Thresholds of nitric oxide-mediated toxicity in human lymphoblastoid cells

A novel delivery system was used to study NO-mediated cyto- and genotoxicity in two human lymphoblastoid cell lines, TK6 (wild-type p53) and NH32 (p53-null but isogenic to TK6). The delivery system, which supplied NO and O(2) continuously by diffusion through gas permeable tubing, was found to maintain the NO and O(2) concentrations at constant, predictable values. Cellular rates of NO and O(2) consumption and mass transfer coefficients for the two gases were measured in separate experiments and used to calculate the NO concentrations during exposure experiments. The TK6 and NH32 cells were each exposed to several steady state NO concentrations for varying lengths of time, so that the total dose (area under the concentration-time curve) covered a wide range. End point assays, including lethality, apoptosis, mitochondrial damage, and mutation rate in the thymidine kinase (TK1) gene locus, were performed at different posttreatment times. Control experiments using Ar instead of NO resulted in normal cell proliferation for all exposure times tested (up to 36 h). As compared to those controls, significant cell death, apoptosis, and mitochondrial membrane depolarization were observed in NO-treated TK6 cells, and the TK1 mutation rate was elevated. Of particular importance, toxic effects were observed only when the NO concentration and dose were greater than threshold values of approximately 0.5 micro M and approximately 150 micro M min, respectively. If neither or only one threshold was exceeded, the effects were insignificant; when both were exceeded, total cell survival and the number of nonapoptotic cells both decreased exponentially with increasing NO dose. In general, the NH32 cells were much more resistant to NO-induced damage and death than TK6 cells, demonstrating that p53 status is an important determinant of NO-induced cytotoxicity.     

Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol 3-kinase-Akt/PKB survival pathway

Synthetic alkyl-lysophospholipids (ALPs) represent a new class of anti-tumor agents that target cell membranes and induce apoptosis. However, the exact mechanisms by which ALPs exert these effects remain unclear. Here, we investigated in the epithelial carcinoma cell lines A431 and HeLa the effect of three clinically relevant ALPs [Et-18-OCH3 (Edelfosine), HePC (Miltefosine) and D-21266 (Perifosine)] on the phosphatidylinositol 3-kinase (PI3K)-Akt/PKB survival pathway. We found that growth factor-induced Akt/PKB activation in these cells is dependent on PI3K and that all three ALPs inhibited this pathway in a dose-dependent manner. We further showed that inhibition of the PI3K-Akt/PKB pathway by wortmannin or ALPs is associated with activation of the pro-apoptotic SAPK/JNK pathway. Inhibition of the PI3K-Akt/PKB survival pathway represents a novel mode of action of ALPs that may significantly contribute to the induction of apoptosis.     

Eosinophilic granuloma of the orbit: understanding the paradox of aggressive destruction responsive to minimal intervention

PURPOSE: To describe the findings and outcomes in eosinophilic granuloma (unifocal Langerhans-cell histiocytosis [LCH]) of the orbit and to explain the paradox of aggressive bone destruction responsive to minimal intervention. METHODS: Retrospective, consecutive, interventional case series of patients treated from 1985 to 2001. Minimum inclusion criteria were demonstration of CD1a positivity or Birbeck granules, treatment by a single surgeon, systemic evaluation by a pediatric oncologist, and follow-up of 12 months. A pathogenetic construct was assembled from general LCH concepts and the specific orbital findings. RESULTS: Seven patients met study criteria. All were male, 2 to 16 years of age. All had eyelid or forehead swelling and osteolytic defects, with symptoms of 2 to 6 weeks' duration. All underwent incisional biopsy, with frozen-section examination suggestive of LCH in 6 of 7 cases. The 2 earliest patients received low-dose irradiation after simple biopsy. The 5 most recent patients had subtotal curettage at the time of biopsy; 4 of 5 received simultaneous intralesional corticosteroid injection. In all cases, systemic evaluation showed no other focus of LCH, reossification was timely, and no local recurrence or additional focus was noted in follow-up of 1 to 17 years. CONCLUSIONS: Transient immune dysfunction may provoke the cytokine-mediated proliferation of pathologic Langerhans cells within the hematopoietic marrow of the anterolateral frontal bone. These cells cause osteolysis through elaboration of interleukin-1 and prostaglandin E2. Corticosteroids can inhibit the mediators. We recommend incisional biopsy, frozen-section provisional diagnosis, subtotal curettage, intralesional corticosteroid instillation, postoperative systemic evaluation, and long-term surveillance.     

