The use of naturally occurring botanicals with substantial antioxidant activity to afford protection to human skin against UV damage is receiving increasing attention. The green tea constituent (-)-epigallocatechin-3-gallate (EGCG) is a potent antioxidant and has shown remarkable preventive effects against photocarcinogenesis and phototoxicity in mouse models. In this study we have investigated the effects of topical application of EGCG, the major polyphenol present in green tea, to human skin before UV irradiation on UV-induced markers of oxidative stress and antioxidant enzymes. Using immunohistochemistry and analytical enzyme assays, we found that application of EGCG (mg/cm(2) skin) before a single UV exposure of 4x minimal erythema dose (MED) markedly decreases UV-induced production of hydrogen peroxide (68-90%, P < 0.025-0.005) and nitric oxide (30-100%, P < 0.025-0.005) in both epidermis and dermis in a time-dependent manner. EGCG pretreatment also inhibits UV-induced infiltration of inflammatory leukocytes, particularly CD11b(+) cells (a surface marker of monocytes/macrophages and neutrophils), into the skin, which are considered to be the major producers of reactive oxygen species. EGCG treatment was also found to inhibit UV-induced epidermal lipid peroxidation at each time point studied (41-84%, P < 0.05). A single UV exposure of 4x MED to human skin was found to increase catalase activity (109-145%) and decrease glutathione peroxidase (GPx) activity (36-54%) and total glutathione (GSH) level (13-36%) at different time points studied. Pretreatment with EGCG was found to restore the UV-induced decrease in GSH level and afforded protection to the antioxidant enzyme GPx. Further studies are warranted to study the preventive effects of EGCG against multiple exposures to UV light of human skin.

Vaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) from Mycobacterium tuberculosis is a powerful technique for generating robust specific Thl helper T-cell responses, CD8+-mediated cytotoxicity, and protection against M. tuberculosis challenge (K. Huygen et al., Nat. Med. 2:893-898, 1996). We have now analyzed in more detail the antigen-specific immune CD4+- and CD8+-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection with M. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than did M. tuberculosis infection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not following M. tuberculosis infection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.

OBJECTIVE

The purpose of this study was to conduct a large-scale investigation with adult recipients of the Clarion, Med-El, and Nucleus cochlear implant systems to (1) determine average scores and ranges of performance for word and sentence stimuli presented at three intensity levels (70, 60, and 50 dB SPL); (2) provide information on the variability of scores for each subject by obtaining test-retest measures for all test conditions; and (3) further evaluate the potential use of lower speech presentation levels (i.e., 60 and/or 50 dB SPL) in cochlear implant candidacy assessment.

METHODS

Seventy-eight adult cochlear implant recipients, 26 with each of the three cochlear implant systems, participated in the study. To ensure that the data collected reflect the range of performance of adult recipients using recent technology for the three implant systems (Clarion HiFocus I or II, Med-El Combi 40+, Nucleus 24M or 24R), a composite range and distribution of consonant-nucleus-consonant (CNC) monosyllabic word scores was determined. Subjects using each device were selected to closely represent this range and distribution of CNC performance. During test sessions, subjects were administered the Hearing in Noise Test (HINT) sentence test and the CNC word test at three presentation levels (70, 60, and 50 dB SPL). HINT sentences also were administered at 60 dB SPL with a signal-to-noise ratio (SNR) of +8 dB. Warble tones were used to determine sound-field threshold levels from 250 to 4000 Hz. Test-retest measures were obtained for each of the speech recognition tests as well as for warble-tone sound-field thresholds.

