Evaluation of the Biodistribution,Persistence, Toxicity,and Potential of Germ-Line Transmission of a Replication-Competent Human Adenovirus Following Intraprostatic Administration in the Mouse

Adenovirus-mediated gene transfer may hold much promise in the treatment of human cancer. However, concerns regarding vector dissemination beyond the target tissue, particularly with replication-competent viruses, require an evaluation of the persistence of viral infection in collateral tissue and vector-associated toxicities. In addition, for indications such as prostate cancer, the proximity of the point of viral administration to organs of the male reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus. To address these concerns, the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent adenovirus (Ad5-CD/TKrep) following intraprostatic administration in the mouse was examined. Ad5-CD/TKrep (10¹⁰ vp, 5 × 10¹¹ vp/kg) was injected intraprostatically on Day 1 of the study and its presence in the major organs of the male urogenital tract (prostate, testes, seminal vesicles, and urinary bladder) and liver was determined on Days 8 and 29. For comparison, a parallel group of animals was injected with the same dose of a related replication-defective Ad5-FGNR virus. To evaluate germ-line transmission, Ad5-CD/TKrep-injected males were mated to females on Days 8 and 29 and resulting embryos were examined for Ad5-CD/TKrep viral DNA. Ad5-CD/TKrep viral DNA was detected in all major organs of the adult male urogenital tract and liver 7 and 28 Days postinjection. Interestingly, relative to the replication-defective Ad5-FGNR adenovirus, the replication-competent Ad5-CD/TKrep virus accumulated to a much greater level (∼300-fold) and persisted for a longer period of time in prostate, testes, and liver. This difference could not be explained on the basis of differences in viral infectivity, suggesting that the Ad5-CD/TKrep virus may be capable of replicating in mouse tissues in vivo. In vitro infection of six mouse cell lines representing prostate, testes, and liver demonstrated that the Ad5-CD/TKrep virus was indeed capable of replicating in these mouse cell types, albeit with reduced efficiencies relative to human cells. Despite the fact that the Ad5-CD/TKrep vector persisted in the adult male gonads and may have replicated in vivo, we observed no evidence of germ-line transmission in 149 offspring examined. To evaluate the toxicity of combining Ad5-CD/TKrep viral therapy with CD/5-FC and HSV-1 TK/GCV suicide gene therapies as a prerequisite for a human trial, an escalating dose (10⁸, 10⁹, 10¹⁰ vp) of Ad5-CD/TKrep was administered intraprostatically followed by 7 days of 5-FC and GCV double prodrug therapy. Although the virus persisted in the mouse urogenital tract and liver for up to 28 days postinjection, most of the toxicities observed were expected, minimal, and self-limiting. These results lead us to believe that intraprostatic administration of the Ad5-CD/TKrep virus to humans concomitant with double suicide gene therapy will be associated with acceptable toxicities and will not result in vertical transmission of viral-encoded genes through the germ line.     

Design considerations for incorporating sodium iodide symporter reporter gene imaging into prostate cancer gene therapy trials

This study was done to aid in the design of a phase I gene therapy trial in patients with prostate cancer. We determined the dosimetric characteristics of our reporter gene system when coupled with intravenous administration of radioactive sodium pertechnetate (Na(99m) TcO(4)) and determined the feasibility of using human sodium iodide symporter (hNIS) as a reporter gene to study the dynamics of adenoviral transgene expression in a large animal tumor. A replication-competent Ad5-yCD/mutTK(SR39) rep-hNIS adenovirus was injected into the prostate gland of dogs for dosimetry purposes, and into a canine soft tissue sarcoma (STS) for imaging purposes. After resection of the prostate, the amount of (99m)TcO(4)() sequestered in the prostate was determined, the radiation dose absorbed by the prostate and nontarget critical organs was calculated, and hNIS reporter gene expression was imaged in the STS by single-photon emission computed tomography (SPECT). On the basis of the findings from 25 dogs, the amount of (99m)TcO (4)() sequestered in the prostate ranged from 13 to 276 muCi. Using the highest value observed, absorbed radiation dose to critical organs was calculated and found to be below U.S. Food and Drug Administration limits for diagnostic imaging. Also, (99m)TcO (4)() uptake was readily detected by SPECT and found to persist in vivo for at least 4 days. On the basis of our dosimetry calculations, up to five imaging procedures can be safely performed in humans after intraprostatic injection of the Ad5-yCD/mutTK(SR39)rep-hNIS adenovirus and the hNIS reporter gene system can be used to study the dynamics of adenoviral gene therapy vectors in large animal tumors.     

Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer.

In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer.     

Second-generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity.

Replication-competent adenovirus-mediated suicide gene therapy has proven to be safe in humans when delivered intraprostatically. Although signs of efficacy are emerging, it is likely that further improvements will be needed before this technology will have widespread applicability in the clinic. Toward this end, we have developed a second-generation, replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-ADP) containing an improved yeast cytosine deaminase (yCD)/mutant(SR39) herpes simplex virus thymidine kinase fusion (yCD/mutTK(SR39)) gene and the adenovirus death protein (ADP) gene. Relative to the first-generation Ad5-CD/TKrep adenovirus, Ad5-yCD/mutTK(SR39)rep-ADP demonstrated greater tumor cell kill in vitro and significantly greater tumor control in preclinical models of human cancer. Quantification of transgene volume following direct injection of fadenovirus into human tumor xenografts and the na?ve canine prostate demonstrated that ADP enhanced adenoviral spread in vivo. Toxicology studies were performed to determine whether the improved yCD/mutTK(SR39) fusion and ADP genes increased toxicity. Intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-ADP did not result in significantly increased toxicity relative to the parental Ad5-CD/TKrep adenovirus, the latter of which has proven to be safe in two Phase I prostate cancer clinical trials. Together, these results provide the scientific basis for evaluating the safety and efficacy of the second-generation Ad5-yCD/mutTK(SR39)rep-ADP adenovirus in humans.     

A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells

Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 × 10⁴ purified rAAV particles per cell (range: 3.7 × 10⁴ to 9.6 × 10⁴) from suspension cultures of recombinant baculovirus–infected insect cells.     

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 (¹²⁵I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of ¹²³I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 × 10⁶ TCID₅₀) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of ¹³¹I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by ¹³¹I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.     

Inherited Methylmalonyl CoA Mutase Apoenzyme Deficiency in Human Fibroblasts: EVIDENCE FOR ALLELIC HETEROGENEITY, GENETIC COMPOUNDS, AND CODOMINANT EXPRESSION

We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut° mutants. Those with detectable residual activity in cell extracts (~0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut⁻ mutants. The mutant enzyme in the mut⁻ mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (K_m) for adenosylcobalamin in vitro, a normal K_m for methylmalonyl CoA, and a strikingly reduced thermal stability at 45°C relative to control. Mutase from one mut⁻ mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.     

Balloon Catheter Delivery of Helper-dependent Adenoviral Vector Results in Sustained,Therapeutic hFIX Expression in Rhesus Macaques

Hemophilia B is an excellent candidate for gene therapy because low levels of factor IX (FIX) (≥1%) result in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors can mediate long-term transgene expression without chronic toxicity. To determine the potential for HDAd-mediated liver-directed hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a novel and minimally invasive balloon occlusion catheter-based method that permits preferential, high-efficiency hepatocyte transduction with low, subtoxic vector doses. Animals given 1 × 10¹² and 1 × 10¹¹ virus particle (vp)/kg achieved therapeutic hFIX levels for the entire observation period (up to 1,029 days). At 3 × 10¹⁰ and 1 × 10¹⁰ vp/kg, only subtherapeutic hFIX levels were achieved which were not sustained long-term. Balloon occlusion administration of HDAd was well tolerated with negligible toxicity. Five of six animals developed inhibitors to hFIX. These results provide important information in assessing the clinical utility of HDAd for hemophilia B gene therapy.     

