Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine resides with S-methylthioacetimidate (SMTA) across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of Brome Mosaic Virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from six to twelve, correlating well with the known pH dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein’s lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.
Brome Mosaic Virus; virion structure; pH dependence; chemical modification; mass spectrometry; Lysine; Conformational Change
The emergence of hepatitis C virus (HCV) and its associated sequelae in Africa is a cause for significant concern. Human immunodeficiency virus (HIV) positive patients are at an increased risk of contracting HCV infection due to similar risk factors and modes of transmission. We investigated the seroprevalence of hepatitis C in hospitalized HIV-positive and HIV-negative patients in Mulago Hospital, an academic hospital in Uganda. Blood samples were first tested for HCV antibodies, and positive tests were confirmed with HCV RNA PCR. We enrolled five hundred patients, half HIV-positive and half HIV negative. Overall, 13/500 patients (2.6%) tested positive for HCV antibodies. There was no difference in HCV antibody detection among HIV-positive and HIV-negative patients. Out of all risk factors examined, only an age greater than 50 years was associated with HCV infection. Traditional risk factors for concurrent HIV and HCV transmission, such as intravenous drug use, were exceedingly rare in Uganda. Only 3 of 13 patients with detectable HCV antibodies were confirmed by HCV RNA detection. This result concurs with recent studies noting poor performance of HCV antibody testing when using African sera. These tests should be validated in the local population before implementation.
Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. The present study evaluated cell killing effects of ATRA-enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA-ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N-acetylcysteine, inhibited ATRA-ND induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 growth arrest, up-regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor-α (RARα) and retinoid X receptor-γ (RXRγ) were increased. Compared to naked ATRA, ATRA-ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41-5253, inhibited ATRA-ND induced ROS generation and prevented ATRA-ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.
Nanodisks; Mantle Cell Lymphoma; ATRA; Reactive Oxygen Species; Apoptosis
Perspective on the paper by Rudolf et al (see page 736)
adolescence; overweight; prevention
A global epidemic of paediatric obesity occurred in recent years, and prevalence of obesity is continuing to rise. In the developed world obesity is now the most common disease of childhood and adolescence. Paediatric obesity is not a cosmetic issue, being associated with a significant burden of ill health both for obese children and for adults who were obese as children. Health professionals tend to underestimate the impact of paediatric obesity, and lack the skills, knowledge, and time to treat it effectively. This short review aims to summarise recent systematic reviews on the origins, consequences, prevention, and treatment of paediatric obesity.
energy metabolism; nutrition; obesity; physical activity; child health
Adoptive T cell therapy involving the use of ex vivo generated antigen-specific cytotoxic T lymphocytes provides a promising approach to immunotherapy. It has become increasingly apparent that anti-tumor efficacy using adoptively transferred T cells is linked to their duration of in vivo persistence and can only be achieved when combined with some form of pre-infusion patient conditioning regimen. An optimal conditioning regimen that provides a positive benefit without serious toxicities has yet to be defined. We have established a unique clinical model that allows for evaluation of a given conditioning regimen on adoptively transferred T cells in humans. In this first-in-human study (FHCRC #1796), we evaluate the use of fludarabine, an FDA-approved reagent with predictable lymphodepleting kinetics and duration of action, as a conditioning regimen that promotes homeostatic upregulation of cytokines and growth signals contributing to in vivo T cell persistence.
We conducted a phase I study in patients with refractory metastatic melanoma. Patients received two infusions of a single tumor-reactive antigen-specific CTL clone expanded to 1010/m2; the first infusion was given without fludarabine conditioning, and the second CTL infusion was given after a course of fludarabine (25 mg/m2/day×5 days). This design permits intra-patient comparison of in vivo T cell persistence pre- and post-fludarabine. Nineteen CTL infusions were administered to ten patients. No serious toxicities were observed. Three of nine evaluable patients experienced minor response or stable disease for periods of 5.8–11.0 months with two additional patients demonstrating delayed disease stabilization. The median overall survival in this heavily pre-treated population was 9.7 months. Fludarabine led to a 2.9 fold improvement in the in vivo persistence of transferred CTL clones from a median of 4.5 days (range 0–38+) to 13.0 days (range 2–63+) (p<0.05). Fludarabine lymphodepletion increased plasma levels of the homeostatic cytokines IL-7 and IL-15. Surprisingly, fludarabine also increased the relative percentage of CD4+ T cells expressing the regulatory protein Foxp3.
