Intravesical pH: a potentially important variable affecting efficacy and the further development of anthracycline chemotherapy for superficial bladder cancer.

OBJECTIVE: To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro, the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy. MATERIALS AND METHODS: Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate. The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay. Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy. Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0. The MDR-reversing agent verapamil was added at 100 micro g/mL to some incubations. RESULTS: Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0. Flow cytometry results showed striking and consistent differences in epirubicin handling with pH. Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour. Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH. Adding verapamil to resistant cells restored the sensitive phenotype of drug handling. CONCLUSION: Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity. The potential for synergy at certain drug combinations will be enhanced by applying these findings. MDR reversal and fatty acid augmentation of drug uptake are discussed as examples.

Atypical hyperplasia, proliferative fibrocystic change, and exogenous hormone use.

BACKGROUND: The association between breast cancer development and exogenous hormone use (EHU) is suggested by indirect clinical evidence. We undertook this study to better define the relationship that EHU has with proliferative fibrocystic change (PFC) and atypical hyperplasia (AH). METHODS: Women diagnosed with AH without associated carcinoma from January 1990 to December 1999 were compared with control subjects who underwent breast biopsy procedures during the same interval and who were diagnosed with either a proliferative fibrocystic change (PFC) or a nonproliferative fibrocystic change (NPFC). EHU was defined as the use of estrogen or progesterone taken together or separately within 3 months of biopsy. RESULTS: EHU was significantly higher in patients with AH compared with women with NPFC (P =.01). This observation was also significant if all proliferative change (both AH and PFC) was compared with NPFC (P =.03); it was not significant when PFC alone was compared with NPFC. No significant difference in EHU was demonstrated between women with AH and those with PFC. CONCLUSIONS: There is strong association between AH and EHU. These results support the theory that a continuum exists between hyperplasia and carcinoma and that EHU may influence the transition from one to the other in an undefined subset of women. We encourage our patients with AH to discontinue EHU.

Phylogeny of Malpighiaceae: evidence from chloroplast ndhF and trnl-F nucleotide sequences.

The Malpighiaceae are a family of ∼1250 species of predominantly New World tropical flowering plants. Infrafamilial classification has long been based on fruit characters. Phylogenetic analyses of chloroplast DNA nucleotide sequences were analyzed to help resolve the phylogeny of Malpighiaceae. A total of 79 species, representing 58 of the 65 currently recognized genera, were studied. The 3' region of the gene ndhF was sequenced for 77 species and the noncoding intergenic spacer region trnL-F was sequenced for 65 species; both sequences were obtained for the outgroup, Humiria (Humiriaceae). Phylogenetic relationships inferred from these data sets are largely congruent with one another and with results from combined analyses. The family is divided into two major clades, recognized here as the subfamilies Byrsonimoideae (New World only) and Malpighioideae (New World and Old World). Niedenzu's tribes are all polyphyletic, suggesting extensive convergence on similar fruit types; only de Jussieu's tribe Gaudichaudieae and Anderson's tribes Acmanthereae and Galphimieae are monophyletic. Fleshy fruits evolved three times in the family and bristly fruits at least three times. Among the wing-fruited vines, which constitute more than half the diversity in the family, genera with dorsal-winged samaras are fairly well resolved, while the resolution of taxa with lateral-winged samaras is poor. The trees suggest a shift from radially symmetrical pollen arrangement to globally symmetrical pollen at the base of one of the clades within the Malpighioideae. The Old World taxa fall into at least six and as many as nine clades.

Molecular systematics of Malpighiaceae: evidence from plastid rbcL and matK sequences.

