Immune responses in resting T cells are initiated by the presentation of antigen by bone marrow-derived dendritic cells (DC). Normal DC are susceptible to infection with human immunodeficiency virus (HIV) in vitro (Patterson & Knight, 1987) and this blocks their capacity to stimulate T-cell responses to other antigens (Macatonia, Patterson & Knight, 1989a). To study the relationship between HIV and DC in patients and its relevance to the pathogenesis of disease, DC have been isolated from the blood of individuals in the different clinical categories, counted, examined for the presence of virus genome and their antigen-presenting capacity measured. Infection, depletion and impaired function of DC occur in early HIV infection. HIV seropositive patients who were asymptomatic and those with symptoms of disease had significantly reduced numbers of DC, but patients with persistent generalized lymphadenopathy had normal numbers. Between 3% and 21% of DC, identified as large low-density cells not bearing monocyte, lymphocyte or natural killer cell markers, were infected with HIV, as indicated by in situ hybridization. Less than 0.12% of the lymphocytes or monocytes were infected. The DC from infected individuals were poor at enhancing responses to the mitogen concanavalin A (Con A). They also caused low levels of stimulation in allogeneic lymphocytes in mixed leucocyte cultures. By contrast, T cells from asymptomatic patients gave normal T-cell responses to uninfected allogeneic DC, although those from acquired immunodeficiency syndrome (AIDS) patients did show reduced responsiveness. Defects in DC thus precede both the appearance of symptoms and changes in T cells and may be instrumental in the development of AIDS. Furthermore, since DC numbers and function differ at different stages of disease, monitoring these may contribute to clinical assessment and lead to new therapeutic approaches.

BACKGROUND

Previous studies have demonstrated that genital infection with high-risk types of human papillomavirus (HPV), most often HPV16, is the most significant risk factor for the development of cervical cancer. However, serologic assays that have been developed to identify high-risk HPV infection have either failed to associate serum reactivity with other indicators of HPV infection or have identified only a minority of HPV-infected individuals.

OBJECTIVE

Our purpose was to determine whether a specifically developed enzyme-linked immunosorbent assay (ELISA) could detect IgG anti-HPV16 virion antibodies in the sera of women who had tested positive for genital HPV16 infection by DNA-based methods.

METHODS

An ELISA was developed using newly developed HPV16 virus-like particles as antigens to detect anti-HPV16 virion IgG antibodies. These particles are comprised of HPV16 structural proteins that are self-assembled in insect cells after expression by recombinant baculoviruses. The sera of 122 women, whose HPV status had been previously evaluated by nucleic acid-based methods, were tested by this ELISA.

RESULTS

The sera of 59% of women (32 of 54) positive for genital HPV16 DNA by polymerase chain reaction (PCR) were positive in the ELISA assay compared with sera from women who had tested negative for HPV DNA (P < .0005). In contrast, 6% of HPV DNA-negative women (two of 31) and 9% of women positive for low-risk HPV6/11 DNA (one of 11) were ELISA positive by this criterion. The sera of women who were DNA positive for two additional high-risk HPV types were evaluated; the sera of 31% of HPV18-positive (four of 13) and 38% of HPV31-positive women (five of 13) were positive in the HPV16 particle ELISA. The sera of 75% of HPV16 DNA-positive women with severe dysplasias (12 of 16) gave positive ELISA results. The sera of 67% of women (28 of 42) who tested positive for HPV16 DNA by both PCR and the less sensitive ViraType assay tested positive in the ELISA compared with 33% of women (four of 12) who were positive by PCR but negative by ViraType (P < .05).

CONCLUSIONS

The majority of women with cervical HPV16 infection generate an IgG antibody response to conformationally dependent epitopes of HPV16 L1 that can be detected by ELISA.

CONCLUSIONS

This particular ELISA, or a similar one incorporating virus-like particles of additional HPV types, may be useful in determining the natural history of high-risk HPV infection and perhaps help to identify women at risk for developing cervical cancer.

In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.

