Bill Chiu, MD

Abstract

PURPOSE: Tumor-associated macrophages are present within neuroblastoma, and interferon-gamma (IFN-gamma) can polarize macrophages into cancer-inhibiting M1 type. We hypothesize that treating neuroblastoma with interferon-gamma (IFN-gamma) can suppress tumor growth, and the concurrent treatment with IFN-gamma and vincristine can lead to enhanced tumor killing as compared to vincristine alone.METHODS: We loaded IFN-gamma or vincristine into silk biomaterials and recorded the amount released over time. Orthotopic, syngeneic neuroblastoma xenografts were generated by injecting 9464D cells into adrenal gland of C57BL/6 mice, and IFN-gamma-loaded and/or vincristine-loaded silk biomaterials were implanted into the tumor once the tumors reached 100mm3. Drug release at different timepoints was measured and tumor growth after different treatments were compared.RESULTS: 1-2% of IFN-gamma and 70% of vincristine were released from the biomaterials by the fifth day. Combining IFN-gamma and vincristine significantly slowed tumor growth as compared to the controls (12.22.7days to reach 800mm3 versus 5.71.2days, p=0.01), and IFN-gamma alone also delayed tumor growth as compared to the controls (10.91.5days versus 5.71.2days, p=0.001). Hematoxylin and eosin staining demonstrated tumor necrosis adjacent to the drug-loaded silk biomaterials.CONCLUSION: Local delivery of sustained release IFN-gamma can inhibit neuroblastoma tumor growth by itself and in combination with vincristine.

View details for DOI 10.1007/s00383-023-05523-w

View details for PubMedID 37500800

Abstract

BACKGROUND: Hair at the gluteal cleft plays a key role in the development and recurrence of pilonidal disease (PD). We hypothesized that more hair reduction achieved using laser could correlate with lower chance of PD recurrence.METHODS: PD patients who underwent laser epilation (LE) were categorized by Fitzpatrick skin type, hair color, and hair thickness. Photos taken at LE sessions were compared to determine hair reduction amount. LE sessions completed prior to the recurrences were recorded. Groups were compared using multivariate T-test.RESULTS: 198 PD patients had mean age 18.13.6 years. 21, 156, and 21 patients had skin types 1/2, 3/4, and 5/6, respectively. 47 patients had light- and 151 had dark-colored hair. 29 patients had fine hair, 129 medium, and 40 thick. Median follow-up was 217 days. 95%, 70%, 40%, and 19% of patients reached 20%, 50%, 75%, and 90% hair reduction after mean LE sessions of 2.6, 4.3, 6.6, 7.8 sessions, respectively. To reach 75% hair reduction, patients require a mean of 4.8-6.8 LE sessions, depending on different skin/hair characteristics. PD recurrence rate was 6%. Probability of recurrence after 20%, 50%, 75% hair reduction was decreased by 50%, 78%, 100%, respectively. Dark hair and skin type 5/6 were associated with higher recurrence rates.CONCLUSION: Patients with dark-color and thick hair require more LE sessions to achieve certain degree of hair reduction. Patients with dark hair and skin type 5/6 were more likely to recur; more hair reduction correlated with lower chance of recurrence.LEVEL OF EVIDENCE: Level IV.

View details for DOI 10.1016/j.jpedsurg.2023.02.054

View details for PubMedID 36934004

Abstract

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the nicotinamide adenine dinucleotide (NAD)+/NADH and pyruvate/lactate ratios and also the alpha-ketoglutarate (alpha-KG)/2- hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and beta-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and beta-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma.

View details for DOI 10.1158/0008-5472.CAN-22-1029

View details for PubMedID 36318118

View details for DOI 10.1097/01.XCS.0000894496.27882.d8

View details for Web of Science ID 000867889300377

Abstract

BACKGROUND: The majority of high-risk neuroblastomas harbor telomerase activity, and telomerase-interacting compounds, such as 6-thio-2'-deoxyguanosine (6-thio-dG), have been found to impair the growth of telomerase-positive neuroblastoma cell lines. It has remained unclear, however, how such drugs can be combined with other compounds used in current treatment concepts for neuroblastoma patients.METHODS: Growth-inhibitory effects of varying concentrations of 6-thio-dG in combination with etoposide, doxorubicin or ceritinib were determined in eight telomerase-positive neuroblastoma cell lines with distinct genetic backgrounds. Tumor growth inhibition of subcutaneous xenografts from three different cell lines was assessed upon treatment with 6-thio-dG, the competitive telomerase inhibitor imetelstat, etoposide, or combinations of these compounds.RESULTS: Robust synergistic anti-tumor effects were observed for combinations of 6-thio-dG and etoposide or doxorubicin, but not for 6-thio-dG and ceritinib, in telomerase-positive neuroblastoma cell lines in vitro. Treatment of mouse xenografts with combinations of 6-thio-dG and etoposide significantly attenuated tumor growth and improved mouse survival over etoposide alone in two of three cell line models. Treatment of xenograft tumors by imetelstat monotherapy decreased telomerase activity by roughly 50% and significantly improved survival over control in all three models, whereas treatment with imetelstat plus etoposide led to enhanced survival over etoposide monotherapy in one model. Mechanistically, the synergistic effect was found to be due to both increased apoptosis and cell cycle arrest.CONCLUSION: Our study indicates that telomerase is an actionable target in telomerase-positive neuroblastoma, and demonstrates that combination therapies including telomerase-interacting compounds may improve the efficacy of established cytotoxic drugs. Targeting telomerase may thus represent a therapeutic option in high-risk neuroblastoma patients.

View details for DOI 10.1007/s13402-022-00702-8

View details for PubMedID 35953764

Abstract

PURPOSE: Pilonidal Disease (PD) affects adolescents in different aspects. We hypothesized that patients with different gender, ethnicity, and age have different quality of life (QOL) measurements which could improve with minimally invasive treatment (MIT).METHODS: 131 PD patients underwent MIT (laser epilationtrephination) from 2019 to 2021. Patients' demographics were recorded. Before and after MIT, patients received QOL questionnaire consisting of four categories: daily activities, sports participation, school/work attendance, and socializing. Data were analyzed using Student and multivariate t test. P<0.05 was considered statistically significant.RESULTS: 101 (51 male, 50 female) patients were included. 30 patients with incomplete data were excluded. 54% of patients were<18years old. 47.5% were Hispanic. Median symptom duration prior to presentation was 5.4 (1.3-15) months. Prior to MIT, patients' ability to perform daily activities, participate in sports, attend school/work, and socialize was moderately or severely impacted in 66%, 57%, 45%, and 23% of respondents, respectively; after MIT, only 7%, 8%, 2%, and 4% were affected (p<0.01). Recurrence rate was 6%. Pre-MIT, older patients and non-Hispanics reported worse impact on their QOL. Symptom duration or PD recurrence did not correlate with patient's pre- or post-MIT QOL.CONCLUSION: Patients' ethnicity and age impacted QOL in PD. All patients' QOL significantly improved with MIT. Considering the importance of socializing, playing sports, and school/work attendance in adolescents, our study highlights importance of early treatment of PD.

View details for DOI 10.1007/s00383-022-05175-2

View details for PubMedID 35842877

Abstract

BACKGROUND: Pilonidal disease may present as acute abscesses or chronic draining sinuses. There is no standardized treatment and recurrence rates can be as high as 30%. Within our five-hospital network we have established a standardized treatment protocol including minimally invasive surgical trephination and aggressive epilation. We hypothesize that such a treatment protocol can be established across different hospital settings and lead to low overall recurrence.METHODS: Patients with pilonidal disease were enrolled in the study on presentation to our hospital network. Those that underwent initial surgery outside our hospital system or were noncompliant with our treatment protocol were excluded. Patients were grouped based on surgeon and treating facility. Frequency of recurrence per surgeon and per hospital was calculated and compared.RESULTS: Out of 132 patients, 80 patients were included (45 female, 35 male) while 52 were excluded because of initial surgery at a non-network hospital or for protocol noncompliance. Median age was 17 (16-19) years and median length of follow-up was 352 (261-496) days. There were 6 patients who experienced at least one recurrence. There was an overall 8% recurrence rate with no significant difference noted between surgeons or hospitals (p=0.15, p=0.64, respectively).CONCLUSIONS: We have successfully implemented a standardized treatment protocol for pilonidal disease across different hospital settings and by different surgeons, with an overall low recurrence rate. Our findings suggest that adoption of a standardized protocol for treatment of pilonidal disease can lead to low recurrence.LEVEL OF EVIDENCE: Level IV.