Lymphodepletion with fludarabine enhances transferred T cell persistence but suggest that additional improvements to optimize T cell survival and address regulatory T cells are critical in providing anti-tumor efficacy.
A socioeconomic gradient in childhood obesity is known to be present by the age of school entry in the UK. The origin of this gradient is unclear at present, but must lie in socioeconomic differences in habitual physical activity, sedentary behaviour, or dietary intake.
To test the hypothesis that habitual physical activity and/or sedentary behaviour are associated with socioeconomic status (SES) in young Scottish children.
Observational study of 339 children (mean age 4.2 years, SD 0.3) in which habitual physical activity and sedentary behaviour were measured by accelerometry over six days (study 1). In a second study, 39 pairs of children of distinctly different SES (mean age 5.6 years, SD 0.3) were tested for differences in habitual physical activity and sedentary behaviour by accelerometry over seven days.
In study 1, SES was not a significant factor in explaining the amount of time spent in physical activity or sedentary behaviour once gender and month of measurement were taken into account. In study 2, there were no significant differences in time spent in physical activity or sedentary behaviour between affluent and deprived groups.
Results do not support the hypothesis that low SES in young Scottish children is associated with lower habitual physical activity or higher engagement in sedentary behaviour.
social inequalities; sedentary behaviour; energy metabolism; obesity
Aims: (a) To measure infant angiotensin converting enzyme (ACE) activity in healthy term infants at birth and during the first three months of life. (b) To determine the relation between serum ACE activity and infant feeding practice during this period. (c) To investigate the relation between serum ACE activity and birth weight and other potential contributing factors including acid-base status at birth, gestation, and maternal ACE genotype.
Methods: Prospective study of term infants, with clinical and feeding data collected from parents and medical records, and serum ACE measured in the infant at birth and 1 and 3 months of age, and in the mother at the time of birth and one to three months after birth.
Results: At birth and 1 and 3 months of age, infant serum ACE activity was twice that of maternal ACE activity. Infant ACE activity at birth and 1 and 3 months did not significantly differ between breast and formula fed infants. There was a highly significant negative correlation between infant ACE activity at 3 months and birth weight (r = –0.52; p < 0.001). This persisted after the conversion of birth weights to z scores (r = –0.34; p = 0.03). ACE activity at 3 months was also related to placental weight (r = –0.30; p = 0.02) and maternal age (r = –0.30; p = 0.05). The strong correlation between serum ACE activity and birth weight z score persisted after adjustment for maternal age and placental weight (r = –0.34; p = 0.03).
Conclusion: As ACE is increasingly identified as a risk factor for cardiovascular disease, serum ACE activity in infancy may contribute to the link between low birth weight and later cardiovascular events.
Background: The Boston Early-Onset COPD study showed that current or ex-smoking first degree relatives of severe early onset COPD probands have significantly lower forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity (FVC) values than current or ex-smoking control subjects, which suggests the existence of genetic risk factors for the development of COPD in response to cigarette smoking. We hypothesised that first degree relatives of early onset COPD probands may also have lower values of spirometric parameters such as forced expiratory flow at the mid-portion of forced vital capacity (FEF25–75) and FEF25–75/FVC.
Methods: Using generalised estimating equations, FEF25–75 and FEF25–75/FVC were analysed in 333 first degree relatives of probands with severe early onset COPD and 83 population based controls; analyses were also performed on data stratified by smoking status. Narrow sense heritability estimates were calculated using a variance component approach.
Results: Significantly lower FEF25–75 and FEF25–75/FVC were observed in smoking (FEF25–75: ß –0.788 l/s (95% CI –1.118 to –0.457), FEF25–75/FVC: ß –20.4% (95% CI –29.3 to –11.6, p<0.0001 for both phenotypes) and non-smoking (FEF25–75: ß –0.357 l/s (95% CI –0.673 to –0.041, p = 0.0271), FEF25–75/FVC: ß –9.5% (95% CI –17.1 to –1.9, p = 0.0145)) first degree relatives of early onset COPD probands. Narrow sense heritability estimates for FEF25–75 (h2 = 0.38) and FEF25–75/FVC (h2 = 0.45) were similar to those for FEV1 and FEV1/FVC.