RESULTS

Cochlear implant recipients using the Clarion, Med-El, or Nucleus devices performed on average equally as well at 60 compared with 70 dB SPL when listening for words and sentences. Additionally, subjects had substantial open-set speech perception performance at the softer level of 50 dB SPL for the same stimuli; however, subjects' ability to understand speech was poorer when listening in noise to signals of greater intensity (60 dB SPL + 8 SNR) than when listening to signals presented at a soft presentation level (50 dB SPL) in quiet. A significant correlation was found between sound-field thresholds and speech recognition scores for presentation levels below 70 dB SPL. The results demonstrated a high test-retest reliability with cochlear implant users for these presentation levels and stimuli. Average sound-field thresholds were between 24 and 29 dB HL for frequencies of 250 to 4000 Hz, and results across sessions were essentially the same.

CONCLUSIONS

Speech perception measures used with cochlear implant candidates and recipients should reflect the listening challenges that individuals encounter in natural communication situations. These data provide the basis for recommending new candidacy criteria based on speech recognition tests presented at 60 and/or 50 dB SPL, intensity levels that reflect real-life listening, rather than 70 dB SPL.

Focal cerebral ischemia elicits an inflammatory response characterized by the infiltration and accumulation of leukocytes, as well as the secretion of inflammatory mediators (Clark et al., Brain Res. Bull., 35 (1994) 387-392; Garcia et al., Am. J. Pathol., 144 (1994) 188-199; Wang et al., J. Neurochem. 71 (1998) 1194-1204). Leukocytes eliminate microbial invaders and necrotizing tissue debris, and can also turn against surrounding healthy tissue and exacerbate tissue injury (Furie and Randolph, Am. J. Pathol., 146 (1995) 1287-1301; Kochanek and Hallenbeck, Stroke 23 (1992) 1367-1379). Inflammatory mediators are considered to play an important role in attracting and stimulating leukocytes (Weiss, N. Engl. J. Med., 320 (1989) 365-376). Monocyte chemoattractant protein-1 (MCP-1) functions as an inflammatory mediator, whose source and role in focal cerebral ischemia is worth studying. MCP-1, a potent chemoattractant factor, may play an important role in ischemia-induced inflammatory response. The aim of the present study is to determine the time course and cell type of MCP-1 protein expression after permanent focal ischemia in mice. ELISA and immunohistochemical staining were used to detect the expression of MCP-1 protein after 0 h, 2 h, 4 h, 12 h, 1 day, 2 days, 3 days, 5 days and 7 days of middle cerebral artery occlusion (n=3-5 in each group). Double-labeled fluorescent staining was used to examine the cellular localization of MCP-1. The results demonstrated that MCP-1 expression was mainly observed in the ischemic core after 12 h of middle cerebral artery occlusion, then gradually increased and extended to the ischemic perifocal area. MCP-1 expression peaked at 2 days and 3 days, and gradually decreased after 5 days of MCAO. Double-labeled immunostaining for MCP-1 and neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP) showed that MCP-1 positive neurons were observed as early as 12 h of ischemia, while MCP-1 positive astrocytes were observed after 2 days of ischemia. These results support the functional role of MCP-1 in ischemic brain injury and reveal a distinct temporal and spatial expression of MCP-1 in cells believed to be neurons and astrocytes.

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.

Owing to its highly polymorphic nature and major contribution to the metabolism and bioactivation of numerous clinically used drugs, CYP2D6 is one of the most extensively studied drug-metabolizing enzymes and pharmacogenes. CYP2D6 alleles confer no, decreased, normal, or increased activity and cause a wide range of activity among individuals and between populations. However, there is no standard approach to translate diplotypes into predicted phenotype.

We exploited CYP2D6 allele-frequency data that have been compiled for Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (>60,000 subjects, 173 reports) in order to estimate genotype-predicted phenotype status across major world populations based on activity score (AS) assignments.

Allele frequencies vary considerably across the major ethnic groups predicting poor metabolizer status (AS = 0) between 0.4 and 5.4% across world populations. The prevalence of genotypic intermediate (AS = 0.5) and normal (AS = 1, 1.5, or 2) metabolizers ranges between 0.4 and 11% and between 67 and 90%, respectively. Finally, 1 to 21% of subjects (AS >2) are predicted to have ultrarapid metabolizer status.

