Assessing capacity for diagnosing tuberculosis in children in sub-Saharan African HIV care settings.

Research on the prevalence of pediatric-specific tuberculosis (TB) diagnostics in sub-Saharan Africa is scarce. We assessed the availability of pediatric TB diagnostic tests at 651 pediatric human immunodeficiency virus care and treatment sites across nine African countries: 54% of the sites had access to sputum culture capacity and 51% to chest X-ray services. While 87% of sites had access to smear microscopy, only 6% had the capacity to perform sputum induction and 5% to perform gastric aspirate. These findings confirm that diagnostic resources for the accurate diagnosis of pediatric TB are limited. Capacity-building initiatives to improve sputum collection in children are urgently required.

The role of mast cells in the structural alterations of the airways as a potential mechanism in the pathogenesis of severe asthma.

Mast cells, traditionally regarded as effector cells of the immune system, have more recently been demonstrated to be key figures in initiating, developing and sustaining complex pathophysiological processes underlying asthma and other allergic diseases. Asthma is characterised by airway inflammation alongside a disturbance to airway physiology manifesting as variable airflow obstruction and airway hyper-responsiveness (AHR). Evidence has emerged that mast cells influence airway function by forming close intercellular relationships with different structural components of the airway wall. In asthma, mast cells are seen to localise to the airway epithelium, to mucous glands and to the airway smooth muscle (ASM). It is mast cell-ASM interaction that is most fundamental to the asthma phenotype and many mast cell mediators have been demonstrated to have important effects on ASM function. In asthma, alongside the inflammatory and physiological changes, structural changes occur to the airway wall in the form of denudation of the epithelium, goblet cell and mucous gland hyperplasia, subepithelial fibrosis, abnormal extracellular matrix (ECM) deposition, vascular proliferation and increased ASM mass. There are many ways in which mast cells can contribute to these structural changes through direct cell to cell communication and more indirectly through mediator release. Mast cells exhibit an array of diverse functions and roles and are fundamental to our current understanding of asthma pathogenesis including severe asthma. Novel targeting of mast cells and their mediators therefore should offer significant therapeutic potential in the treatment of asthma.

A computational approach to identify genes for functional RNAs in genomic sequences.

Currently there is no successful computational approach for identification of genes encoding novel functional RNAs (fRNAs) in genomic sequences. We have developed a machine learning approach using neural networks and support vector machines to extract common features among known RNAs for prediction of new RNA genes in the unannotated regions of prokaryotic and archaeal genomes. The Escherichia coli genome was used for development, but we have applied this method to several other bacterial and archaeal genomes. Networks based on nucleotide composition were 80-90% accurate in jackknife testing experiments for bacteria and 90-99% for hyperthermophilic archaea. We also achieved a significant improvement in accuracy by combining these predictions with those obtained using a second set of parameters consisting of known RNA sequence motifs and the calculated free energy of folding. Several known fRNAs not included in the training datasets were identified as well as several hundred predicted novel RNAs. These studies indicate that there are many unidentified RNAs in simple genomes that can be predicted computationally as a precursor to experimental study. Public access to our RNA gene predictions and an interface for user predictions is available via the web.

Motor coordination and balance in rodents.

Measurement of motor coordination and balance can be used not only to assess the effect of drugs or other experimental manipulations on mice and rats, but also to characterize the motor phenotype of transgenic or knock-out animals. Three well established and widely used protocols for measuring motor coordination and balance in mice and rats (rotarod, beam walking and footprint analysis) are described in this unit. The tests can be used equally well for rats and mice, and have been used both for the phenotypic characterization of transgenic mice and for evaluating the effects of lesions and aging in rats. The protocols are described in the primary context of testing mice, but modifications of the test apparatus or variations in the test parameters for assessment of rats are noted.

Environmental stimulation increases survival in mice transgenic for exon 1 of the Huntington's disease gene.