Formation of 7-carboxyheptyl radical induced by singlet oxygen in the reaction mixture of oleic acid, riboflavin and ferrous ion under the UVA irradiation

Identification of the radicals was performed for the standard reaction mixtures, which contained 4.3 mM oleic acid, 25 µM riboflavin, 1 mM FeSO₄(NH₄)₂SO₄, 10 mM cholic acid, 40 mM phosphate buffer (pH 7.4) and 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone under the UVA irradiation (365 nm), using an electron spin resonance, an high performance liquid chromatography-electron spin resonance and an high performance liquid chromatography-electron spin resonance-mass spectrometry. The electron spin resonance and high performance liquid chromatography-electron spin resonance measurements of the standard reaction mixtures showed prominent signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) and peaks 1 and 3 (retention times, 37.0 min and 49.0 min). Since the peak 3 was not observed for the standard reaction mixture without oleic acid, the radical of the peak 3 seems to be derived from oleic acid. Singlet oxygens seem to participate in the formation of the oleic acid-derived radicals because the peak height of the peak 3 observed in the standard reaction mixture of D₂O increased to 308 ± 72% of the control. The high performance liquid chromatography-electron spin resonance-mass spectrometry analysis showed that 7-carboxyheptyl radical forms in the standard reaction mixture.     

Enhancement of Adenovirus Delivery after Ultrasound-Stimulated Therapy in a Cancer Model

Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. In vitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for in vivo analysis to quantify Ad infection within the tumor. In vitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p = 0.80). In vivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1 × 10⁶ plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p = 0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues.     

Polished rice as natural sources of cancer-preventing geranylgeranoic acid

Geranylgeranoic acid, a 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were previously developed as synthetic “acyclic retinoids” for cancer chemoprevention. Recently, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji and Ogawa, 2004). In this present study, we present several lines of evidence to demonstrate that geranylgeranyl diphosphate taken in foods could be metabolized to GGA through geranylgeraniol and geranylgeranyl aldehyde via the following steps: 1) The conversion from geranylgeranyl diphosphate to geranylgeraniol was demonstrated to occur by the action of bovine intestinal alkaline phosphatase, with a K_m of 46.1 µM. 2) Geranylgeraniol oxidase-mediated conversion of geranylgeraniol to geranylgeranyl aldehyde was revealed in rat liver homogenates, which activity was mainly localized in the mitochondrial fraction. The mitochondrial enzyme showed a K_m of 92.9 µM. 3) The conversion of geranylgeranyl aldehyde to geranylgeranoic acid by geranylgeranyl aldehyde dehydrogenase in rat liver homogenates was absolutely dependent on exogenously added NAD⁺ or NADP⁺. The K_m of the mitochondrial geranylgeranyl aldehyde dehydrogenase was 27.5 µM for geranylgeranyl aldehyde. Taken together, our data suggest that cancer preventive geranylgeranoic acid could be a physiological metabolite from commonly consumed foods.     

Therapeutic Effect of Sodium Iodide Symporter Gene Therapy Combined With External Beam Radiotherapy and Targeted Drugs That Inhibit DNA Repair

Adenoviral (AdV) transfer of sodium iodide symporter (NIS) gene has translational potential, but relatively low levels of transduction and subsequent radioisotope uptake limit the efficacy of the approach. In previous studies, we showed that combining NIS gene delivery with external beam radiotherapy (EBRT) and DNA damage repair inhibitors increased viral gene expression and radioiodide uptake. Here, we report the therapeutic efficacy of this strategy. An adenovirus expressing NIS from a telomerase promoter (Ad-hTR-NIS) was cytotoxic combined with relatively high-dose (50 µCi) ¹³¹I therapy and enhanced the efficacy of EBRT combined with low-dose (10 and 25 µCi) ¹³¹I therapy in colorectal and head and neck cancer cells. Combining this approach with ataxia-telangiectasia mutated (ATM) or DNA-dependent protein kinase (DNA-PK) inhibition caused maintenance of double-stranded DNA breaks (DSBs) at 24 hours and increased cytotoxicity on clonogenic assay. When the triplet of NIS-mediated ¹³¹I therapy, EBRT, and DNA-PKi was used in vivo, 90% of mice were tumor-free at 5 weeks. Acute radiation toxicity in the EBRT field was not exacerbated. In contrast, DNA-PKi did not enhance the therapeutic efficacy of EBRT plus adenovirus-mediated HSVtk/ganciclovir (GCV). Therefore, combining NIS gene therapy and EBRT represents an ideal strategy to exploit the therapeutic benefits of novel radiosensitizers.     