View details for DOI 10.1016/j.jpedsurg.2022.06.014

View details for PubMedID 35868880

View details for Web of Science ID 000892509506053

View details for Web of Science ID 000718303100356

Abstract

INTRODUCTION: High-risk neuroblastoma is a deadly disease; poor prognosticators are MYCN-amplification and TERT-overexpression. We hypothesized that Gene Set Enrichment Analysis (GSEA) could identify pathways associated with MYCN-amplification and that inhibition of these pathways could decrease tumor growth.METHODS: We analyzed the Neuroblastoma-Kocak dataset (GSE45547, n=649) and identified pathways associated with MYCN-amplification. Inhibitors were selected from upregulated gene sets for in vitro cytotoxicity testing using ST16-patient-derived primary neuroblastoma cells and in vivo testing using orthotopic ST16-patient-derived xenografts (PDX) in mice. Tumor volume was measured with ultrasound and tumor sections examined after H&E staining.RESULTS: GSEA identified significantly overexpressed gene sets in MYCN-amplified tumors including MYC targets, cell cycle mitotic genes, TERT associated genes, loss of RB1 gene sets, and E2Fs targets. Several genes were potential Bromodomain-containing protein 4 (Brd4) targets, making Brd4 inhibitors - JQ1, AZD5153 - and cyclin-dependent kinase (Brd4's binding partner) inhibitors - dinaciclib - potential therapeutic agents. JQ1 and dinaciclib were synergistic in inducing cytotoxicity in vitro. Dinaciclib-AZD5153 in vivo decreased tumor size compared to control, and increased tumor lymphocyte infiltration and necrosis on histology.CONCLUSIONS: GSEA is a powerful approach to identify upregulated genes and potential therapeutic targets. Dinaciclib-AZD5153 combination therapy can be effective against MYCN-amplified and TERT-overexpressing neuroblastoma tumors.

View details for DOI 10.1016/j.jpedsurg.2021.03.037

View details for PubMedID 33838899

Abstract

Stage 4S neuroblastoma (4SNB) is associated with spontaneous tumor regression and an excellent prognosis. However, a small group of the patients have a poor prognosis. One hundred eighty-five 4SNB cases filed at the Children's Oncology Group Neuroblastoma Pathology Reference Laboratory were studied. MYCN oncogene status [non-amplified (NA) vs. Amplified (A)] determined by fluorescence in situ hybridization, MYC-family (MYCN/MYC) protein expression [no-overexpression(-)/(+/-) vs. overexpression(+)] by immunohistochemistry and histopathology by International Neuroblastoma Pathology Classification [Favorable Histology (FH) vs. Unfavorable Histology (UH)] with particular attention to nucleolar hypertrophy [NH(-) vs. (+)] were assessed with patient survival. One hundred forty-seven (79.5%) tumors were MYCN-NA, FH, MYC-family protein(-)/(+/-), and NH(-) with a good prognosis [88.5%+3.1% 5-y event-free survival (EFS); 94.1%+2.3% 5-y overall survival (OS)]. Among MYCN-NA tumors, 11 demonstrated MYCN protein(+) with a moderate and uniform (M/U) staining pattern: they were FH(10/11), NH(-), 1 showed MYC protein(+) simultaneously, and all patients are alive. Also found were 5 MYC protein(+) and MYCN(-)/(+/-) tumors; they were FH without NH (4/5), and all patients are alive. Among MYCN-A tumors, 18 had MYCN protein(+) with a strong and heterogeneous (S/H) staining pattern, 9 had UH (44.4%+23.4% EFS/OS) and 9 had FH (68.6%+19.2% EFS/OS), and 15 showed NH(+). Two tumors had MYCN protein(-)/(+/-) despite MYCN-A; both were FH and NH(-), and 1 patient died. S/H staining pattern of MYCN protein overexpression by immunohistochemistry was associated with MYCN amplification, NH(+) and a poor prognosis. In contrast, the M/U staining pattern was associated with MYCN nonamplification and NH(-), and had no adverse prognostic effects for the 4SNB patients.

View details for DOI 10.1097/PAS.0000000000001647

View details for PubMedID 33739795

Abstract

Neuroblastoma is the most common extracranial solid tumor of childhood and is associated with poor survival in high risk patients. Recently, dinutuximab (DNX) has emerged as an effective immunotherapy to treat patients with high risk neuroblastoma. DNX works through the induction of cell lysis via complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). However, one third of patients who undergo DNX treatment exhibit tumor relapse and the therapy is dose limited by side effects such as severe pain. To overcome delivery challenges of DNX, including large size and dose limiting side effects, we fabricated a delivery system capable of sustained local delivery of bioactive DNX utilizing silk fibroin. We evaluated the impact of silk properties (MW, crystallinity, and concentration) on release properties and confirmed the bioactivity of the release product. Additionally, we observed that the effectiveness of CDC induction by DNX could be correlated to the GD2 expression level of the target cells, with both the intravenous DNX formulation and the released DNX. Collectively, this data highlights a strategy to overcome delivery challenges and potentially improve therapeutic efficacy in cells expressing heterogenous levels of GD2. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/jbm.a.37131

View details for PubMedID 33252182

View details for Web of Science ID 000582792300362

Abstract

BACKGROUND: Neuroblastoma is the most common pediatric extracranial solid malignancy with limited effective treatment. We have shown that sustained-release, single drugs delivered locally through a silk-based biomaterial are effective in decreasing orthotopic neuroblastoma xenograft growth. We further optimized this approach and hypothesized that increasing doses of local chemotherapy or delivering 2 chemotherapeutic agents simultaneously inhibit additional tumor growth.METHODS: MYCN-amplified and non-MYCN-amplified neuroblastoma cells were treated with combinations of cisplatin, vincristine, doxorubicin, and etoposide to determine cytotoxicity and synergy. Drug-loaded silk material was created, and the amounts of drug released from the material over time were recorded. Murine orthotopic neuroblastoma xenografts were generated; tumors were implanted with single- or dual-agent chemotherapy-loaded silk. Ultrasound was used to monitor tumor growth, and tumor histology was evaluated.RESULTS: Invitro, vincristine/cisplatin combination was synergistic and significantly decreased cell viability relative to other combinations. Both drugs loaded into silk could be released effectively for over 2 weeks. Locally implanted vincristine/cisplatin silk induced increased tumor growth suppression compared with either agent alone in MYCN-amplified tumors (P < .05). The dose-dependent effect seen in MYCN-amplified tumors treated with combination therapy diminished at higher doses in non-MYCN-amplified tumors, with little benefit with doses >50 mug to 500 mug for vincristine-cisplatin, respectively. Tumor histology demonstrated tumor cell necrosis adjacent to drug-loaded silk material and presence of large cell neuroblastoma.CONCLUSION: Local delivery of sustained release chemotherapy can suppress tumor growth especially at high doses or with 2 synergistic drugs. Locally delivered dual therapy is a promising approach for future clinical testing.

View details for DOI 10.1016/j.surg.2020.01.012

View details for PubMedID 32122657

Abstract

Immunotherapy targeting GD2 is a primary treatment for patients with high-risk neuroblastoma. Dinutuximab is a monoclonal antibody with great clinical promise but is limited by side effects such as severe pain. Local delivery has emerged as a potential mechanism to deliver higher doses of therapeutics into the tumor bed, while limiting systemic toxicity. We aim to deliver dinutuximab locally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Dinutuximab-loaded silk fibroin foams were fabricated through lyophilization. In vitro release profile and bioactivity of the release through complement-dependent cytotoxicity were characterized. MYCN-amplified neuroblastoma cells (KELLY) were injected into the left gland of mice to generate an orthotopic neuroblastoma model. Once the tumor volume reached 100mm3 , dinutuximab-, human IgG-, or buffer-loaded foams were implanted into the tumor and growth was monitored using high-resolution ultrasound. Post-resection histology was performed on tumors. Dinutuximab-loaded silk fibroin foams exhibited a burst release, with slow release thereafter in vitro with maintenance of bioactivity. The dinutuximab-loaded foam significantly inhibited xenograft tumor growth compared to IgG- and buffer-loaded foams. Histological analysis revealed the presence of dinutuximab within the tumor and neutrophils and macrophages infiltrating into dinutuximab-loaded silk foam. Tumors treated with local dinutuximab had decreased MYCN expression on histology compared to control or IgG-treated tumors. Silk fibroin foams offer a mechanism for local release of dinutuximab within the neuroblastoma tumor. This local delivery achieved a significant decrease in tumor growth rate in a mouse orthotopic tumor model.

