Epidemiologic and Clinical Outcomes of Pediatric Renal Tumors in Korea: A Retrospective Analysis of The Korean Pediatric Hematology and Oncology Group (KPHOG) Data.

PURPOSE: Renal tumors account for approximately 7% of all childhood cancers. These include Wilms tumor (WT), clear cell sarcoma of the kidney (CCSK), malignant rhabdoid tumor of the kidney (MRTK), renal cell carcinoma (RCC), congenital mesoblastic nephroma (CMN) and other rare tumors. We investigated the epidemiology of pediatric renal tumors in Korea. MATERIALS AND METHODS: From January 2001 to December 2015, data of pediatric patients (0-18 years) newly-diagnosed with renal tumors at 26 hospitals were retrospectively analyzed. RESULTS: Among 439 patients (male, 240), the most common tumor was WT (n=342, 77.9%), followed by RCC (n=36, 8.2%), CCSK (n=24, 5.5%), MRTK (n=16, 3.6%), CMN (n=12, 2.7%), and others (n=9, 2.1%). Median age at diagnosis was 27.1 months (range 0-225.5) and median follow-up duration was 88.5 months (range 0-211.6). Overall, 32 patients died, of whom 17, 11, 1, and 3 died of relapse, progressive disease, second malignant neoplasm, and treatment-related mortality. Five-year overall survival and event free survival were 97.2% and 84.8% in WT, 90.6% and 82.1% in RCC, 81.1% and 63.6% in CCSK, 60.3% and 56.2% in MRTK, and 100% and 91.7% in CMN, respectively (p < 0.001). CONCLUSION: The pediatric renal tumor types in Korea are similar to those previously reported in other countries. WT accounted for a large proportion and survival was excellent. Non-Wilms renal tumors included a variety of tumors and showed inferior outcome, especially MRTK. Further efforts are necessary to optimize the treatment and analyze the genetic characteristics of pediatric renal tumors in Korea.

Characteristics of pediatric rhabdomyolysis and the associated risk factors for acute kidney injury: a retrospective multicenter study in Korea.

BACKGROUND: The clinical features of pediatric rhabdomyolysis differ from those of the adults with rhabdomyolysis; however, multicenter studies are lacking. This study aimed to investigate the characteristics of pediatric rhabdomyolysis and reveal the risk factors for acute kidney injury (AKI) in such cases. METHODS: This retrospective study analyzed the medical records of children and adolescents diagnosed with rhabdomyolysis at 23 hospitals in South Korea between January 2007 and December 2016. RESULTS: Among 880 patients, those aged 3 to 5 years old composed the largest subgroup (19.4%), and all age subgroups were predominantly male. The incidence of AKI was 11.3%. Neurological disorders (53.6%) and infection (39.0%) were the most common underlying disorder and cause of rhabdomyolysis, respectively. The median age at diagnosis in the AKI subgroup was older than that in the non-AKI subgroup (12.2 years vs. 8.0 years). There were no significant differences in body mass index, myalgia, dark-colored urine, or the number of causal factors between the two AKI-status subgroups. The multivariate logistic regression model indicated that the following factors were independently associated with AKI: multiorgan failure, presence of an underlying disorder, strong positive urine occult blood, increased aspartate aminotransferase and uric acid levels, and reduced calcium levels. CONCLUSIONS: Our study revealed characteristic clinical and laboratory features of rhabdomyolysis in a Korean pediatric population and highlighted the risk factors for AKI in these cases. Our findings will contribute to a greater understanding of pediatric rhabdomyolysis and may enable early intervention against rhabdomyolysis-induced AKI.

Lower albumin level and longer disease duration are risk factors of acute kidney injury in hospitalized children with nephrotic syndrome.