A marked increase in gastric fluid volume during cardiopulmonary bypass

Major physiological stress occurs during cardiac surgery with cardiopulmonary bypass. This is related to hypothermia and artificial organ perfusion. Thus, serious gastrointestinal complications, particularly upper gastrointestinal bleeding, sometimes follow cardiac surgery. We have compared the antisecretory effects of a preanesthetic H₂ antagonist (roxatidine, cardiopulmonary bypass-H₂ group, n = 15) and a proton pump inhibitor (rabeprazole, cardiopulmonary bypass-PPI group, n = 15) in patients undergoing cardiac surgery with cardiopulmonary bypass, and also compared in patients undergoing a off-pump coronary artery bypass graft surgery (off-pump cardiopulmonary bypass-H₂ group, n = 15). Gastric pH (5.14 ± 0.61) and gastric fluid volume (13.2 ± 2.4 mL) at the end of surgery in off-pump cardiopulmonary bypass-H₂ groups was significantly lower and higher than those in both cardiopulmonary bypass-H₂ (6.25 ± 0.54, 51.3 ± 8.0 mL) and cardiopulmonary bypass-PPI (7.29 ± 0.13, 63.5 ± 14.8 mL) groups, respectively although those variables did not differ between groups after the induction of anesthesia. Plasma gastrin (142 ± 7 pg/mL) at the end of surgery and maximal blood lactate levels (1.50 ± 0.61 mM) in off-pump cardiopulmonary bypass-H₂ group were also significantly lower than those in both cardiopulmonary bypass-H₂ (455 ± 96 pg/mL, 3.97 ± 0.80 mM) and cardiopulmonary bypass-PPI (525 ± 27 pg/mL, 3.15 ± 0.44 mM) groups, respectively. In addition, there was a significant correlation between gastric fluid volume and maximal blood lactate (r = 0.596). In conclusion, cardiopulmonary bypass may cause an increase in gastric fluid volume which neither H₂ antagonist nor PPI suppresses. A significant correlation between gastric fluid volume and maximal blood lactate suggests that gastric fluid volume may predict degree of gastrointestinal tract hypoperfusion.     

The effect of irradiation wavelengths and the crystal structures of titanium dioxide on the formation of singlet oxygen for bacterial killing

Safe and effective methods for oral bacterial disinfection have been desired, since bacteria cause many infectious diseases such as dental caries, periodontal disease, and endodontic infections. Singlet oxygen (¹O₂) is attractive, because it is toxic to prokaryotic cells, but not to eukaryotic cells. We selected irradiation of titanium dioxide (TiO₂) as a source of ¹O₂, because it has been used in sunscreens and cosmetic products without complications. In order to establish the optimal oral photodynamic therapy conditions, we measured the rate of ¹O₂ formation from the irradiated anatase or rutile forms of TiO₂ using 365 or 405 nm lamps. The rate of ¹O₂ formation decreased in the following order: anatase, 365 nm > rutile, 405 nm > rutile, 365 nm > anatase, 405 nm. Therefore, we concluded that irradiation of the rutile form of TiO₂ by a 405 nm lamp is the most favorable photodynamic therapy condition, because visible light is more desirable than UV light from the viewpoint of patient safety. We also confirmed that there was no direct HO^• formation from the irradiated TiO₂.     

