitter data of 4986 tweets (between August 2015 and January 2016) and 1429 tweets (between April 2016 and May 2016) to analyze public discourse on heart transplantation in Japan and identify the reasons for low organ donation rates. We manually categorized all tweets relevant to heart transplantation into nine categories and counted the number of tweets in each category per month. During the study period, the most popular category of tweets was related to the media, followed by money (tweets questioning or even criticizing the high price of fundraising goals to go overseas for heart transplantations), while some tweets were misconceptions. We also conducted a sentiment analysis, which revealed that the most popular negative tweets were related to money, while the most positive tweets were related to reports on the favorable outcomes of recipients. Our results suggest that listening to concerns, providing correct information (particularly for some misconceptions), and emphasizing the outcomes of recipients will facilitate an increase in the number of people contemplating heart transplantation and organ donation., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

CIC-rearranged undifferentiated small round cell sarcoma (CIC-rearranged USRCS) is a recently established type of Ewing-like small round cell sarcomas, characterized by CIC gene rearrangement, most commonly CIC-DUX4 fusion. This report presents the second case of CIC-rearranged USRCS arising primarily in the cerebrum. A 64-year-old otherwise healthy woman presented with a 1 × 1 cm sized hemorrhagic subcortical tumor in the left temporo-parietal lobe. The tumor repeatedly recurred, and the patient underwent three surgeries, chemotherapy with doxorubicin and ifosfamide, and radiotherapy, as well as gamma knife surgery. Systemic examination revealed no other extracranial masses. Imprint cytology revealed small to moderate-sized round-to-ovoid tumor cells with mild pleomorphism and variations in size and shape. The nuclei contained finely granular chromatin, and some had easily-recognizable nucleoli. The tumor exhibited a mainly cytoplasmic pattern of CD99 immunostaining, rather than a diffuse membranous pattern. The tumor also exhibited diffuse positivity for calretinin and p16, as well as partial positivity for WT1 (nuclear and cytoplasmic staining pattern) and D2-40. FISH assessment showed CIC split signals. In conclusion, CIC-rearranged USRCSs can occur primarily in the cerebrum. It would be impossible to diagnose them through cytology alone, but cytology would be useful to rule out other small round cell brain tumors including gliomas, lymphomas, carcinomas, and germinoma. Immunohistochemical analysis including tests for CD99, calretinin, and WT1 would help to suggest CIC-rearranged USRCSs and distinguish them from Ewing sarcomas. Additionally, immunohistochemistry for p16 might be useful in the diagnosis. Diagn. Cytopathol. 2016;44:828-832. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

We recently found a constitutively active mutant of natriuretic peptide receptor 2 (caNPR2; V883M), which synthesizes larger amounts of cyclic guanosine monophosphate (cGMP) intracellularly without any ligand stimulation than existing drugs. The aim of this study was to investigate the therapeutic effects of gene transduction using caNPR2 for pulmonary arterial hypertension (PAH). In vitro gene transduction into human pulmonary arterial smooth muscle cells using Sendai virus (SeV) vectors carrying caNPR2 induced 10,000-fold increases in the synthesis of cGMP without ligand stimulation, and the proliferation of caNPR2-expressing cells was significantly attenuated. The PAH model rats generated by hypoxia and the administration of SU5416 were then treated with SeV vectors through a direct injection into the left pulmonary artery. Right ventricular systolic pressure was significantly decreased 2 weeks after the treatment, while systemic blood pressure remained unchanged. Histological analyses revealed that the medial wall thickness and occlusion rate of pulmonary arterioles were significantly improved in caNPR2-treated lungs. Neither the systemic integration of virus vectors nor side effects were observed. The massive stimulation of cGMP synthesis by gene therapy with caNPR2 was safe and effective in a PAH rat model and, thus, has potential as a novel therapy for patients with severe progressive PAH.

