Risk of Major Adverse Cardiovascular Events After Emergency Department Visits for Hypertensive Urgency.

BACKGROUND: Chronic hypertension is an established long-term risk factor for major adverse cardiovascular events (MACEs). However, little is known about short-term MACE risk after hypertensive urgency, defined as an episode of acute severe hypertension without evidence of target-organ damage. We sought to evaluate the short-term risk of MACE after an emergency department (ED) visit for hypertensive urgency resulting in discharge to home. METHODS: We performed a case-crossover study using deidentified administrative claims data. Our case periods were 1-week intervals from 0 to 12 weeks before hospitalization for MACE. We compared ED visits for hypertensive urgency during these case periods versus equivalent control periods 1 year earlier. Hypertensive urgency and MACE components were all ascertained using previously validated International Classification of Diseases, Tenth Revision Clinical Modification codes. We used McNemar test for matched data to calculate risk ratios. RESULTS: Among 2 225 722 patients with MACE, 1 893 401 (85.1%) had a prior diagnosis of hypertension. There were 4644 (0.2%) patients who had at least 1 ED visit for hypertensive urgency during the 12 weeks preceding their MACE hospitalization. An ED visit for hypertensive urgency was significantly more common in the first week before MACE compared with the same chronological week 1 year earlier (risk ratio, 3.5 [95% CI, 2.9-4.2]). The association between hypertensive urgency and MACE decreased in magnitude with increasing temporal distance from MACE and was no longer significant by 11 weeks before MACE (risk ratio, 1.2 [95% CI, 0.99-1.6]). CONCLUSIONS: ED visits for hypertensive urgency were associated with a substantially increased short-term risk of subsequent MACE.

Liver Severity Score-Based Modeling to Predict Six-Week Mortality Risk Among Hospitalized Cirrhosis Patients With Upper Gastrointestinal Bleeding.

Background: Patients with cirrhosis who have gastrointestinal bleeding have high short-term mortality, but the best modality for risk calculation remains in debate. Liver severity indices, such as Child-Turcotte-Pugh (CTP) and Model-for-End-Stage-Liver Disease (MELD) score, are well-studied in portal hypertensive bleeding, but there is a paucity of data confirming their accuracy in non-portal hypertensive bleeding and overall acute upper gastrointestinal bleeding (UGIB), unrelated to portal hypertension. Aims: This study aims to better understand the accuracy of current mortality risk calculators in predicting mortality for patients with any type of UGIB, which could allow for earlier risk stratification and targeted intervention prior to endoscopy to identify the bleeding source. Methods: In a large US single-center cohort, we investigated and recalibrated the model performance of CTP and MELD scores to predict six-week mortality risk for both sources of UGIB (portal hypertensive and non-portal hypertensive). Results: Both CTP- and MELD-based models have excellent discrimination in predicting six-week mortality for all types of bleeding sources. However, only a CTP-based model demonstrates calibration for all bleeding, regardless of bleeding etiology. Median predicted 6-week mortality by CTP class A, B, and C estimates a risk of 1%, 7%, and 35% respectively. Conclusions: Our study corroborates findings in the literature that CTP- and MELD-based models have similar discriminative abilities for predicting 6-week mortality in hospitalized cirrhosis patients presenting with either portal hypertensive or non-portal hypertensive UGIB. CTP class is an effective clinical decision tool that can be used, even prior to endoscopy, to accurately risk stratify a patient with known cirrhosis presenting with any UGIB into low, moderate, and severe risk groupings.

Impact of Race and Neighborhood Socioeconomic Characteristics on Liver Cancer Diagnosis in Patients with Viral Hepatitis and Cirrhosis.