View details for DOI 10.1002/cam4.2936

View details for PubMedID 32096344

Abstract

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-seq data from 498 neuroblastoma patients and revealed a differentially overexpressed gene signature in MYCN non-amplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDKs) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.

View details for DOI 10.1158/0008-5472.CAN-19-2560

View details for PubMedID 31900258

View details for DOI 10.1200/JCO.2019.37.31_suppl.147

View details for Web of Science ID 000518224200145

View details for Web of Science ID 000492740900401

Abstract

PURPOSE: Large cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system.METHODS: Orthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN.RESULTS: Image analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC >0.90 and 100% accuracy when >30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%.CONCLUSIONS: We recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.

View details for DOI 10.1016/j.jpedsurg.2019.08.022

View details for PubMedID 31519361

View details for DOI 10.1016/j.xphs.2019.03.019

View details for Web of Science ID 000477754400028

View details for DOI 10.1016/j.jpedsurg.2019.02.028

View details for Web of Science ID 000469332500020

Abstract

Neuroblastoma is the most common extra-cranial childhood tumor and current treatment requires surgical resection and multi-drug chemotherapy. Local, perioperative delivery of chemotherapeutics is a promising treatment method for solid tumors that require surgical removal. In this study we have aimed to develop a controlled release implant system to deliver cisplatin in tumor/tumor resection area. Silk fibroin, a biodegradable, non-immunogenic biopolymer was employed to encapsulate different doses of cisplatin in a reservoir system. The physical integrity of the reservoirs was characterized by evaluating the crystalline structure of silk secondary structure using Fourier Transform Infrared (FTIR) spectroscopy. The in vitro release of cisplatin was evaluated in phosphate buffered saline at 37C and the reservoirs were able to release the drug up to 30 days. The cytotoxicity of cisplatin and cisplatin reservoirs were tested on KELLY cells. Cytotoxicity data showed 3.2 mug/mL cisplatin was required to kill 50 percent of the cell population and the released cisplatin from the silk reservoirs showed significant cytotoxicity up to 21 days. Intra-tumoral implantation of silk reservoirs into an orthotopic neuroblastoma mouse model decreased tumor growth significantly when compared to controls. These results suggest that silk reservoirs are promising carriers for cisplatin delivery to the tumor site.

View details for PubMedID 30905702

Abstract

PURPOSE: MYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461.METHODS: MYCN-amplified (KELLY, IMR5) and nonamplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24-48 h was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed.RESULTS: IC50 for KELLY, IMR5, SY5Y, and SKNAS cells to CX-5461 was 0.75 muM, 0.02 muM, 0.8 muM, and 1.7 muM, respectively. CX-5461 down-regulated MYCN and MYC proteins at 0.25-1.0 muM on Western blot analysis. CX-5461-loaded silk film released 23.73 mug of the drug in 24 h and 48.23.9 mug at 120 h. KELLY tumors treated with CX-5461-loaded film reached 800 mm3 after 7.81.4 days, while those treated with control film reached the same size on 5.10.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation.CONCLUSION: We demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.

View details for PubMedID 30879743

View details for DOI 10.1016/j.jamcollsurg.2018.08.523

View details for Web of Science ID 000447772500463

Abstract

High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0.1N NaOH for 24h. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37C. Silk coated 6% wafers released the drug up to 45days, while uncoated wafers released the drug for 30days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1g/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intra-tumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.

View details for PubMedID 30018030

View details for PubMedCentralID PMC6098973

Abstract

PURPOSE: Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted.METHODS: Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin.RESULTS: Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology.CONCLUSIONS: Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

View details for PubMedID 30244940

Abstract

BACKGROUND: Advanced-stage neuroblastoma patients require multiagent chemotherapy. Intratumoral implantation of vincristine-loaded silk gel uses local diffusion to decrease orthotopic neuroblastoma tumor growth in mice. We hypothesize that injecting vincristine-loaded silk gel into 8 locations within the tumor, instead of only centrally, decreases the diffusion distance and improves tumor growth suppression.METHODS: Human neuroblastoma cells, KELLY, were injected into mouse adrenal glands to create orthotopic tumors. After the tumors reached 100mm3 by ultrasound, silk gels loaded with 50 g vincristine were injected centrally or in 8 areas throughout the tumor. Drug-release profile was measured in vitro. Endpoints were tumor size >1,000 mm3 and histologic examination.RESULTS: Vincristine-loaded silk gels suppressed tumor growth up to an inflection point (458.7 234.4mm3 for central, 514.3 165.8 mm3 for 8-point injection) before tumor growth accelerated >200mm3 over 3 days. The time to inflection point was 6.6 days for central, 13.3 days for 8-point injection (P < .05). Using the sphere volume equation to approximate tumor volume, splitting the volume into 1/8 decreased the diffusion radius by 1/2. Histologic examination confirmed tumor necrosis adjacent to vincristine-loaded silk gel.CONCLUSION: Injecting vincristine-loaded sustained release silk gel at 8 separate locations halved the diffusion distance and doubled the time for the tumor to reach the growth inflexion point.

View details for PubMedID 30061039

View details for DOI 10.1158/1538-7445.AM2018-2497

View details for Web of Science ID 000468818905357

Abstract

Cardiotoxicity is a dose-limiting and potentially lethal complication of anthracycline administration. Previous studies failed to determine definitive toxic doses or cardioprotective factors. Current dosing strategies may utilize unnecessarily high anthracycline doses, such that survival benefit may not outweigh increased toxicity rates. A systematic review of randomized controlled trials and prospective/retrospective studies investigating anthracycline treatment in pediatric solid tumors was performed from PubMed/MEDLINE and Cochrane databases. Generalized linear models mapping survival, cardiotoxicity, and cardiotoxicity-free survival adjusted for male-to-female ratio, follow-up time, and concomitant chemotherapeutic drugs or cardioprotective agents (dexrazoxane) were generated using R. Survival rose linearly with increasing cumulative anthracycline dose whereas cardiotoxicity demonstrated exponential increases both without (dose, >200mg/m) and with (dose, >400mg/m) dexrazoxane. Maximum cardiotoxicity-free survival was 268.2mg/m without and 431.8mg/m with dexrazoxane. Despite increasing cardiotoxicity-free dose by >150mg/m, dexrazoxane minimally improved projected survival (71.9% vs. 75.4%). Cardiotoxicity increased linearly as a function of follow-up time with rates doubling from 5 to 20 years, without evidence of plateau. On the basis of our model, current dosing regimens-doxorubicin doses >375mg/m without dexrazoxane-overvalue increased anthracycline administration and may contribute to devastating cardiotoxicity. The linear increase of cardiotoxicity without evidence of plateau confirms the necessity for lifelong cardiac monitoring.

View details for PubMedID 29432315

Abstract

Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(-)/(+/-), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(-)/(+/-) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.

