In vitro assessment of nucleoside analogs in multiple myeloma

Purpose To identify nucleoside analogs that may be effective for multiple myeloma (MM), we tested fludarabine, clofarabine, arabinosylguanine, cytarabine, troxacitabine, and gemcitabine in MM cell lines.Methods We employed biologic and biochemical assays in MM cell lines to evaluate the clinical potential of these nucleoside analogs.Results Among these purine and pyrimidine nucleoside analogs, fludarabine, clofarabine and gemcitabine were the most potent. MM cell lines, resistant to commonly used chemotherapeutic agents for this disease, were more sensitive to gemcitabine with an IC₅₀ in the nanomolar range. The greater cytotoxicity of gemcitabine in MM cells was consistent with greater accumulation of gemcitabine triphosphate, the major cytotoxic metabolite of this drug. MM.1S cells accumulated >100 M gemcitabine triphosphate but accumulated <20 M of the other analogs as the respective triphosphates. In addition incubation with gemcitabine resulted in inhibition of DNA synthesis. Incubation with 25, 50 or 100 nM gemcitabine resulted in a dose- and time-dependent increase in the cell population with a subG₁ DNA content indicative of apoptosis.Conclusions These results suggest that gemcitabine is a potent nucleoside analog in MM cell lines including cell types resistant to other chemotherapeutic agents. The greater activity of gemcitabine compared to other analogs seems to be due to favorable metabolism of this agent.This work was supported in part by grants CA57629 and CA85915 from the National Cancer Institute, Department of Health and Human Services and a Translational Research Award #6506-00 from the Leukemia and Lymphoma Society of America. Steven T. Rosen is the Dr. Ralph and Marion Falk Research Trust Translational Researcher of the Lymphoma Society of America.     

Zafirlukast for severe recurrent vulvovaginal candidiasis: an open label pilot study

BACKGROUND: Recurrent vulvovaginal candidiasis (VVC) has been linked to allergic disease, particularly allergic rhinitis. OBJECTIVE: A pilot study to assess the possible use of the leukotriene receptor antagonist zafirlukast as a treatment for recurrent VVC. METHODS: 20 women with six or more symptomatic attacks of VVC in the past year (at least four proved microbiologically). Clinical atopy determined by the International Study for Asthma and Allergies in Childhood (ISAAC) questionnaire assessed blindly. Monitoring by daily symptom diary and self taken vaginal swabs. Treatment with zafirlukast 20 mg twice daily for 24 weeks or until three microbiologically confirmed episodes of VVC. Response assessed by daily symptom diary and self taken vaginal swabs. Subjective response scales for improvement, side effects, and change in other allergic disease completed when stopping treatment. Semistructured telephone interview 1 year after stopping medication. RESULTS: 14 patients (70%) reported a subjective response on the improvement response scale. Six (30%) showed a complete response with no further symptomatic attacks of VVC or negative swabs when symptomatic. Seven (37%) remained symptom free 18 months after entering the study-that is, 12 months after stopping therapy. 11 (58%) remained symptom free for at least 3 months after stopping therapy. This does not include one patient who remained symptom free but continued on zafirlukast because of an improvement in her asthma. There was no clear relation between response and atopic status. Six of nine atopic subjective responders reported improvements in other allergic symptoms. Side effects were minimal; one seemed clearly attributable to the drug. CONCLUSION: Zafirlukast offers a potential new treatment for recurrent VVC that requires confirmation in controlled studies.     

Comparative subchronic inhalation study of smoke from the 1R4F and 2R4F reference cigarettes

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a 1R4F research cigarette to that of a 2R4F research cigarette. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, pulmonary plethysmography, gross pathology, and histopathology were determined. When histological changes resulting from exposure to smoke from the two types of cigarettes were compared, no biologically significant differences were observed. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the 1R4F and the 2R4F cigarettes. The complete toxicological assessment in this comparative inhalation study of 1R4F and 2R4F cigarettes suggests no overall biologically significant differences between the rats exposed to the two cigarettes.     