BACKGROUND: Children with nephrotic syndrome (NS) are at an increased risk of acute kidney injury (AKI) and the incidence of AKI in this population is reportedly increasing. This study aimed to investigate the incidence, clinical profiles, and risk factors of AKI in hospitalized children with NS through a nationwide study. METHODS: This retrospective multicenter study included 14 pediatric nephrology centers in Korea. From 2013 to 2017, a total of 814 patients with idiopathic NS were cared for at participating centers. Among them, 363 patients were hospitalized for NS and investigated in this study. RESULTS: A total of 363 children with NS were hospitalized 574 times. AKI occurred in 93 admissions (16.2%) of 89 patients: 30 (32.3%) stage 1; 24 (25.8%) stage 2; and 39 (41.9%) stage 3. Multivariate logistic regression analysis showed that longer disease duration, lower albumin level, and methylprednisolone pulse treatment were significantly associated with AKI development in hospitalized children with NS. AKI was associated with a longer hospital stay than non-AKI (median 10 vs. 7 days, P = 0.001). Among 93 admissions, 85 (91.4%) episodes recovered from AKI without complication, whereas 6 (6.5%) progressed to advanced chronic kidney disease (CKD). CONCLUSIONS: AKI is not uncommon in hospitalized children with NS, and its incidence in this nationwide study was 16.2%. Risk factors for AKI in hospitalized children with NS include longer disease duration, lower albumin level, and methylprednisolone pulse therapy. Pediatric NS patients with these characteristics should be under more strict scrutiny for the occurrence of AKI. Graphical abstract.

Serum cytokine profiles in infants with infantile hemangiomas on oral propranolol treatment: VEGF and bFGF, potential biomarkers predicting clinical outcomes.

BACKGROUND: Oral propranolol has become first-line treatment for infantile hemangiomas (IHs). This study focused on identifying cytokines related to the biology of IH and early regression indicators of IH after propranolol treatment. METHODS: For inclusion, the patients had to be aged less than 1 year and have an IH with a largest diameter ≥2 cm. Patients were scheduled to receive 1 year of propranolol treatment. Serum cytokines involved in angiogenesis, vasculogenesis, and/or chronic inflammation were analyzed at 0, 1, and/or 12 months after treatment using Multiplex Luminex assays. RESULTS: Among the 49 evaluable patients, 33 completed the 1-year treatment: 16 showed excellent response and 12 had good response to propranolol. Significant decreases in serum MMP-2, bFGF, VEGF-α, and MCP-1 levels were observed after 1 year of treatment compared to pretreatment values. The maximal diameters of the lesions significantly correlated with pretreatment serum VEGF-α, bFGF, and MMP-9. Patients with higher bFGF and VEGF levels showed better response to propranolol at 1 year. CONCLUSION: MMP-2, VEGF-α, bFGF, and MCP-1 may involve in the biology of IH and their downregulation may be associated with involution processes of IH. Pretreatment bFGF and VEGF could be novel biomarkers for predicting response to propranolol. IMPACT: We found that decreases in the concentrations of MMP-2, bFGF, VEGF, and MCP-1 were associated with regression of the hemangioma, which indicates that one of the mechanisms of propranolol in the treatment of proliferative hemangiomas may involve downregulation of those cytokines. Patients with higher bFGF and VEGF levels showed better response to propranolol at 1 year. Importantly, serum bFGF higher than 37.07 pg/mL may predict an excellent response to propranolol. Therefore, along with the patient's age and the size and visual characteristics of the lesion, bFGF levels could help determine the viability of propranolol use in the treatment of IHs. Our study represented extensive serum profiling in IH, reporting the indicators and molecules clearly related to IH regression with propranolol treatment. The authors believe that monitoring serum cytokines, including MMP-2, bFGF, VEGF, and MCP-1, in IH patients could be important, in addition to clinical follow-up, for determining when to start and end propranolol treatment.

Korean parents' perceptions of the challenges and needs on school re-entry during or after childhood and adolescent cancer: a multi-institutional survey by Korean Society of Pediatric Hematology and Oncology.