View details for DOI 10.18632/oncotarget.23740

View details for PubMedID 29464082

View details for PubMedCentralID PMC5814222

View details for DOI 10.1016/j.jpedsurg.2017.08.028

View details for Web of Science ID 000417641500036

Abstract

Local drug delivery minimizes systemic toxicity while delivering high-dose chemotherapy for neuroblastoma patients. We hypothesized that varying burst and maintenance dosing of implanted silk platforms would improve survival.Platforms were loaded with vincristine 25g, 50g, 100g, and 200g varying burst (released 1-4days postimplantation) and maintenance (over the next 20days) dosing. Orthotopic tumors were created in mice using human neuroblastoma KELLY cells. Silk platforms were implanted into tumors when volumewas >300mm3. Tumor volume was monitored weekly with ultrasound. Experimental endpoints were tumor volumewas >1000mm3 or weight losswas >25%.Drug release ranged from burst dosing of 18.2 to 80.9g, maintenance of 5.0 to 111.6g, and cumulative of 23.3 to 177.4g. Animals treated with 200g platform died 9-13days postimplantation, corresponding to 128.1-141.2g released (toxic dose). Animals received 30.23.4gday-one survived longer than those that received 10.11.1g (p=0.03), suggesting <10.1gday-one was insufficient. Tumors treated with 100g or 50g silk platform took longer to reach 1000 mm3 compared to those treated with control, 44.89.5days (p<0.001) and 26.76.7days (p<0.05), respectively, versus 7.01.7days. Overall survival correlated with higher burst (r=0.446, p=0.004) and maintenance dosing (r=0.353, p=0.02), Animal survival days=30.314+0.626 (dose on day-one) - 0.020(tumor volume at day-ten) (p<0.05).Platform formulations can be manipulated to vary burst and maintenance dosing, summarized by an equation consisting of these variables.

View details for DOI 10.1016/j.jpedsurg.2017.08.028

View details for PubMedID 28927981

View details for PubMedCentralID PMC5723549

Abstract

Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1), a scaffold protein involved in phosphoinositide 3-kinase (PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector (pSR), vectors expressing scrambled shRNA (pSCR), or shRNAs targeting ITSN1 (sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000mm. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6247.7mm, pSCR was 1180159.9mm, sh#1 was 526.3212.8mm, and sh#2 was 589.274.91mm. sh#1-tumors and sh#2-tumors were smaller than pSCR (P=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors (P=0.02), trended towards improved survival in sh#1-tumors (P=0.09), compared with pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared with pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.

View details for PubMedID 28787396

View details for PubMedCentralID PMC5656511

Abstract

A term male baby, after delivery, was found to have a 3-centimeter beefy-red mass protruding from the left chest wall, adjacent to the left nipple. Radiological imaging suggested it's origin from the left lateral liver segment. A diagnostic laparoscopy confirmed the isolated connection to the liver, elevated left hemidiaphragm, and protrusion between the ribs. The mass was excised using electrocautery, and pathologic examination showed normal liver tissue.

View details for PubMedID 28920025

Abstract

Neuroblastoma is the most common extracranial childhood solid tumor. Treatment of high risk tumors require intense multicycle chemotherapies, resulting in short- and long-term toxicities. Here, we present treatment of an orthotopic neuroblastoma mouse model, with silk fibroin materials loaded with vincristine, doxorubicin or the combination as a intratumoral, sustained release system. The materials, loaded with vincristine with or without doxorubicin, significantly decreased neuroblastoma tumor growth compared to materials loaded without drug or doxorubicin only as well as intravenous (IV) drug treatment. The intratumoral drug concentration was significantly higher with intratumoral delivery versus IV. Furthermore, intratumor delivery decreased the maximum plasma concentration compared to IV delivery, reducing systemic exposure and possibly reduing long-term side effects of chemotherapy exposure. Histopathologically, tumors with remission periods >25 days before recurrence transformed from a "small-round-blue cell" (SBRC) to predominantly "large cell" neuroblastoma (LCN) histopathology, a more aggressive tumor subtype with unfavorable clinical outcomes. These results show that intratumoral chemotherapy delivery may be a treatment strategy for pediatric neuroblastoma, potentially translatable to other focal tumors types. Furthermore, this treatment modality allows for a clinically relevant mouse model of tumor transformation that may be used for studying the phenotypical tumor recurrence and developing more effective treatment strategies for recurrent tumors.

View details for PubMedID 27770551

Abstract

According to the US Center for Disease Control, cancer deaths are the second most common cause of mortality in both adults and children. Definitive treatment of solid tumors involves surgical resection with or without systemic chemotherapy and radiation. The advent of local drug delivery presents a unique treatment modality that can offer substantial benefits in cancer management. Three main phases in solid tumor management exist for the treating physician: initial diagnosis with tissue biopsy, surgical resection with or without chemotherapy, and management of metastatic disease.A literature review of both basic science as well as clinical trials using local drug delivery strategies in the management of solid tumors was done on PubMed. These were then further divided into the categories of initial tissue biopsy intervention, surgical resection, and management of metastatic disease.A total of 27 articles were review that included both pre-clinical as well as clinical investigation of local drug delivery therapies in the treatment of solid tumors. Treatments such as MRI guided therapies, FDA approved local therapies for intracranial gliomas as well as local therapy for single site metastatic disease were identified.This review focuses the current state of local drug delivery in the treatment of solid tumors in both the pre-clinical as well as clinical investigation settings. Local drug delivery therapy offers an exciting new treatment modality for solid malignancies.

View details for PubMedID 28240175

Abstract

Sarcoma accounts for 20% of solid tumors in children. Surgery has significant morbidity. We hypothesized that delivering chemotherapy directly into tumors through sustained release silk systems could slow tumor growth.Human Ewing sarcoma cells A673 were cultured with vincristine and doxorubicin to determine half maximal inhibitory concentration (IC50). Cells were injected into mouse hind leg to create orthotopic tumors. Tumor volumes were measured using ultrasound. When volume reached >250mm3, interventions included: implantation of drug-free silk foam (Control-F), doxorubicin 400g foam (Dox400-F), vincristine 50g foam (Vin50-F), drug-free silk gel (Control-G), vincristine 50g gel (Vin50-G), or single dose intravenous vincristine 50g (Vin50-IV). End-point was volume>1000mm3. Kaplan Meier and ANOVA were used.IC50 for vincristine and doxorubicin was 0.5ng/mL and 200ng/mL, respectively. There was no difference between Dox400-F [61days to end point (DTEP)] and Control-F (51.3 DTEP). Vin50-F (12.43.5 DTEP) had slower growth compared to Control-F (p<0.001), and there was no difference between Vin50-F and Vin50-IV (140 DTEP). Growth was slowest with Vin50-G, 2810.3 DTEP compared to all other treatment groups (p<0.05).Sustained delivery of vincristine inside the sarcoma tumor with silk gel decreased tumor growth. Applying this intratumoral treatment strategy may potentially decrease the extent of surgical excision.

View details for PubMedID 27680598

View details for PubMedCentralID PMC5138133

Abstract

Irreversible electroporation (IRE) induces apoptosis in tumor cells with electric energy, allowing treatment of unresectable tumors. One potential application is metastatic osteosarcoma (OS) in the pediatric population. A 12-year-old underwent thoracotomy with resection of metastatic OS. IRE was applied to 1 resected tumor section. Using 2 probes, 100 pulses with width of 90 ms were delivered. Efficacy was measured by increase in current draw during treatment. The treated sample was analyzed with hematoxylin and eosin and transmission electron microscopy. Default voltage of 1800 kV was ineffective. Voltage of 2700 kV caused excessive current draw and was aborted to prevent thermal injury. At 2200 kV, current draw rise was 9 amps, signifying successful treatment. Untreated specimen showed viable OS, normal surrounding lung tissue. Treated tumor had edema within the tumor and in surrounding lung tissue, with intra-alveolar hemorrhage and cellular architecture destruction. There was also evidence for cellular destruction such as disruption of lipid bilayer and release of intracellular fluid. Optimal voltage for treatment was 2200 kV, likely higher due to electrical conduction variation in the aerated lung. IRE may be an option for pediatric patients with unresectable metastatic OS.

View details for PubMedID 26950088

View details for DOI 10.4103/2249-4847.179911

View details for Web of Science ID 000383839600007

Abstract

AIM: To create an orthotopic hepatoma model with local metastasis monitored with ultrasound could be created as a platform for testing new treatments.BACKGROUND: Hepatoma accounts for 25% of liver tumors in children with poor overall survival. Intraabdominal metastasis are present in 35% of patients at time of diagnosis. We hypothesized that an orthotopic tumor model with local metastasis could be created as a platform for testing treatment modalities and could be monitored with ultrasound.PATIENTS AND METHODS: One million human hepatoma cells (Hep3B) were injected into the left lobe of the liver of immunocompromised mice. Tumor volume was monitored with high frequency-ultrasound until it reached 1,000mm(3). At that time animals were sacrificed and examined for gross metastatic disease. Tumor sections were analyzed with hematoxylin and eosin (H&E) staining.RESULTS: Tumor formed in 8/15 mice. The tumor was detected as small as 19.59mm(3) on ultrasound. Of the forming tumors, tumor size was 145177.93mm(3) at 60 days post-injection, 665650.39mm(3) at 67 days, and reached >1000mm(3) by 76.69.9 days. At necropsy, four mice (50%) had tumor only within the liver, four (50%) had additional tumors in omentum, pelvis and peritoneum. H&E showed tumor within the normal liver parenchyma, with multiple mitotic figures, small areas of necrosis, and hemorrhage within the tumor.CONCLUSION: We have successfully established an orthotopic hepatoma murine model, with a local metastatic rate of 50%. Non-invasive tumor monitoring is feasible via ultrasound.