Background: Elucidating public concerns regarding vaccinations is important for successful immunization programs. The objective of the present study was to categorize public concerns regarding influenza vaccinations in Japan by analyzing a massive web-based question dataset., Methods: The Yahoo! Chiebukuro (Japanese Yahoo! Answers) Dataset, which includes more than 16 million questions collected between April 2004 and April 2009, was used in this study. We sequentially filtered data to obtain questions on influenza vaccinations. Any questions that met our exclusion criteria concerning veterinary vaccines or computer virus vaccines were removed from the analysis. Filtered questions and their answers were manually analyzed for their content by a team of board-certified pediatricians., Results: After filtering data, we obtained 1950 questions regarding influenza vaccinations. The three most frequently asked questions were regarding the vaccination schedule, safety, and effectiveness. When we analyzed monthly trends in question contents, we noted the emergence of similar questions in the same period every year. Therefore, we classified the time periods of each year into three parts: (1) from April to the commencement of seasonal influenza vaccinations (September), (2) from October until the epidemic period, and (3) the epidemic period. Two interesting results were obtained: concerns regarding effectiveness abruptly increased during the epidemic period, and pregnant or breastfeeding women increasingly asked questions regarding feasibility between October and the epidemic period., Conclusions: The questions and concerns collected and analyzed in this study illustrate that the public have questions about the influenza vaccine and also that questions changed with periodical consistency. These results highlight the possible usefulness of providing the public with the latest and correct information to their questions in a timely manner, for example via an official health website., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nucleus ruber is known as an important supraspinal center that controls forelimb movements in tetrapods, and the rubral homologue may serve similar functions in fishes (motor control of pectoral fin). However, two apparently different structures have been identified as 'nucleus ruber' in actinopterygians. One is nucleus ruber of Goldstein (1905) (NRg), and the other nucleus ruber of Nieuwenhuys and Pouwels (1983) (NRnp). It remains unclear whether one of these nuclei (or perhaps both) is homologous to tetrapod nucleus ruber. To resolve this issue from a phylogenetic point of view, we have investigated the distribution of tegmental neurons retrogradely labeled from the spinal cord in eight actinopterygian species. We also investigated the presence/absence of the two nuclei with Nissl- or Bodian-stained brain section series of an additional 28 actinopterygian species by comparing the morphological features of candidate rubral neurons with those of neurons revealed by the tracer studies. Based on these analyses, the NRg was identified in all actinopterygians investigated in the present study, while the NRnp appears to be absent in basal actinopterygians. The phylogenetic distribution pattern indicates that the NRg is the more likely homologue of nucleus ruber, and the NRnp may be a derived nucleus that emerged during the course of actinopterygian evolution., (© 2016 S. Karger AG, Basel.)

Objectives: To review the fetal echocardiograms of patients with total anomalous pulmonary venous connection (TAPVC) in order to determine whether the distance between the left atrium and the descending aorta would be useful in the prenatal diagnosis of fetal TAPVC., Methods: We reviewed the fetal echocardiograms of eight cases of TAPVC (five supracardiac type and three infracardiac type) with no other cardiac malformations. We evaluated the ratio of the left atrium-descending aorta distance to the diameter of the descending aorta ('post-LA space index') in 101 normal and eight TAPVC fetuses, and compared the values between groups. In addition, we examined the tricuspid valve/mitral valve diameter ratio (TVD/MVD) and the right ventricular end-diastolic diameter/left ventricular end-diastolic diameter ratio (RVDd/LVDd)., Results: The echocardiograms for fetuses with TAPVC and normal fetuses were performed at mean gestational ages of 27.5 weeks and 29.6 weeks, respectively. There were no significant differences in the TVD/MVD and RVDd/LVDd ratios between the groups. However, the post-LA space index was significantly higher in the TAPVC cases (mean, 1.51) than it was in the normal fetuses (mean, 0.71 ± 0.23) (P < 0.0001). On an analysis of the receiver-operating characteristics curve, a post-LA space index cut-off of 1.27 was found to be optimal for distinguishing between TAPVC and normal hearts, with a sensitivity of 100% and specificity of 99%., Conclusions: The novel post-LA space index could potentially be used for the prenatal diagnosis of TAPVC. A diagnosis of TAPVC is very likely in cases with a post-LA space index of > 1.27., (Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.)

Background: To investigate the possible role of sex hormones in the pathogenesis of pulmonary arterial hypertension (PAH), the effect of β-estradiol (E2) on bone morphogenetic protein (BMP) signaling, a key signaling pathway involved in PAH, was studied in human pulmonary arterial endothelial cells (HPAEC)., Methods and Results: BMP signaling molecules, including BMP receptor, Smad1/5/8 and Id1, were studied in HPAEC under 1% O2 (hypoxia) and 21% O2 (normoxia) as well as the effect of hypoxia-inducible factor (HIF)-1α expression in the presence of E2 on BMP signaling. The effects of an estrogen receptor (ER) antagonist (ICI 182,780) and cycloheximide, and the interaction of ER with Smad or HIF-1α were also studied. In the presence of E2, BMP signaling was augmented under normoxia but suppressed under hypoxia. HIF-1α accumulation suppressed BMP signaling, whereas HIF-1α inhibition augmented signaling. These effects were cancelled by ICI 182,780. Moreover, binding between ER, HIF-1α and phosphorylated (p)-Smad1/5/8 proteins occurred only under hypoxia. On inhibition of de novo synthesis with cycloheximide, however, p-Smad1/5/8 expression was suppressed only under normoxia., Conclusions: The effects of E2 on BMP signaling in HPAEC altered depending on O2 concentration and different mechanisms may be involved. BMP and sex hormones may play an important role in PAH development.