Phylogenetic analyses of DNA nucleotide sequences from the plastid genes rbcL and matK were employed to investigate intergeneric relationships within Malpighiaceae. Cladistic relationships generated from the independent data matrices for the family are generally in agreement with those from the combined matrix. At the base of Malpighiaceae are several clades mostly representing genera from a paraphyletic subfamily Byrsonimoideae. Intergeneric relationships among these byrsonimoid malpighs are well supported by the bootstrap, and the tribe Galphimeae is monophyletic. There is also a well-supported clade of genera corresponding to tribes Banisterieae plus Gaudichaudieae present in all trees, and many of the relationships among these banisterioid malpighs are well supported by the bootstrap. However, tribes Hiraeae and Tricomarieae (the hiraeoid malpighs) are paraphyletic and largely unresolved. Species of Mascagnia are distributed throughout these hiraeoid clades, confirming the suspected polyphyly of this large genus. Optimization of selected morphological characters on these trees demonstrates clear phylogenetic trends such as the evolution of globally symmetrical from radially symmetrical pollen, increased modification and sterilization of stamens, and switch from base chromosome number n = 6 to n = 10.

Sleep disturbance and nonmalignant chronic pain: a comprehensive review of the literature.

Sleep disturbance is an important clinical complaint for individuals with nonmalignant pain conditions. This review is a broad introduction to the literature on sleep disturbance and chronic pain conditions. The article critically reviews studies of sleep disturbance in musculoskeletal pain, arthritis, headache, and fibromyalgia. Current neurobiological hypotheses regarding the pathogenesis of sleep disturbance and chronic pain, common comorbid disorders, and pharmacologic and non-pharmacologic treatments for sleep disturbance are reviewed.

Alpha 2 mRNA of Na+K+ ATPase is increased in astroctyes of rat hippocampus after treatment with kainic acid.

The Na+K+ ATPase (Na+ pump) plays a central role in regulating cation homeostasis and is thought to have an important role in cell proliferation. The multitude of subunit isoforms comprising the functional Na+K+ ATPase has raised the possibility that specific subunit isoform combinations may be involved in different cellular processes. We have investigated the involvement of the specific isoforms in neurons and glia at the site of a CNS lesion. Intracerebroventricular injection of kainic acid was used to induce neuronal cell loss and reactive gliosis in rat hippocampus and levels of Na+K+ ATPase subunit isoform mRNA levels were determined in cells of rat hippocampus using in situ hybridization. alpha 2 mRNA levels increased 35-40% in CA1 and CA3 astrocytes between 1-3 weeks after KA injection with no significant change in other subunit isoform mRNA levels. In addition alpha 3 mRNA levels in CA1 pyramidal neurons were decreased by approx. 35%. Small neurons in the CA1 and CA3 region showed no changes in mRNA levels for any of the Na+K+ ATPase subunit isoforms. These results may indicate a possible role for alpha 2 subunit isoform in the conversion of glial cells from a normal phenotype to the reactive phenotype characteristic in this model of CNS injury.

Macrognathia of renal osteodystrophy in dialysis patients.

A multiinstitutional study of macrognathia secondary to renal osteodystrophy in dialysis patients is presented. The nine cases reviewed reveal a variety of radiographic and histopathologic features, some of which resemble fibrous dysplasia and others suggestive of Paget's disease of bone. This article contains diagnostic criteria for differentiating renal osteodystrophy from similar fibro-osseous proliferations along with a discussion of the underlying cause and appropriate therapeutic interventions.

Effect of sustained estradiol release in the intact male rat: correlation of estradiol serum levels with actions on body weight, serum testosterone, and peripheral androgen-dependent tissues.