View details for PubMedID 27458509

Abstract

OBJECTIVES: Gastroschisis is a congenital anomaly affecting 2.3-4.4/10,000 births. Previous studies show initiation of early enteral feeds predicts improved outcomes. We hypothesize that earlier definitive closure after silo placement; can lead to earlier enteral feed initiation. Design/ Setting/ Duration: Retrospective review of patients with gastroschisis from 2005 and 2014 at a single institution.MATERIAL AND METHODS: The data, including ethnicity, gestational age, birth weight, time to definitive closure, and time of first and full feeds, were analyzed using both Spearman's rho and the Kruskal-Wallis rank sum test where appropriate; a p value less than 0.05 was considered significant.RESULTS: Forty-three patients (24 males, 19 females) born with gastroschisis were identified. Overall survival rate was 88% (38/43). Forty of the 43 patients had a silo placed prior to definitive closure. Median days to closure were 6 (0 to 85) days. First feeds on average began on day of life (DOL) 17, and full feeds on DOL 25. Earlier closure of gastroschisis correlated with early initiation of feeds (p=0.0001) and shorter time to full feeds (p=0.018), closure by DOL4 showed a trend toward earlier feeding (p=0.13).CONCLUSION: Earlier closure of gastroschisis after silo placement was associated with earlier feed initiation and shorter time to full feeds.

View details for PubMedID 26290810

Abstract

Current methods for treatment of high-risk neuroblastoma patients include surgical intervention, in addition to systemic chemotherapy. However, only limited therapeutic tools are available to pediatric surgeons involved in neuroblastoma care, so the development of intraoperative treatment modalities is highly desirable. This study presents a silk film library generated for focal therapy of neuroblastoma; these films were loaded with either the chemotherapeutic agent doxorubicin or the targeted drug crizotinib. Drug release kinetics from the silk films were fine-tuned by changing the amount and physical crosslinking of silk; doxorubicin loaded films were further refined by applying a gold nanocoating. Doxorubicin-loaded, physically crosslinked silk films showed the best in vitro activity and superior in vivo activity in orthotopic neuroblastoma studies when compared to the doxorubicin-equivalent dose administered intravenously. Silk films were also suitable for delivery of the targeted drug crizotinib, as crizotinib-loaded silk films showed an extended release profile and an improved response both in vitro and in vivo when compared to freely diffusible crizotinib. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based anticancer drug delivery systems.

View details for PubMedID 25861948

View details for PubMedCentralID PMC4428956

Abstract

Neuroblastoma tumour resection goal is maximal tumour removal. We hypothesise that combining surgery with sustained, local doxorubicin application can control tumour growth.We injected human neuroblastoma cells into immunocompromised mouse adrenal gland. When KELLY cell-induced tumour volume was >300 mm(3), 80-90% of tumour was resected and treated as follows: instantaneous-release silk film with 100 g doxorubicin (100IR), controlled-release film with 200 g (200CR) over residual tumour bed; and 100 and 200 g intravenous doxorubicin (100IV and 200IV). Tumour volume was measured and histology analysed.Orthotopic tumours formed with KELLY, SK-N-AS, IMR-32, SH-SY5Y cells. Tumours reached 1800180 mm(3) after 28 days, 2200290 mm(3) after 35 days, 1280260 mm(3) after 63 days, and 1700360 mm(3) after 84 days, respectively. At 3 days post KELLY tumour resection, tumour volumes were similar across all groups (P=0.6210). Tumour growth rate was similar in untreated vs control film, 100IV vs 100IR, and 100IV vs 200IV. There was significant difference in 100IR vs 200CR (P=0.0004) and 200IV vs 200CR (P=0.0003). Tumour growth with all doxorubicin groups was slower than that of control (P: <0.0001-0.0069). At the interface of the 200CR film and tumour, there was cellular necrosis, surrounded by apoptotic cells before reaching viable tumour cells.Combining surgical resection and sustained local doxorubicin treatment is effective in tumour control. Administering doxorubicin in a local, controlled manner is superior to giving an equivalent intravenous dose in tumour control.

View details for DOI 10.1038/bjc.2014.324

View details for Web of Science ID 000341024100011

View details for PubMedID 24921912

View details for PubMedCentralID PMC4134491

Abstract

INTRODUCTION: Patients with combined esophageal atresia (EA), tracheoesophageal fistula (TEF), and duodenal atresia (DA) pose a rare management challenge.PRESENTATION OF CASE: Three patients with combined esophageal atresia (EA), tracheoesophageal fistula (TEF), and duodenal atresia safely underwent a staged approach inserting a gastrostomy tube and repairing the EA/TEF first followed by a duodenoduodenostomy within one week. None of the patients suffered significant pre- or post-operative complications and our follow-up data (between 12 and 24 months) suggest that all patients eventually outgrow their reflux and respiratory symptoms.DISCUSSION: While some authors support repair of all defects in one surgery, we recommend a staged approach. A gastrostomy tube is placed first for gastric decompression before TEF ligation and EA repair can be safely undertaken. The repair of the DA can then be performed within 3-7 days under controlled circumstances.CONCLUSION: A staged approach of inserting a gastrostomy tube and repairing the EA/TEF first followed by a duodenoduodenostomy within one week resulted in excellent outcomes.

View details for PubMedID 25460495

View details for DOI 10.1007/978-1-4419-6643-8_37

View details for Web of Science ID 000288294500037

Abstract

We hypothesized that a higher frequency of multigland disease and higher cure rate would result if routine four-gland exploration (4GL) was used as compared with focused parathyroidectomy (FP) for treatment of primary hyperparathyroidism.During a 5-year period, data from two academic endocrine surgical practices were retrospectively reviewed for patients having an operation for primary hyperparathyroidism. Three hundred ninety-five consecutive patients underwent 4GL at one institution (A), and 405 consecutive patients underwent FP with selective use of 4GL at the other institution (B). The main outcomes measures were gender, preoperative imaging, surgical findings, gland weight, and operative success.Three hundred ten (78%) patients at institution A were women, and 292 (72%) at institution B were women (p < 0.05). Routine 4GL strategy at institution A yielded a 16.5% frequency of multigland disease; and an FP strategy at institution B yielded 11.1% multigland disease (p = 0.028). At both institutions, single adenomas weighed more than multigland disease. Gland weights were not significantly different between the two institutions. Nine of 395 (2.3%) patients at institution A remained hypercalcemic postoperatively compared with 15 of 405 (3.7%) at B (p = 0.24; not significant).A greater frequency of multigland disease was found with routine 4GL. There was no statistically significant difference in operative success between the two approaches. Sound surgical technique and intraoperative judgment, including interpretation of intraoperative parathyroid hormone values, will result in a high success rate, regardless of the operative strategy chosen for primary hyperparathyroidism.

View details for PubMedID 18656054

Abstract

A 3-year-old girl developed extrahepatic portal vein obstruction (EHPVO) after a liver transplant. She had sequelae of portal hypertension that required another transplantation. The circumstances allowed for comparison of liver-dependent coagulation factor production between the second donor liver and the explanted liver with EHPVO.Liver samples from the explanted first graft and the second transplant were obtained. Fresh tissue was used to perform reverse transcription-polymerase chain reaction with primers against factors V, VII, as well as VIII, protein C, and paraffin-embedded sections for hepatocyte proliferation using Ki-67 antibody as well as for apoptosis using TUNEL assay.The transcription of factor VII and that of protein C were decreased in the explant as compared with the newly transplanted liver (factor VII, 77% of the donor; protein C, 88% of the donor). The transcription of factor V and that of factor VIII were unchanged. The explant had a greater percentage of proliferating hepatocytes than the new organ (0.85% +/- 0.75% vs 0.11% +/- 0.21%). The percentage of apoptotic cells was similar between the 2 livers (0.09% +/- 0.13% vs 0.09% +/- 0.13%).Idiopathic EHPVO is associated with a reduction in liver-dependent coagulation factor transcription and an increase in hepatocyte proliferation. Portal blood flow deprivation alters hepatic homeostasis and initiates mechanisms that attempt to restore liver-dependent coagulation factors.