This comprehensive study summarizes allele frequencies, diplotypes, and predicted phenotype across major populations, providing a rich data resource for clinicians and researchers. Challenges of phenotype prediction from genotype data are highlighted and discussed.Genet Med 19 1, 69-76.

In humans, phencyclidine (PCP) is known to produce a syndrome of behavioral effects which have many characteristics in common with schizophrenia. Therefore, antagonism of PCP effects might be evidence for antipsychotic efficacy of a compound. In the present studies, the effects of the D2-like antagonist haloperidol, the mixed D2-like/5-HT2 antagonists olanzapine and clozapine, and a series of 5-HT receptor subtype selective antagonists on the hyperlocomotion produced by PCP were evaluated in mice. PCP (0.3-10 mg/kg) produced a dose-related increase in locomotor activity, with a peak effect at 3.0 mg/kg. The D2-like antagonist haloperidol produced a dose-related decrease in locomotor activity when administered alone, and blocked the hyperactivity effects of PCP over the same dose-range (minimal effective dose, MED = 0.3 mg/kg for both effects). In contrast, olanzapine and clozapine reversed the hyperlocomotion effects of PCP at doses (MED = 0.03 and 0.3 mg/kg, respectively) approximately 30- and 10-fold, respectively, below those that decreased activity when administered alone (MED = 1.0 and 3.0 mg/kg, respectively). The selective 5-HT2 antagonist LY53857 (0.3-3.0 mg/kg) administered alone had no effect on locomotor activity but reversed (MED = 0.1 mg/kg) the effects of PCP. Similarly, the selective 5-HT2A/2C antagonist ritanserin (0.001-1.0 mg/kg) alone had no effect on locomotor activity, but reversed (MED = 0.01 mg/kg) the effects of PCP. The selective 5-HT2A antagonists ketanserin (MED = 3.0 mg/kg) and MDL 100,907 (MED = 0.3 mg/kg) produced dose-related decreases in locomotor activity and ketanserin (MED = 0.1 mg/kg) and MDL 100,907 (MED = 0.003 mg/kg) reversed the effects of PCP. The selective 5-HT3 antagonist zatosetron (0.01-10 mg/kg) and the selective 5-HT1A antagonist WAY 100,635 (0.001-3 mg/kg) were without effects on spontaneous locomotor activity. Zatosetron reversed the effects of 3.0 mg/kg PCP at the nonselective dose of 10 mg/kg whereas WAY 100,635 (0.001-1 mg/kg) did not affect PCP-induced hyperlocomotion. The present results indicate that PCP increases locomotor activity, at least in part, due to actions at 5-HT2A, but not 5-HT3 or 5-HT1A, receptors. Further, the present findings support the hypothesis that antagonism at 5-HT2A receptors contributes to the in vivo actions of atypical antipsychotics such as olanzapine and clozapine.

Interferon-producing killer dendritic cells (IKDCs) are a recently described subset of CD11c(lo)B220(+) cells that share phenotypic and functional properties of DCs and natural killer (NK) cells (Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, et al. 2006. Nat. Med. 12:207-213; Taieb, J., N. Chaput, C. Menard, L. Apetoh, E. Ullrich, M. Bonmort, M. Pequignot, N. Casares, M. Terme, C. Flament, et al. 2006. Nat. Med. 12:214-219). IKDC development appears unusual in that cytokines using the interleukin (IL)-2 receptor beta (IL-2Rbeta) chain but not those using the common gamma chain (gamma(c)) are necessary for their generation. By directly comparing Rag2(-/-)gamma(c)(-/y), Rag2(-/-)IL-2Rbeta(-/-), Rag2(-/-)IL-15(-/-), and Rag2(-/-)IL-2(-/-) mice, we demonstrate that IKDC development parallels NK cell development in its strict IL-15 dependence. Moreover, IKDCs uniformly express NK-specific Ncr-1 transcripts (encoding NKp46), whereas NKp46(+) cells are absent in Ncr1(gfp/+)gamma(c)(-/y) mice. Distinguishing features of IKDCs (CD11c(lo)B220(+)MHC-II(+)) were carefully examined on developing NK cells in the bone marrow and on peripheral NK cells. As B220 expression was heterogeneous, defining B220(lo) versus B220(hi) NK1.1(+) NK cells could be considered as arbitrary, and few phenotypic differences were noted between NK1.1(+) NK cells bearing different levels of B220. CD11c expression did not correlate with B220 or major histocompatibility complex (MHC) class II (MHC-II) expression, and most MHC-II(+) NK1.1(+) cells did not express B220 and were thus not IKDCs. Finally, CD11c, MHC-II, and B220 levels were up-regulated on NK1.1(+) cells upon activation in vitro or in vivo in a proliferation-dependent fashion. Our data suggest that the majority of CD11c(lo)B220(+) "IKDC-like" cells represent activated NK cells.

