Dyspnoea: Pathophysiology and a clinical approach.

Dyspnoea, also known as shortness of breath or breathlessness, is a subjective awareness of the sensation of uncomfortable breathing. It may be of physiological, pathological or social origin. The pathophysiology of dyspnoea is complex, and involves the activation of several pathways that lead to increased work of breathing, stimulation of the receptors of the upper or lower airway, lung parenchyma, or chest wall, and excessive stimulation of the respiratory centre by central and peripheral chemoreceptors. Activation of these pathways is relayed to the central nervous system via respiratory muscle and vagal afferents, which are consequently interpreted by the individual in the context of the affective state, attention, and prior experience, resulting in the awareness of breathing. The clinical evaluation and approach to the management of dyspnoea are directed by the clinical presentation and underlying cause. The causes of dyspnoea are manifold, and include a spectrum of disorders, from benign to serious and life-threatening entities. The pathophysiology, aetiology, clinical presentation and management of dyspnoea are reviewed.

Postoperative pain assessment in children: a pilot study of the usefulness of the analgesia nociception index.

BACKGROUND: The ability to perform objective pain assessment is very important in paediatric patients. The goal of this study was to investigate the relationship between the analgesia nociception index (ANI), which is based on the heart rate variability, and objective measurements of pain intensity in young or cognitively impaired children, after surgical or imaging procedures (control group) under general anaesthesia. METHODS: On arrival in the recovery room and subsequently at 5-10 min intervals, the level of pain was rated using the FLACC pain scale (0-10). The ANI values (0-100; 0 indicating the worst pain) were recorded simultaneously. The area under the receiver operating characteristic curve (AUC) and grey zone approach were used to evaluate the performance of the ANI to detect patients with FLACC >4. Instantaneous ANI values were compared with ANI values averaged over 256 s periods of time. RESULTS: All children in the surgical group (n=32) developed moderate-to-severe pain (FLACC >4). Children in the control group (n=30) exhibited minimal pain. Instantaneous ANI values were lower in children of the surgical group than in the control group [52 (sd16) vs 69 (16), P<0.001]. The AUC for the 256 s ANI recording period [0.94 (95% confidence interval 0.85-0.99)] was significantly higher than for instantaneous ANI (P<0.05). When measured for a period of 256 s, an ANI cut-off value of 56 (grey zone [58-60]) was most predictive of a FLACC ≥4. CONCLUSIONS: The ANI may provide an objective measurement of acute postoperative pain, which is correlated with that measured on a FLACC scale in young or cognitively impaired children.

Effects of ß2 agonists on post-thoracotomy pain incidence.

BACKGROUND: Pre-clinical research has shown ß2 -adrenoceptors to be essential for the antiallodynic action of antidepressant drugs in murine models of neuropathic pain and that sustained treatment with ß2 -agonists has an antiallodynic action. Here, we clinically investigated whether chronic ß2 -agonist treatments may influence the incidence of post-thoracotomy chronic pain, defined as pain that recurs or persists along a thoracotomy scar more than 2 months after surgery, either neuropathic or non-neuropathic. METHODS: We conducted an epidemiological study on patients operated by thoracotomy. Demographic data, medical history and treatments concomitant to the surgery were recorded at a follow-up visit. Information on perioperative treatments was collected from the anaesthesia records and confirmed by the patients. In patients with pain at the surgery level, post-thoracotomy chronic pain was assessed by clinical examination and numeric scale. Physical examination and DN4 questionnaire were used to discriminate neuropathic and non-neuropathic chronic pain at scar level. RESULTS: One hundred and eighty-nine patients were included. Eighty-one patients reported persisting thoracic pain, with neuropathic characteristics in 58 of them (30% of the 189 patients). The most common chronic drugs during the perioperative period were inhaled ß2 -agonists (28.6%). The chronic use of ß2 -agonists was an independent predictor of thoracic neuropathic pain (but not of non-neuropathic pain) and was associated with a five-fold decrease in the relative incidence of neuropathic pain [OR = 0.19 (0.06-0.45)]. CONCLUSIONS: These data suggest a possible influence of chronic ß2 -agonist treatments on neuropathic pain secondary to thoracotomy. This apparent preventive effect of ß2 -agonist treatments should warrant controlled clinical trials.

A socio-ecological approach promoting physical activity and limiting sedentary behavior in adolescence showed weight benefits maintained 2.5 years after intervention cessation.