The Collaborative Study on the Genetics of Alcoholism (COGA) is a multicenter research program to detect and map susceptibility genes for alcohol dependence and related phenotypes. The measure M of "maximum number of drinks consumed in a 24-hour period" is closely related to alcoholism diagnosis in this dataset and provides a quantitative measure to grade nonalcoholic individuals. Twin studies have shown log(M) to have a heritability of approximately 50%. Genome screens for this trait were performed in two distinct genotyped samples (wave 1 and wave 2), and in the combined sample. MAPMAKER/SIBS was used to carry out Haseman-Elston based regression analyses. On chromosome 4, an unweighted all-pairs multipoint LOD of 2.2 was obtained between D4S2407 and D4S1628 in wave 1; in wave 2, the region flanked by D4S2404 and D4S2407 gave a LOD of 1.5. In the combined sample, the maximal LOD was 3.5 very close to D4S2407. This evidence for linkage is in the region of the alcohol dehydrogenase gene cluster on chromosome 4. These findings on chromosome 4 are consistent with a prior report from COGA in which strictly defined nonalcoholic subjects in wave 1 were analyzed. The present analysis on log(M) allows more individuals to be included and thus is potentially more powerful.

Microinjection of opioid agonists, such as morphine, into the nucleus accumbens shell produces increases in eating behavior (i.e. 'wanting' for food). This study (1) reports direct evidence that activation of accumbens opioid receptors in rats also augments food 'liking', or the hedonic impact of taste, and (2) identified a neural site that definitely contains receptors capable of increasing food intake. Morphine microinjections (0.5 microgram) into accumbens shell, which caused rats to increase eating, were found also to cause selective increases in positive hedonic patterns of behavioral affective reaction elicited by oral sucrose, using the 'taste reactivity' test of hedonic palatability. This positive shift indicated that morphine microinjections enhanced the hedonic impact of food palatability. The accumbens site mediating morphine-induced increases in food 'wanting' and 'liking' was identified using a novel method based on local expression of Fos induced directly by drug microinjections. The plume-shaped region of drug-induced increase in Fos immunoreactivity immediately surrounding a morphine microinjection site (Fos plume) was objectively mapped. A point-sampling procedure was used to measure the shape and size of 'positive' plumes of Fos expression triggered by microinjections of morphine at locations that caused increases in eating behavior. This revealed a functionally 'positive' neural region, containing receptors directly activated by behaviorally-effective drug microinjections. A subtraction mapping procedure was then used to eliminate all surrounding regions containing any 'negative' Fos plumes that failed to increase food intake. The subtraction produced a conservative map of the positive site, by eliminating regions that gave mixed effects, and leaving only a positive region that must contain receptors capable of mediating increases in food intake. The resulting mapped 'opioid eating site' was contained primarily within the medial caudal subregion of the nucleus accumbens shell, and did not substantially penetrate either into the accumbens core or into other subregions of the shell. Several other structures outside the nucleus accumbens (such as rostral ventral pallidum), immediately medial and adjacent to the shell, also appeared to be included in the functional site. Opioid receptors within this site thus are capable of mediating morphine-induced increases in eating, in part by enhancing the hedonic reward properties of food.