Mice transgenic for the first exon of the human Huntington's disease (HD) gene carrying an expanded CAG repeat expansion (R6/2 line) develop a progressive neurologic phenotype with symptoms resembling those seen in HD. The overt symptoms of R6/2 mice worsen with age, resulting in a rapid decline in health and premature death between 13 and 18 weeks of age. In this study, we characterized the onset and progression of the overt phenotype in R6/2 mice and examined factors that affect the phenotype and life expectancy of these mice. In particular, the effects of altering home cage environment, through changing feeding regimes and providing environmental stimulation, were investigated. We show that changes in feeding regimes significantly improved the general well-being and life expectancy of R6/2 mice. Furthermore, we find that various forms of environmental stimulation, including regular behavioral testing, significantly improved the survival of R6/2 mice over and above that resulting from the enhanced feeding regime. The fact that environmental stimulation improves the health and life expectancy in R6/2 mice not only enables the mice to serve as more useful research tools, but also suggests that environmental stimulation may have a beneficial impact on the progression of HD in patients.

Abnormal synaptic plasticity and impaired spatial cognition in mice transgenic for exon 1 of the human Huntington's disease mutation.

Huntington's disease (HD) is an autosomal dominant progressive and fatal neurodegenerative brain disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the gene. Presymptomatic Huntington's disease patients often exhibit cognitive deficits before the onset of classical symptoms. To investigate the possibility that changes in synaptic plasticity might underlie cognitive impairment in HD, we examined hippocampal synaptic plasticity and spatial cognition in a transgenic mouse (R6/2 line) expressing exon 1 of the human Huntington's disease gene containing an expanded CAG repeat. This mouse exhibits a progressive and fatal neurological phenotype that resembles Huntington's disease. We report that R6/2 mice show marked alterations in synaptic plasticity at both CA1 and dentate granule cell synapses, and impaired spatial cognitive performance in the Morris water maze. The changes in hippocampal plasticity were age dependent, appearing at CA1 synapses several weeks before they were observed in the dentate gyrus. Deficits in synaptic plasticity at CA1 synapses occurred before an overt phenotype. This suggests that altered synaptic plasticity contributes to the pre-symptomatic changes in cognition reported in human carriers of the Huntington' disease gene. The temporal and regional changes in synaptic plasticity within the hippocampus mirror the appearance of neuronal intranuclear inclusions, suggesting a relationship between polyglutamine aggregation and dysfunction.

Selective discrimination learning impairments in mice expressing the human Huntington's disease mutation.

Cognitive decline is apparent in the early stages of Huntington's disease and progressively worsens throughout the course of the disease. Expression of the human Huntington's disease mutation in mice (R6/2 line) causes a progressive neurological phenotype with motor symptoms resembling those seen in Huntington's disease. Here we describe the cognitive performance of R6/2 mice using four different tests (Morris water maze, visual cliff avoidance, two-choice swim tank, and T-maze). Behavioral testing was performed on R6/2 transgenic mice and their wild-type littermates between 3 and 14.5 weeks of age, using separate groups of mice for each test. R6/2 mice did not show an overt motor phenotype until approximately 8 weeks of age. However, between 3.5 and 8 weeks of age, R6/2 mice displayed progressive deterioration in specific aspects of learning in the Morris water maze, visual cliff, two-choice swim tank, and T-maze tasks. The age of onset and progression of the deficits in the individual tasks differed depending on the particular task demands. Thus, as seen in humans with Huntington's disease, R6/2 mice develop progressive learning impairments on cognitive tasks sensitive to frontostriatal and hippocampal function. We suggest that R6/2 mice provide not only a model for studying cognitive and motor changes in trinucleotide repeat disorders, but also a framework within which the functional efficacy of therapeutic strategies aimed at treating such diseases can be tested.

Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.