The differential effect of increasing serum estradiol on various parameters in the intact male rat was assessed through the use of subcutaneously implanted, hormone-laden pellets. The delivery systems were designed to release drug through bioerosion at a zero-order rate over a 12-day time-course. Male Sprague-Dawley rats (190 to 220 g) were given estrogen pellets at increasing labeled strenghts (0, 0.001, 0.01, 0.1, 1.0, 10, 50, and 100 mg). Animals were weighed at various intervals before and after implantation. At Day 6, 12, and 26 after drug administration, rats were examined for 4 additional parameters, including serum estradiol and testoterone concentrations and accessory organ weights (i.e., ventral prostate and seminal vesicles). Serum estradiol levels were consistent with pellet potency and lifetime. Increases in body weight were suppressed 50% by circulating estradiol levels of approximately 200 pg/mL at Day 6,250 pg/mL at Day 12, and 285 pg/mL at Day 26. On the other hand, suppression of serum testosterone was more sensitive and was decreased 50% by peripheral estrogen levels of 36, 43, and 51 pg/mL at Days 6, 12, and 26, respectively. Accessory organ weights essentially reflected serum testosterone levels as indicated by their similar ED50 values: 50.5, 50.5, and 44.3 pg/mL for the ventral prostate at Day 6, 12, and 26, respectively, and 48, 56, and 51.5 pg/mL for the seminal vesicle regression at Day 6, 12, and 26, respectively. The data indicate the pellet used provided sustained plasma levels of hormone and these constant peripheral levels exerted potent pharmacological action. Initial body weight changes seemed to be less sensitive to the action of estradiol than serum testosterone or derivative properties, such as accessory organ weight.

Correlative transmission and scanning electron microscopy study of microglia activated by interferon-gamma and tumor necrosis factor-alpha in vitro.

Microglia, in response to cytokines, demonstrate a number of enhanced biochemical and functional properties which reflect a state of activation. In this study, we evaluated the ultrastructural alterations of murine microglia that were associated with activation by interferon-gamma (IFN-gamma) plus tumor necrosis factor-alpha (TNF-alpha). Microglial cell culture treated with these cytokines generated significant amounts of the free radical nitric oxide (NO), a biochemical marker of activation. Correlative transmission (TEM) and scanning (SEM) electron microscopic analyses of these cytokine-activated microglia demonstrated two prominent features: proliferation of cell processes and increased formation of membrane bound dense bodies typical of lysosomes. Since activated microglia have been implicated in the pathogenesis of a number of neurodegenerative diseases, application of the ultrastructural findings in the in vitro study may prove useful in determining the state of activation of microglia in brain specimens from patients with these neurological disorders.

Improved brain delivery and in vitro activity of zidovudine through the use of a redox chemical delivery system.

OBJECTIVE: Improved therapy for AIDS dementia and related encephalopathies may be achieved through enhanced delivery of effective antiretroviral agents to the central nervous system (CNS). DESIGN: A novel chemical delivery system (CDS) was used, which utilized redox trapping of drugs in the brain. This study was aimed at defining the pharmacokinetics of a zidovudine (ZDV)-CDS as well as establishing its in vitro antiviral efficacy against HIV in both lymphocytes and in a neural cell line. RESULTS: ZDV-CDS administered parenterally to rats produced significantly higher brain levels of ZDV [area under the curve (AUC), 425 micrograms x min/g] than equimolar ZDV (AUC, 13.5 micrograms x min/g). Native ZDV uptake was minimal after 1 h when analyzed in CEM lymphocytes and in SKNMC neuroblastoma cell line. By contrast, marked uptake of ZDV-CDS was followed by biochemical conversion of ZDV-CDS to its main metabolites (ZDV-CDS quaternary salt, ZDV-Q+, and native ZDV). These improved uptake profiles were associated with greater in vitro virucidal effect. ZDV-CDS at 0.5 microM was 80% more effective than ZDV in suppressing p24 production in a lymphocyte culture infected with 6000 median tissue culture infective doses (TCID50) of the HIV N1T strain and 50% more effective at 0.05 microM. Furthermore, syncytia formation was completely suppressed at a ZDV-CDS dose of 0.5 microM (600 TCID50) but native ZDV at the same dose was ineffective. Finally, while ZDV (at 0.5 microM) is not active in reducing viral replication in an SKNMC neural cell line, the ZDV-CDS complex significantly suppressed p24 synthesis. CONCLUSION: The ZDV-CDS complex is capable of delivering higher ZDV doses to lymphocytes and neural cells, with improved antiretroviral activity.

Efficacy of a 3-substituted versus 17-substituted chemical delivery system for estradiol brain targeting.