In 1993, the Health Services Research Committee of the American Society of Clinical Oncology (ASCO) charged an Outcomes Working Group with defining the outcomes of adult and pediatric cancer treatment to be used for technology assessment and development of cancer treatment guidelines. The Working Group defined by consensus outcomes for technology assessment and guideline development, focusing on cancer treatments. The Working Group considered a variety of perspectives on outcomes, including those of patients, physicians, clinical investigators, ASCO, and policy makers. Because ASCO's guidelines will define what constitutes the best treatment and not whether that treatment should be paid for, the Working Group gave higher priority to the clinical and clinical research perspectives than to the health policy perspective. Survival is the most important outcome of cancer treatment. An improvement in at least disease-free survival is a prerequisite for recommending adjuvant therapy. In the case of metastatic cancer, treatment can be recommended even without an improvement in survival, if it improves quality of life. Quality of life includes global quality of life, as well as its physical, psychologic, and social dimensions. To be an outcome of cancer treatment, quality-of-life measures must be sensitive to clinically meaningful changes produced by treatment; evaluations must control for placebo effects and determinants of quality of life not related to cancer or its treatment. Toxicity, both short and long term, is vitally important, with the latter being particularly critical in children. The value of cancer outcomes like tumor response (eg, complete or partial response) and biomarkers (eg, CA-125) for technology assessment and guideline development depends on their ability to predict patient outcomes (survival and quality of life) or to influence decisions about treatment. Complete response is an important outcome when it predicts survival. Progression is important because it signals the need to change or stop treatment. Cost-effectiveness is an especially important outcome to consider when the benefits of treatment are modest or the costs are high. Patient outcomes (eg, survival and quality of life) should receive higher priority than cancer outcomes (eg, response rate), but both types of outcomes are important in technology assessment and guideline development. Multiple outcomes should be considered because no single outcome adequately describes the results of cancer treatment. In general, there is no minimum benefit above which treatments are justified; rather, benefits should be balanced against toxicity and cost.

Meth-A sarcoma cells were stable transfected to overexpress (sense construct) or underexpress (antisense construct) tissue factor. In vitro, there was no difference in plating efficiency or growth between these cell lines. In vivo, tumor cells transfected to overexpress tissue factor grew more rapidly, and established larger and more vascularized tumors than control transfectants. Antisense transfectants grew the slowest and were the least vascularized. Anticoagulation of mice with warfarin did not alter the difference between these tumor lines. Tumor cells over-expressing tissue factor released more (compared with control transfectants) mitogenic activity for endothelial cells in parallel with enhanced transcription of vascular permeability factor/vascular endothelial cell growth factor (VEGF/VPF), and diminished transcription of thrombospondin (TSP2), a molecule with anti-angiogenic properties. Antisense tissue factor transfectants, while releasing the lowest amount of mitogenic activity, had increased thrombospondin and decreased VEGF/VPF transcription compared with control transfectants or wild-type cells. Experiments with these sense, antisense, truncated sense, or vector tumor lines gave comparable results in complete medium, serum free medium or in the presence of hirudin, indicating that the activation of the coagulation mechanism was not likely to be responsible for changes in tumor cell properties. These results suggest that tissue factor regulates angiogenic properties of tumor cells by altering the production of growth regulatory molecules of endothelium by a mechanism distinct from tissue factor activation of the coagulation mechanism.

Cardiac complications of influenza infection, such as myocarditis, are well recognised, but the role of influenza as a trigger of acute myocardial infarction is less clear. We did a systematic review of the evidence that influenza (including influenza-like illness and acute respiratory infection) triggers acute myocardial infarction or cardiovascular death. We examined the effectiveness of influenza vaccines at protecting against cardiac events and did a meta-analysis of data from randomised controlled trials. 42 publications describing 39 studies were identified. Many observational studies in different settings with a range of methods reported consistent associations between influenza and acute myocardial infarction. There was weaker evidence of an association with cardiovascular death. Two small randomised trials assessed the protection provided by influenza vaccine against cardiac events in people with existing cardiovascular disease. Whereas one trial found that influenza vaccination gave significant protection against cardiovascular death, the other trial was inconclusive. A pooled estimate from a random-effects model suggests a protective, though non-significant, effect (relative risk 0.51, 95% CI 0.15-1.76). We believe influenza vaccination should be encouraged wherever indicated, especially in people with existing cardiovascular disease, among whom there is often suboptimum vaccine uptake. Further evidence is needed on the effectiveness of influenza vaccines to reduce the risk of cardiac events in people without established vascular disease.