View details for PubMedID 17923213

Abstract

Extrahepatic portal vein obstruction (EHPVO) is clinically associated with decreased liver-dependent coagulation factors and smaller than normal liver volumes. We developed a rodent EHPVO model to describe changes in coagulation factor regulation and liver homeostasis.Fifteen rats underwent controlled narrowing of the portal vein (PV) at the hilum, and 15 underwent sham operations. Three animals from each group were sacrificed at postoperative days (PODs) 1, 2, 4, 7, and 21. Their livers were studied for apoptosis, proliferation, and hepatocyte growth factor (HGF) expression using terminal transferase dUTP nick end labeling (TUNEL) assays, Ki-67, and hepatocyte growth factor antibody. Liver total RNA was harvested for reverse transcriptase-polymerase chain reaction (RT-PCR), using primers for factors V, VII, VIII, X, protein C, and antithrombin. Appropriate statistical analysis was applied.Ligation of the portal vein in the experimental group resulted in a 59+/-17% (mean +/- standard deviation) decrease in distal portal vein diameter. Proportional body weight was equivalent in both groups, but spleen weight was higher in the experimental group at PODs 4, 7, 21, and liver weight was higher at POD 7 (p < 0.05). The percentage of apoptotic cells in the experimental group increased from that of the control group at POD 4 (p < 0.05). The percentages of proliferating cells, HGF expression, and factor VII transcription in the experimental group were lower than those of controls at POD 2 (p < 0.05) but higher at POD 7 (p < 0.05). Ki-67/TUNEL double staining showed no grouping of apoptotic and proliferating cells. Factor V transcription in the experimental group was increased compared with that in controls at POD 2 (p < 0.05). Transcription of factors VIII, X, protein C, and antithrombin was unchanged.Our model demonstrated an early increase in hepatocyte apoptosis, impairment of factor VII transcription, and decrease in proliferative indices. These data support the hypothesis that EHPVO disrupts hepatic homeostasis and leads to dysregulation of coagulation factor synthesis.

View details for PubMedID 17660074

Abstract

We previously reported that epidermal growth factor (EGF) induced cleaved caspase 3 expression and apoptosis in human neuroblastoma cells. We hypothesized that EGF upregulates total caspase 3 expression, thereby enhancing the cytotoxic potency of chemotherapeutic agents.Wild-type (WT) and doxorubicin-resistant (Dox-R) SK-N-SH neuroblastoma cells were incubated with EGF 0 to 250 ng/mL for 24 hours or with 5 ng/mL for 0 to 5 days, and total caspase 3 expression and transcription were determined by immunoblots and reverse transcription and polymerase chain reaction, respectively. Cell proliferation was determined after EGF (5 ng/mL) incubation for 1 to 5 days. Cells incubated with EGF (5 ng/mL) for 24 hours before doxorubicin treatment were used to determine the effect of EGF on cell replication and cleaved caspase 3 expression.Wild-type and Dox-R cells had maximal total caspase 3 expression after incubation with EGF (5 ng/mL) for 3 and 5 days, respectively, and a corresponding increase in DNA transcription. Treating the cells with or without EGF (5 ng/mL) resulted in similar replication rate. However, cell death was increased by EGF pretreatment and doxorubicin when compared with that by doxorubicin alone (WT, 53% +/- 8%; Dox-R, 44% +/- 9%; P < .05). Cleaved caspase 3 expression was upregulated when cell death was increased.Epidermal growth factor upregulates the expression and transcription of total caspase 3 in WT and Dox-R cells in a concentration- and time-dependent manner. Epidermal growth factor pretreatment augments the cytotoxic effect of doxorubicin.

View details for PubMedID 17706502

Abstract

The meso-Rex bypass procedure has been used to treat patients with portal hypertension from extrahepatic portal vein obstruction. This report describes modifications of this procedure in 5 patients. Either the splenic or coronary vein was used as the venous inflow point, and the bypass was performed either directly through transposition of the vein or with the use of a venous conduit.

View details for PubMedID 17618880

Abstract

Extrahepatic portal vein obstruction (EHPVO) results in decreased levels of liver-dependent coagulation factors in children. We developed a rat model to test the hypothesis that lower factor levels associated with EHPVO were from diminished synthesis rather than increased consumption.A total of 8 rats (experimental group) underwent narrowing of portal vein (PV) and 8 underwent sham operations. Liver and spleen mass, serum alanine aminotransferase, bilirubin, ammonia, prothrombin time, factor VII, and protein-C were measured before and 3 months after PV narrowing. Hepatocyte proliferation and apoptosis were quantified using Ki-67 and TUNEL assays.Portal vein diameter was 71% +/- 13% narrower in experimental animals. Liver mass was unchanged, but proportional spleen mass was higher in the experimental group at 3 months (0.31% +/- 0.05% vs 0.26% +/- 0.04%; P < .05). Percent apoptotic cells at 3 months was similar in both groups (0.14% +/- 0.08% vs 0.13% +/- 0.07%), but percent proliferating cells was higher in the experimental group (0.63% +/- 0.17% vs 0.34% +/- 0.11%; P < .05). Three-month protein-C levels decreased significantly only in the experimental group compared with preoperative values (12.8% +/- 4.4% vs 7.6% +/- 5.1%; P < .05). Changes in other parameters were not significant.Our EHPVO model consistently produced PV narrowing. The increase in hepatocyte proliferation seen after EHPVO suggests a liver repair response that is insufficient to maintain normal protein-C synthesis and serum levels.

View details for PubMedID 17502186

Abstract

In previous studies, incubation of doxorubicin-resistant neuroblastoma SK-N-SH (Dox-R) cells with epidermal growth factor (EGF) decreased extracellular signal-regulated kinase activation. Because extracellular signal-regulated kinase activation is associated with cell proliferation, we hypothesized that EGF could induce apoptosis and decrease the rate of cell growth in these cells.The growth of wild-type (WT) SK-N-SH and Dox-R cells after incubation with EGF concentrations ranging from 1 to 100 ng/mL was determined by a colorimetric assay. Apoptosis was assessed by Hoechst staining and DNA laddering in WT, Dox-R, and cisplatin-resistant cells treated with EGF (100 ng/mL). Cleaved caspase-3 and EGF receptor (human epidermal growth factor receptor [HER1-HER4]) expression were verified by Western blot and reverse transcriptase-polymerase chain reaction.Epidermal growth factor decreased WT cell growth at concentrations between 50 and 100 ng/mL; Dox-R cell growth was attenuated at all EGF concentrations. Apoptosis was observed in WT and Dox-R cells incubated with EGF. Maximal cleaved caspase-3 expression occurred in WT cells treated with EGF 100 ng/mL and in Dox-R treated with EGF 5 to 10 ng/mL. Epidermal growth factor did not induce apoptosis in cisplatin-resistant cells. HER2 and HER3 transcription was maximal in WT and Dox-R cells, respectively.Wild-type and Dox-R cells exhibited decreased cell growth after EGF treatment because of apoptosis. This involved caspase-3 activation and could work through HER2 and HER3 receptors.