BACKGROUND: For children and adolescents with cancer, going back to school is a key milestone in returning to "normal life." PURPOSE: To identify the support vital for a successful transition, we evaluated the parents' needs and the challenges they face when their children return to school. METHODS: This multi-institutional study was conducted by the Korean Society of Pediatric Hematology and Oncology. The written survey comprised 24 questions and was completed by 210 parents without an interviewer. RESULTS: Most parents (165 of 206) reported that their children experienced difficulties with physical status (n=60), peer relationships (n=30), academic performance (n=27), emotional/behavioral issues (n=11), and relationships with teachers (n=4) on reentering school. Parents wanted to be kept informed about and remain involved in their children's school lives and reported good parent-teacher communication (88 of 209, 42.1%). Parents reported that 83.1% and 44.9% of teachers and peers, respectively, displayed an adequate understanding of their children's condition. Most parents (197 of 208) answered that a special program is necessary to facilitate return to school after cancer therapy that offers emotional support (n=85), facilitates social adaptation (n=61), and provides tutoring to accelerate catch up (n=56), and continued health care by hospital outreach and school personnel (n=50). CONCLUSION: In addition to scholastic aptitude-oriented programs, emotional and psychosocial support is necessary for a successful return to school. Pediatric oncologists should actively improve oncology practices to better integrate individualized school plans and educate peers and teachers to improve health literacy to aid them in understanding the needs of children with cancer.

Serum Hepcidin to Prohepcidin Ratio in Children with Iron Deficiency Anemia.

BACKGROUND: Hepcidin is produced in hepatocytes as a precursor form that needs to be converted to a mature hepcidin to be active. In iron deficiency anemia (IDA), the synthesis of hepcidin is inhibited; however, it is not clear how the maturation of hepcidin precursor is affected. To assess the relative maturity of serum hepcidin in the setting of IDA, we compared the ratio of mature hepcidin to prohepcidin in children with different iron statuses. METHODS: A total of 51 children (age: 0.6~18.2 yr) with normal renal function and C-reactive protein levels were enrolled. Based on hemoglobin levels and iron status, the subjects were classified as control (n=29), iron deficiency without anemia (n=6), and IDA (n=16). Serum concentrations of hepcidin and prohepcidin were measured by enzyme-linked immunosorbent assays. RESULTS: Hepcidin was positively correlated with hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, iron, transferrin saturation, and ferritin, and negatively correlated with total iron binding capacity and transferrin. IDA patients had lower levels of prohepcidin [4.7(1.9~21.7) vs 34.6(11.0~140.4) ng/mL, p<0.001] and hepcidin [9.6(3.3~39.5) vs 75.2(19.9~256.4) ng/mL, p<0.001] than control subjects. Hepcidin was strongly correlated with prohepcidin (r=0.991, p<0.001). As compared with control subjects, the hepcidin to prohepcidin ratio was lower in those with IDA (1.89±0.24 vs 2.11±0.18, p=0.009) or low serum ferritin. CONCLUSION: These findings suggest that inhibited maturation, as well as inhibited synthesis, may contribute to low hepcidin level in IDA.

Bone marrow recovery of hematopoietic stem cells and microenvironment after chemotherapy in childhood acute lymphoblastic leukemia: consecutive observations according to chemotherapy schedule.

