Modulator of heme biosynthesis induces apoptosis in leukemia cells.

OBJECTIVE: The purpose of this research is the investigation of the possible cause(s) of the dark-cell death phenomenon induced by 1,10-phenanthroline (Oph), a porphyrin biosynthesis modulator. SUMMARY BACKGROUND DATA: We have previously shown that porphyrin biosynthesis modulators, such as Oph, which is also an iron-chelating agent, enhance protoporphyrin IX (Proto) accumulation in mammalian neoplastic cells treated with delta-aminolevulinic acid (ALA). As a result of the enhanced Proto accumulation, a significant increase in photodynamic damage was observed under illumination. Also tetrapyrrole and heme-biosynthesis modulators have been shown to cause death in treated insect larvae in darkness, a phenomenon referred to as dark-cell death. Dark-cell death was also observed in Oph + ALA-treated transformed mammalian cells. METHODS: Neoplastic cells were treated with ALA, Oph, and ALA + Oph, and the following cell properties were investigated: growth arrest, membrane permeability, cell survival, nucleosomal cleavage, and cell cycle alterations. RESULTS: It was observed that Oph but not ALA induced growth arrest, in a T-cell leukemia line (MLA 144) as assessed by reduction in DNA synthesis. Exogenous Proto and isomers of Oph lacking the iron-chelating property of Oph also caused a dose-dependent inhibition of proliferation in MLA 144 cells. Although the plasma membrane of Oph-treated cells remained intact following 3 h of dark-incubation, the cells exhibited DNA internucleosomal cleavage, characteristic of cells undergoing apoptosis. Cell cycle analysis using the DNA intercalating dye propidium iodide (PI) coupled to flow cytometry, indicated that 81 +/- 5.6% of Oph-treated MLA 144 cells were apoptotic, with the majority of the cells arrested in the early S phase. On the other hand, treatment with either ALA or Proto did not alter the cell cycle. Also, using a double-labeling protocol with Hoechst 33342, and PI, and analysis by flow cytometry, Oph-treated cells were found to be 82% apoptotic after 3 h of dark-incubation. Apoptosis was reduced by 75% (p < 0.05) by the cytoplasmic protein synthesis inhibitor cycloheximide. CONCLUSIONS: These results indicate that in addition to enhancing Proto accumulation, the heme biosynthesis modulator Oph also induces growth arrest and apoptosis in transformed cells in darkness.     

Effect of reconstruction algorithms on myocardial blood flow measurement with 13N-ammonia PET.

Filtered backprojection (FBP) is the traditional method for 13N-NH3 PET studies. Ordered-subsets expectation maximization (OSEM) is popular for PET studies because of better noise properties. Scant data exist on the effect of reconstruction algorithms on quantitative myocardial blood flow (MBF) estimation. METHODS: Twenty patients underwent dynamic acquisition rest/stress 13N-NH3 studies. In Part 1, 19 rest/stress image pairs were reconstructed by FBP (10-mm Hanning filter) and by OSEM with 28 subsets and 2 (OSEM2), 6 (OSEM6), or 8 iterations (OSEM8), and a 10-mm postreconstruction smoothing gaussian filter. In Part 2, 9 image pairs were reconstructed by FBP (10-mm Hanning filter) and by OSEM with 28 subsets, 8 iterations, and a gaussian 5-, 10-, or 15-mm postreconstruction smoothing filter. Average MBF (mL/min/mL of myocardium) was calculated using a 3-compartment model. RESULTS: Part 1: For rest MBF, the correlations between FBP and each of the OSEM algorithms were r2 = 0.71, 0.73, and 0.77, respectively. MBF by OSEM6 (0.98 +/- 0.48 [mean +/- SD]) and OSEM8 (0.96 +/- 0.46) was not significantly different from FBP (1.02 +/- 0.39), but OSEM2 (0.80 +/- 0.37) was significantly lower (P < 0.0003). With stress, the correlations were high between FBP and OSEM6 and OSEM8 (r2 = 0.85 and 0.90), and MBF by OSEM6 and OSEM8 was not significantly different from FBP. Part 2: Resting MBF correlated well between FBP and all OSEM smoothing filters (r2 = 0.82, 0.85, and 0.88). Rest MBF using postsmoothing 5- or 10-mm filters was not different from FBP but was significantly lower with the 15-mm filter (P < 0.05). With stress, the correlations were good between FBP and OSEM regardless of smoothing (r2 = 0.76, 0.77, and 0.79). However, MBF with postsmoothing 10- and 15-mm filters was significantly lower than by FBP (P < 0.05). CONCLUSION: Reconstruction algorithms significantly affect the estimation of quantitative blood flow data and should not be assumed to be interchangeable. Although aggressive smoothing may produce visually appealing images with reduced noise levels, it may cause an underestimation of absolute quantitative MBF. In selecting a reconstruction algorithm, an optimal balance between noise properties and diagnostic accuracy must be emphasized.     