Conclusion: Lower values of FEF25–75 and FEF25–75/FVC in non-smoking first degree relatives of early onset COPD probands than in controls suggest a genetic susceptibility to develop obstructive lung disease, independent of smoking, which is magnified by exposure to deleterious environments as suggested by the further decrements in FEF25–75 and FEF25–75/FVC seen in smoking first degree relatives. FEF25–75 and FEF25–75/FVC have high heritability and are important intermediate phenotypes for inclusion in genetic epidemiological studies of COPD.
Aims: To test for the coexistence of social inequalities in undernutrition and obesity in preschool children.
Methods: Retrospective, cross sectional, study of routinely collected data from 74 500 children aged 39–42 months in 1998/99. Main outcome measures were weight, height, sex, and age routinely recorded by health visitors. Body mass index (BMI) standardised for age and sex, relative to UK 1990 reference data, was used to define undernutrition (BMI <2nd centile) and obesity (BMI >95th centile; BMI >98th centile). Social deprivation was assessed as Carstairs deprivation category (1 = most affluent to 7 = most deprived).
Results: Both undernutrition (3.3%) and obesity (8.5% above 95th centile; 4.3% above 98th centile) significantly exceeded expected frequencies from UK 1990 reference data. Undernutrition and obesity were significantly more common in the more deprived families. Odds ratios in deprivation category 7 relative to category 1 were 1.51 (95% CI 1.22 to 1.87) for undernutrition (BMI <2nd centile) and 1.30 (95% CI 1.05 to 1.60) for obesity (BMI >98th centile). The cumulative prevalence of under and overnutrition (malnutrition) in the most deprived group was 9.5% compared to 6.9% in the least deprived group.
Conclusions: Undernutrition and obesity are significantly more common than expected in young children and strongly associated with social deprivation. Both undernutrition and obesity have adverse short and long term health effects. Public health strategies need to tackle malnutrition (both undernutrition and obesity) in children and take into consideration the association with social deprivation.
Since the introduction of new growth charts in the mid 1990s, there has been confusion about which charts should be used, with many districts using more than one version. Because of this uncertainty, an expert working party, the Growth Reference Review Group, was convened by the Royal College of Paediatrics and Child Health to provide guidance on the validity and comparability of the different charts currently in use. This paper describes the technical background to the construction and evaluation of growth charts and outlines the group's findings on the validity of each growth reference in relation to contemporary British children. The group concluded that for most clinical purposes the UK90 reference is superior and for many measures is the only usable reference that can be recommended, while the original Tanner–Whitehouse and the Gairdner–Pearson charts are no longer reliable for use at any age. After the age of 2 the revised Buckler–Tanner references are still suitable for assessing height. There are presently no reliable head circumference reference charts for use beyond infancy. The group propose that apart from refinements of chart design and layout, the new UK90 reference should now be "frozen", with any future revisions only undertaken after careful planning and widespread consultation
In the past decade, cellular immunophenotyping has become a new discipline in diagnostic haematology and immunology, and is invaluable in the rapid diagnosis of leukaemia and monitoring disease progression in human immunodeficiency virus infected individuals. The introduction of bench top flow cytometers has meant that immunophenotyping is now also used for the quantitation of CD34+ peripheral blood stem cells (PBSCs) to ensure the correct timing and adequacy of haematopoietic progenitor cell harvests. Furthermore, flow cytometry has become an important tool for the counting of leucocytes in blood components after leucocyte depletion. Because this new discipline is now such a major diagnostic and prognostic tool in the clinical arena, its use must be subject to both internal and external quality control. Such a requirement was first recognised as early as 1986 when an Inter-Regional Quality Assessment Scheme (IRQAS) was initiated for laboratories that undertook the immunocytochemical diagnosis of leukaemia using the alkaline phosphates anti-alkaline phosphatase technique. This programme began with around 25 UK laboratories. In 1990, after the introduction of two more programmes (one for leukaemia diagnosis using UV microscopy and latterly flow cytometry, and one for the enumeration of CD4+ T cells) the IRQAS achieved UK National External Quality Assessment Scheme (UK NEQAS) status and changed its title to UK NEQAS for Leucocyte Immunophenotyping. In the past decade the once small IRQAS programme has evolved into the largest international scheme of its kind, providing EQA to over 650 laboratories world wide for leukaemia immunophenotyping, lymphocyte subset analysis, PBSCs, and more recently low level leucocyte counting. Over the years, this EQA programme has highlighted important problems, such as the inappropriate use of fluorochromes and antibody titre, and the identification of effective gating strategies, all of which have contributed directly to the high interlaboratory variations seen in cellular immunophenotyping. Furthermore, particularly in absolute counting of lymphocyte subsets, PBSCs, and the enumeration of low numbers of leucocytes, UK NEQAS for Leucocyte Immunophenotyping programmes have been instrumental in highlighting the differences that occur between single and dual platform flow cytometric technologies. As a result of these findings, UK NEQAS for Leucocyte Immunophenotyping has helped to reduce the variation seen on an interlaboratory basis and enabled greater standardisation both in the UK and internationally. These advances have been attributable to the development, by UK NEQAS for Leucocyte Immunophenotyping, of a unique whole blood stabilising process that ensures the retention of the physical characteristics (both light scatter and antigenic profile) required of cells to ensure successful cellular immunophenotyping. This major technological advancement has enabled the distribution of specimens for EQA purposes on a global scale that have minimal matrix effect and behave in a manner identical to fresh blood for several months after stabilisation.