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.

BACKGROUND

Conflicting findings with regard to the teratogenic risks of first trimester use of paroxetine have prompted the FDA, Health Canada, and the manufacturer of the drug to issue warnings against its use during pregnancy. Given that untreated depression during pregnancy can lead to deleterious effect on the mother and her unborn fetus, data on the relationship between the dose and the range of malformations is warranted. This study attempts to quantify the association between first trimester exposure to paroxetine and congenital cardiac malformations, adjusting for possible confounders, and to quantify the dose-response relationship between paroxetine use and cardiac defects.

METHODS

The Medication and Pregnancy registry was used. This population-based registry was built by linking three administrative databases (RAMQ, Med-Echo, and ISQ), and includes all pregnancies in Quebec between 01/01/1997 and 06/30/2003. Date of entry in the registry is the date of the first day of the last menstrual period. To be eligible for this study, women had to: 1) be 15-45 years of age at entry; 2) be covered by the RAMQ drug plan>>or=12 months before and during pregnancy; 3) be using only one type of antidepressant during the first trimester; and 4) have a live birth. Two nested case-control studies were carried out comparing the prevalence of paroxetine use in the first trimester of pregnancy to the prevalence of other antidepressant exposures during the same time period. Cases were defined as: 1) any major malformations; or 2) any cardiac malformations diagnosed in the first year of life; controls were defined as no major or minor malformations. Multivariate logistic regression techniques were used to analyze data.

RESULTS

Among the 1,403 women meeting inclusion criteria, 101 infants with major congenital malformations were identified; 24 had cardiac malformations. Adjusting for possible confounders, the use of paroxetine (odds ratio [OR] = 1.38, 95% confidence interval [CI] = 0.49-3.92), and the use of other SSRIs (OR = 0.89, 95% CI = 0.28-2.84) during the first trimester of pregnancy did not increase the risk of congenital cardiac malformations compared with the use of non-SSRI antidepressants. When considering the dose, however, a dose-response relationship was observed, thus women exposed to >25 mg/day of paroxetine during the first trimester of pregnancy were at increased risk of having an infant with major congenital malformations (adjusted [adj] OR = 2.23, 95% CI = 1.19, 4.17), or major cardiac malformations (adj OR = 3.07, 95% CI = 1.00, 9.42).

CONCLUSIONS

Gestational exposure to paroxetine is associated with major congenital malformations and major cardiac malformations for only first trimester exposure above 25 mg/day.

BACKGROUND

Age-related decline in muscle power may be an early indicator of balance deficits and fall risk, even in nonfrail adults. This study examined the dose-dependent effect of power training on balance performance in healthy older adults.

METHODS

One hundred twelve community-dwelling healthy older adults (69 +/- 6 years) were randomized to 8-12 weeks of power training at 20% (LOW), 50% (MED), or 80% (HIGH) of maximal strength, or a nontraining control (CON) group. Participants trained twice weekly (five exercises; three sets of eight rapid concentric/slow eccentric repetitions) using pneumatic resistance machines. Balance, muscle performance (strength, power, endurance, contraction velocity), and body composition were measured.