Provider attitudes and self-reported behaviors related to hormone replacement therapy.

OBJECTIVE: The purpose of this study was to survey providers within a large health maintenance organization regarding their attitudes and practice patterns related to counseling women about hormone replacement therapy (HRT). DESIGN: A total of 260 providers from gynecology (n = 81), family practice (n = 96), and internal medicine (n = 83) from owned and contracted clinics were surveyed. Each was asked about prescribing philosophies, behaviors, and barriers to providing counseling regarding HRT. RESULTS: Respondents reported HRT's greatest benefit to be in the prevention of osteoporosis (99%) and cardiovascular conditions (96%). Gynecologists were more likely to report the benefits of HRT for Alzheimer's than were clinicians in internal medicine or family practice (p < 0.05), and women providers were more likely than men to report this (p < 0.01). There was no statistical difference based on years in practice. Providers did not vary significantly by specialty or sex in their concerns of risk for breast cancer of endometrial cancer. However, those in family practice and internal medicine were significantly more likely to report concern about thromboembolism (p < 0.01). Only 42% of physicians claimed to initiate discussion with their patients more than 75% of the time. The two factors most often mentioned as barriers to counseling were time and lack of adequate knowledge. CONCLUSIONS: Providers want to be an integral part of their patient's education regarding HRT; however, time constrains and a need for adequate information make this difficult. Now health systems must examine models of education for both providers and patients to ensure that women have access to current information with which to make informed decisions.     

Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions

PURPOSE: The objective of this study was to develop a scalable and broadly applicable active immunotherapy approach against cancer, circumventing the limitations typically encountered with autologous vaccination strategies. We hypothesized that human embryonic stem cells (hESC) can serve as a virtually unlimited source for generating dendritic cells (DC) with potent antigen-presenting function. Here, we investigated the developmental processes and requirements for generating large numbers of mature, antigen-presenting DC from pluripotent hESC. EXPERIMENTAL DESIGN: A feeder cell-free culture system was developed to differentiate hESC into mature DC sequentially through hematopoietic and myeloid precursor stages. RESULTS: Using this method, we were able to yield large numbers of mature immunostimulatory DC from hESC to enable clinical investigation. Upon activation, the hESC-derived DC secreted interleukin-12p70, migrated in response to MIP-3beta, and exhibited allostimulatory capacity. Most importantly, antigen-loaded, hESC-derived DC were capable of stimulating potent antigen-specific CD8(+) T-cell responses in an HLA class I-matched semiallogeneic assay system. Moreover, HLA class II-mismatched hESC-derived DC induced a potent Th1-type cytokine response without expanding FOXP3(+) regulatory T cells in vitro. CONCLUSIONS: These data suggest the development of a novel active immunotherapy platform to stimulate potent T-cell immunity in patients with intractable diseases, such as cancer or viral infection.     

Oral Candida dubliniensis as a clinically important species in HIV-seropositive patients in the United States.

OBJECTIVE: Interest in Candida dubliniensis has led to renewed clinical investigations regarding incidence, drug resistance, pathogenesis, and epidemiology of fungal infections in patients with HIV. C dubliniensis phenotypically resembles Candida albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of this study was to prospectively evaluate the prevalence of C dubliniensis in clinical isolates and determine the clinical and demographic characteristics of patients harboring C dubliniensis. STUDY DESIGN: Over a 6-week period, 24 yeast-positive isolates from HIV-positive dental patients were screened for C dubliniensis through use of phenotypic criteria. HIV viral load, CD4 count, and complete oral health evaluations were performed on each patient at the same visit during which the oral fungal surveillance culture was taken. RESULTS: Six isolates from 24 HIV-seropositive and yeast-positive patients were shown to be consistent phenotypically and by electrophoretic karyotyping with the European reference strain of C dubliniensis. Dose-dependent susceptibility to fluconazole was shown in one of the C dubliniensis isolates. Five of the 6 patients demonstrated moderate to high viral loads. General oral health, as evidenced by the presence of advanced periodontal lesions and a high decayed, missing, and filled teeth index (>20), was poor in 3 of the 6 patients with C dubliniensis and 7 of the 18 patients with C albicans. A history of intravenous drug abuse was present in 50% of the C dubliniensis -positive patients, which is representative of the HIV-positive population at the hospital. CONCLUSIONS: In this small sample, C dubliniensis represented 25% of the yeast-positive cultures. The clinical significance of this interesting species in the United States may be related to high viral load, rapid AIDS progression, and/or concomitant oral disease, such as a high caries index or periodontal disease.