Background: Concerning data have revealed that viral hepatitis and hepatocellular carcinoma (HCC) disproportionally impact non-White patients and those from lower socioeconomic status. A recent study found that HCC clusters were more likely to be in high poverty areas in New York City. Aims: We aim to investigate the impacts of neighborhood characteristics on those with viral hepatitis and cirrhosis, particularly with advanced HCC diagnosis. Methods: Patients with cirrhosis and viral hepatitis admitted to a New York City health system between 2012 and 2019 were included. Those with prior liver transplants were excluded. Neighborhood characteristics were obtained from US Census. Our primary outcome was HCC and advanced HCC diagnosis. Results: This study included 348 patients; 209 without history of HCC, 20 with early HCC, 98 with advanced HCC, and 21 patients with HCC but no staging information. Patients with advanced HCC were more likely to be older, male, Asian, history of HBV, and increased mortality. They were more likely to live in areas with more foreign-born, limited English speakers, and less than high school education. After adjusting for age, sex, and payor type, Asian race and low income were independent risk factors for advanced HCC. Neighborhood factors were not associated with mortality or readmissions. Conclusion: We observed that in addition to age and sex, Asian race, lower household income, lower education, and lower English proficiency were associated with increased risk of advanced HCC. These disparities likely reflect suboptimal screening programs and linkage to care among vulnerable populations. Further efforts are crucial to validate and address these concerning disparities.

The International Classification of Diseases, 10 th Edition, Clinical Modification (ICD-10-CM) Code I16.0 Accurately Identifies Patients with Hypertensive Urgency.

Objective: Hypertensive urgency, defined as acutely elevated BP without target organ damage, is associated with an increased risk of adverse cardiovascular events and accounts for a substantial proportion of national emergency department (ED) visits. To advance research in this space, we sought to validate the new ICD-10-CM diagnostic code for hypertensive urgency within a single healthcare system. Methods: We performed a retrospective chart-review study of ED encounters at Weill Cornell Medicine from 2016 â€" 2021. We randomly selected 25 encounters with the ICD-10-CM code I16.0 as the primary discharge diagnosis and 25 encounters with primary ICD-10-CM discharge diagnosis codes for benign headache disorders. A single board-certified vascular neurologist reviewed all 50 encounters while blinded to the assigned ICD-10-CM codes to identify cases of hypertensive urgency. We calculated the sensitivity, specificity, and positive predictive values of the ICD-10-CM code I16.0 with 95% confidence intervals (CI). Results: Out of 50 randomly selected ED encounters, 24 were adjudicated as hypertensive urgency. All encounters adjudicated as hypertensive urgency had been assigned the ICD-10-CM discharge diagnosis code of I16.0. All 25 of the encounters adjudicated as headache were assigned an ICD-10-CM discharge diagnosis code for a benign headache disorder. The ICD-10-CM code for hypertensive urgency, I16.0, was thus found to have a sensitivity of 100% (95% CI: 86-100%), specificity of 96% (95% CI: 80-100%), and positive predictive value of 96% (95% CI: 78-99%). Conclusion: We found that the new ICD-10-CM code for hypertensive urgency, I16.0, can reliably identify patients with this condition.

Body-Temperature Programmable Soft-Shape Memory Hybrid Sponges for Comfort Fitting.

Porous shape memory hybrids are fabricated with different matrix (silicone) hardness and different inclusion (polycaprolactone, PCL) ratios. They are characterized to obtain their mechanical response to cyclic loads (with/without pre-straining/programming) and their shape memory performances after body-temperature programming are investigated. These materials are lightweight due to their porous structures. Wetted hydrogels used in the fabrication process for creating pores are reusable and hence this process is eco-friendly. These porous shape memory hybrids exhibit the good shape memory effect of around 90% with higher inclusion (PCL) ratios, which is better than the solid versions reported in the literature. Hence, it is concluded that these materials have great potential to be used in, for instance, insoles and soles for comfort fitting, as demonstrated.

Anticancer mechanisms of Strobilanthes crispa Blume hexane extract on liver and breast cancer cell lines.