The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective

Current regulatory guidances do not address specific study designs for in vitro and in vivo drug-drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (CYP) probe substrates, inhibitors, and inducers and for the development of classification systems to improve the communication of risk to health care providers and patients. While existing guidances cover mainly CYP-mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently and should also be addressed. This paper was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug-drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.     

Genomic analysis of a recently identified virus (SEN virus) and genotypes D and H by polymerase chain reaction

BACKGROUND/AIMS: SEN virus (SENV) was discovered in 1999 as a DNA virus with hepatotropic properties. Nine genotypes (A-I) have been identified with genotypes D and H being more prevalent in cases of chronic hepatitis. Attempts to determine whether SENV causes liver disease have been hampered by limited diagnostic testing. METHODS: In the present study, we developed two PCR based assays; a general SENV screening and genotype-specific assay. RESULTS: By screening PCR, the specificity for all SENV genotypes and SENV-related sequences was 20/20 (100%) with confirmation of the results being provided by genomic sequencing. With the genotype-specific PCR, specificities for SENV-D and SENV-H were 7/7 (100%) and 7/11 (64%), respectively. All screening PCR products were cloned and sequenced. The results of sequencing showed high genetic diversity in representative SENV genotypes. Five of twenty patients (25%) had mixed infections with several SENV genotypes. CONCLUSIONS: The screening PCR was useful for identifying cases of SENV infection. However, because of high genetic divergence and mixed co-infection, it was difficult to establish a specific method for genotype distinction. Hence, sequencing is still required for further investigations of SENV as a potential cause of liver disease.     

Using reproductive endpoints in small forage fish species to evaluate the effects of Athabasca Oil Sands activities

The main objective of this study was to evaluate the influence of naturally occurring oil sands-related compounds (OSRC) on reproductive function in fish in order to assess the impacts of anthropogenic point-source inputs. The health of slimy sculpin (Cottus cognatus) and pearl dace (Semotilus margarita) collected from the Alberta Athabasca Oil Sands (Canada) watershed were examined. Two rivers were selected for study: the Steepbank and the Ells. These rivers originate outside the oil sands formation, where fish are unexposed (Ref), exposed to naturally occurring oil sands-related compounds (Nat), or exposed to naturally occurring compounds as well as adjacent to surface mining activity (Dev). Assessment endpoints included gonadosomatic indices (GSI), fecundity, and in vitro gonadal steroid production. In vitro gonadal incubations demonstrated lower levels of steroid production at sites along the Steepbank River within the oil sands deposit. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity, an indicator of exposure to OSRC, was elevated twofold at the site with natural compounds and up to 10-fold at the site adjacent to development compared to EROD activity in fish from the reference site. Fish collected in the Ells River had a threefold induction in EROD activity but no significant reduction in steroid production when compared to reference fish. No consistent alterations in gonadal development were seen in fish collected from sites within the oil sands deposit. This research in the Athabasca River basin provides baseline information of the health of fish populations within the oil sands deposit prior to further development in the area.     

The struggle over employee benefits: the role of labor in influencing modern health policy

After organized labor failed to institute national health insurance in the mid-twentieth century, its influence on health care policy diminished even further. This article proposes an alternative interpretation of the development of health care policy in the United States, by examining the association of health policy with the relationships between employers and employees. The social welfare and health insurance systems that resulted were a direct outcome of the pressures brought by organized and unorganized labor movements. The greater dependency created by industrial and demographic changes, conflicts between labor and capital over the political meaning of disease and accidents, and attempts by the political system to mitigate the impending social crisis all helped determine new health policy options.     

Surgical outcome in 85 patients with primary cardiac tumors

BACKGROUND: We present a large, single institution experience with adult cardiac tumors and address factors affecting outcome. METHODS: A retrospective review was made of all patients who underwent surgery for primary cardiac tumors from April 1975 through August 2002. RESULTS: Eighty-five patients (33 male and 52 female) with a mean age of 54 years were identified with follow-up available for 80 (94%) patients. There were 68 (80%) benign tumors and 17 (20%) malignant tumors. Three tumors recurred and were resected giving a total of 88 surgeries. All benign tumors were grossly resected and the extent of resection for malignant disease ranged from 14 (78%) gross resections and 3 (17%) debulkings to 1 (5%) biopsy. There were 4 (5%) early hospital deaths. Median survival was 9.6 months and 322 months for patients with malignant and benign diseases, respectively. Significant predictors of long-term mortality were malignant disease (P <0.0001) and New York Heart Association class (P <0.03). CONCLUSIONS: Surgical resection provides excellent outcome in patients with benign cardiac tumors. Malignant tumors continue to pose a challenge with good local tumor control but limited survival owing to metastatic disease.