RESULTS

Power training significantly improved balance performance (p =.006) in participants who underwent power training compared to controls. Low intensity power training produced the greatest improvement in balance performance (p =.048). Average contraction velocity at low load (40% one repetition maximum [1RM]) at baseline independently predicted improvement in balance following training (r = -.29, p =.004).

CONCLUSIONS

Power training improves balance, particularly using a low load, high velocity regimen, in older adults with initial lower muscle power and slower contraction. Further studies are warranted to define the mechanisms underlying this adaptation, as well as the optimum power training intensity for a range of physiological and clinical outcomes in older adults with varying levels of health status and functional independence.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756-760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins.

The fatty-acid ethanolamide, oleoylethanolamide (OEA), is a naturally occurring lipid that regulates feeding and body weight [Rodriguez de Fonseca, F., Navarro, M., Gomez, R., Escuredo, L., Nava, F., Fu, J., Murillo-Rodriguez, E., Giuffrida, A., LoVerme, J., Gaetani, S., Kathuria, S., Gall, C., Piomelli, D., 2001. An anorexic lipid mediator regulated by feeding. Nature 414, 209-212], and serves as an endogenous agonist of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) [Fu, J., Gaetani, S., Oveisi, F., Lo Verme, J., Serrano, A., Rodriguez De Fonseca, F., Rosengarth., A., Luecke, H., Di Giacomo, B., Tarzia, G., Piomelli, D., 2003. Oleoylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 425, 90-93], a ligand-activated transcription factor that regulates several aspects of lipid metabolism [. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev. 20, 649-688]). OEA reduces food intake in wild-type mice, but not in mice deficient in PPAR-alpha (PPAR-alpha(-/-)), an effect that is also observed with the PPAR-alpha agonists Wy-14643 and GW7647 [Brown, P.J., Chapman, J.M., Oplinger, J.A., Stuart, L.W., Willson, T.M. and Wu, Z., 2000. Chemical compounds as selective activators of PPAR-alpha. PCT Int. Appl., 32; . The PPARs: from orphan receptors to drug discovery. J. Med. Chem. 43, 527-550]. By contrast, specific agonists of PPAR-delta/beta (GW501516) or PPAR-gamma (ciglitazone) have no such effect. In obese Zucker rats, which lack functional leptin receptors, OEA reduces food intake and lowers body-weight gain along with plasma lipid levels. Similar effects are seen in diet-induced obese rats and mice. In the present study, we report that subchronic OEA treatment (5mgkg(-1), intraperitoneally, i.p., once daily for two weeks) in Zucker rats initiates transcription of PPAR-alpha and other PPAR-alpha target genes, including fatty-acid translocase (FAT/CD36), liver fatty-acid binding protein (L-FABP), and uncoupling protein-2 (UCP-2). Moreover, OEA decreases neutral lipid content in hepatocytes, as assessed by Oil red O staining, as well as serum cholesterol and triglyceride levels. The results suggest that OEA regulates lipid metabolism and that this effect may contribute to its anti-obesity properties.

Ribavirin is administered in combination with interferon-alpha for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection.

T lymphocytes recirculate continually through the T cell areas of peripheral lymph nodes. During each passage, the T cells survey the surface of large dendritic cells (DCs), also known as interdigitating cells. However, these DCs have been difficult to release from the lymph node. By emphasizing the use of calcium-free media, as shown by Vremec et al. (Vremec, D., M. Zorbas, R. Scollay, D.J. Saunders, C.F. Ardavin, L. Wu, and K. Shortman. 1992. J. Exp. Med. 176:47-58.), we have been able to release and enrich DCs from the T cell areas. The DCs express the CD11c leukocyte integrin, the DEC-205 multilectin receptor for antigen presentation, the intracellular granule antigens which are recognized by monoclonal antibodies M342, 2A1, and MIDC-8, very high levels of MHC I and MHC II, and abundant accessory molecules such as CD40, CD54, and CD86. When examined with the Y-Ae monoclonal which recognizes complexes formed between I-Ab and a peptide derived from I-Ealpha, the T cell area DCs expressed the highest levels. The enriched DCs also stimulated a T-T hybridoma specific for this MHC II-peptide complex, and the hybridoma underwent apoptosis. Therefore DCs within the T cell areas can be isolated. Because they present very high levels of self peptides, these DCs should be considered in the regulation of self reactivity in the periphery.

