Supplementary Material for: Use of Sotrovimab for COVID-19 in a patient with IGCCC Poor Prognosis testicular germ cell tumor

A 35-year-old man was diagnosed with stage IIIC non-seminoma with paralysis of the lower half of his body by 8th thoracic spine metastasis. The patient received bleomycin, etoposide, and cisplatin (BEP) therapy. On day 4 of the second course of BEP, the patient developed a fever and was diagnosed with coronavirus disease (COVID-19). COVID-19 was suspected to worsen because of cancer and chemotherapy-induced immunosuppression. However, the benefits of continuing BEP therapy outweighed these risks. After obtaining fully informed consent, BEP therapy was continued from day 5, while sotrovimab (anti-COVID-19 drug) was administered. The second course of BEP was completed without worsening severe COVID-19 or bleomycin-induced lung injury. The patient completed four courses of BEP, with normalization of tumor markers, partial response on imaging, and improvement in lower body paralysis. In this case, we successfully treated a patient with testicular germ cell tumor with chemotherapy while COVID-19 without treatment delay. During the COVID-19 pandemic, concomitant chemotherapy and COVID-19 treatment is warranted because delaying treatment will decrease the efficacy of highly curative diseases such as germ cell tumors

Supplementary Material for: Administration of Umbilical Cord Blood Cells Transiently Decreased Hypoxic-Ischemic Brain Injury in Neonatal Rats

This study aimed to investigate whether the administration of mononuclear cells derived from human umbilical cord blood cells (UCBCs) could ameliorate hypoxic-ischemic brain injury in a neonatal rat model. The left carotid arteries of 7-day-old rats were ligated, and the rats were then exposed to 8% oxygen for 60 min. Mononuclear cells derived from UCBCs using the Ficoll-Hypaque technique were injected intraperitoneally 6 h after the insult (1.0 × 10⁷ cells). Twenty-four hours after the insult, the number of cells positive for the oxidative stress markers 4-hydroxy-2-nonenal and nitrotyrosine, in the dentate gyrus of the hippocampus in the UCBC-treated group, decreased by 36 and 42%, respectively, compared with those in the control group. In addition, the number of cells positive for the apoptosis markers active caspase-3 and apoptosis-inducing factor decreased by 53 and 58%, respectively. The number of activated microglia (ED1-positive cells) was 51% lower in the UCBC group compared with the control group. In a gait analysis performed 2 weeks after the insult, there were no significant differences among the sham-operated, control and UCBC groups. An active avoidance test using a shuttle box the following week also revealed no significant differences among the groups. Neither the volumes of the hippocampi, corpus callosum and cortices nor the numbers of neurons in the hippocampus were different between the UCBC and control groups. In summary, a single intraperitoneal injection of UCBC-derived mononuclear cells 6 h after an ischemic insult was associated with a transient reduction in numbers of apoptosis and oxidative stress marker-positive cells, but it did not induce long-term morphological or functional protection. Repeated administration or a combination treatment may be required to achieve sustained protection

Supplementary Material for: The balance of CD8-positive T cells and PD-L1 expression in the myocardium predicts prognosis in lymphocytic fulminant myocarditis

Introduction: The clinical significance and prognostic value of T cell involvement and programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) have not been established in lymphocytic fulminant myocarditis (FM). We investigated the prognostic impact of the number of CD4+, CD8+, FoxP3+, and PD-1+ T cells, as well as PD-L1 expression, in cardiomyocytes in lymphocytic FM. Methods: This is a single-center observational cohort study. Myocardial tissue was obtained from 16 consecutive patients at lymphocytic FM onset. The median follow-up was 140 days. Cardiac events were defined as a composite of cardiac death and left ventricular-assist device implantation. CD4, CD8, FoxP3, PD-1, and PD-L1 immunostaining was performed on myocardial specimens. Results: The median age of the patients was 52 years (seven men and nine women). There was no significant difference in the number of CD4+ cells. The number of CD8+ cells and the CD8+/CD4+ T cell ratio were higher in the cardiac event group (Event+) than in the group without cardiac events (Event−) (P = 0.048 and P = 0.022, respectively). The number of FoxP3+ T cells was higher in the Event+ group (P = 0.049). Although there was no difference in the number of PD-1+ cells, cardiomyocyte PD-L1 expression was higher in the Event+ group (P = 0.112). Event-free survival was worse in the group with a high CD8+ cell count (P = 0.012) and high PD-L1 expression (P = 0.049). When divided into three groups based on the number of CD8+ cells and PD-L1 expression (CD8highPD-L1high [n = 8], CD8lowPD-L1high [n = 1], and CD8lowPD-L1low [n = 7]), the CD8highPD-L1high group demonstrated the worst event-free survival, while the CD8lowPD-L1high group had a favorable prognosis without cardiac events (P = 0.041). Conclusion: High myocardial expression of CD8+ T cells and PD-L1 may predict a poor prognosis in lymphocytic FM

Supplementary Material for: The balance of CD8-positive T cells and PD-L1 expression in the myocardium predicts prognosis in lymphocytic fulminant myocarditis