BACKGROUND: Obesity in youth remains a major public health issue. Yet no effective long-term preventive strategy exists. We previously showed that a school-based socio-ecological approach targeting behavior and social/environmental influences on physical activity (PA) prevented 4-year excessive weight gain in 12-year olds. In this study, we investigated if this efficacy persists 30 months after intervention cessation. METHODS AND FINDINGS: The program targeted students, family, school and the living environment to promote/support PA and prevent sedentary behavior (SB). A total of 732 students from eight randomized middle schools completed the 4-year trial. At the 30-month post-trial follow-up, body mass index (BMI), fat mass index (FMI), leisure PA (LPA), home/school/workplace active commuting, TV/video time (TVT), and attitudes toward PA were measured in 531 adolescents. The beneficial effects of the intervention on the excess BMI increase (+0.01 vs +0.34 kg m(-2) in the intervention and control groups, respectively) and on the overweight incidence in initially non-overweight students (4.3% vs 8.6%; odds ratio=0.48 (95% confidence interval: 0.23-1.01)) were maintained at the post-trial follow-up. LPA was not maintained at the level achieved during the trial. However, we still observed a prevention of the age-related decrease of the adolescents' percentage reporting regular LPA (-14.4% vs -26.5%) and a higher intention to exercise in the intervention group. The intervention promoted lower TVT (-14.0 vs +13.6 min per day) and higher active commuting changes (+11.7% vs -4.8%). Trends in higher BMI reduction in students with high initial TVT and in the least wealthy group were noted. TVT changes throughout the follow-up predicted excess BMI and FMI changes. CONCLUSIONS: Long-term multilevel approach targeting PA and SB prevents excessive weight gain up to 30 months after intervention cessation. The efficacy may be higher in the most sedentary and least wealthy adolescents. Healthy PA-related behavior inducing long-lasting weight effects can be promoted in youth providing that an ecological approach is introduced in the prevention strategy.

Spatial accessibility to physical activity facilities and to food outlets and overweight in French youth.

OBJECTIVE: Some characteristics of the built environment have been associated with obesity in youth. Our aim was to determine whether individual and environmental socio-economic characteristics modulate the relation between youth overweight and spatial accessibility to physical activity (PA) facilities and to food outlets. DESIGN: Cross-sectional study. SUBJECTS: 3293 students, aged 12 ± 0.6 years, randomly selected from eastern France middle schools. MEASUREMENTS AND METHODS: Using geographical information systems (GIS), spatial accessibility to PA facilities (urban and nature) was assessed using the distance to PA facilities at the municipality level; spatial accessibility to food outlets (general food outlets, bakeries and fast-food outlets) was calculated at individual level using the student home address and the food outlets addresses. Relations of weight status with spatial accessibility to PA facilities and to food outlets were analysed using mixed logistic models, testing potential direct and interaction effects of individual and environmental socio-economic characteristics. RESULTS: Individual socio-economic status modulated the relation between spatial accessibility to PA facilities and to general food outlets and overweight. The likelihood of being overweight was higher when spatial accessibility to urban PA facilities and to general food outlets was low, but in children of blue-collar-workers only. The odds ratio (OR) (95% confidence interval) for being overweight of blue-collar-workers children compared with non-blue-collar-workers children was 1.76 (1.25-2.49) when spatial accessibility to urban PA facilities was low. This OR was 1.86 (1.20-2.86) when spatial accessibility to general food outlets was low. There was no significant relationship of overweight with either nature PA facilities or other food outlets (bakeries and fast-food outlets). CONCLUSION: These results indicate that disparities in spatial accessibility to PA facilities and to general food outlets may amplify the risk of overweight in socio-economically disadvantaged youth. These data should be relevant for influencing health policies and urban planning at both a national and local level.

Perceived barriers and facilitators for general practitioner-patient communication in palliative care: a systematic review.

While effective general practitioner (GP)-patient communication is required for the provision of good palliative care, barriers and facilitators for this communication are largely unknown. We aimed to identify barriers and facilitators for GP-patient communication in palliative care. In a systematic review seven computerized databases were searched to find empirical studies on GP-patient communication in palliative care. Fifteen qualitative studies and seven quantitative questionnaire studies were included. The main perceived barriers were GPs' lack of availability, and patients' and GPs' ambivalence to discuss 'bad prognosis'. Main perceived facilitators were GPs being available, initiating discussion about several end-of-life issues and anticipating various scenarios. Lack of availability and failure to discuss former mistakes appear to be blind spots of GPs. GPs should be more forthcoming to initiate discussions with palliative care patients about prognosis and end-of-life issues. Empirical studies are needed to investigate the effectiveness of the perceived barriers and facilitators.