Analytical advantages of using multiple enzymes for sample digestion (MED), primarily an increase of sequence coverage, have been reported in several studies. However, this approach is only rarely used, mainly because it requires additional sample and mass spectrometric measurement time. We have previously described Filter Aided Sample Preparation (FASP), a type of proteomic reactor, in which samples dissolved in sodium dodecyl sulfate (SDS) are digested in an ultrafiltration unit. In FASP, such as in any other preparation protocol, a portion of sample remains after digestion and peptide elution. Making use of this fact, we here develop a protocol enabling consecutive digestion of the sample with two or three enzymes. By use of the FASP method, peptides are liberated after each digestion step and remaining material is subsequently cleaved with the next proteinase. We observed excellent performance of the ultrafiltration devices in this mode, allowing efficient separation of orthogonal populations of peptides, resulting in an increase in the numbers of identified peptides and proteins. At the low microgram level, we found that the consecutive use of endoproteinases LysC and trypsin enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.

SSR180711 (4-bromophenyl 1,4diazabicyclo(3.2.2) nonane-4-carboxylate, monohydrochloride) is a selective alpha7 nicotinic receptor (n-AChR) partial agonist. Based on the purported implication of this receptor in cognitive deficits associated with schizophrenia, the present study assessed efficacy of SSR180711 (i.p. and p.o.) in different types of learning and memory involved in this pathology. SSR180711 enhanced episodic memory in the object recognition task in rats and mice (MED: 0.3 mg/kg), an effect mediated by the alpha7 n-AChR, as it was no longer seen in mice lacking this receptor. Efficacy was retained after repeated treatment (eight administrations over 5 days, 1 mg/kg), indicating lack of tachyphylaxia. SSR180711 also reversed (MED: 0.3 mg/kg) MK-801-induced deficits in retention of episodic memory in rats (object recognition). The drug reversed (MED: 0.3 mg/kg) selective attention impaired by neonatal phencyclidine (PCP) treatment and restored MK-801- or PCP-induced memory deficits in the Morris or linear maze (MED: 1-3 mg/kg). In neurochemical and electrophysiological correlates of antipsychotic drug action, SSR180711 increased extracellular levels of dopamine in the prefrontal cortex (MED: 1 mg/kg) and enhanced (3 mg/kg) spontaneous firing of retrosplenial cortex neurons in rats. Selectivity of SSR180711 was confirmed as these effects were abolished by methyllycaconitine (3 mg/kg, i.p. and 1 mg/kg, i.v., respectively), a selective alpha7 n-AChR antagonist. Additional antidepressant-like properties of SSR180711 were demonstrated in the forced-swimming test in rats (MED: 1 mg/kg), the maternal separation-induced ultrasonic vocalization paradigm in rat pups (MED: 3 mg/kg) and the chronic mild stress procedure in mice (10 mg/kg o.d. for 3 weeks). Taken together, these findings characterize SSR180711 as a promising new agent for the treatment of cognitive symptoms of schizophrenia. The antidepressant-like properties of SSR180711 are of added interest, considering the high prevalence of depressive symptoms in schizophrenic patients.

OBJECTIVE

This review attempted to examine the validity and clinical utility of the DSM-IV binge eating disorder (BED) diagnosis across a wide range of validating strategies.