To elucidate the risk factors for anal cancer, we interviewed and obtained blood specimens from 148 persons with anal cancer and from 166 controls with colon cancer in whom these diseases were diagnosed during 1978-1985. We found that in men, a history of receptive anal intercourse (related to homosexual behavior) was strongly associated with the occurrence of anal cancer (relative risk, 33.1; 95 percent confidence interval, 4.0 to 272.1). Anal intercourse was only weakly associated with the risk of anal cancer in women (relative risk, 1.8; 95 percent confidence interval, 0.7 to 4.2). Among the subjects with squamous-cell anal cancer, 47.1 percent of homosexual men, 28.6 percent of heterosexual men, and 28.3 percent of women gave a history of genital warts, as compared with only 1 to 2 percent of controls and no patients with transitional-cell anal cancer. In patients without a history of warts, anal cancer was associated with a history of gonorrhea in heterosexual men (relative risk, 17.2; 95 percent confidence interval, 2.0 to 149.4) and with seropositivity for herpes simplex type 2 (relative risk, 4.1; 95 percent confidence interval, 1.9 to 8.8) and Chlamydia trachomatis (relative risk, 2.3; 95 percent confidence interval, 1.1 to 4.8) in women. Current cigarette smoking was a substantial risk factor in both women (relative risk, 7.7; 95 percent confidence interval, 3.5 to 17.2) and men (relative risk, 9.4; 95 percent confidence interval, 2.3 to 38.5). We conclude that homosexual behavior in men is a risk factor for anal cancer, and that squamous-cell anal cancer is also associated with a history of genital warts, an association suggesting that papillomavirus infection is a cause of anal cancer. Certain other genital infections and cigarette smoking are also associated with anal cancer.

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.

BACKGROUND

For typing of Staphylococcus aureus, DNA sequencing of the repeat region of the protein A (spa) gene is a well established discriminatory method for outbreak investigations. Recently, it was hypothesized that this region also reflects long-term epidemiology. However, no automated and objective algorithm existed to cluster different repeat regions. In this study, the Based Upon Repeat Pattern (BURP) implementation that is a heuristic variant of the newly described EDSI algorithm was investigated to infer the clonal relatedness of different spa types. For calibration of BURP parameters, 400 representative S. aureus strains with different spa types were characterized by MLST and clustered using eBURST as "gold standard" for their phylogeny. Typing concordance analysis between eBURST and BURP clustering (spa-CC) were performed using all possible BURP parameters to determine their optimal combination. BURP was subsequently evaluated with a strain collection reflecting the breadth of diversity of S. aureus (JCM 2002; 40:4544).

RESULTS

In total, the 400 strains exhibited 122 different MLST types. eBURST grouped them into 23 clonal complexes (CC; 354 isolates) and 33 singletons (46 isolates). BURP clustering of spa types using all possible parameter combinations and subsequent comparison with eBURST CCs resulted in concordances ranging from 8.2 to 96.2%. However, 96.2% concordance was reached only if spa types shorter than 8 repeats were excluded, which resulted in 37% excluded spa types. Therefore, the optimal combination of the BURP parameters was "exclude spa types shorter than 5 repeats" and "cluster spa types into spa-CC if cost distances are less than 4" exhibiting 95.3% concordance to eBURST. This algorithm identified 24 spa-CCs, 40 singletons, and excluded only 7.8% spa types. Analyzing the natural population with these parameters, the comparison of whole-genome micro-array groupings (at the level of 0.31 Pearson correlation index) and spa-CCs gave a concordance of 87.1%; BURP spa-CCs vs. manually grouped spa types resulted in 95.7% concordance.

CONCLUSIONS

BURP is the first automated and objective tool to infer clonal relatedness from spa repeat regions. It is able to extract an evolutionary signal rather congruent to MLST and micro-array data.

The strength of male-driven evolution - that is, the magnitude of the sex ratio of mutation rate - has been a controversial issue, particularly in primates. While earlier studies estimated the male-to-female ratio (alpha) of mutation rate to be about 4-6 in higher primates, two recent studies claimed that alpha is only about 2 in humans. However, a more recent comparison of mutation rates between a noncoding fragment on Y and a homologous region on chromosome 3 gave an estimate of alpha = 5.3, reinstating strong male-driven evolution in hominoids. Several studies investigated variation in mutation rates among genomic regions that may not be related to sex differences and found strong evidence for such variation. The causes for regional variation in mutation rate are not clear but GC content and recombination are two possible causes. Thus, while the strong male-driven evolution in higher primates suggests that errors during DNA replication in the germ cells are the major source of mutation, the contribution of some replication-independent factors such as recombination may also be important.