Cancer is a major public health concern not only in developed countries, but also in developing countries. It is one of the leading causes of mortality worldwide. However, current treatments may cause severe side effects and harm. Therefore, recent research has been focused on identifying alternative therapeutic agents extracted from plant-based sources in order to develop novel treatment options for cancer. Strobilanthes crispa Blume is a plant native to countries including Madagascar and Indonesia. It has been used as an anti-diabetic, diuretic and laxative in traditional folk medicine. Furthermore, S. crispa has potential in treating cancer, as evidenced in previous studies. In the present study, the cytotoxic and apoptotic activities of S. crispa crude extracts were investigated in liver and breast cancer cell lines. Hexane, ethyl acetate, chloroform, methanol and water extracts prepared from the leaves, and stems of S. crispa were evaluated for their cytotoxicity on HepG-2 and MDA-MB-231 cells using an MTT assay. The anti-proliferative properties of stem hexane (SH) extract on both cell lines were analysed using cell doubling time determination and cell cycle analysis, while the apoptogenic properties was determined through the detection of caspase-8. Among the extracts tested, SH extract exhibited the lowest half maximal inhibitory concentrations in both the cell lines. The SH extract induced morphological changes in HepG-2 and MDA-MB-231 cells, and significantly delayed cell population doubling time. Furthermore, it altered cell cycle profile and significantly increased caspase-8 activity in HepG-2 cells, but not in MDA-MB-231 cells. In conclusion, the SH extract of S. crispa possesses potent anticancer properties and may be a suitable chemotherapeutic target.

A Pilot Trial of Humanized Anti-GD2 Monoclonal Antibody (hu14.18K322A) with Chemotherapy and Natural Killer Cells in Children with Recurrent/Refractory Neuroblastoma.

Purpose: Anti-GD2 mAbs, acting via antibody-dependent cell-mediated cytotoxicity, may enhance the effects of chemotherapy. This pilot trial investigated a fixed dose of a unique anti-GD2 mAb, hu14.18K322A, combined with chemotherapy, cytokines, and haploidentical natural killer (NK) cells.Experimental Design: Children with recurrent/refractory neuroblastoma received up to six courses of hu14.18K322A (40 mg/m2/dose, days 2-5), GM-CSF, and IL2 with chemotherapy: cyclophosphamide/topotecan (courses 1,2), irinotecan/temozolomide (courses 3,4), and ifosfamide/carboplatin/etoposide (courses 5,6). Parentally derived NK cells were administered with courses 2, 4, and 6. Serum for pharmacokinetic studies of hu14.18K322A, soluble IL2 receptor alpha (sIL2Rα) levels, and human antihuman antibodies (HAHA) were obtained.Results: Thirteen heavily pretreated patients (9 with prior anti-GD2 therapy) completed 65 courses. One patient developed an unacceptable toxicity (grade 4 thrombocytopenia >35 days). Four patients discontinued treatment for adverse events (hu14.18K322A allergic reaction, viral infection, surgical death, second malignancy). Common toxicities included grade 3/4 myelosuppression (13/13 patients) and grade 1/2 pain (13/13 patients). Eleven patients received 29 NK-cell infusions. The response rate was 61.5% (4 complete responses, 1 very good partial response, 3 partial responses) and five had stable disease. The median time to progression was 274 days (range, 239-568 days); 10 of 13 patients (77%) survived 1 year. Hu14.18K322A pharmacokinetics was not affected by chemotherapy or HAHA. All patients had increased sIL2Rα levels, indicating immune activation.Conclusions: Chemotherapy plus hu14.18K322A, cytokines, and NK cells is feasible and resulted in clinically meaningful responses in patients with refractory/recurrent neuroblastoma. Further studies of this approach are warranted in patients with relapsed and newly diagnosed neuroblastoma. Clin Cancer Res; 23(21); 6441-9. ©2017 AACR.

DNA modification study of major depressive disorder: beyond locus-by-locus comparisons.