Introduction: The clinical significance and prognostic value of T cell involvement and programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) have not been established in lymphocytic fulminant myocarditis (FM). We investigated the prognostic impact of the number of CD4+, CD8+, FoxP3+, and PD-1+ T cells, as well as PD-L1 expression, in cardiomyocytes in lymphocytic FM. Methods: This is a single-center observational cohort study. Myocardial tissue was obtained from 16 consecutive patients at lymphocytic FM onset. The median follow-up was 140 days. Cardiac events were defined as a composite of cardiac death and left ventricular-assist device implantation. CD4, CD8, FoxP3, PD-1, and PD-L1 immunostaining was performed on myocardial specimens. Results: The median age of the patients was 52 years (seven men and nine women). There was no significant difference in the number of CD4+ cells. The number of CD8+ cells and the CD8+/CD4+ T cell ratio were higher in the cardiac event group (Event+) than in the group without cardiac events (Event−) (P = 0.048 and P = 0.022, respectively). The number of FoxP3+ T cells was higher in the Event+ group (P = 0.049). Although there was no difference in the number of PD-1+ cells, cardiomyocyte PD-L1 expression was higher in the Event+ group (P = 0.112). Event-free survival was worse in the group with a high CD8+ cell count (P = 0.012) and high PD-L1 expression (P = 0.049). When divided into three groups based on the number of CD8+ cells and PD-L1 expression (CD8highPD-L1high [n = 8], CD8lowPD-L1high [n = 1], and CD8lowPD-L1low [n = 7]), the CD8highPD-L1high group demonstrated the worst event-free survival, while the CD8lowPD-L1high group had a favorable prognosis without cardiac events (P = 0.041). Conclusion: High myocardial expression of CD8+ T cells and PD-L1 may predict a poor prognosis in lymphocytic FM

Supplementary Material for: The balance of CD8-positive T cells and PD-L1 expression in the myocardium predicts prognosis in lymphocytic fulminant myocarditis

Introduction: The clinical significance and prognostic value of T cell involvement and programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) have not been established in lymphocytic fulminant myocarditis (FM). We investigated the prognostic impact of the number of CD4+, CD8+, FoxP3+, and PD-1+ T cells, as well as PD-L1 expression, in cardiomyocytes in lymphocytic FM. Methods: This is a single-center observational cohort study. Myocardial tissue was obtained from 16 consecutive patients at lymphocytic FM onset. The median follow-up was 140 days. Cardiac events were defined as a composite of cardiac death and left ventricular-assist device implantation. CD4, CD8, FoxP3, PD-1, and PD-L1 immunostaining was performed on myocardial specimens. Results: The median age of the patients was 52 years (seven men and nine women). There was no significant difference in the number of CD4+ cells. The number of CD8+ cells and the CD8+/CD4+ T cell ratio were higher in the cardiac event group (Event+) than in the group without cardiac events (Event−) (P = 0.048 and P = 0.022, respectively). The number of FoxP3+ T cells was higher in the Event+ group (P = 0.049). Although there was no difference in the number of PD-1+ cells, cardiomyocyte PD-L1 expression was higher in the Event+ group (P = 0.112). Event-free survival was worse in the group with a high CD8+ cell count (P = 0.012) and high PD-L1 expression (P = 0.049). When divided into three groups based on the number of CD8+ cells and PD-L1 expression (CD8highPD-L1high [n = 8], CD8lowPD-L1high [n = 1], and CD8lowPD-L1low [n = 7]), the CD8highPD-L1high group demonstrated the worst event-free survival, while the CD8lowPD-L1high group had a favorable prognosis without cardiac events (P = 0.041). Conclusion: High myocardial expression of CD8+ T cells and PD-L1 may predict a poor prognosis in lymphocytic FM

Supplementary Material for: Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy

<p>Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major cause of mortality and persistent neurological disabilities in affected individuals. At present, hypothermia is considered to be the only applicable treatment option, although growing evidence suggests that cell-based therapy might achieve better outcomes. Dedifferentiated fat (DFAT) cells are derived from mature adipocytes via a dedifferentiation strategy called ceiling culture. Their abundance and ready availability might make them an ideal therapeutic tool for the treatment of HIE. In the present study, we aimed to determine whether the outcome of HIE can be improved by DFAT cell treatment. HI injury was achieved by ligating the left common carotid artery in 7-day-old rat pups, followed by 1-h exposure to 8% O₂. Subsequently, the severity of damage was assessed by diffusion-weighted magnetic resonance imaging to assign animals to equivalent groups. 24 h after hypoxia, DFAT cells were injected at 10⁵ cells/pup into the right external jugular vein. To evaluate brain damage in the acute phase, a group of animals was sacrificed 48 h after the insult, and paraffin sections of the brain were stained to assess several acute injury markers. In the chronic phase, the behavioral outcome was measured by performing a series of behavioral tests. From the 24th day of age, the sensorimotor function was examined by evaluating the initial forepaw placement on a cylinder wall and the latency to falling from a rotarod treadmill. The cognitive function was tested with the novel object recognition (NOR) test. In vitro conditioned medium (CM) prepared from cultured DFAT cells was added at various concentrations to neuronal cell cultures, which were then exposed to oxygen-glucose deprivation (OGD). The number of cells that stained positive for the apoptosis marker active caspase-3 decreased by 73 and 52% in the hippocampus and temporal cortex areas of the brain, respectively, in the DFAT-treated pups. Similarly, the numbers of ED-1-positive cells (activated microglia) decreased by 66 and 44%, respectively, in the same areas in the DFAT-treated group. The number of cells positive for the oxidative stress marker 4-hydroxyl-2-nonenal decreased by 68 and 50% in the hippocampus and the parietal cortex areas, respectively, in the DFAT-treated group. The HI insult led to a motor deficit according to the rotarod treadmill and cylinder test, where it significantly affected the vehicle group, whereas no difference was confirmed between the DFAT and sham groups. However, the NOR test indicated no significant differences between any of the groups. DFAT treatment did not reduce the infarct volume, which was confirmed immunohistochemically. According to in vitro experiments, the cell death rates in the DFAT-CM-treated cells were significantly lower than those in the controls when DFAT-CM was added 48 h prior to OGD. The treatment effect of adding DFAT-CM 24 h prior to OGD was also significant. Our results indicate that intravenous injection with DFAT cells is effective for ameliorating HI brain injury, possibly via paracrine effects.</p