Changes in Prandial Glucagon Levels After a 2-Year Treatment With Vildagliptin or Glimepiride in Patients With Type 2 Diabetes Inadequately Controlled With Metformin Monotherapy

OBJECTIVE

To determine if the dipeptidyl peptidase-4 inhibitor vildagliptin more effectively inhibits glucagon levels than the sulfonylurea glimepiride during a meal.

RESEARCH DESIGN AND METHODS

Glucagon responses to a standard meal were measured at baseline and study end point (mean 1.8 years) in a trial evaluating add-on therapy to metformin with 50 mg vildagliptin b.i.d. compared with glimepiride up to 6 mg q.d. in type 2 diabetes (baseline A1C 7.3 ± 0.6%).

RESULTS

A1C and prandial glucose area under the curve (AUC)_{0–2 h} were reduced similarly in both groups, whereas prandial insulin AUC_{0–2 h} increased to a greater extent by glimepiride. Prandial glucagon AUC_{0–2 h} (baseline 66.6 ± 2.3 pmol · h⁻¹ · l⁻¹) decreased by 3.4 ± 1.6 pmol · h⁻¹ · l⁻¹ by vildagliptin (n = 137) and increased by 3.8 ± 1.7 pmol · h⁻¹ · l⁻¹ by glimepiride (n = 121). The between-group difference was 7.3 ± 2.1 pmol · h⁻¹ · l⁻¹ (P < 0.001).

CONCLUSIONS

Vildagliptin therapy but not glimepiride improves postprandial α-cell function, which persists for at least 2 years.Glucagon levels are increased in type 2 diabetes because of impaired glucose-mediated suppression of glucagon secretion resulting in increased hepatic glucose output with subsequent hyperglycemia (1). Improved glycemia by the dipeptidyl peptidase-4 inhibitor, vildagliptin (2), is mediated primarily by improved β- and α-cell sensitivity to glucose (3). As an add-on to metformin, vildagliptin displays equal efficacy as glimepiride, with the added benefits of a much lower risk of hypoglycemia and no weight gain (4). Here we report prandial assessments of glucagon levels and insulin secretion rates after up to ∼2 years of therapy with the two drugs.     

Brainstem 1H-MR spectroscopy in episodic and chronic migraine

The pathogenesis of evolution from episodic migraine (EM) to chronic migraine (CM) has not yet been clearly determined. Some studies revealed that dysfunction of the brainstem may play a role. We aimed to determine the brainstem ¹H-MR spectroscopic (MRS) findings in episodic and chronic migraine. We recruited patients with EM, CM and controls. Patients with CM were divided into subgroups with and without medication overuse (MO). The ¹H-MRS metabolite ratios at the periaqueductal gray (PAG) and bilateral dorsal pons were measured and compared with those in controls. A total of 19 patients with EM, 53 patients with CM (with MO n = 30, without MO n = 23) and 16 control subjects completed the study. Patients with EM had the highest N-acetylaspartate (NAA)/creatine (Cr) ratio at the dorsal pons (right, P = 0.014; left, P = 0.034) in comparison with those of CM and controls. The latter two groups did not differ. Among migraine patients, NAA/Cr ratios at dorsal pons were inversely correlated with headache frequency (right, r = −0.350, P = 0.004; left, r = −0.284, P = 0.019) and intensity (right, r = −0.286, P = 0.019; left, r = −0.244, P = 0.045), but not disease duration. In contrast, the metabolite ratios did not differ at the PAG among the study groups. Of note, MO was not associated with brainstem MRS ratios in patients with CM. The increased NAA/Cr levels may suggest neuronal hypertrophy at the dorsal pons in EM. A progressive dysfunction of this region may occur from EM to CM since the levels declined with increasing headache frequency and intensity.     