View details for PubMedID 17336184

Abstract

In previous studies we have shown that epidermal growth factor (EGF) at concentrations between 50 and 100 ng/mL induced apoptosis in wild-type SK-N-SH neuroblastoma cells. We hypothesize that this apoptotic event separates EGF-induced neuroblastoma cell growth into a biphasic concentration-dependent process, due to activation of different signaling cascades.Cells were incubated in concentrations of EGF ranging from 5 to 250 ng/mL for 3 days, and cell proliferation was determined by the MTT assay. Cells incubated with EGF 5, 100, or 250 ng/mL for 17 h were also assayed for apoptosis by DNA laddering. Western immunoblots were performed on whole cell lysates prepared from cells incubated with EGF (5-250 ng/mL) for 17 h. Antibodies against cleaved caspase3, p-AKT, p-GSK-3beta, p-BAD, p-RAF, p-ERK, and p-P38 were used as probes.A triphasic, concentration-dependent response was observed following incubation of cells with EGF. Cell proliferation was increased by EGF 5 ng/mL (P < 0.05), decreased by EGF 100 ng/mL, and increased when incubated with EGF 250 ng/mL (P < 0.05). DNA laddering only occurred after treatment with EGF 100 ng/mL. The expressions of p-ERK, p-RAF, p-BAD, and p-GSK-3beta were increased at EGF concentrations of 5-10 ng/mL. At 50-100 ng/mL EGF, the expression of cleaved caspase3 was increased. Maximal p-P38 expression was at 50 ng/mL EGF. At EGF concentrations of 150-250 ng/mL, the expressions of p-AKT and p-GSK-3beta were elevated.Neuroblastoma cell growth induced by EGF exhibited a triphasic pattern; cell growth was increased at EGF concentrations 5-20 and 150-250 ng/mL, but decreased at 50-100 mg/mL. Apoptosis was induced at 50-100 ng/mL EGF. Each growth phase activated different signaling molecules.

View details for PubMedID 16872636

Abstract

We hypothesized that nonlocalizing sestamibi scans would correlate with multigland disease and persistent primary hyperparathyroidism.We reviewed records for 401 consecutive patients who underwent parathyroidectomy from 1999 to 2004. Gender, age, preoperative imaging, surgical findings, gland weight and volume, and 6-month calcium levels (Ca) were examined.We identified 289 women and 112 men, 297 of whom had a preoperative sestamibi scan localized to a single gland (localized group; LG). Ninety-six percent of the LG were found to have single-gland disease, and 4% had multigland disease (MGD). In the nonlocalized group (NLG), 76% had single-gland disease and 24% MGD. Mean gland weight was greater in the LG than in the NLG (1128 mg vs 699 mg; P < .05). Mean gland volume was larger in the LG (1.34 cc vs 0.89 cc; P < .05). A localizing sestamibi scan had a positive predictive value (PPV) of 96% and a likelihood ratio of 2.29 for predicting "curative" intraoperative parathyroid hormone drop after removal of a single abnormal gland. Patients were stratified into normocalcemic (NCa) and hypercalcemic (HCa) groups based on 6-month postoperative serum calcium data (n = 328). HCa incidence at 6 months did not differ significantly between the LG (5%) and NLG (3%). A localizing scan had a PPV of 95% for normocalcemia at 6 months. A nonlocalizing scan had a PPV of 21% for HCa at 6 months.Nonlocalizing sestamibi scans were more common in primary hyperparathyroidism with MGD and were associated with smaller-volume abnormal glands found at operation. Preoperative sestamibi scan-results did not predict HCa at 6 months.

View details for PubMedID 16934604

Abstract

Injury is one of the leading causes of death for infants younger than 1 year of age. We investigated potential contributing factors for injury among children born to high-risk families.The Fragile Families and Child Wellbeing Study is a longitudinal cohort of approximately 5000 children from mostly unwed parents across the United States. Data from interviews with mothers conducted shortly after birth and follow-up surveys at 1 year were used for this analysis. Injuries sustained in the first year of life that required medical attention were studied. Multivariate regression analysis was used to identify independent risk factors for injury in this population.A total of 13.7% of mothers reported that their child had sustained an injury in the first year of life. We identified 17 potential maternal, paternal, and environmental risk factors for injury, including socioeconomic, mental health, relationship violence, criminal justice system involvement, and substance abuse challenges. Multivariate regression analyses revealed two significant independent risk factors, maternal alcohol use (odds ratio 2.15, P = 0.044) and mother spanking child in the previous month (odds ration 2.32, P = 0.027).Among this higher-risk group, injury in the first year of life is more than twice the national incidence. Predisposing factors to injury often are complex and interrelated, but with focused education and prevention efforts, including discussions of maternal alcohol use and attitudes toward physical discipline, we may decrease the burden of infant injury in this vulnerable population.

View details for PubMedID 16566944

Abstract

Surgically created portosystemic shunts can alleviate the symptoms of bleeding from gastric and esophageal varices and improve the hematologic consequences of hypersplenism in patients with portal hypertension. However, the diversion of mesenteric venous blood away from the liver can result in encephalopathy. In this report, we describe a case in which encephalopathy caused by a proximal splenorenal shunt was reversed by the restoration of portal flow to the liver by a mesenteric-to-left portal vein bypass operation.

View details for DOI 10.1016/j.jpedsurg.2006.01.075

View details for Web of Science ID 000238710900027

View details for PubMedID 16769357

Abstract

The 6 published criteria for predicting curative parathyroid resection by means of intraoperative parathyroid hormone (IOPTH) assay are not equivalent.Retrospective review of 352 patients undergoing parathyroidectomy for primary hyperparathyroidism from January 1, 1999, to December 31, 2004. We evaluated 6-month postoperative IOPTH values and serum calcium levels.Tertiary referral center.The IOPTH values at baseline (preincision and preexcision) and at 5 and 10 minutes after parathyroidectomy were reviewed according to the Miami criterion (>50% drop from highest baseline IOPTH level at 10 minutes after excision), criterion 1 (>50% drop from preincision IOPTH level at 10 minutes), criterion 2 (>50% drop from highest baseline IOPTH level at 10 minutes and final IOPTH level within the reference range), criterion 3 (>50% drop from highest baseline IOPTH level at 10 minutes and final IOPTH level less than the preincision value), criterion 4 (>50% drop from highest baseline IOPTH level at 5 minutes), and criterion 5 (>50% drop from preexcision IOPTH level at 10 minutes).Criterion 2 had sensitivity of 88%, specificity of 22%, positive predictive value of 97%, and negative predictive value of 6%. Criterion 2 had good agreement with criteria 1 and 3. Of patients whose IOPTH level drop satisfied criterion 2 but not criterion 1, 14% had postoperative hypercalcemia at 6 months. When criterion 2 was not satisfied but criteria 1, 3, 4, and 5 and the Miami criterion were, failure rates were 0%, 4%, 7%, 6%, and 9%, respectively.Satisfying criterion 2 had a high operative success but resulted in additional unnecessary surgical exploration. Criterion 1 was better at predicting postoperative normocalcemia than criterion 2.

View details for PubMedID 16702520

Abstract

Chronic pancreatitis requiring surgery is rare in children. We review our experience in treating pediatric chronic pancreatitis with longitudinal pancreaticojejunostomy (LPJ).Records of children with chronic pancreatitis treated with LPJ between 1997 and 2003 were reviewed. Demographic data, associated conditions, endoscopic interventions, operative procedures, postoperative complications, length and costs of hospitalization, and long-term outcome were recorded.Four patients (one girl), 3 to 16 years old, underwent LPJ. Associated conditions included bile duct obstruction (2), single (1) or multiple (1) pancreatic duct strictures, recurrent familial pancreatitis (1), pseudocyst (1), Down's syndrome (1), and duodenal web (1). Preoperative endoscopic stenting was performed in two patients. All were on restricted diets, one on parenteral nutrition. Pre-LPJ, each child had 3 to 6 admissions for pancreatitis with mean total cost of 39,000 dollars, excluding diet charges. At surgery, two patients required biliary diversion for persistent biliary obstruction in addition to LPJ. Postoperatively, no patient developed fistulas or anastomotic leaks. There were no deaths. The median length of hospitalization post-LPJ was 8 days with mean cost of US37,000 dollars. All patients resumed a normal diet post-LPJ. There were no recurrences of pancreatitis with follow-ups between 2 and 6 years.Longitudinal pancreaticojejunostomy is safe and cost-effective for treating pediatric chronic pancreatitis. It has minimal complications and frees patients from pancreatitis-related hospitalizations.