Safety and efficiency of procedural sedation and analgesia (PSA) conducted by medical officers in a level 1 hospital in Cape Town.

OBJECTIVES: To study the efficacy and safety of procedural sedation and analgesia (PSA) administered by medical officers (MOs) without formal anaesthetic training. METHODS: A retrospective descriptive study in the Emergency Department (ED) of False Bay Hospital (FBH), situated in the southern suburbs of the Cape Town Metro Health District. The study included all patients who received PSA at FBH between 1 March 2007 and 31 August 2009. Variables recorded included age, gender, physical status as determined by the American Society of Anesthesiologists (ASA status), procedure, fasting and intoxication status, PSA medications, adverse effects, rescue manoeuvres performed, if any, and time to discharge. Analysis was largely descriptive and clinical and demographic data are presented as means (standard deviations), medians, ranges and proportions as appropriate. Success of sedation and incidence of adverse effects are presented as proportions. RESULTS: Of 166 patients, 140 (84.3%) showed a good level of sedation, 14 (8.4%) were inadequately sedated, 5 (3%) were too deeply sedated but showed no signs of respiratory compromise, and 7 (4.2%) developed respiratory side-effects. Respiratory complications were treated with simple airway manoeuvres; no patient required intubation or experienced respiratory problems after waking up. There was no significant difference in the risk of adverse effects between the fasted and non-fasted groups. Mildly intoxicated patients who received PSA were at a higher risk of adverse effects. CONCLUSION: PSA can be administered safely by medical officers. Future research should expand on PSA research in this setting and focus on safety and patient satisfaction.

Seroprevalence of West Nile virus infection in solid organ transplant recipients.

BACKGROUND: Of people infected with mosquito-borne West Nile virus (WNV), <1% develop neuroinvasive disease (NID). Population studies suggest that people older than 65 years may be at higher risk for neurologic symptoms. It has been suggested that solid organ transplant (SOT) recipients are also at higher risk for WNV NID, but definitive serologic and epidemiologic data are lacking. METHODS: A serologic screening survey, using a US Food & Drug Administration-approved enzyme-linked immunosorbant assay to detect WNV immunoglobulin-G (IgG) antibody responses in cohorts of SOT recipients and non-immunocompromised controls, was undertaken at a large Midwestern university organ transplant center in the aftermath of the summer 2003 WNV regional outbreak. Hemagglutination-inhibition testing was used to confirm WNV IgG-positive results and differentiate them from positive results caused by Saint Louis encephalitis virus, another flavivirus that is endemic in the Midwestern US. FINDINGS: The rate of WNV IgG-seropositive responses did not differ between SOT recipients and non-immunocompromised controls, and were 12% and 10%, respectively. Retrospective chart review showed no documented WNV NID in the seropositive SOT recipients, suggesting an incidence of WNV NID may be as low as 0.7% in this population. INTERPRETATION: Asymptomatic WNV infection is common among immunocompromised SOT patients, occurring as often as it does in non-immunocompromised controls. Our data indicated that severe WNV NID is less frequent in SOT patients, contrary to what has been suggested in other studies.

Adiponectin is associated with lipid profile and insulin sensitivity in French adolescents.

AIM: The favourable relationship of adiponectin with the metabolic profile demonstrated in adults has been less studied in youths. The aim of this study was to examine cross-sectional and longitudinal associations between adiponectin and various metabolic risk factors in 12-year-old adolescents. METHODS: Subjects were participants in a randomized controlled study to promote physical activity (PA). Cross-sectional associations were assessed at entry in 2002 among 647 PA-exposed and control first-level students (49% male, 11.6+/-0.6 years of age). Longitudinal analyses involved 288 control students surveyed in 2002 and 2004. Baseline measurements included fasting serum adiponectin and anthropometric indices (body mass, waist size, body fat [BF] by bioimpedance), insulin concentration, homeostasis model assessment (HOMA), high-density lipoprotein (HDL) cholesterol, triglycerides (TG), soluble TNF-alpha receptor 1 (sTNF-alpha R1) and high-sensitivity C-reactive protein. Analyses were performed with generalized linear mixed-effects models, taking into account correlations among adolescents in the same school. RESULTS: Cross-sectionally, plasma adiponectin was inversely associated with obesity indices, especially waist size (P<10(-2)), HOMA (P<0.03), insulin (P<0.04), TG (P<10(-2)) and sTNF-alpha R1 (P<0.05), and positively related to HDL cholesterol (P<10(-4)), after adjusting for age, gender, sexual maturity, sports participation and adiposity when relevant. Longitudinally, a higher baseline adiponectin level was associated with a more favourable two-year change in TG (P<0.05), even after accounting for baseline TG, and two-year BF and insulin changes. CONCLUSION: The findings of this study suggest a favourable relationship between adiponectin and both metabolic profile and subsequent changes in TG level in young adolescents.