We investigated bone marrow (BM) recovery of hematopoietic stem cells (HSC) and hematopoietic microenvironment after chemotherapy in childhood acute lymphoblastic leukemia (ALL). Twenty-nine de novo childhood ALL patients were enrolled and BM biopsy sections at diagnosis (BM0), after induction (BM1), consolidation (BM3), interim maintenance (BM5) and delayed intensification (BM7) chemotherapy were obtained. Expressions of CD133, CD34, CD117, osteopontin, osteonectin, CXCL12, and CXCR4 were evaluated by semiquantitative immunohistochemical stains. All markers recovered significantly following chemotherapy while highest values at BM3 (for CD133/CD117/CXCL12/CXCR4), BM5 (for CXCL12/CD34/osteonectin), and BM7 (for osteopontin). Patients with cytogenetic good risk expressed significantly more CD133+/CD34+ cells than those with standard and poor risk in BM5. Patients without aberrant immunophenotype expressed significantly more CD133+ cells in BM1, and more CD117+ cells in BM5 than those with aberrant immunophenotype. Patients treated with standard risk-average chemotherapeutic protocol expressed significantly more CXCR4+ cells than those treated with other protocols in BM7. Patients who showed lowest ANC ≥ 200/µL during induction chemotherapy expressed significantly more CXCR4+ cells at from BM1 to BM5, and more CD133+ cells in BM3 than those who did not. Early and full recovery of BM HSC is most vigorous at BM3 and BM5, respectively. Reconstruction of BM niche and stromal cell recovery is mostly active at BM5, and hematopoietic activity of BM niche recovers mostly at BM7. Patients with cytogenetic good risk, nonaberrant immunophenotype, standard risk-average chemotherapeutic protocol and less BM suppression during induction chemotherapy show prompt recovery of some BM HSC and microenvironment markers compared to others.

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte.

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.

Arginine vasopressin attenuates the effects of TNF-α in aortic endothelial cells by inducing ectodomain shedding of TNF receptor 1.

In septic shock, arginine vasopressin (AVP) is commonly used as a vasopressor to restore blood pressure. Exogenous AVP may have anti-inflammatory effects as well. We investigated whether AVP modulates the effects of tumor necrosis factor-α (TNF-α) in human aortic endothelial cells (HAECs). TNF-α stimulated intercellular adhesion molecule-1 expression, while AVP pretreatment attenuated this effect of TNF-α. Upon treatment with AVP, extracellular Ca2+ entered the cells rapidly through L-type calcium channels, which in turn induced cell surface translocation of a disintegrin and metalloprotease 10 (ADAM10) and ectodomain shedding of tumor necrosis factor receptor 1 (TNFR1). On the other hand, siRNA depletion of ADAM10 suppressed AVP-induced ectodomain shedding of TNFR1 and eliminated the inhibitory effect of AVP against TNF-α. Depletion of oxytocin receptor also abolished AVP-induced extracellular Ca2+ influx, AVP-induced ectodomain shedding of TNFR1 and the inhibitory effect of AVP against TNF-α. These findings suggest that AVP decreases the responsiveness of HAECs to TNF-α by inducing ADAM10-dependent ectodomain shedding of TNFR1. Extracellular Ca2+ influx through L-type calcium channels was essential for ADAM10 activation. This effect of AVP was mediated through the oxytocin receptor.

Risk Factor Analysis for Secondary Malignancy in Dexrazoxane-Treated Pediatric Cancer Patients.

PURPOSE: Dexrazoxane has been used as an effective cardioprotector against anthracycline cardiotoxicity. This study intended to analyze cardioprotective efficacy and secondary malignancy development, and elucidate risk factors for secondary malignancies in dexrazoxane-treated pediatric patients. MATERIALS AND METHODS: Data was collected from 15 hospitals in Korea. Patients who received any anthracyclines, and completed treatment without stem cell transplantation were included. For efficacy evaluation, the incidence of cardiac events and cardiac event-free survival rates were compared. Data about risk factors of secondary malignancies were collected. RESULTS: Data of total 1,453 cases were analyzed; dexrazoxane with every anthracyclines group (D group, 1,035 patients) and no dexrazoxane group (non-D group, 418 patients). Incidence of the reported cardiac events was not statistically different between two groups; however, the cardiac event-free survival rate of patients with more than 400 mg/m2 of anthracyclines was significantly higher in D group (91.2% vs. 80.1%, p=0.04). The 6-year cumulative incidence of secondary malignancy was not different between both groups after considering follow-up duration difference (non-D, 0.52%±0.37%; D, 0.60%±0.28%; p=0.55). The most influential risk factor for secondary malignancy was the duration of anthracycline administration according to multivariate analysis. CONCLUSION: Dexrazoxane had an efficacy in lowering cardiac event-free survival rates in patients with higher cumulative anthracyclines. As a result of multivariate analysis for assessing risk factors of secondary malignancy, the occurrence of secondary malignancy was not related to dexrazoxane administration.