Like nuclear premessenger introns, group II self-splicing introns are excised from primary transcripts as branched molecules, containing a 2'-5' phosphodiester bond. For this reason, it is widely believed that the ribozyme (catalytic RNA) core of group II introns, or some evolutionarily related molecule, gave rise to the RNA components of the spliceosomal splicing machinery of the eukaryotic nucleus. One difficulty with this hypothesis has been the restricted distribution of group II introns. Unlike group I self-splicing introns, which interrupt not only organelle primary transcripts, but also some bacterial and nuclear genes, group II introns seemed to be confined to mitochondrial and chloroplast genomes (reviewed in ref. 6). We now report the discovery of group II introns both in cyanobacteria (the ancestors of chloroplasts) and the gamma subdivision of purple bacteria, or proteobacteria, whose alpha subdivision probably gave rise to mitochondria. At least one of these introns actually self-splices in vitro.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.

Around the world, adults with serious illnesses or chronic conditions account for a disproportionate share of national health care spending. We surveyed patients with complex care needs in eleven countries (Australia, Canada, France, Germany, the Netherlands, New Zealand, Norway, Sweden, Switzerland, the United Kingdom, and the United States) and found that in all of them, care is often poorly coordinated. However, adults seen at primary practices with attributes of a patient-centered medical home--where clinicians are accessible, know patients' medical history, and help coordinate care--gave higher ratings to the care they received and were less likely to experience coordination gaps or report medical errors. Throughout the survey, patients in Switzerland and the United Kingdom reported significantly more positive experiences than did patients in the other countries surveyed. Reported improvements in the United Kingdom tracked with recent reforms there in health care delivery. Patients in the United States reported difficulty paying medical bills and forgoing care because of costs. Our study indicates a need for improvement in all countries through redesigning primary care, developing care teams accountable across sites of care, and managing transitions and medications well. The United States in particular has opportunities to learn from diverse payment innovations and care redesign efforts under way in the other study countries.

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.

Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150(+)CD48(-)CD244(-) cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150(+)CD48(-)CD244(-), and the vast majority of colony-forming progenitors were CD48(+)CD244(-)CD150(-) or CD48(+)CD244(+)CD150(-), just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 Thy(low)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150(+)CD48(-)CD41(-) cells and 1 (37%) of every 2.7 CD150(+)CD48(-)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.

Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.

Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.

It is well accepted that pain is a multidimensional experience, but little is known of how the brain represents these dimensions. We used positron emission tomography (PET) to indirectly measure pain-evoked cerebral activity before and after hypnotic suggestions were given to modulate the perceived intensity of a painful stimulus. These techniques were similar to those of a previous study in which we gave suggestions to modulate the perceived unpleasantness of a noxious stimulus. Ten volunteers were scanned while tonic warm and noxious heat stimuli were presented to the hand during four experimental conditions: alert control, hypnosis control, hypnotic suggestions for increased-pain intensity and hypnotic suggestions for decreased-pain intensity. As shown in previous brain imaging studies, noxious thermal stimuli presented during the alert and hypnosis-control conditions reliably activated contralateral structures, including primary somatosensory cortex (S1), secondary somatosensory cortex (S2), anterior cingulate cortex, and insular cortex. Hypnotic modulation of the intensity of the pain sensation led to significant changes in pain-evoked activity within S1 in contrast to our previous study in which specific modulation of pain unpleasantness (affect), independent of pain intensity, produced specific changes within the ACC. This double dissociation of cortical modulation indicates a relative specialization of the sensory and the classical limbic cortical areas in the processing of the sensory and affective dimensions of pain.