BACKGROUND: Major depressive disorder (MDD) exhibits numerous clinical and molecular features that are consistent with putative epigenetic misregulation. Despite growing interest in epigenetic studies of psychiatric diseases, the methodologies guiding such studies have not been well defined. METHODS: We performed DNA modification analysis in white blood cells from monozygotic twins discordant for MDD, in brain prefrontal cortex, and germline (sperm) samples from affected individuals and control subjects (total N = 304) using 8.1K CpG island microarrays and fine mapping. In addition to the traditional locus-by-locus comparisons, we explored the potential of new analytical approaches in epigenomic studies. RESULTS: In the microarray experiment, we detected a number of nominally significant DNA modification differences in MDD and validated selected targets using bisulfite pyrosequencing. Some MDD epigenetic changes, however, overlapped across brain, blood, and sperm more often than expected by chance. We also demonstrated that stratification for disease severity and age may increase the statistical power of epimutation detection. Finally, a series of new analytical approaches, such as DNA modification networks and machine-learning algorithms using binary and quantitative depression phenotypes, provided additional insights on the epigenetic contributions to MDD. CONCLUSIONS: Mapping epigenetic differences in MDD (and other psychiatric diseases) is a complex task. However, combining traditional and innovative analytical strategies may lead to identification of disease-specific etiopathogenic epimutations.

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Curcumin potentiates rhabdomyosarcoma radiosensitivity by suppressing NF-κB activity.

Ionizing radiation (IR) is an essential component of therapy for alveolar rhabdomyosarcoma. Nuclear factor-kappaB (NF-κΒ) transcription factors are upregulated by IR and have been implicated in radioresistance. We evaluated the ability of curcumin, a putative NF-κΒ inhibitor, and cells expressing genetic NF- κΒ inhibitors (IκBα and p100 super-repressor constructs) to function as a radiosensitizer. Ionizing radiation induced NF-κΒ activity in the ARMS cells in vitro in a dose- and time-dependent manner, and upregulated expression of NF-κΒ target proteins. Pretreatment of the cells with curcumin inhibited radiation-induced NF-κΒ activity and target protein expression. In vivo, the combination of curcumin and IR had synergistic antitumor activity against Rh30 and Rh41 ARMS xenografts. The greatest effect occurred when tumor-bearing mice were treated with curcumin prior to IR. Immunohistochemistry revealed that combination therapy significantly decreased tumor cell proliferation and endothelial cell count, and increased tumor cell apoptosis. Stable expression of the super-repressor, SR-IκBα, that blocks the classical NF-κB pathway, increased sensitivity to IR, while expression of SR-p100, that blocks the alternative pathway, did not. Our results demonstrate that curcumin can potentiate the antitumor activity of IR in ARMS xenografts by suppressing a classical NF-κΒ activation pathway induced by ionizing radiation. These data support testing of curcumin as a radiosensitizer for the clinical treatment of alveolar rhabdomyosarcoma. IMPACT OF WORK: The NF-κΒ protein complex has been linked to radioresistance in several cancers. In this study, we have demonstrated that inhibiting radiation-induced NF-κΒ activity by either pharmacologic (curcumin) or genetic (SR-IκBα) means significantly enhanced the efficacy of radiation therapy in the treatment of alveolar rhabdomyosarcoma cells and xenografts. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of ionizing radiation and warrants clinical trials.

HIF-1α activation mediates resistance to anti-angiogenic therapy in neuroblastoma xenografts.

INTRODUCTION: The anti-tumor activity of angiogenesis inhibitors is often limited by the development of resistance to these drugs. Here we establish HIF-1α as a major factor in the development of this resistance in neuroblastoma xenografts. METHODS: Neuroblastoma xenografts were established by injecting unmodified SKNAS or NB-1691 cells (2 × 10(6) cells), or cells in which HIF-1α expression had been knocked down with shRNA, into the retroperitoneal space of SCID mice. Treatment of established tumors included bevacizumab (5mg/kg q2wk), sunitinib (40 mg/kg qd), or topotecan (0.5mg/kg qd) alone or in combination for a total of two weeks. RESULTS: NB-1691 xenografts showed no difference in relative growth in HIF-1α knockdowns compared to control tumors (73.33 ± 7.90 vs 79.94 ± 6.15, p=0.528). However, HIF-1α knockdowns demonstrated relative final volumes that were significantly lower than unmodified tumors when both were treated with bevacizumab (35.88 ± 4.24 vs 53.57 ± 6.61, p=0.0544) or sunitinib (12.46 ± 2.59 vs 36.36 ± 4.82, p=0.0024). Monotherapy of unmodified xenografts with bevacizumab, sunitinib, or topotecan was largely ineffective. Relative final volumes of NB-1691 xenografts were significantly less in cohorts treated with sunitinib+topotecan (4.78 ± 0.77 vs 39.17 ± 2.44 [sunitinib alone], p=0.011) and bevacizumab+topotecan (13.63 ± 1.55 vs 48.16 ± 9.94 [bevacizumab alone], p=0.014). CONCLUSION: Upregulation of HIF-1α appears to be a significant mechanism of resistance to antiangiogenic therapies in neuroblastoma. Suppressing HIF-1α with low-dose topotecan potentiates the effects of the antiangiogenic drugs in a mouse model.