Successful overweight prevention in adolescents by increasing physical activity: a 4-year randomized controlled intervention.

BACKGROUND: Population-based studies directed at promoting physical activity in youth have shown limited success in obesity prevention. OBJECTIVE: To assess whether an intervention integrating environmental changes to induce sustained changes in physical activity, prevents overweight in adolescents. DESIGN: Four-year randomized trial started in 2002 in eight middle schools of Eastern France. The intervention, randomized at school level, was designed to promote physical activity by changing attitudes through debates and attractive activities, and by providing social support and environmental changes encouraging physical activity. SUBJECTS: Nine hundred and fifty four 12-year-old six-graders. MEASUREMENTS: Body mass index (BMI), body composition, physical activity by questionnaire, plasma lipids and glucose, insulin resistance. RESULTS: Intervention students had a lower increase in BMI (P=0.01) and age- and gender-adjusted BMI (P<0.02) over time than controls. The differences across groups of the age- and gender-adjusted BMI changes (95% confidence interval (CI)) were -0.29 (-0.51; -0.07) kg/m2 at 3 years, -0.25 (-0.51; 0.01) kg/m2 at 4 years. An interaction with baseline weight status was noted. The intervention had a significant effect throughout the study in initially non-overweight adolescents (-0.36 (-0.60;-0.11) kg/m2 for adjusted BMI at 4 years), corresponding to a lower increase in fat mass index (P<0.001). In initially overweight adolescents, the differences observed across groups at 2 years (-0.40 (-0.94; 0.13) kg/m2 for adjusted BMI) did not persist over time. At 4 years, 4.2% of the initially non-overweight adolescents were overweight in the intervention schools, 9.8% in the controls (odds ratio=0.41 (0.22; 0.75); P<0.01). Independent of initial weight status, compared with controls, intervention adolescents had an increase in supervised physical activity (P<0.0001), a decrease of TV/video viewing (P<0.01) and an increase of high-density cholesterol concentrations (P<0.0001). CONCLUSION: Enhancing physical activity with a multilevel program prevents excessive weight gain in non-overweight adolescents. Our study provides evidence that prevention of obesity in youth is feasible.

Relationships of physical activity with metabolic syndrome features and low-grade inflammation in adolescents.

AIMS/HYPOTHESIS: Physical activity has beneficial effects on symptoms of the metabolic syndrome and low-grade inflammation in adults. These associations have rarely been studied in adolescents. Moreover, it has not been established whether they depend on adiposity, fat localisation and adipokines. SUBJECTS, MATERIALS AND METHODS: We used cross-sectional data of 640 12-year-old adolescents participating in the Intervention Centred on Adolescents' Physical Activity and Sedentary Behaviour Study (ICAPS). Weight, height, body fat mass and WHR were measured. Metabolic syndrome components, two inflammatory markers (IL-6 and C-reactive protein), plasma leptin, adiponectin and soluble TNF-alpha receptor 1 (sTNF-alpha R1) were determined. Insulin resistance was estimated by homeostasis model assessment (HOMA) and energy expenditure due to organised leisure-time physical activity (PAE) assessed by questionnaire. RESULTS: The metabolic syndrome was present in 5.8% of the adolescents. After adjustment for sex, sexual maturity and socio-economic status, a beneficial relationship between PAE and all metabolic syndrome features was found, but only the associations with HOMA and IL-6 were independent of body fat mass and WHR. Adjusted means from the lowest to the highest tertile of PAE were 1.99, 1.80 and 1.78 for HOMA (p=0.04), and 0.88, 0.69 and 0.70 pg/ml for IL-6 (p=0.02). PAE was inversely associated with leptin, independently of body fat mass and WHR (p<10(-2)), but not with adiponectin or sTNF-alpha R1. Further adjustment for adipokines did not change the relationships of PAE with HOMA and IL-6. CONCLUSIONS/INTERPRETATION: In adolescents, physical activity is inversely related to HOMA and IL-6, independently of adiposity and fat localisation. These relationships are not accounted for by adipokines.