Transgenic mice expressing exon 1 of the human Huntington's disease (HD) gene carrying a 141-157 CAG repeat (line R6/2) develop a progressive neurological phenotype with motor symptoms resembling those seen in HD. We have characterized the motor deficits in R6/2 mice using a battery of behavioral tests selected to measure motor aspects of swimming, fore- and hindlimb coordination, balance, and sensorimotor gating [swimming tank, rotarod, raised beam, fore- and hindpaw footprinting, and acoustic startle/prepulse inhibition (PPI)]. Behavioral testing was performed on female hemizygotic R6/2 transgenic mice (n = 9) and female wild-type littermates (n = 22) between 5 and 14 weeks of age. Transgenic mice did not show an overt behavioral phenotype until around 8 weeks of age. However, as early as 5-6 weeks of age they had significant difficulty swimming, traversing the narrowest square (5 mm) raised beam, and maintaining balance on the rotarod at rotation speeds of 33-44 rpm. Furthermore, they showed significant impairment in prepulse inhibition (an impairment also seen in patients with HD). Between 8 and 15 weeks, R6/2 transgenic mice showed a progressive deterioration in performance on all of the motor tests. Thus R6/2 mice show measurable deficits in motor behavior that begin subtly and increase progressively until death. Our data support the use of R6/2 mice as a model of HD and indicate that they may be useful for evaluating therapeutic strategies for HD, particularly those aimed at reducing the severity of motor symptoms or slowing the course of the disease.

Dopamine modulates the susceptibility of striatal neurons to 3-nitropropionic acid in the rat model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by chorea, psychiatric disturbances, and dementia. The striatum is the primary site of neuronal loss in HD; however, neither the mechanism of neurodegeneration nor the underlying cause of the selectivity for the striatum is understood. Chronic systemic injection of 3-nitropropionic acid (3-NP) into rats induces bilateral striatal lesions with many neuropathological features of HD and is widely used as a model of HD. In this study we examine the role striatal dopamine plays in 3-NP-induced striatal toxicity. The effect of elevated striatal dopamine levels on 3-NP toxicity was examined by using acute administration of methamphetamine. After 7 d of 3-NP treatment, a single low dose of methamphetamine markedly increased the frequency of striatal lesion formation. This effect was mediated via dopamine receptors because it could be blocked by the administration of dopamine receptor antagonists. The effect of decreased striatal dopamine on 3-NP toxicity was examined by lesioning the nigrostriatal dopamine input to one striatum 7 d before 3-NP treatment was started. Removal of the dopamine input protected the denervated striatum from the neurotoxic effects of systemic 3-NP but did not prevent the formation of lesions in the intact striatum. Thus the formation of 3-NP lesions is critically dependent on an intact dopamine input. Our data show that dopamine plays an important role in the formation of 3-NP lesions. We suggest that modulation of the dopaminergic system should be reevaluated as a potential drug target in the treatment for HD.

Structure of an RNA internal loop consisting of tandem C-A+ base pairs.

The crystal structure of the RNA octamer 5'-CGC(CA)GCG-3' has been determined from X-ray diffraction data to 2.3 A resolution. In the crystal, this oligomer forms a self-complementary double helix in the asymmetric unit. Tandem non-Watson-Crick C-A and A-C base pairs comprise an internal loop in the middle of the duplex, which is incorporated with little distortion of the A-form double helix. From the geometry of the C-A base pairs, it is inferred that the adenosine imino group is protonated and donates a hydrogen bond to the carbonyl group of the cytosine. The wobble geometry of the C-A+ base pairs is very similar to that of the common U-G non-Watson-Crick pair.

Striatal transplantation in a transgenic mouse model of Huntington's disease.

Striatal grafts have been proposed as a potential strategy for striatal repair in Huntington's disease, but it is unknown whether the diseased brain will compromise graft survival. A transgenic mouse line has recently been described in which hemizygotes with an expanded CAG repeat in exon 1 of the HD gene exhibit a progressive neurological phenotype similar to the motor symptoms of Huntington's disease. We have therefore evaluated the effects of the transgenic brain environment on the survival, differentiation, and function of intrastriatal striatal grafts and undertaken a preliminary analysis of the effects of the grafts on the development of neurological deficits in the host mice. Hemizygote transgenic and wild-type littermate female mice received striatal grafts at 10 weeks of age and were allowed to survive 6 weeks. Normal healthy grafts were seen to survive and differentiate within the striatum of transgenic mice in a manner comparable to that seen in control mice. The transgenic mice exhibited a progressive decline in body weight from 9 weeks of age and a progressive hypoactivity in an open field test of general locomotor behavior. Although striatal grafts exerted a statistically significant influence on several indices of this impairment, all behavioral effects were small and did not exert any clinically relevant effect on the profound neurological deficiency of the transgenic mice.

The crystal structure of an RNA oligomer incorporating tandem adenosine-inosine mismatches.