Key Words: leucocyte immunophenotyping • leukaemia • human immunodeficiency virus infection • flow cytometry • guidelines
clinical formulae (CF conference formula and estimation based on 120%
of average requirement for energy) have been recommended for the
estimation of energy requirements in cystic fibrosis but their accuracy
AIM—To compare the
accuracy of estimates of energy requirement derived from the two formulae.
requirement, defined as total daily energy expenditure, was measured
using the doubly labelled water method in 15 patients (six girls, nine
boys; mean (SD) age, 10.0 (2.4) years) who were well and clinically
stable. The accuracy of the formulae was assessed using calculation of
biases and limits of agreement relative to measured energy requirement.
the CF conference formula were lower than measured values (mean paired
difference, 0.52 MJ/day; 95% confidence interval (CI), −1.10 to
0.10), but this bias was not significant, and was smaller than that
from the alternative formula (mean paired difference, 0.77 MJ/day; 95%
CI, −0.20 to 1.74). Limits of agreement relative to measured total
daily energy expenditure were narrower for the CF conference formula
(−2.72 to 1.68 MJ/day) than for that based on 120% of estimated
average requirement (−2.75 to 4.29 MJ/day), but with both formulae
errors in estimation at the individual level were large.
conference formula offers improved prediction of energy requirements,
but the accuracy of both formulae at the individual level is not
sufficiently good for clinical purposes.
1990" data have been proposed for assessing growth and nutritional
status in infancy and childhood. These are still largely untried in
clinical practice. There is also doubt about the applicability of more
traditional reference data, which are still widely used, in assessing
length, weight, skinfold thicknesses, and head circumference.
AIMS—To determine the
suitability of new and traditional reference data for the assessment of
growth and nutritional status in infancy and early childhood.
were recruited at birth and assessed monthly to 6 months of age then at
9, 12, 18, and 24 months. Length, weight, head circumference, and
triceps and subscapular skinfold thicknesses were measured. Body mass
index (BMI) was calculated. Measurements were expressed as standard
deviation scores relative to "revised UK 1990" data for weight,
length, head circumference and BMI; relative to Tanner-Whitehouse data
for skinfold thicknesses; and relative to Gairdner-Pearson standards
for head circumference. Agreement at the extremes of the distribution
was assessed by comparison of observed and expected frequencies above
the 90th and below the 10th centile.
the revised UK 1990 references small differences were found for weight,
length, head circumference, and BMI. Mean head circumference exceeded
Gairdner-Pearson standards at all ages. Triceps and subscapular
skinfold thicknesses were substantially below Tanner-Whitehouse
reference data at all ages and in both sexes.
revised UK 1990 reference data are small and not clinically important.
The new standards are considerably more appropriate than older
reference data. Use of older reference data for head circumference and
skinfold thicknesses is inappropriate.
AIMS—Visual outcome of 66 eyes in 37 patients who had undergone treatment with either cryotherapy or diode laser for threshold retinopathy of prematurity was assessed.