Brain-targeted delivery of estrogens has been achieved by a chemical delivery system (CDS) in which a molecular targetor (1-methyl-1,4-dihydronicotinate) was attached to the 17-alcohol of estradiol. Optimization of this effect was attempted with the isomeric 3-phenol ester. Estradiol 3-nicotinate was prepared with nicotinic anhydride, which selectively acylated the phenol position. Methylation and reduction gave estradiol 3-(1-methyl-1,4-dihydronicotinate) of the 3-E2-CDS. Theoretical and electrochemical investigation indicated that the 3-E2-CDS was more stable to oxidation than was the prototype 17-ester (17-E2-CDS). Systemic administration of the 17-E2-CDS produced high levels of the corresponding quaternary salt in the brain of rats, which disappeared with an estimated half-life of > 2 days, but 3-E2-CDS dosing resulted in no significant quaternary salt trapping. Pharmacological activity was potent and sustained after 17-E2-CDS dosing but transient after 3-E2-CDS administration. Thus, the 3-E2-CDS reduced the rate of weight gain in male rats but to a lesser extent and for a shorter duration than did the 17-E2-CDS. Similar effects were seen on pituitary hypertrophy, reduction in serum androgen concentrations, and involution of prostate and seminal vesicles. The results of these studies suggest that placement of the targeting ester at the phenol position increases dihydropyridine stability but, at the same time, reduces brain sequestration.

Effect of molecular manipulation on the estrogenic activity of a brain-targeting estradiol chemical delivery system.

The structural parameters important for biological efficacy of an estradiol chemical delivery system (CDS), a brain-targeting approach based on redox trapping, were examined by molecular manipulation of a prototype derivative, estradiol 17-(1-methyl-1, 4-dihydronicotinate) (E2-CDS). Seven E2-CDS analogs in which the N-methyl substituent was altered were prepared including N-substituted short and medium straight chain alkyl, short branched chain alkyl, and aralkyl derivatives. Chemical and in vitro testing indicated that the most stable derivative was the N-benzyl E2-CDS. The analogs were tested in an intact male rat model to assess various central estrogenic manifestations including the rate of body weight gain, serum E2 and testosterone concentrations, and seminal vesicle, prostate and pituitary weight changes. Results indicated that all prepared CDS derivatives exerted some degree of central estrogenization with the most potent compounds being the parent E2-CDS and its ethyl homologue. Importantly, while the ethyl E2-CDS was equipotent to E2-CDS in various biological assays, it did not significantly elevate serum E2 compared to vehicle control at day 14.

Transforming growth factor-beta protects human neurons against beta-amyloid-induced injury.

Deposition of amyloid fibrils in the brain is a histopathologic hallmark of Alzheimer disease (AD) and beta-amyloid protein (A beta), the principal component of amyloid fibrils, has been implicated in the neuropathogenesis of AD. In the present study, we first developed an in vitro model of A beta-induced neurodegeneration using human fetal brain-cell cultures and then tested the hypothesis that cytokines modulate A beta-induced neurodegeneration. When brain-cell cultures were exposed to A beta, marked neuronal loss (60% of neurons by microscopic assessment) and functional impairment (i.e., reduction in uptake of [3H]gamma-aminobutyric acid) were observed after 6 d of incubation. A beta-induced neurodegeneration was dose-dependent with maximal effect at 100 microM. Although interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha had a nominal effect, both the beta 1 and beta 2 isoforms of transforming growth factor-beta dose-dependently protected > 50% of neurons against A beta-induced injury. IL-4 also proved to be neuro-protective. A beta-induced neurodegeneration was accompanied by microglial cell proliferation and enhanced release of IL-1, IL-6, and TNF-alpha. These findings are consistent with the emerging concept that AD is an inflammatory disease and may lead to new therapeutic strategies aimed at reducing A beta-induced neurotoxicity.

Nitric oxide production and neurotoxicity mediated by activated microglia from human versus mouse brain.