Novel chewable sustained-release tablet containing verapamil hydrochloride

The aim of this research is to produce a compactable self-sealing chewable tablet of verapamil hydrochloride. Tablets were prepared by compressing beads coated with multiple layers including drug, hydroxypropyl methylcellulose, polyethylene oxide, ethylcellulose, lactose, and sodium starch glycolate. Dissolution studies were carried out according to the USP XXII paddle method for 14 h. A new tablet formulation was evaluated in three different forms: 1) whole tablet, 2) crushed tablet using a commercial tablet crusher, and 3) tablet chewed in the mouth and then expelled into dissolution fluid. Sustained release from the new formulation was maintained and was similar in all three different treatments, and similar to drug release from intact commercially available Isoptin SR, but crushing or chewing destroyed the sustained release property of Isoptin SR (as expected). This new formulation can be administered either by swallowing the whole tablet or by first crushing or chewing the tablet. Controlled release properties of this new formulation do not change by chewing or crushing the tablet first. Such a tablet could be valuable for all patients including those who have difficulty swallowing, such as pediatrics and geriatrics.     

Expression and intracellular localization of protein phosphatases 2A and 2B, protein kinase a, A-Kinase anchoring protein (AKAP79), and binding of the regulatory (RII) subunit of protein kinase a to AKAP79 in human myometrium

OBJECTIVE: To determine the expression and intracellular localization of protein phosphatases 2A (PP2A) and 2B (PP2B), protein kinase A (PKA), and A-kinase anchoring protein (AKAP79), and expression of PKA (RII subunit) binding to AKAP79 in human postmenopausal and pregnant myometrium and to correlate their expressions to blood levels of estradiol, progesterone, and oxytocin. METHODS: Myometrial samples were taken from postmenopausal hysterectomy specimens (group 1, n = 5), from pregnant nonlaboring women (group 2, n = 7) and pregnant laboring women (group 3, n = 5) at cesarean. Western immunoblotting, immunohistochemical, and RII overlay assays were performed. Blood samples were assayed for estradiol, progesterone, and oxytocin levels. RESULTS: There were no significant differences in expression of PP2A, PKA, AKAP79, or PKA(RII) binding to AKAP79 between the three groups. Expression of PP2B was significantly greater in the nonlabor group (group 2) compared with groups 1 and 3. Protein phosphatase 2B, PKA, and AKAP79 expressions were localized in myometrial cytoplasm, but PP2A was localized in blood vessel endothelium. There was no significant correlation between the protein expression and the hormone level in the three groups. CONCLUSION: Human postmenopausal and pregnant (nonlabor and labor) myometrium expressed PP2A, PP2B, PKA, AKAP79, and PKA (RII)-AKAP79 binding. Levels of PP2A, PKA, and AKAP79 expression did not appear to be determinants of human myometrial contractility at parturition. Expression of PP2B may play a role in uterine quiescence. No association was found between protein expression and hormone level.     

Genetic differentiation of Aedes aegypti (Diptera: Culicidae), the major dengue vector in Brazil

In 2000, Brazil reported 180,137 cases of dengue, approximately 80% of the total in the Americas. However, little is known about gene flow among the vector populations in Brazil. Random amplified polymorphic DNA (RAPD) was used to study the genetic structure of Aedes aegypti in 15 populations from five states, with a range extending 2,800 km. An analysis of 47 polymorphic RAPD loci estimated gene flow at the macro- (different states) and micro- (different cities) geographical levels. Genetic polymorphism was high (H(S) = 0.274), and high levels of genetic differentiation existed both between different states (G(ST) = 0.317) and between cities or neighborhoods in each state (G(ST) = 0.085-0.265). These values are higher than those described for any other populations of A. aegypti.     