The X-ray crystallographic structure of the RNA duplex [r(CGCAIGCG)]2 has been refined to 2.5 A. It shows a symmetric internal loop of two non-Watson-Crick base pairs which form in the middle of the duplex. The tandem A-I/I-A pairs are related by a crystallographic two-fold axis. Both A(anti)-I(anti) mismatches are in a head-to-head conformation forming hydrogen bonds using the Watson-Crick positions. The octamer duplexes stack above one another in the cell forming a pseudo-infinite helix throughout the crystal. A hydrated calcium ion bridges between the 3'-terminal of one molecule and the backbone of another. The tandem A-I mismatches are incorporated with only minor distortion to the backbone. This is in contrast to the large helical perturbations often produced by sheared G-A pairs in RNA oligonucleotides.

New crystals of the histone core octamer diffract to higher resolution, 2.65 A.

A new crystal form of the histone octamer, crystallized in 1.6 M KCl, 1.6 M phosphate, diffracts to appreciably better than 2.6 A resolution. The crystals have space group P6(1) or P6(5) and lattice parameters a = b = 158.29, c = 103.27 A, alpha = beta = 90, gamma = 120 degrees, with one molecule per asymmetric unit. The new crystals promise to yield more detail of the histone basic domains and a higher resolution structure for the histone octamer.

Evaluation of 37 AIDS prevention projects: successful approaches and barriers to program effectiveness.

In 1988, the Robert Wood Johnson Foundation awarded grants to 54 AIDS prevention and service projects. This article presents the results from a survey of the 37 projects that contained a substantial prevention effort and embellishes these findings with qualitative data from in-depth site visits to 12 projects. Survey respondents reported conducting a mean of 19 different intervention activities. Small-group discussion, outreach to populations engaged in high-risk behaviors, and training peers and volunteers were the intervention activities rated most effective by project staff. Qualitative analysis identified eight factors facilitating intervention effectiveness. Three site-visited projects were chosen to exemplify the ways in which these facilitating factors contributed to the perceived effectiveness of small-group discussions, outreach, and the training of peer educators. Recommendations to guide the development and delivery of future community-based AIDS prevention projects are presented.

Outbreak of Legionnaires' disease among cruise ship passengers exposed to a contaminated whirlpool spa.

BACKGROUND: Outbreaks of travel-related Legionnaires' disease present a public-health challenge since rapid, sensitive, and specific diagnostic tests are not widely used and because detection of clusters of disease among travellers is difficult. We report an outbreak of Legionnaires' disease among cruise ship passengers that occurred in April, 1994, but that went unrecognised until July, 1994. METHODS: After rapid diagnosis of Legionnaires' disease in three passengers by urine antigen testing, we searched for additional cases of either confirmed (laboratory evidence of infection) or probable Legionnaires' disease (pneumonia of undetermined cause). A case-control study was conducted to compare exposures and activities on the ship and in ports of call between each case-passenger and two or three matched control-passengers. Water samples from the ship, from sites on Bermuda, and from the ship's water source in New York City were cultured for legionellae and examined with PCR. FINDINGS: 50 passengers with Legionnaires' disease (16 confirmed, 34 probable) were identified from nine cruises embarking between April 30 and July 9, 1994. Exposure to whirlpool spas was strongly associated with disease (odds ratio 16.2, 95% Cl 2.8-351:7); risk of acquiring Legionnaires' disease increased by 64% (95% Cl 12-140) for every hour spent in the spa water. Passengers spending time around the whirlpool spas, but not in the water, were also significantly more likely to have acquired infection. Legionella pneumophila serogroup 1 was isolated only from the sand filter in the ship's whirlpool spa. This isolate matched a clinical isolate from the respiratory secretions of a case-passenger as judged by monoclonal antibody subtyping and by arbitrarily primed PCR. INTERPRETATION: This investigation shows the benefit of obtaining a recent travel history, the usefulness or urine antigen testing for rapid diagnosis of legionella infection, and the need for improved surveillance for travel-related Legionnaires' disease. New strategies for whirlpool spa maintenance and decontamination may help to minimise transmission of legionellae from these aerosol-producing devices.