ICAPS: a multilevel program to improve physical activity in adolescents.

OBJECTIVES: ICAPS (Intervention Centred on Adolescents' Physical activity and Sedentary behaviour) is aimed at preventing excessive weight gain and cardiovascular risk in adolescents by promoting physical activity (PA) with an emphasis on recreational and daily-life PA, with a lifelong perspective. DESIGN: Randomized study designed to last for four years. Study cohort constituted of 954 first-level students (91% of eligible pupils), aged 11.7 +/- 0.6 y (mean +/- SD) from four pairs of schools randomly selected in eastern France, after sociogeographical stratification. In each pair, intervention status was randomised at school-level. The program, not limited to school settings, involves multiple partners with three objectives: 1) changing attitudes through debates and access to attractive activities during breaks and after-school hours, 2) encouraging social support, 3) providing environmental conditions that enable PA. Adapted times and places, open participation, emphasis on fun, meeting with others and absence of competitive aspects are used to reduce usual barriers to PA. Accessibility and safety are permanent concerns. RESULTS: Prevalence of overweight was 23.7%. High participation rates were attained (50% participated in at least one weekly activity). At six-month, the proportion of intervention adolescents not performing supervised PA out of academic PA was reduced by half (36% to 17% vs 42% to 42% in controls P < 10-4); the proportion of those spending > 3 h/day in sedentary occupations decreased (34% to 28% vs 27% to 36%; P < 10-4). CONCLUSION: These data demonstrate the feasibility of implementing a multilevel PA intervention program in adolescents. Six-month results document increased PA and decreased sedentary behaviour.

Intervention centred on adolescents' physical activity and sedentary behaviour (ICAPS): concept and 6-month results.

OBJECTIVE: To evaluate the 6-month impact of a physical activity (PA) multilevel intervention on activity patterns and psychological predictors of PA among adolescents. The intervention was directed at changing knowledge and attitudes and at providing social support and environmental conditions that encourage PA of adolescents inside and outside school. SUBJECTS AND DESIGN: Randomised, controlled ongoing field trial (ICAPS) in middle-school's first-level adolescents from eight schools selected in the department of the Bas-Rhin (Eastern France) with a cohort of 954 adolescents (92% of the eligible students) initially aged 11.7+/-0.6 y. The 6-month changes in participation in leisure organised PA (LOPA), high sedentary (SED) behaviour (>3 h/day), self-efficacy (SELF) and intention (INTENT) towards PA were analysed after controlling for baseline measures and different covariables (age, overweight, socioprofessional occupation), taking into account the cluster randomisation design. RESULTS: The proportion of intervention adolescents not engaged in organised PA was reduced by 50% whereas it was unchanged among control students. After adjustment for baseline covariables, LOPA participation significantly increased among the intervention adolescents (odds ratio (95% confidence interval) (OR)=3.38 (1.42-8.05) in girls; 1.73 (1.12-2.66) in boys), while high SED was reduced (OR=0.54 (0.38-0.77) in girls; 0.52 (0.35-0.76) in boys). The intervention improved SELF in girls, whatever their baseline LOPA (P<10(-4)) and INTENT in girls with no baseline LOPA (P=0.04). SELF tended to improve in boys with no baseline LOPA, without reaching statistical significance. When included in the regression, follow-up LOPA was associated with improvement of SELF in girls (P=0.02) and of INTENT in girls with no baseline PA (P<0.02). The intervention effect was then attenuated. CONCLUSION: After 6 months of intervention, ICAPS was associated with a significant improvement of activity patterns and psychological predictors, indicating a promising approach for modifying the long-term PA level of adolescents.

[Relationship between asthma or asthma-related symptoms and school problems among French children attending kindergarden]. / Relation entre l'asthme ou les signes respiratoires évocateurs d'asthme et les difficultés scolaires chez les enfants d'école maternelle.