Rat subchronic inhalation study of smoke from cigarettes containing flue-cured tobacco cured either by direct-fired or heat-exchanger curing processes

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a cigarette containing 100% flue-cured tobacco cured by a direct-fired process to that of a cigarette containing 100% flue-cured tobacco cured by a heat exchanger process. The tobaccos and mainstream smoke from tobaccos cured by the heat exchanger process have been shown to have significantly lower levels of tobacco-specific nitrosamines than tobaccos cured by a direct-fired process. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. When histologic changes resulting from exposure to smoke from the two types of cigarettes were compared, the only significant difference was increased epithelial hyperplasia of the anterior nasal cavity in males in the high-exposure group for the heat-exchanger cigarette. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the heat-exchanger-cured tobacco cigarette when compared to the direct-fired cured tobacco cigarette. The complete toxicological assessment in this study of heat exchanger and direct-fired tobaccos suggests no overall biologically significant differences between the two cigarettes.     

Linkage disequilibrium assessment via log-linear modeling of SNP haplotype frequencies

Analyses of high-density single-nucleotide polymorphism (SNP) data, such as genetic mapping and linkage disequilibrium (LD) studies, require phase-known haplotypes to allow for the correlation between tightly linked loci. However, current SNP genotyping technology cannot determine phase, which must be inferred statistically. In this paper, we present a new Bayesian Markov chain Monte Carlo (MCMC) algorithm for population haplotype frequency estimation, particularly in the context of LD assessment. The novel feature of the method is the incorporation of a log-linear prior model for population haplotype frequencies. We present simulations to suggest that 1) the log-linear prior model is more appropriate than the standard coalescent process in the presence of recombination (>0.02 cM between adjacent loci), and 2) there is substantial inflation in measures of LD obtained by a "two-stage" approach to the analysis by treating the "best" haplotype configuration as correct, without regard to uncertainty in the recombination process.     

Cytoplasmic expression of EGFP in dendritic cells transfected with in vitro transcribed mRNA or cellular total RNA extracted from EGFP expressing leukemia cells

The present study was designed for identifying the protein synthesis in cytoplasm of dendritic cells transfected with in vitro transcribed mRNA and cellular total RNA extracted from tumor cells. Dendritic cells were generated from cord blood-CD34⁺ cells by culture with GM-CSF, SCF, and TNF-α, or from peripheral blood adherent cells or CD14⁺ cells by culture with GM-CSF and IL-4. Dendritic cells were transfected with in vitro transcribed EGFP mRNA or cellular total RNA, which was isolated from EGFP expressing K562, by electroporation using a square-wave pulse. Optimal in vitro transcribed EGFP mRNA transfection efficiency (>90%) was observed in a single electroporation of 1.75 kV/cm (electric field strength) with a pulse width of 250 μs. Although the intensity of EGFP expression in dendritic cells transfected with cellular total RNA was less compared with that in dendritic cells transfected with in vitro transcribed EGFP mRNA, a definite cytoplasmic synthesis of EGFP was demonstrated in dendritic cells transfected with cellular total RNA. The visual identification of cytoplasmic expression of cellular total RNA in dendritic cells revealed that electroporation of tumor cell-derived RNA could be a useful tool to load dendritic cells with tumor antigens for establishing an efficient dendritic cell-based tumor immunotherapy.     

Brittle asthma: a separate clinical phenotype of asthma?

There is now good evidence that brittle asthma should be regarded as a separate clinical phenotype of asthma at the severe end of the spectrum. Two types of brittle asthma can be identified. Type I is characterized by wide swings in peak expiratory flow (PEF) despite maximal therapy and type II by very sudden attacks out of the blue. Type I brittle asthma is more common in females and although the exact aetio-pathogenic mechanisms are not yet known, several factors including allergen sensitization (with exposure) and psychosocial factors may be important. Peak expiratory flow monitoring is essential for recognising these patients. Treatment of type I brittle asthma is difficult and needs to be holistic, with particular attention being paid to psychosocial factors where required. Continuous subcutaneous infusion of terbutaline (or salbutamol)) and dietary exclusion of relevant foods to which the patient may be allergic may be helpful in selected patients. Type II brittle asthma is less difficult to manage and includes the use of self-administered subcutaneous adrenaline to abort the rapidly developing exacerbations.