METHODS—17 patients, representing 30 eyes treated with cryotherapy, were examined at between 56 and 98 months corrected age (median 68 months). 20 patients representing 36 eyes treated with diode laser, were examined at between 30 and 66 months corrected age (median 51 months). Structural outcome was categorised as: optimal—flat posterior pole; suboptimal—macular ectopia, optic nerve hypoplasia, retinal fold involving the macula, and retinal detachment involving the macula.
RESULTS—Optimal structural outcome was, in the absence of amblyopia, associated with optimal visual acuity (of 6/12 or better) in all cases, with most eyes achieving a visual acuity of 6/9 or 6/6. Suboptimal structural outcome was invariably associated with suboptimal visual acuity. Amblyopia was present in eight out of 20 cryotherapy treated eyes and in five out of 26 laser treated eyes with an optimal structural outcome. Refractive errors were significantly less in laser treated eyes as was the incidence of anisometropic amblyopia.
CONCLUSION—Eyes treated with either cryotherapy or diode laser for threshold retinopathy of prematurity with optimal structural outcome are associated with development of optimal visual acuity—that is, 6/12 or better. Treatment with either cryotherapy or laser does not in itself reduce the visual potential of these eyes.
Keywords: retinopathy of prematurity; cryotherapy; laser; visual acuity
compliance with Department of Health guidelines on weaning practice in
a representative sample of 127 infants from Glasgow, and to identify
factors influencing timing of weaning.
on feeding and weaning were completed during home visits. Ninety eight
mothers completed a further questionnaire on attitudes to weaning
age at introduction of solid food was 11 weeks (range 4-35 weeks);
only 7% of infants had not been weaned before age 4 months. There was
no difference in timing of weaning between boys and girls. Younger
mothers (< 20 years old), those of lower socioeconomic status, and
those who formula fed their infants tended to introduce solids earlier.
Infants who were heaviest before weaning were weaned earlier. Seventy
three of 98 mothers reported that they weaned their babies because they
felt that they required more food. Sources of information influencing
time of weaning were previous experience (53/98), books and leaflets (43/98), advice from the health visitor (31/98), and family and friends
(15/98). Sixty five of 98 mothers reported receiving formal information
on weaning, in most cases (54) this was from the health visitor.
Mothers who received formal information tended to wean their infants
later. Two per cent of infants had been given cow's milk as a main
drink by age 6 months, 17% by 9 months, and 45% by the end of the
with recommendations on timing of weaning (not before 4 months),
weaning foods, and cow's milk consumption in Glasgow is poor, although
no poorer than in many other areas of the UK as found by Office of
Population, Censuses and Surveys. Public health messages in relation to
weaning may not be reaching their target audience.
BACKGROUND: Many women consult general practitioners each year, seeking treatment for premenstrual syndrome. This qualitative study presents evidence of women's own perceptions of this problem, which may assist in the provision of individualized health care. AIM: To explore women's constructions of premenstrual syndrome using grounded analysis. METHOD: A qualitative, semi-structured interview study carried out in Northern Ireland. Thirteen women were interviewed individually. Thereafter, 33 women participated in group discussions. Five health visitors then commented individually on the findings. RESULTS: Seven themes emerged from the analysis. These themes suggested that women tend to view the menstrual cycle holistically and that premenstrual syndrome is regarded as debilitating by only a small minority of women. Participants indicated an awareness of the intra- and inter-personal variability of menstrual experience. They were ambivalent about menstruation, viewing it as natural but, at the same time, unnatural in terms of day-to-day existence. Talking to other women served two functions, first by providing a yardstick against which to evaluate their own experiences, and secondly by providing support and advice. In contrast, women tended to talk about menstruation only to selected men, mainly partners, primarily in the interests of educating them. Women viewed menstruation as potentially disempowering by virtue of its uncontrollability, and felt that both a positive attitude and the use of a range of remedies were important for women wishing to become empowered with respect to this aspect of their lives. CONCLUSIONS: Women's own constructions of premenstrual syndrome differ markedly from those as presented in medical textbooks and research literature: secondary sources that have significantly impacted upon general practitioners' attitudes towards this condition. The provision of a range of treatment options, including support groups, is suggested, on the basis of evidence gathered using qualitative methods, as likely to be viewed by women as more appropriate than offering treatment based on the evidence provided by traditional randomized controlled trials.