BACKGROUND: The impact of chronic respiratory symptoms on school performance has been studied less than its impact on children's health. Our survey explored the relationship between chronic respiratory symptoms and school problems among children attending kindergarten. It assessed the impact of school absenteeism, sport practise in a club and health care access for school problems among pupils with chronic respiratory symptoms. METHODS: A population-based study was carried out by school physicians on a representative sample of pupils attending third-year kindergarten, in Alsace - France. Assessments included school problems, sociodemographic and health care access characteristics. Health conditions, notably chronic respiratory symptoms, were asked to include the pupils in one of the three study groups: physician-diagnosed asthma, asthma-related symptoms not associated with diagnosed asthma, and without current health problems (controls). Logistic regression was used to compare the odds ratio of school problems for the three study groups, overall and stratified by income. RESULTS: The 2,632 included pupils aged 5 to 6 Years, were assigned to the three groups according to their respiratory status: 179 with diagnosed asthma, 176 with asthma related symptoms and 2,277 healthy pupils. Diagnosed asthma was not linked with school problems. The overall association between asthma related symptoms and school problems was significant (OR=1.5, 95% CI: 1.0-2.2). After adjustment, this association persisted among pupils whose mother's education level was intermediary (technical or vocational diploma) (OR=2.3, 95% CI: 1.2-4.6). Among the diagnosed asthma group, pupils practising a sport in a club had less school problems than those not taking part in sports (ORadjusted=0.2, 95% CI: 0.03-0.8). CONCLUSION: In our study, asthmatic children were not at higher risk of school problems. So developing programs preventing school problems which focus on asthmatic children is not needed. Among the pupils having a mother with an intermediary education level, asthma-related symptoms were associated with school problems. It is important to identify unknown asthmatics to institute to treatment for their respiratory symptoms in order to prevent the consequences of the functional impact of asthma on their scholarship.

Signal amplification by rolling circle amplification on DNA microarrays.

While microarrays hold considerable promise in large-scale biology on account of their massively parallel analytical nature, there is a need for compatible signal amplification procedures to increase sensitivity without loss of multiplexing. Rolling circle amplification (RCA) is a molecular amplification method with the unique property of product localization. This report describes the application of RCA signal amplification for multiplexed, direct detection and quantitation of nucleic acid targets on planar glass and gel-coated microarrays. As few as 150 molecules bound to the surface of microarrays can be detected using RCA. Because of the linear kinetics of RCA, nucleic acid target molecules may be measured with a dynamic range of four orders of magnitude. Consequently, RCA is a promising technology for the direct measurement of nucleic acids on microarrays without the need for a potentially biasing preamplification step.

SpL201: a conditionally immortalized Schwann cell precursor line that generates myelin.

Dramatic progress has been made over recent years toward the elucidation of the mechanisms regulating lineage determination and cell survival in the developing peripheral nervous system. However, our understanding of Schwann cell development is limited. This is partly due to the difficulties in culturing primary Schwann cell precursor cells, the earliest developmental stage of the Schwann cell lineage defined to date. Both the inability to maintain cultured Schwann cell precursor cells in an undifferentiated state and the technical difficulties involved in their isolation have hampered progress. We have conditionally immortalized rat Schwann cell precursor cells using a retrovirally encoded EGFR/neu fusion protein to circumvent these problems and to generate a source of homogeneous cells. The resulting SpL201 cell line expresses p75 and nestin, two proteins expressed by neural crest-derived cells, as well as peripheral myelin protein 22, protein zero, and Oct-6 as markers of the Schwann cell lineage. When cultured in EGF-containing medium, the SpL201 cells proliferate and maintain an undifferentiated, Schwann cell precursor cell-like state. The cell line is dependent on EGF for survival but can differentiate into early Schwann cell-like cells in response to exogenous factors. Like primary rat Schwann cells, SpL201 cells upregulate Oct-6 and myelin gene expression in response to forskolin treatment. Furthermore, the SpL201 cell line can form myelin in the presence of axons in vitro and is capable of extensively remyelinating a CNS white matter lesion in vivo. Thus, this cell line provides a valuable and unique tool to study the Schwann cell lineage, including differentiation from the Schwann cell precursor cell stage through to myelination.

Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.

Combining nucleic acid amplification and detection.

Major recent advances in molecular amplification in the past year were initial validation of two new amplification technologies (rolling circle amplification and Invader), a significant increase in the number of molecular diagnostic assays, achievement of amplification directly on microarrays (by strand displacement amplification and rolling circle amplification), and description of two new read-out probes (Scorpions and nanoparticles).

Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates.

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.