BACKGROUND

Access to mobile phones continues to increase exponentially globally, outstripping access to fixed telephone lines, fixed computers and the Internet. Mobile phones are an appropriate and effective option for the delivery of smoking cessation support in some contexts. This review updates the evidence on the effectiveness of mobile phone-based smoking cessation interventions.

OBJECTIVE

To determine whether mobile phone-based smoking cessation interventions increase smoking cessation in people who smoke and want to quit.

METHODS

For the most recent update, we searched the Cochrane Tobacco Addiction Group Specialised Register in April 2015. We also searched the UK Clinical Research Network Portfolio for current projects in the UK, and the ClinicalTrials.gov register for ongoing or recently completed studies. We searched through the reference lists of identified studies and attempted to contact the authors of ongoing studies. We applied no restrictions on language or publication date.

METHODS

We included randomised or quasi-randomised trials. Participants were smokers of any age who wanted to quit. Studies were those examining any type of mobile phone-based intervention for smoking cessation. This included any intervention aimed at mobile phone users, based around delivery via mobile phone, and using any functions or applications that can be used or sent via a mobile phone.

METHODS

Review authors extracted information on risk of bias and methodological details using a standardised form. We considered participants who dropped out of the trials or were lost to follow-up to be smoking. We calculated risk ratios (RR) and 95% confidence intervals (CI) for each included study. Meta-analysis of the included studies used the Mantel-Haenszel fixed-effect method. Where meta-analysis was not possible, we presented a narrative summary and descriptive statistics.

RESULTS

This updated search identified 12 studies with six-month smoking cessation outcomes, including seven studies completed since the previous review. The interventions were predominantly text messaging-based, although several paired text messaging with in-person visits or initial assessments. Two studies gave pre-paid mobile phones to low-income human immunodeficiency virus (HIV)-positive populations - one solely for phone counselling, the other also included text messaging. One study used text messages to link to video messages. Control programmes varied widely. Studies were pooled according to outcomes - some providing measures of continuous abstinence or repeated measures of point prevalence; others only providing 7-day point prevalence abstinence. All 12 studies pooled using their most rigorous 26-week measures of abstinence provided an RR of 1.67 (95% CI 1.46 to 1.90; I(2) = 59%). Six studies verified quitting biochemically at six months (RR 1.83; 95% CI 1.54 to 2.19).

CONCLUSIONS

The current evidence supports a beneficial impact of mobile phone-based smoking cessation interventions on six-month cessation outcomes. While all studies were good quality, the fact that those studies with biochemical verification of quitting status demonstrated an even higher chance of quitting further supports the positive findings. However, it should be noted that most included studies were of text message interventions in high-income countries with good tobacco control policies. Therefore, caution should be taken in generalising these results outside of this type of intervention and context.

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na+ + K+]coupled L-glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the L-glutamate transport activity--as assayed upon reconstitution--from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al. (1990) Biochemistry 29, 6734-6740] closely correlated with the L-glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the L-glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate L-glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.

Although missing outcome data are an important problem in randomized trials and observational studies, methods to address this issue can be difficult to apply. Using simulated data, the authors compared 3 methods to handle missing outcome data: 1) complete case analysis; 2) single imputation; and 3) multiple imputation (all 3 with and without covariate adjustment). Simulated scenarios focused on continuous or dichotomous missing outcome data from randomized trials or observational studies. When outcomes were missing at random, single and multiple imputations yielded unbiased estimates after covariate adjustment. Estimates obtained by complete case analysis with covariate adjustment were unbiased as well, with coverage close to 95%. When outcome data were missing not at random, all methods gave biased estimates, but handling missing outcome data by means of 1 of the 3 methods reduced bias compared with a complete case analysis without covariate adjustment. Complete case analysis with covariate adjustment and multiple imputation yield similar estimates in the event of missing outcome data, as long as the same predictors of missingness are included. Hence, complete case analysis with covariate adjustment can and should be used as the analysis of choice more often. Multiple imputation, in addition, can accommodate the missing-not-at-random scenario more flexibly, making it especially suited for sensitivity analyses.