Ectodomain Shedding of RAGE and TLR4 as a Negative Feedback Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells.

BACKGROUND/AIMS: High-mobility group box 1 (HMGB1) elicits inflammatory responses through interactions with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). We investigated how RAGE and TLR4 expressions are regulated after HMGB1 stimulation in cultured human aortic endothelial cells (HAECs). METHODS: RAGE and TLR4 expressions were analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorogenic substrate. RESULTS: Upon treatment with HMGB1, both RAGE and TLR4 began to decrease in cell lysate and remained decreased up to 24 h. The decrease in cellular RAGE and TLR4 was accompanied by an increase of N-terminal fragment of RAGE and TLR4 in culture supernatant, indicating ectodomain shedding of the receptors. HMGB1 activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while HMGB1-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. HMGB1-induced ectodomain shedding of RAGE and TLR4 was prevented by siRNA depletion of ADAM17 as well as TAPI-2, an inhibitor of ADAM family, and SB203580. HMGB1 pretreatment abolished p38 MAPK activation in response to 2nd HMGB1 stimulation. In the cells depleted of ADAM17, HMGB1-induced p38 MAPK activation was prolonged. siRNA depletion of RAGE, but not TLR4, suppressed HMGB1-induced p38 MAPK activation. CONCLUSION: In response to HMGB1 stimulation, HAECs rapidly undergo ectodomain shedding of RAGE and TLR4, and thereby become insensitive to further HMGB1 stimulation. ADAM17, activated through RAGE-p38 MAPK pathway, is implicated in the ectodomain cleavage of the receptors.

School performance of childhood cancer survivors in Korea: A multi-institutional study on behalf of the Korean Society of Pediatric Hematology and Oncology.

OBJECTIVE: To investigate school performance of childhood cancer survivors focusing on the child's functioning, including peer relationships, school attendance, and academic achievement. METHODS: We studied 241 children from 15 institutions in Korea between 2015 and 2016. The self-reported paper-and-pencil questionnaires were used. RESULTS: Approximately 22% of the survivors suffered from lack of friends. Bullying was reported by 30% of survivors. Survivors who returned to primary school reported a higher incidence of bullying compared with survivors who returned to middle or high school (P = 0.03). The percentage of children who missed classes more than 4 days in a month was higher in survivors with brain tumors than those with other tumors (P = 0.04). Approximately 41% of children reported learning difficulty. After returning to school, 53% of the patients reported that they had lower overall mark averages than they had before. Patients who returned to high school showed the highest rate of repeating a grade and the lowest rate of achieving high academic marks. The school marks in the Korean (P = 0.03), English (P = 0.04), and physical education (P = 0.04) were worse for the children with brain tumors than for the children with other tumors. CONCLUSION: We found that 20% to 25% of survivors experienced peer-related difficulties upon returning to school. Patients who return to school, especially high school, should be provided more educational support to overcome low academic achievement. Particular concern is needed to the patients with brain tumors, who are at risk for significant academic and social difficulties and therefore may require more intensive support in school.

ADAM17-Mediated Ectodomain Shedding of Toll-Like Receptor 4 as a Negative Feedback Regulation in Lipopolysaccharide-Activated Aortic Endothelial Cells.