Recent studies indicate that human macrophages lack a high-output inducible nitric oxide synthase (NOS) antimicrobial system. In the present study, microglial cells derived from fetal human versus neonatal mouse brain were compared in a coculture assay of human and murine neuronal cell injury. Neurotoxicity (reflected by lactate dehydrogenase release and impaired neuronal uptake of [3H] gamma-amino butyric acid) and nitric oxide (NO) production (assessed by measurement of nitrite) were observed only in cocultures containing interferon (IFN)-gamma-lipopolysaccharide (LPS)-stimulated murine microglia. Cultures of purified human fetal microglia, however, did produce low levels of NO upon stimulation with IFN-gamma-LPS. These findings support the proposal that human macrophages have an inefficient IFN-gamma-inducible NOS and suggest that in tissues, such as brain, this deficiency could be advantageous for neighboring cells.

Na,K-ATPase is decreased in hippocampus of kainate-lesioned rats.

The effects of intraventricular injection of kainic acid on the Na,K-ATPase (Na,K pump) were examined in discrete pyramidal cell regions of rat hippocampus. [3H]Ouabain binding was used to quantitate Na,K-ATPase catalytic subunits and in situ hybridization was used to determine Na,K-ATPase mRNA levels. Large decreases were found in both [3H]ouabain binding and alpha 3 isoform mRNA in hippocampus areas, especially in the CA3 pyramidal cell layer, which sustains heavy cell losses as a result of bilateral, intraventricular injection of kainic acid. Substantial decreases in the high affinity component of ouabain binding and in the alpha 3 isoform mRNA (but not isoforms for other Na,K-ATPase subunits) were also observed in the CA1 region of hippocampus, an area preserved in this model. High affinity [3H]ouabain binding was decreased 25-33% in the stratum pyramidale and stratum radiatum after treatment with kainic acid, and alpha 3 mRNA was decreased by 26-50%. To further characterize the decrease in alpha 3 mRNA, animals were killed at 1, 2, and 3 weeks after injection of kainate and results show a large decrease in alpha 3 mRNA only at 2 weeks recovery time. While the pathology underlying temporal lobe epilepsy is unclear, changes in the Na,K-ATPase may be involved in abnormal firing characteristics of cells in epileptic tissue.

Role of cytokines in lipopolysaccharide-induced functional and structural abnormalities of astrocytes.

The mechanism underlying meningitis-associated brain injury is unclear. This study investigated the hypothesis that lipopolysaccharide (LPS) alters astrocyte function and structure via the release of proinflammatory cytokines. In enriched murine astrocyte cultures, LPS inhibited (P < 0.05) glutamine synthetase activity, 3H-gamma aminobutyric acid uptake, and DNA synthesis; LPS also induced ultrastructural changes. Antibodies to tumor necrosis factor-alpha, interleukin-1, and interleukin-6 blocked (P < 0.05) in part the LPS-induced inhibition of astrocyte function. Also, treatment of astrocyte cultures with cytokines significantly altered these astrocyte functions and ultrastructure. Taken together, the present findings support the hypothesis that LPS affects astrocyte function and structure via the release of proinflammatory cytokines, especially tumor necrosis factor-alpha.

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism.

The role of microglia in host defense against Toxoplasma gondii is unknown. In the present study, we investigated the multiplication of T. gondii tachyzoites in murine microglial cell cultures. T. gondii multiplied readily in these cells; multiplication was prevented when microglia were activated with interferon-gamma plus lipopolysaccharide, a treatment that also upregulates nitric oxide (NO) synthase activity. Simultaneous treatment of microglial cell cultures with activation signals and the NO synthase inhibitor NG-monomethyl-L-arginine (NGMA) prevented the antitoxoplasmic activity. Transmission electron microscopic analysis demonstrated degenerative tachyzoites in activated microglia but not in control or NGMA groups. These findings support the view that the host defense function of activated microglia against T. gondii involves generation of the free radical NO.