View details for DOI 10.1016/j.jpedsurg.2006.01.015

View details for Web of Science ID 000238390700016

View details for PubMedID 16677890

Abstract

There are two approaches to close gastroschisis. Primary closure (PC) is reduction and fascial closure; silo closure (SC) places viscera in a preformed-silo and reduces the contents over time. We have shifted from PC to SC. This study compared the outcomes of these two techniques.Records of babies with gastroschisis from 1994-2004 were reviewed. Closure type, ventilator days, days to full-feeds, hospital days, complications, and mortality were recorded.Twenty-eight patients underwent PC; 20 patients had SC. Differences in ventilator days, days to full-feeds, and hospital days were not statistically significant. Nine PC patients developed closure-related complications vs. none in SC (P < 0.05). Eight PC vs. two SC patients had non-closure-related complications (P < 0.05). Four PC vs. zero SC patients developed necrotizing enterocolitis (P < 0.05). Five PC vs. one SC patients had ventral hernia (P < 0.05). No patient died.PC resulted in higher incidence of reclosure, non-closure-related complications, and necrotizing enterocolitis. Consequently, we recommend SC as the preferred treatment.

View details for DOI 10.1515/JPM.2006.043

View details for Web of Science ID 000236957200011

View details for PubMedID 16602846

Abstract

The optimal surgical treatment for extremely-low-birth-weight (ELBW) neonates with pneumoperitoneum is controversial. This study aimed to identify clinical factors associated with two known causes of pneumoperitoneum-necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP), and assesses the treatment outcome with primary peritoneal drainage (PPD) vs. laparotomy.We reviewed and analyzed clinical characteristics and outcome from records of neonates with pneumoperitoneum treated at our institution from January 1999 to January 2003.Forty-six neonates (31 NEC, 15 SIP) were treated with either PPD (20 with NEC, 13 with SIP) or laparotomy (11 with NEC, 2 with SIP). In neonates who underwent PPD, those with NEC (vs. SIP) were less likely to have a patent ductus arteriosus, but were more likely to have been fed, have drains placed later in life, have a subsequent laparotomy, a longer total parental nutrition course, a higher 30-day mortality, and to take more days to begin enteral feeds.The etiology of pneumoperitoneum (NEC vs. SIP) in ELBW neonates can usually be determined preoperatively. Neonates with SIP should have a drain placed while those with NEC should undergo laparotomy.

View details for DOI 10.1515/JPM.2006.065

View details for Web of Science ID 000239497400015

View details for PubMedID 16856827

Abstract

Epidermoid cysts of the biliary tree have not previously been described. A baby boy presented with a prenatally diagnosed echolucent intrahepatic cyst. Postnatal radioisotope study of the liver demonstrated that the cyst communicated with the biliary tree. Follow-up ultrasound at 6 months demonstrated that the cyst was filled with echogenic material consistent with either blood or biliary debris. Due to the potential for obstruction and cholangitis, surgery was planned. The cyst was located at the confluence of the right and left hepatic ducts and involved all of the common hepatic duct. The entire cyst was resected except for the patch containing 3 duct orifices: the opening of both hepatic ducts as well as the orifice leading to the common bile duct. A Roux-en-Y cyst jejunostomy was created to allow drainage of both left and right hepatic ducts. The connection also provided access to the cyst remnant through the common duct for future endoscopic monitoring of potential malignant transformation.

View details for DOI 10.1016/j.jpedsurg.2005.08.038

View details for Web of Science ID 000235189900046

View details for PubMedID 16338291

View details for DOI 10.1016/j.surg.2004.05.045

View details for Web of Science ID 000233653000023

View details for DOI 10.1016/j.surg.2004.05.045

View details for PubMedID 16291400

Abstract

The effect of portal flow deprivation to the liver on bile composition and the biliary system remains undefined in children. This report catalogues the authors' experience with biliary tract problems in children with extrahepatic portal vein thrombosis (EHPVT).Twenty-nine children with symptomatic idiopathic EHPVT were evaluated for the Rex shunt procedure (mesenterico-left portal bypass) over a 4-year period. The authors retrospectively reviewed all operative reports and pre- and postoperative abdominal ultrasound findings with regard to associated congenital anomalies and abnormal biliary tract findings.Seven of the 29 patients with EHPVT (24%) had associated nonbiliary congenital abnormalities. Twenty-four of 29 (83%) patients had detectable biliary tract pathology by ultrasound examination. Biliary symptoms developed in 3 of the 9 (33%) patients with either stones or sludge (10.3% of all patients). Two patients were treated by cholecystectomy. There was no statistical correlation between biliary tract pathology and the age of presentation, symptoms of portal hypertension, gender, or underlying medical condition.The authors have noted a high incidence of biliary tract pathology in patients with EHPVT compared with the normal population and a 10% incidence of symptomatic biliary pathology in this series.

View details for DOI 10.1016/j.jpedsurg.2004.03.051

View details for Web of Science ID 000222802700012

View details for PubMedID 15213899

Abstract

Periodic alternating nystagmus is a rare central nervous system disorder in which the eyes undergo a horizontal jerk nystagmus that periodically reverses direction. A patient with a hypoplastic cerebellum and enlarged cisterna magna exhibited transient periodic alternating nystagmus following an attack of Mnire's disease. We hypothesize that in susceptible individuals with cerebellar disturbances, periodic alternating nystagmus may be transiently induced by vestibular stimuli.

View details for PubMedID 12131470

Abstract

A murine abdominal aortic aneurysm model was developed by applying calcium chloride periarterially. A 13.6 mEq/10 ml calcium chloride solution was applied to the abdominal aorta of nine mice. Three mice were randomly selected at the end of the first, second, and third weeks postoperatively, and their vessel diameters were measured. The vessel diameter at the end of the first week postoperatively was 0.39 +/- 0.03 mm (mean +/- SD) pretreatment and 0.41 +/- 0.03 mm posttreatment (5.3% increase, P > 0.05). The vessel diameter at the end of the second week postoperatively was 0.48 +/- 0.03 mm pretreatment and 0.78 +/- 0.20 mm posttreatment (64% increase, P < 0.05). The vessel diameter at the end of the third week postoperatively was 0.57 +/- 0.14 mm pretreatment and 1.16 +/- 0.43 mm posttreatment (110% increase, P < 0.05). Nine other murine abdominal aortas were treated with sodium chloride, and their vessel diameters were measured in similar 7-day intervals. No measurements in this group were statistically significant when comparing pretreatment to posttreatment vessel diameters. A larger number of inflammatory infiltrates was observed in the intima and media layers of calcium-chloride-treated mice. Underlying mechanisms for this model include disrupting the elastic network within the media by calcium precipitations and activating the inflammatory response. We conclude that periarterial application of calcium chloride is a convenient and reliable model for creating abdominal aortic aneurysms in mice.

View details for DOI 10.1006/jsre.2001.6207

View details for Web of Science ID 000170299200032

View details for PubMedID 11469913

Abstract

As a rare postoperative complication, renal artery aneurysm has been reported in 0.95% of kidney transplants. A renal artery aneurysm was repaired prior to transplantation of the kidney.

View details for PubMedID 11586459

Abstract

Recent research in arterial aneurysm formation has focused on animal model development. Mice are an ideal experimental organism due to their short life cycle, prolific progeny, and extensively studied genome. Most experiments require the sacrifice of the mice to observe and assess any morphological changes. Noninvasive or minimally invasive imaging is limited due to the relatively small size of the structures. The development of such a technique, therefore, is especially useful for allowing repeated measurement without sacrificing the mice. We introduce a novel technique of imaging and measuring the aorta, the aorta/inferior vena cava complex, and the right and the left common iliac artery/vein complex by the use of an intravascular ultrasound catheter. The catheter is inserted through the anus and rectum and into the sigmoid and left colon, where the aorta can be observed to fluctuate at approximately 500 beats/min. The aortic bifurcation can also be observed. The diameters of the aorta and the inferior vena cava were measured first with the transrectal ultrasound technique and then with direct visualization upon laparotomy for 10 mice. This revealed a percentage error between 13.7 and 14.2% for this novel technique. Fifteen more sets of vessel measurements were also made with 8 male and 7 female mice. The results demonstrated a correlation between vessel size and body weight in male but not female mice and suggested an intersex difference in vessel growth rate. We conclude that transrectal ultrasound is a useful tool in imaging and measuring the murine aorta and its bifurcation.

View details for DOI 10.1006/jsre.1999.5790

View details for Web of Science ID 000085065400020

View details for PubMedID 10644488