BACKGROUND/AIMS: Lipopolysaccharide (LPS)-activated monocytes/macrophages develop endotoxin tolerance in part by reducing cell surface toll-like receptor 4 (TLR4) through cluster of differentiation 14 (CD14)-dependent endocytosis. In case of endothelial cells, CD14 is expressed in low copy numbers as compared with monocytes/macrophages. Thus, we explored how endothelial cells regulate TLR4 expression after LPS stimulation. METHODS: Cultured human aortic endothelial cells (HAECs) were treated with LPS. TLR4 expression was analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorescent substrate. RESULTS: TLR4 in cell lysate began to decrease within 30 min of LPS treatment with a maximal reduction at 2 h, and it was accompanied by an increase of N-terminal fragment of TLR4 in culture supernatant, indicating ectodomain shedding of the receptor. LPS activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while LPS-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. LPS-induced ectodomain shedding of TLR4 was attenuated by siRNA depletion of ADAM17 as well as TAPI-2 (an inhibitor of ADAM family) and SB203580. LPS pretreatment resulted in a blunted response of p38 MAPK activation to further LPS stimulation. In the cells depleted of ADAM17, LPS-induced p38 MAPK activation was prolonged and LPS-induced intercellular adhesion molecule-1 expression was potentiated. CONCLUSION: HAECs respond to LPS by rapid shedding of the ectodomain of TLR4 and thereby reduce the responsiveness to subsequent LPS exposure. ADAM17, downstream of p38 MAPK, is implicated in the ectodomain cleavage of TLR4.

Diosgenin, an Activator of 1,25D3-MARRS Receptor/ERp57, Attenuates the Effects of TNF-α by Causing ADAM10-Dependent Ectodomain Shedding of TNF Receptor 1.

BACKGROUND/AIMS: We investigated how diosgenin, a steroidal sapogenin, has anti-tumor necrosis factor-α (TNF-α) effects in human aortic endothelial cells (HAECs). METHODS: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Intracellular Ca2+ was measured using Fluo-4 AM. Immunofluorescence staining was performed for a disintegrin and metalloprotease 10 (ADAM10). RESULTS: Diosgenin (1 ∼ 100 nM) induced ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) expression. Upon treatment with diosgenin, extracellular Ca2+ entered into the cells via L-type calcium channels, whereas diosgenin-induced ectodomain shedding of TNFR1 was almost completely inhibited by BAPTA-AM (intracellular Ca2+ chelator), verapamil (L-type calcium channel antagonist) and the absence of extracellular Ca2+. Diosgenin caused translocation of ADAM10 to the cell surface, which was mediated by extracellular Ca2+ influx. Depletion of ADAM10 prevented diosgenin-induced ectodomain shedding of TNFR1 and abolished the inhibitory effect of diosgenin on TNF-α-induced ICAM-1 expression. Diosgenin did not induce extracellular Ca2+ influx and ectodomain shedding of TNFR1 in cells depleted of 1,25D3-membrane associated rapid response steroid-binding receptor (1,25D3-MARRS receptor/ERp57). CONCLUSION: Diosgenin elicits L-type calcium channel-mediated extracellular Ca2+ influx, and thereby induces ADAM10-mediated ectodomain shedding of TNFR1. This effect of diosgenin was exerted through 1,25D3-MARRS receptor/ERp57.

Epigallocatechin-3-Gallate Attenuates the Effects of TNF-α in Vascular Endothelial Cells by Causing Ectodomain Shedding of TNF Receptor 1.

BACKGROUND/AIMS: We investigated the mechanism underlying anti-tumor necrosis factor-α (TNF-α) effects of epigallocatechin-3-gallate (EGCG) in human aortic endothelial cells. METHODS: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Cytosolic Ca2+ was measured using Fluo-4 AM. A disintegrin and metalloprotease 10 (ADAM10) was localized by immunofluorescence staining. RESULTS: EGCG caused ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced endothelin-1 (ET-1) expression. EGCG-induced TNFR1 ectodomain shedding was prevented by BAPTA-AM (intracellular Ca2+ chelator), but not by the absence of extracellular Ca2+. In physiologic extracellular Ca2+ concentration, EGCG markedly increased cytosolic Ca2+. Even in the absence of extracellular Ca2+, EGCG raised cytosolic Ca2+, though less potently. siRNA depletion of ADAM10 prevented EGCG-induced ectodomain shedding of TNFR1 and also diminished the inhibitory effect of EGCG on TNF-α-induced ET-1 expression. EGCG caused translocation of ADAM10 to the plasma membrane, and this effect was prevented by BAPTA-AM. Besides extracellular Ca2+ influx, release of intracellular stored Ca2+ caused ADAM10-dependent ectodomain shedding of TNFR1. CONCLUSION: EGCG decreases the responsiveness of cells to TNF-α by causing ADAM10-dependent ectodomain shedding of TNFR1. This effect was attributed to its property to increase cytosolic Ca2+ through both extracellular Ca2+ influx and release of stored Ca2+.

TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells.

BACKGROUND/AIMS: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. METHODS: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. RESULTS: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. CONCLUSION: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.

Vitamin D-induced ectodomain shedding of TNF receptor 1 as a nongenomic action: D3 vs D2 derivatives.

As a nongenomic action, 1,25-dihydroxyvitamin D3 (1,25D3) induces L-type Ca(2+) channel-mediated extracellular Ca(2+) influx in human aortic smooth muscle cells (HASMCs), which activates a disintegrin and metalloprotease 10 (ADAM10) to cleave and shed the ectodomain of tumor necrosis factor receptor 1 (TNFR1). In this study, we examined the potencies of other vitamin D3 and D2 analogs to stimulate the ectodomain shedding of TNFR1 in HASMCs. 25-Hydroxyvitamin D3 (25D3), a precursor of 1,25D3, and elocalcitol, an analog of 1,25D3, caused ectodomain shedding of TNFR1 within 30 min, whereas 1,25-dihydroxyvitamin D2 (1,25D2) and paricalcitol, a derivative of 1,25D2, did not. Both 25D3 and elocalcitol rapidly induced extracellular Ca(2+) influx and markedly increased intracellular Ca(2+), while 1,25D2 and paricalcitol caused only small increases in intracellular Ca(2+). 25D3- and elocalcitol-induced TNFR1 ectodomain sheddings were abolished by verapamil and in Ca(2+)-free media. Both 25D3 and elocalcitol caused the translocation of ADAM10 to the cell surface, which was inhibited by verapamil, while 1,25D2 and paricalcitol did not cause ADAM10 translocation. When ADAM10 was depleted by ADAM10-siRNA, 25D3 and elocalcitol could not induce ectodomain shedding of TNFR1. The plasma membrane receptor, endoplasmic reticulum stress protein 57 (ERp57), but not the classic vitamin D receptor, mediated the nongenomic action of vitamin D to induce ectodomain shedding of TNFR1. In summary, like 1,25D3, 25D3 and elocalcitol caused ADAM10-mediated ectodomain shedding of TNFR1, whereas 1,25D2 and paricalcitol did not. The difference may depend on their affinities to ERp57 through which extracellular Ca(2+) influx is induced.

Early Spontaneous Recanalization of Sigmoid Sinus Thrombosis Following a Closed Head Injury in a Pediatric Patient : A Case Report and Review of Literature.

Cerebral venous sinus thrombosis (CVST) following a closed head injury in pediatric patients is a rare condition, and an early spontaneous recanalization of this condition is extremely rare. A 10-year-old boy was admitted with a mild, intermittent headache and nausea five days after a bicycle accident. The brain computed tomography showed an epidural hematoma at the right occipital area with pneumocephalus due to a fracture of the occipital skull bone. The brain magnetic resonance imaging and the magnetic resonance venography demonstrated a flow signal loss from the right sigmoid sinus to the right jugular vein. The diagnosis was sigmoid sinus thrombosis, so close observations were selected as a treatment for the patient because of his gradually improving symptoms; however, he complained of vomiting 14 days the after conservative treatment. The patient was readmitted for a further examination of his symptoms. The laboratory and the gastroenterological examinations were normal. Due to concern regarding the worsening of the sigmoid sinus thrombosis, the brain magnetic resonance venography was rechecked and it revealed the recanalization of the venous flow in the sigmoid sinus and in the jugular vein.