Rapamycin increases neuroblastoma xenograft and host stromal derived osteoprotegerin inhibiting osteolytic bone disease in a bone metastasis model.

PURPOSE: Osteoprotegerin (OPG) is a decoy receptor for the Receptor of NF-κB (RANK) ligand that can inhibit osteoclastogenesis. Previous studies have suggested that Mammalian Target of Rapamycin (mTOR) inhibition upregulates OPG production. We tested the hypothesis that the mTOR inhibitor rapamycin could inhibit neuroblastoma bone metastases through its action on OPG. EXPERIMENTAL DESIGN: An orthotopic model of bone metastasis was established. Mice with established disease were subsequently treated with rapamycin (5mg/kg IP daily) or vehicle control (DMSO 1:1000). X-rays were obtained twice a week to detect pathologic fractures. Serum OPG levels were measured by ELISA after two weeks of treatment. RESULTS: Mice with bone disease receiving rapamycin had increased serum levels of OPG in the CHLA-20 mice compared to controls (36.89 pg/mL ± 3.90 vs 18.4 pg/mL ± 1.67, p=0.004) and NB1691 tumor-bearing groups (46.03 ± 2.67 pg/mL vs 17.96 ± 1.84pg/mL, p=0.001), and a significantly longer median time to pathologic fractures with CHLA-20 (103 days vs 74.5 days, p=0.014) and NB1691 xenografts. CONCLUSION: In a xenograft model, increased OPG expression correlated with a delay to pathologic fracture suggesting a potential role for mTOR inhibitors in the treatment of neuroblastoma bone metastases.

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

BACKGROUND: Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery. PRINCIPAL FINDINGS: Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. SIGNIFICANCE: These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Rapamycin-induced tumor vasculature remodeling in rhabdomyosarcoma xenografts increases the effectiveness of adjuvant ionizing radiation.

PURPOSE: Rapamycin inhibits vascular endothelial growth factor expression. Vascular endothelial growth factor is a tumor-elaborated protein that stimulates neovascularization. This inhibition can cause transient "normalization" of the generally dysfunctional tumor vasculature, resulting in improved tumor perfusion and oxygenation. We hypothesized that this may potentiate the antitumor effects of adjuvant ionizing radiation. METHODS: Mice bearing orthotopic Rh30 alveolar rhabdomyosarcomas were treated with rapamycin (5 mg/kg intraperitoneally daily ×5). Tumors were then evaluated for changes in intratumoral oxygenation, perfusion, vessel permeability, and microvessel density. Additional tumor-bearing mice were treated with 5 doses of rapamycin, irradiation (4 Gy), or 5 doses of rapamycin with irradiation administered on the first or sixth day of rapamycin treatment. RESULTS: Although tumor vessel permeability changed only minimally, microvessel density decreased (3153 ± 932 vs 20,477 ± 3717.9 pixels per high-power field), whereas intratumoral oxygenation increased significantly (0.0385 ± 0.0141 vs 0.0043 ± 0.0023 mm Hg/mm(3)) after 5 doses of rapamycin. Contrast-enhanced ultrasound demonstrated a significantly increased rate of change of signal intensity after 5 days of rapamycin, suggesting improved intratumoral perfusion. Tumor volume 14 days after treatment was smallest in mice treated with the combination of rapamycin given before irradiation. CONCLUSION: Combination therapy with rapamycin given before irradiation to normalize the tumor vasculature, thereby improving tumor oxygenation, increased the sensitivity of alveolar rhabdomyosarcoma xenografts to adjuvant irradiation.