The use of an oxygen bomb as an alternative method for digestion of fish tissue for total mercury analysis.

One of the major time consuming steps in many of the methods used for the analysis of fish tissue for total mercury is the wet oxidation by concentrated acids. This paper compares the use of an oxygen bomb with the nitric/sulfuric acid wet oxidation technique for the determination of total mercury in fish tissue. No significant differences existed in the concentrations obtained by wet oxidation and bomb oxidation with absorption by deionised distilled water. When only a small number of samples (< 5) is to be analysed the oxidation bomb is quicker than published techniques. The oxygen bomb technique lacks the hazardous problems associated with the use of concentrated acids and produces a digest suitable for the determination of other elements but has a requirement for added capital cost.

Multiple partners and partner choice as risk factors for sexually transmitted disease among female college students.

Multiple sexual partners and partner choice are believed to increase the risk of sexually transmitted disease (STD), but these behaviors had not previously been assessed outside of clinical populations. In this study, a cross-sectional survey among single, white, female students in their senior year of college was conducted to measure the association between behavioral risk factors and the acquisition of self-reported STDs during college. The usable response rate was 47.2% (n = 467). The combined prevalence of chlamydial infection, gonorrhea, genital herpes, human papillomavirus (HPV) infection, syphilis, and trichomoniasis during a 3.5-year period was 11.7%. There was a strong association between number of sexual partners and having an STD: those women with 5 or more sexual partners were 8 times more likely to report having an STD than those with only 1 partner, even after adjusting for age at first intercourse (odds ratio = 8.1; 95% confidence interval = 1.99, 32.64). The prevalence of a history of STDs increased with more causal partner choice and earlier age at first intercourse, but neither factor was independently associated with a history of STDs. Of the respondents, 23% always used condoms. Future research should focus on identifying ways of effectively changing high-risk sexual behavior.

PIP: In 1990, researchers analyzed data on 467 19-22 year old white female seniors at the University of Michigan to examine partner choice and other behavioral risk factors in relation to sexually transmitted diseases (STDs). 369 (79%) had had sexual intercourse. 43 (11.7%) had at least 1 STD while at the university. The most frequently reported STDs were chlamydia infection (6.3%) and genital warts (5.2%). Other STDs made up 2% of women with at least 1 STD: trichomoniasis, human papillomavirus infection, genital herpes, gonorrhea, and syphilis. Mean age at 1st intercourse stood at 17.7 years. Number of sexual partners while attending university ranged from 1 to 107 (mean 4.4). 70% had 1 partner and 33% =or5 partners. 44% had sex only within a steady relationship. 34% had sex with at least 1 casual partner and 23% with at least 1 nonsteady partner but no casual partners. 77% used condoms at least sometimes. Just 23% always used them. Students who had had an abnormal PAP smear were 8.36 times more likely to have had an STD than those without an abnormal PAP smear. The prevalence of STDs increased significantly with number of sexual partners (p.001). This was also true for chlamydia and genital warts (p.001). Prevalence of these 2 STDs rose as did the number of nonsteady and casual partners (p=.003 and p=.007, respectively). The odds ratio (OR) for women with at least 5 partners was 8.07. STD prevalence rose as the age at 1st intercourse fell (OR=2.19 for =or15 years; p=.035). The trend was similar for chlamydia (p=.002), but not so for genital warts. Even though the percentage of students who had had an STD was greatest among those with at least 1 casual partner (21.5% vs. 3.2% for only steady partners), the association was not significant. Women who always used condoms had the lowest STD prevalence (7.1%), but those who never used condoms had STDs (10.9%) less often than those who did some of the time (15.8%) or most of the time (12.3%). More research is needed to determine means to effectively change high risk sexual behavior.

Effects of medium-chain triglyceride feeding on energy balance in adult humans.