METHODS

Various electronic databases (Pub Med, Psych Info) were searched for terms relevant to the diagnosis of BED (e.g., binge eating disorder, binge eating) in order to identify papers. Additionally, published papers were reviewed in order to locate additional manuscripts and papers that were presented at meetings.

RESULTS

The validity and utility of BED varied substantially according to the validator chosen. There is reasonable evidence that BED can be differentiated from other existing eating disorders and is associated with significant impairment and clinical levels of eating disorder psychopathology. The relationship of BED to obesity is complex, and in spite of some positive findings, further research examining the predictive power of BED, beyond the simple presence of obesity and associated psychopathology, in relationship to clinically relevant outcomes is needed.

CONCLUSIONS

Binge eating disorder is being considered for inclusion in the DSM-V and various options regarding this decision are reviewed based upon the empirical findings in the paper.

A series of bifunctional alkylators were tested against the genotypically and phenotypically heterogeneous continuous human medulloblastoma cell lines, TE-671, Daoy, and D283 Med in vitro and against TE-671 and Daoy growing as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (in vivo), and busulfan (in vivo). Melphalan and phenylketocyclophosphamide were the most active agents in vitro with drug doses at which there is a 90% reduction in the number of colonies in comparison to controls of 2.13, 5.29, and 4.72 microM for melphalan and 4.60, 5.01, and 4.34 microM for phenylketocyclophosphamide against TE-671, D283 Med, and Daoy, respectively. Melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, and thiotepa produced significant growth delays against s.c. TE-671 and Daoy xenografts, while no activity could be demonstrated for 1,3-bis(2-chloroethyl)-1-nitrosourea or busulfan. Melphalan, cyclophosphamide, iphosphamide, and thiotepa also produced significant increases in median survival in mice bearing intracranial TE-671 and Daoy xenografts. These results extend our previous studies demonstrating the antitumor activity of nitrogen and phosphoramide mustard-based bifunctional alkylating agents in the treatment of human medulloblastoma continuous cell lines and transplantable xenografts, and support the continued use of these agents in clinical trials.

In the immunocompetent host, primary cytomegalovirus (CMV) infection is resolved by the immune response without causing overt disease. The viral genome, however, is not cleared but is maintained in a latent state that entails a risk of virus recurrence and consequent organ disease. By using murine CMV as a model, we have shown previously that multiple organs harbor latent CMV and that reactivation occurs with an incidence that is determined by the viral DNA load in the respective organ (M. J. Reddehase, M. Balthesen, M. Rapp, S. Jonjic, I. Pavic, and U. H. Koszinowski. J. Exp. Med. 179:185-193, 1994). This predicts that a therapeutic intervention capable of limiting the load of latent viral genome should also reduce the risk of virus recurrence. Here we demonstrate the benefits and the limits of a preemptive CD8 T-cell immunotherapy of CMV infection in the immunocompromised bone marrow transplantation recipient. Antiviral CD8 T cells prevented CMV disease and accelerated the resolution of productive infection. The therapy also resulted in a lower load of latent CMV DNA in organs and consequently reduced the incidence of recurrence. The data thus provide a further supporting argument for clinical trials of preemptive cytoimmunotherapy of human CMV disease with CD8 T cells. However, CD8 T cells failed to clear the viral DNA. The therapy-susceptible portion of the DNA load differed between organs and was highest in the lungs. The existence of an invariant, therapy-resistant load suggests a role for immune system evasion mechanisms in the establishment of CMV latency.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