Background: Endothelin-1 (ET-1), a potent vasoconstrictor, is considered to be implicated in failing Fontan circulation, however the expressions of ET-1 and endothelin receptor type A (ET(A)R) and type B (ET(B)R) in the pulmonary arteries of failed Fontan patients were not elucidated., Methods: Immunohistochemistry and quantitative real-time PCR were used to analyse the expression levels of ET-1 and its receptors in the pulmonary arteries of the autopsy lung tissues of the patients who died after the Fontan procedure (n=10). We divided these patients into 3 groups, failed Fontan (n=4), heart failure (n=3) and non-failed Fontan (n=3), and then compared those to the age-matched normal controls (n=4)., Results: The intra-acinar pulmonary arteries of failed Fontan patients showed significant medial hypertrophy. Computational optical density analyses of the immunostaining revealed that the expressions of ET-1, ET(A)R, and ET(B)R in the intra-acinar pulmonary arteries were significantly increased in the failed Fontan patients (P<0.05 vs. normal controls), however no significant difference was observed between the non-failed Fontan patients and the normal controls. Quantitative real-time PCR analyses confirmed that the mRNA expressions of ET-1, ET(A)R, and ET(B)R were significantly increased in the failed Fontan patients (P<0.05 vs. normal controls)., Conclusion: The overexpression of ET-1 and its receptors in the pulmonary arteries can cause pulmonary vasoconstriction and vascular remodelling, leading to failed Fontan circulation. This study suggests a histopathological rationale for the potential benefits of endothelin receptor antagonists in patients with failing Fontan circulation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.

Objective: Rheumatoid arthritis (RA) is associated with numerous pulmonary manifestations. However, the inflammatory mechanism remains undetermined. We studied the features of inflammatory cells in bronchoalveolar lavage (BAL) fluid and biopsy lung tissue from patients with RA-associated lung disease., Methods: BAL findings were statistically compared between diseases. We divided RA patients into two groups, airway lesion group (AW) and interstitial lesion group (INT) according to predominant HRCT findings and compared the BAL findings. We immunohistochemically stained lung tissue for CD4, CD8, CD20, and CD163 and counted the immunopositive cells in five different regions., Patients: Twenty patients fulfilling the Japanese criteria for RA, 13 patients with systemic sclerosis (SSc), and 21 patients with polymyositis and dermatomyositis (PM-DM) with pulmonary disease detected by high-resolution CT (HRCT) were enrolled in this study., Results: As for BAL in RA, we found a lower lymphocyte frequency with higher CD4/8 ratio compared with PM-DM and a higher neutrophil percentage than both PM-DM and SSc. Nine and eleven patients with RA were classified into AW and INT groups, respectively. BAL findings did not differ between the two groups. Immunohistochemically, most CD4(+) and CD20(+) lymphocytes were accumulated in lymphoid follicles and in the alveolar wall and T-lymphocytes; in particular CD8(+) lymphocytes were predominant in lung interstitium., Conclusion: These results suggest that 1) neutrophils may play an important role, 2) the inflammatory mechanism may be similar between airway lesion and interstitial pneumonia, and 3) CD8(+) lymphocytes may be major inflammatory cells in lung interstitium in RA-associated interstitial lung disease.

A-76-year-old woman consulted for open biopsy for a pulmonary mass. Thoracoscopic wedge resection was performed. The lesion was histologically diagnosed as nonspecific inflammation. On the first postoperative day (POD1), the patient lost consciousness transiently. Eleven hours after the first stroke, the patient experienced a second stroke together with hypoxia. Pulmonary perfusion scan on POD2 showed multiple perfusion defects, and the patient was diagnosed with pulmonary embolism (PE). Thrombolitic therapy was started. Neurological symptoms didn't improve, and cerebral angiography on POD3 showed delayed perfusion in superficial veins. The patient was diagnosed with cerebral venous thrombosis (CVT). Thrombolytic and anticoagulant therapy had been continued, and the patient was found to have hemorrhagic cerebral infarction on POD11. After persistent therapy, the patient was discharged on POD120. Although both PE and CVT are rare complications after thoracic surgery, we must consider these complications in patients undergoing thoracic operations including thoracoscopic surgery.

Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and alpha-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of alpha-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.