In recent years, the metabolism of triglycerides has attracted much attention. Oxidation of fatty acids is an essential energy supply, especially when glucose supply is limited. In the present study, the effect of a 3-day high medium-chain triglyceride (MCT; 51% of calories), low carbohydrate intake on plasma glucose and amino acid, and urinary organic acid levels, including dicarboxylic and tricarboxylic acid cycle intermediates, was determined in eight normal adult volunteer subjects. Urine was collected at baseline and at 48 to 72 hours for amino acid and organic acid levels, and plasma collected at 0 and 72 hours for glucose and amino acid concentration. The MCT diet increased urinary levels of dicarboxylic acids (adipic 8-, suberic 65-, sebacic 284-fold) and keto acids (acetoacetate and beta-hydroxybutyrate, 67.5-fold); alanine and lactate were decreased 2.5- and 4-fold, respectively, while pyruvate, other amino acids and citric acid intermediates remained unchanged. Plasma amino acid levels were unchanged, while the plasma glucose levels decreased by 8% from baseline. The loss of calories as urinary dicarboxylic acids and keto acids, although increased during the MCT diet, was less than 1% of the daily caloric intake. The data suggest MCT sustain energy expenditure through medium-chain fatty acid (MCFA) oxidation with no decrease in citric acid cycle intermediates, while sparing protein oxidation.

Evaluation of branched-chain amino acid intake in children with maple syrup urine disease and methylmalonic aciduria.

The biochemical and growth responses to dietary branched-chain amino acid (BCAA) intake were studied in two children; one with a disorder of branched-chain amino acid metabolism, maple syrup urine disease (MSUD) (McKusick 24860), and another with methylmalonic aciduria (MMA) (McKusick 25100). Biochemical control of MSUD focussed on plasma leucine levels while measurement of plasma ammonia levels was used in MMA. From 0 to 2.75 years both patients exhibited five episodes of toxicity. In each case toxicity was associated with dietary indiscretion or infection. The quantity of protein tolerated was always less in the MMA patient and was approximately 1 g/kg/day. From 1 to 2.75 years each patient's growth velocity approximated their predicted growth channel except during periods of toxicity. In both cases leucine intake, which gave normal growth without toxicity, was always judged to be less than the FAO/WHO recommendations. The BCAA intake of the MMA patient was remarkably stable from 0.5 to 2.75 years and at 2 years of age isoleucine and valine intake approximated the FAO/WHO recommendations. From 2 to 2.75 years BCAA intake (mg/kg/day) of the MSUD patient was less than that of the MMA patient and well below FAO/WHO recommendations. Neuromotor development in both patients is normal.

Knowledge of HIV antibody status and decisions to continue or terminate pregnancy among intravenous drug users.

We studied decisions to continue or terminate pregnancy among human immunodeficiency virus (HIV)-seropositive (+) and -seronegative (-) intravenous drug users informed of HIV antibody status before 24 weeks' gestation. Fourteen (50%) of 28 HIV+ vs 16 (44%) of 36 HIV- women chose to terminate their pregnancies. In retrospective interviews, HIV+ women were more likely to have perceived their risk of perinatal HIV transmission to be greater than or equal to 50% and, among those who terminated pregnancy, to cite this concern as an important factor in their decision. However, decisions to terminate pregnancy were predicted more readily by pregnancy-related variables such as prior elective abortion, a negative emotional reaction to pregnancy, and whether the pregnancy had been unplanned. Women who were HIV+ and chose to continue their pregnancies cited the desire for a child, religious beliefs, and family pressure as the most important factors in their decisions. Results indicate that while concerns about perinatal transmission of HIV may lead to decisions to terminate pregnancy in certain cases, there may be other determinants of pregnancy decisions in this population. Counseling of HIV-infected women must address not only perinatal transmission but also the sociocultural and behavioral context in which pregnancy decision making takes place.

KIE: Decisions to terminate or continue pregnancy among human immunodeficiency virus (HIV) seropositive (+) and seronegative (-) intravenous drug users in a New York City methadone maintenance treatment program were examined to determine the effect of knowledge of HIV status on decisions about pregnancy in HIV infected and at-risk women. All women were informed of their HIV antibody status prior to 24 weeks gestation. In retrospective interviews, seropositive women who terminated pregnancy cited fear of AIDS for themselves or their infants as an important factor in their decision. However, pregnancy termination for both HIV+ and HIV- women was best predicted by a prior history of elective abortion, whether the pregnancy was unplanned, and whether the woman reported feeling sad or scared upon learning she was pregnant. Desire for a child and religious beliefs were reported as the main reasons to continue pregnancy by both groups.