Liposome-encapsulated curcumin suppresses neuroblastoma growth through nuclear factor-kappa B inhibition.

BACKGROUND: Nuclear factor-κB (NF-κB) has been implicated in tumor cell proliferation and survival and in tumor angiogenesis. We sought to evaluate the effects of curcumin, an inhibitor of NF-κB, on a xenograft model of disseminated neuroblastoma. METHODS: For in vitro studies, neuroblastoma cell lines NB1691, CHLA-20, and SK-N-AS were treated with various doses of liposomal curcumin. Disseminated neuroblastoma was established in vivo by tail vein injection of NB1691-luc cells into SCID mice, which were then treated with 50 mg/kg/day of liposomal curcumin 5 days/week intraperitoneally. RESULTS: Curcumin suppressed NF-κB activation and proliferation of all neuroblastoma cell lines in vitro. In vivo, curcumin treatment resulted in a significant decrease in disseminated tumor burden. Curcumin-treated tumors had decreased NF-κB activity and an associated significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis, as well as a decrease in tumor vascular endothelial growth factor levels and microvessel density. CONCLUSION: Liposomal curcumin suppressed neuroblastoma growth, with treated tumors showing a decrease in NF-κB activity. Our results suggest that liposomal curcumin may be a viable option for the treatment of neuroblastoma that works via inhibiting the NF-κB pathway.

Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Continuous local delivery of interferon-ß stabilizes tumor vasculature in an orthotopic glioblastoma xenograft resection model.

BACKGROUND: High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-ß delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection. METHODS: Xenografts were established by injecting 3 grade IV glioblastoma cell lines (GBM6-luc, MT330-luc, and SJG2-luc) into the cerebral cortex of nude rats. Tumors underwent subtotal resection, and then had gel foam containing an adeno-associated virus vector encoding either hIFN-ß or green fluorescence protein (control) placed in the resection cavity. The primary endpoint was stabilization of tumor vasculature, as evidenced by CD34, α-SMA, and CA IX staining. Overall survival was a secondary endpoint. RESULTS: hIFN-ß treatment altered the tumor vasculature of GBM6-luc and SJG2-luc xenografts, decreasing the density of endothelial cells, stabilizing vessels with pericytes, and decreasing tumor hypoxia. The mean survival for rats with these neoplasms was not improved, however. In rats with MT330-luc xenografts, hIFN-ß resulted in tumor regression with a 6-month survival of 55% (INF-ß group) and 9% (control group). CONCLUSION: The use of AAV hIFN-ß in our orthotopic model of glioblastoma resection stabilized tumor vasculature and improved survival in rats with MT330 xenografts.

Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.

Targeting multiple angiogenic pathways for the treatment of neuroblastoma.

PURPOSE: Resistance to angiogenesis inhibition can occur through the upregulation of alternative mediators of neovascularization. We used a combination of angiogenesis inhibitors with different mechanisms of action, interferon-beta (IFN-beta) and rapamycin, to target multiple angiogenic pathways to treat neuroblastoma xenografts. METHODS: Subcutaneous and retroperitoneal neuroblastoma xenografts (NB-1691 and SK-N-AS) were used. Continuous delivery of IFN-beta was achieved with adeno-associated virus vector-mediated, liver-targeted gene transfer. Rapamycin was delivered intraperitoneally (5 mg/kg per day). After 2 weeks of treatment, tumor size was measured, and tumor vasculature was evaluated with intravital microscopy and immunohistochemistry. RESULTS: Rapamycin and IFN-beta, alone and in combination, had little effect on tumor cell viability in vitro. In vivo, combination therapy led to fewer intratumoral vessels (69% of control), and the remaining vessels had an altered phenotype, being covered with significantly more pericytes (13x control). Final tumor size was significantly less than controls in all tumor models, with combination therapy having a greater antitumor effect than either monotherapy. CONCLUSION: The combination of IFN-beta and rapamycin altered the vasculature of neuroblastoma xenografts and resulted in significant tumor inhibition. The use of combinations of antiangiogenic agents should be further evaluated for the treatment of neuroblastoma and other solid tumors.