Sequestration and ultrasound-induced release of doxorubicin from stabilized Pluronic P105 micelles

Controlled drug delivery from micelles requires that the micelles remain stable when diluted below their critical micelle concentration, such as upon injection into blood. A cross-linked, interpenetrating network of N,N-diethylacrylamide (NNDEA) was polymerized in the core of Pluronic P105 micelles to stabilize temporarily the micelles at concentrations below the critical micellar concentration of free P105. The stabilized Pluronic micelles (called Plurogels) were able to sequester the drug doxorubicin (Dox) and protect HL-60 cells from the drug at concentrations where non-stabilized Pluronic provided no protection. The protection lasted ~ 12 hr, which is similar to the half-life of the particles. Application of low-frequency ultrasound resulted in a synergistic killing effect with Dox and low concentrations of either Pluronic P105 or stabilized Plurogels, most probably due to release of Dox and permeabilization of the cell membrane.     

Improved survival in resected biliary malignancies

BACKGROUND: For many years the prognosis for patients with biliary malignancies has been poor. However, recent advances in radiology and laparoscopy have improved staging, and active biliary stent management may improve outcome in these patients. In the past the goal with surgery was to excise all gross tumor. Now, the surgical goal is to achieve negative microscopic margins even if a major hepatic resection is required. Similarly, chemotherapy or radiation was frequently given in isolation, but chemoradiation has become the standard. Therefore, the aim of this analysis was to determine whether survival has improved with better staging, active stent management, more aggressive surgery, and chemoradiation. METHODS: From 1990 through 2001, 140 patients with biliary malignancies were treated at the Medical College of Wisconsin. One hundred eleven malignancies were cholangiocarcinomas (intrahepatic, 22%; perihilar, 65%; and distal, 13%), and 29 were gallbladder (GB) cancers. Eighty-six of the 140 patients (61%) underwent exploration (intrahepatic, 58%; perihilar, 57%; distal, 67%, and GB, 72%). Forty-four of these 86 patients (51%) underwent resection (intrahepatic, 64%; perihilar, 41%; distal, 70%; and GB, 52%). Chemoradiation with confocal radiation, 5-fluorouracil, and gemcitabine was used more frequently in the patients resected since 1998. RESULTS: Thirty-day operative mortality was 4%. In the resected patients (n = 44) the 5-year actuarial survival was 31% and the median survival was 27.8 months. Patients resected between 1998 and 2001 (n = 25) had a median survival longer than 44 months with a 3-year actuarial survival of 70% as compared to patients resected between 1990 and 1997 (n = 19), who had a median survival of 13 months and a 3-year actuarial survival of 21% (P <.01). CONCLUSIONS: These data suggest that (1) approximately one third of patients with biliary malignancies have resectable disease and (2) surgery in carefully selected patients with adjuvant chemoradiation has improved survival in resected patients. We suspect that a combination of improved staging, active biliary stenting, safe but extensive surgery to obtain negative margins, and newer techniques for chemoradiation have resulted in improved outcomes for patients with biliary malignancies.     

Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (HC)) immune response and protection in the rhesus monkey

Botulinum neurotoxin B (BoNTB) is a distinct protein subtype of a family of neurotoxins with the potential for use in biological warfare or terrorist attacks. This study is one in a series evaluating the immunogenicity and protective effects of recombinant vaccines against the different subtypes of botulinum toxin. The recombinant subunit vaccines encoding the C fragment portion (50 kDa) of the toxins are produced in the yeast, Pichia pastoris. In this study, groups of rhesus monkeys were vaccinated with three doses (1 and 5 μg per dose) of rBoNTB(H_c) vaccine. Total and neutralizing antibody titers were determined at various times during and postvaccination. Two groups of vaccinated monkeys plus non-vaccinated controls were actively challenged with B toxin by aerosol exposure. All monkeys receiving vaccine were protected from the toxin and no clinical signs of disease were observed, while controls displaying classic signs of botulism succumbed to the toxin challenge. Two additional groups of monkeys receiving the same vaccine regiment as the first two groups had significant levels of circulating neutralizing antibody titers up to 24 months postvaccination. This non-human primate study demonstrated the short- and long-term immunity afforded by the rBoNTB(H_c) vaccine.     

Rhinoliths presenting during routine radiography: two cases

Rhinoliths are calcified masses found within the nasal cavity. They are an uncommon finding and usually present to ENT surgeons. This article presents two cases where rhinoliths have been recognized in the dental setting, and discusses their management and treatment.     

Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.     

Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.     

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in heterozygous p53-deficient female mice at the same dose in a 6-month study. To examine whether phenolphthalein carcinogenic potential can be detected in male and female transgenic (Tg) mice carrying the human c-Ha-ras gene (rasH2 mice) and their wild-type littermates (non-Tg mice), a diet containing 3000, 6000 or 12000 ppm was given for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that rasH2 mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.