Bronchoalveolar lavage (BAL) is a technique that samples the inflammatory cells from distal airways and alveoli; however, it is unclear whether or not cellular profiles in the BAL fluid reflect the cellular components of the lung parenchyma in interstitial lung disease (ILD). The aim of this study is to compare immunophenotypes of lymphocytes between BAL fluid and human lung tissue from patients with ILD. Fourteen consecutive patients with ILD who underwent BAL and surgical lung biopsy were enrolled. The diagnosis of ILD was confirmed by the presence of clinical symptoms and impaired respiratory function and on high-resolution computed tomography (CT) of the chest. Mononuclear cells in BAL were immunophenotyped for the expression of CD3, CD4, CD8, CD19, CD45, and CD103 by flow cytometry. Lung tissue obtained by surgical biopsy was digested with collagenase and then centrifuged to extract parenchymal cells. Isolated cells were also immunophenotyped for the same CD expression. Frequencies of positive cells were compared statistically between BAL and different lobes. Seven out of 14 patients were diagnosed clinically as suffering idiopathic interstitial pneumonia. Frequency of CD19+ cells from BAL was significantly lower than that from the upper/middle lobes (P < 0.05). Frequency of CD103+ cells from BAL was significantly higher than that from the upper/middle lobes and the lower lobe (P = 0.01 and P < 0.05, respectively). Comparison between different lobes demonstrated that the frequency of CD4+ cells from the upper/middle lobes was significantly lower than that from the lower lobe (P < 0.05). The results suggest that lymphocyte immunophenotype profiles from BAL may not reflect those in the inflammatory tissue of ILD.

Rationale: Hard metal lung disease is caused by exposure to hard metal, a synthetic compound that combines tungsten carbide with cobalt as well as a number of other metals. Interstitial lung disease caused by hard metal is uniquely characterized by giant cell interstitial pneumonia. The pathogenesis of hard metal lung disease is unclear., Objectives: To elucidate the distribution of inhaled hard metal and reactive inflammatory cells in biopsy lung tissue from patients with hard metal lung disease., Methods: Seventeen patients with interstitial lung disease in which tungsten was detected and five control subjects were studied. Detection and mapping of elements were performed with an electron probe microanalyzer equipped with a wavelength dispersive spectrometer. We immunohistochemically stained mononuclear cells, in tissue samples available from five patients, with anti-human CD4, CD8, CD20, CD68, and CD163 antibodies, and compared the distribution of positive cells with hard metal elements., Measurements and Main Results: Thirteen of 17 patients were pathologically diagnosed as having giant cell interstitial pneumonia. Tungsten and cobalt were accumulated in the centrilobular fibrotic lesions, but were never found in the control lungs. CD8+ lymphocytes and CD163+ monocyte-macrophages were distributed predominantly in centrilobular fibrotic lesions around the hard metal elements. CD163+ colocalized with tungsten. Small numbers of CD8+ and CD163+ cells were also immunohistochemically shown in peribronchiolar areas and alveolar walls., Conclusions: Macrophages may phagocytose inhaled tungsten via CD163 and play an important role in forming the fibrotic lesion of hard metal lung disease with cytotoxic T lymphocytes.

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting alpha-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l(-1) h(-1) lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l(-1) h(-1) lactate). Maximum volumetric lactate productivity was further increased (1.57 g l(-1) h(-1) lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of L: -lactate) was achieved. In this study, we propose a new approach to lactate production by alpha-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

Objective: To quantitatively isolate and immunologically phenotype mononuclear cells contained in human lung tissue., Methods: Normal appearing lung tissue as far distal to the resected lesion as possible was obtained from lung cancer patients. Lung tissue was thoroughly washed and cut into small pieces and digested with collagenase. Peripheral blood mononuclear cells (PBMNC) were prepared from controls using Ficoll gradient. Isolated cells and PBMNC were analyzed by flow cytometry. We immunohistochemically stained snap-frozen lung tissue with anti-CD3, CD4, CD8, CD20, and CD161 antibodies., Participants: Nineteen patients with lung cancer who underwent lobectomy were enrolled. Twelve healthy volunteers also participated as controls for flow cytometric analysis of PBMNC., Results: In forward scatter vs side scatter, 92.1+/-7.8% of isolated cells in the lymphoid population expressed leukocyte common antigen, CD45. The frequency of CD45-positive cells in the lymphoid population from lung tissue was as high as that from PBMNC (p=0.118). CD45-positive cells were successfully further extended by anti-CD3, CD4, CD8, CD19, and CD161 antibodies. Monocyte-macrophages bearing CD68 were also detected. CD68-positive alveolar macrophages disappeared from alveolar spaces after thorough washing by immunohistochemical staining. Mononuclear cells in the interstitium were positively stained by anti-CD3, CD4, CD8, CD20, and CD161 monoclonal antibodies., Conclusions: We could isolate interstitial cells and analyze cell surface markers via flow cytometry from fresh lung specimens by collagenase digestion without further purification. Immunohistochemistry confirmed the presence of the cells detected by flow cytometry in the lung interstitium.

The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.