Cancer development is a multistep process, driven by a series of genetic and environmental alterations, that endows cells with a set of hallmark traits required for tumorigenesis. It is broadly accepted that growth signal autonomy, the first hallmark of malignancies, can be acquired through multiple genetic mutations that activate an array of complex, cancer-specific growth circuits [Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70; Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789-799]. The superfluous nature of these pathways is thought to severely limit therapeutic approaches targeting tumor proliferation, and it has been suggested that this strategy be abandoned in favor of inhibiting more systemic hallmarks, including angiogenesis (Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: Mechanisms of anti-tumor activity. Nat Rev Cancer 8:579-591; Stommel JM, et al. (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318:287-290; Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727-739; Kaiser J (2008) Cancer genetics: A detailed genetic portrait of the deadliest human cancers. Science 321:1280-1281]. Here, we report the unexpected observation that genetically diverse cancers converge at a common and obligatory growth axis instigated by HIF-2alpha, an element of the oxygen-sensing machinery. Inhibition of HIF-2alpha prevents the in vivo growth and tumorigenesis of highly aggressive glioblastoma, colorectal, and non-small-cell lung carcinomas and the in vitro autonomous proliferation of several others, regardless of their mutational status and tissue of origin. The concomitant deactivation of select receptor tyrosine kinases, including the EGFR and IGF1R, as well as downstream ERK/Akt signaling, suggests that HIF-2alpha exerts its proliferative effects by endorsing these major pathways. Consistently, silencing these receptors phenocopies the loss of HIF-2alpha oncogenic activity, abrogating the serum-independent growth of human cancer cells in culture. Based on these data, we propose an alternative to the predominant view that cancers exploit independent autonomous growth pathways and reveal HIF-2alpha as a potentially universal culprit in promoting the persistent proliferation of neoplastic cells.

In a recent study, De Haart et al. (Arch Phys Med Rehabil 85:886-895, 2004) investigated the recovery of balance in stroke patients using traditional analyses of center-of-pressure (COP) trajectories to assess the effects of health status, rehabilitation, and task conditions like standing with eyes open or closed and standing while performing a cognitive dual task. To unravel the underlying control processes, we reanalyzed these data in terms of stochastic dynamics using more advanced analyses. Dimensionality, local stability, regularity, and scaling behavior of COP trajectories were determined and compared with shuffled and phase-randomized surrogate data. The presence of long-range correlations discarded the possibility that the COP trajectories were purely random. Compared to the healthy controls, the COP trajectories of the stroke patients were characterized by increased dimensionality and instability, but greater regularity in the frontal plane. These findings were taken to imply that the stroke patients actively (i.e., cognitively) coped with the stroke-induced impairment of posture, as reflected in the increased regularity and decreased local stability, by recruiting additional control processes (i.e., more degrees of freedom) and/or by tightening the present control structure while releasing non-essential degrees of freedom from postural control. In the course of rehabilitation, dimensionality stayed fairly constant, whereas local stability increased and regularity decreased. The progressively less regular COP trajectories were interpreted to indicate a reduction of cognitive involvement in postural control as recovery from stroke progressed. Consistent with this interpretation, the dual task condition resulted in less regular COP trajectories of greater dimensionality, reflecting a task-related decrease of active, cognitive contributions to postural control. In comparison with conventional posturography, our results show a clear surplus value of dynamical measures in studying postural control.

OBJECTIVE

Interventions that aim to improve child dietary quality and reduce disease risk often involve parents. The most effective methods to engage parents remain unclear. A systematic review of interventions designed to change child and adolescent dietary behavior was conducted to answer whether parent involvement enhanced intervention effectiveness, and what type of involvement was most effective in achieving desired outcomes.

METHODS

In 2008, Pub Med, Medline, Psych Info, and Cochrane Library databases were searched to identify programs designed to change child and adolescent dietary intake that also involved parents. Methods of parental involvement were categorized based on the type and intensity of parental involvement. These methods were compared against intervention design, dietary outcomes, and quality of reporting (evaluated using CONSORT checklist) for each study.

RESULTS

The literature search identified 1774 articles and 24 met review criteria. Four studies systematically evaluated parent involvement with inconsistent results. Indirect methods to engage parents were most commonly used, although direct approaches were more likely to result in positive outcomes. Four studies met >70% of CONSORT items.

CONCLUSIONS

Limited conclusions may be drawn regarding the best method to involve parents in changing child diet to promote health. However, direct methods show promise and warrant further research.