Relationship between dietary folate intake and plasma monocyte chemoattractant protein-1 and interleukin-8 in heart failure patients

This study aimed to examine the association of dietary vitamin intakes with plasma pro-inflammatory cytokine levels in Korean heart failure patients. Stable outpatients with heart failure were recruited and finally 91 patients were included. Dietary intakes were estimated by a developed semi-quantitative food frequency questionnaire. The simultaneous measurement of 17 cytokines was performed along with analysis of plasma C-reactive protein. Plasma C-reactive protein levels significantly correlated with dietary intakes of vitamin C (r = −0.30, p<0.005), β-carotene (r = −0.23, p<0.05), and folate (r = −0.31, p<0.005). However, these associations were no longer significant after adjusting for traditional risk factors for heart failure. On the other hand, plasma levels of monocyte chemoattractant protein-1 significantly correlated with dietary folate intake (r = −0.31, p<0.001), and plasma interleukin-8 levels significantly correlated with dietary intakes of vitamin C (r = −0.38, p<0.001), β-carotene (r = –0.42, p<0.001), and folate (r = −0.38, p<0.001) after the adjustment. Dietary folate intake was found as a primary influencing factor on plasma levels of monocyte chemoattractant protein-1 (p<0.005, R² = 0.20) and interleukin-8 (p<0.001, R² = 0.32) through a stepwise multiple linear regression analysis. Dietary folate intake was significantly associated with plasma levels of monocyte chemoattractant protein-1 and interleukin-8 which indicates dietary folate may have a potentially beneficial role in the prevention and treatment of heart failure.     

Free radical formation from sonolysis of water in the presence of different gases

In the present study by applying electron spin resonance-spin trapping method, when a high frequency (1650 kHz) ultrasound was irradiated to water dissolved with different gas molecules (O₂, N₂, Ar, Ne, He, and H₂) at 25°C of water bulk temperature, free radical generation pattern differed dependently on the dissolved gas molecules. Only ^•OH was detected in the O₂-dissolved water sample, and the amount of the radical was much greater than that determined in any of other gas-dissolved water samples. One of the possible reasons to explain why the ^•H radical was not detected in the O₂-dissolved water is that the ^•H reacts with O₂ to form ^•OOH. However, no electron spin resonance signals related to the adduct of not only 5,5-dimethyl-1-pyrroline-N-oxide but 5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide and ^•OOH were observed. In the H₂-dissolved water, only ^•H was detected, suggesting that H₂ reduces or neutralizes ^•OH. In the N₂-disolved water, both ^•OH and ^•H were detected at comparable level. In the water samples dissolved with rare gases (Ar, Ne, and He), the amount of ^•H was almost double as compared with that of ^•OH, and both ^•OH and ^•H yields increased in the order Ar > Ne > He.     

Use of Adenoviral Vectors to Target Chemotherapy to Tumor Vascular Endothelial Cells Suppresses Growth of Breast Cancer and Melanoma

To target chemotherapy to tumor vascular endothelial cells (TVECs), we created the AdTie2RprCDFib^knob−RGD+ vector by inserting into an AdEasy adenoviral vector (Ad) backbone: (i) the cytosine deaminase (CD) gene driven by the Tie2 receptor promoter (Tie2Rpr) into the E1 region of Ad; (ii) mutations that reduce binding of the fiber knob to the Coxsackie adenovirus receptor (CAR); and (iii) the RGD peptide into the H1 loop of fiber for binding to the α_Vβ₃ integrin receptors on TVECs. To reduce uptake of the AdTie2RprCDFib^knob−RGD+ by reticuloendothelial (RE) and liver cells, we intravenously (i.v.) injected Hetastarch and low-dose Ad (one million vector particles (VPs)) prior to i.v. injection of a therapeutic dose (one billion VPs) of the AdTie2RprCDFib^knob−RGD+ vector. This treatment induced regressions of N202 breast cancer and B16 melanoma without toxicity to normal tissues. We showed that the tumor regression was induced by infection of the TVECs and not by the infection of tumor cells by the AdTie2RprCDFib^knob−RGD+ vector.