The effect of feed processing of novel unheated, low trypsin inhibitor soybeans on the performance of young female turkeys reared from hatch to 21 days of age.

The objective of this study was to evaluate the effects of dietary inclusion of a novel low trypsin inhibitor soybean (LTI) fed as mash or crumbled pellet form on the productive performance and digestibility in turkey poults from hatch to 21 d. A total of 336 Hybrid Converter turkey poults were allocated in groups of 7 to 6 treatments, each with 8 replicate cages. Treatments were arranged as a 3 × 2 factorial with inclusion level of the low trypsin-inhibitor soybean (0, 20, and 40% LTI) and feed form (mash and crumbled pellet). A single batch of feed was mixed for each inclusion level and divided into 2 aliquots: one remaining as mash and the other conditioned at 82°C for approximately 30 s, pelleted and then crumbled. On d 7, 14, and 21 posthatching, BW, and feed intake (FI) were recorded and BW gain (BWG) and feed conversion ratio (FCR) calculated. Excreta samples were collected from d 19 to 21 and pooled by cage. At d 21, intestines were excised, pancreas weights recorded (PRW), and ileal contents collected. There were no interactions (linear, P > 0.05) between LTI inclusion and feed form on BW, BWG, FI, or FCR at d 7 or 21. Increasing LTI resulted in a linear reduction in BW at 7, 14, and 21 d (P < 0.006). Poults fed crumbles were significantly heavier at d 21 than those fed mash feed (P < 0.027) with no interaction of LTI level with feed form. FCR was not significantly greater with increasing LTI. However, poults fed crumbles had a better, lower FCR that those fed mash feed from d 0 to 21 (P < 0.018). There was a significant interaction between feed form and LTI level at 14 d (P < 0.031), but not 7 or 21 d. Pancreatic hypertrophy (PRW) increased linearly with increasing LTI (P < 0.001) with a significant linear interaction with feed form (P < 0.001). Poults fed crumbles had less pancreatic hypertrophy. At 21 d of age, dietary fat digestibility (ALD, %) was linearly reduced with increasing LTI (P < 0.001). However, poults fed crumbles had significantly better fat absorption than poults fed mash (91.2 vs. 85.8%) (P < 0.001), and there was a significant linear interaction between feed form and LTI level (P < 0.001). AMEn was significantly better for the poults fed crumbles compared to mash (3228 vs. 3132 kcal/kg) (P < 0.001), and there was a significant linear interaction between feed form and LTI level (P < 0.001). Based on the results this trial, it is possible to include up to 20% unheated full fat LTI soybeans into poult starter diets after pelleting. Pelleting improves nutrient utilization, allowing for greater incorporation of the LTI soybean in the crumbled diet compared to the mash diet.

Interventions to reduce consumption of sugar-sweetened beverages or increase water intake: evidence from a systematic review and meta-analysis.

A systematic review and meta-analyses were conducted to evaluate the effects of interventions to reduce sugar-sweetened beverages (SSB) or increase water intakes and to examine the impact of behaviour change techniques (BCTs) in consumption patterns. Randomized and nonrandomized controlled trials published after January 1990 and until December 2016 reporting daily changes in intakes of SSB or water in volumetric measurements (mL d-1 ) were included. References were retrieved through searches of electronic databases and quality appraisal followed Cochrane principles. We calculated mean differences (MD) and synthesized data with random-effects models. Forty studies with 16 505 participants were meta-analysed. Interventions significantly decreased consumption of SSB in children by 76 mL d-1 (95% confidence interval [CI] -105 to -46; 23 studies, P < 0.01), and in adolescents (-66 mL d-1 , 95% CI -130 to -2; 5 studies, P = 0.04) but not in adults (-13 mL d-1 , 95% CI -44 to 18; 12 studies, P = 0.16). Pooled estimates of water intakes were only possible for interventions in children, and results were indicative of increases in water intake (MD +67 mL d-1 , 95% CI 6 to 128; 7 studies, P = 0.04). For children, there was evidence to suggest that modelling/demonstrating the behaviour helped to reduce SSB intake and that interventions within the home environment had greater effects than school-based interventions. In conclusion, public health interventions - mainly via nutritional education/counselling - are moderately successful at reducing intakes of SSB and increasing water intakes in children. However, on average, only small reductions in SSBs have been achieved by interventions targeting adolescents and adults. Complementary measures may be needed to achieve greater improvements in both dietary behaviours across all age groups.

The effect of snacking and eating frequency on dietary quality in British adolescents.

PURPOSE: To describe the effects of number of eating occasions and snacks on dietary quality (DQ), defined as adherence to dietary recommendations. METHODS: A sample of 884 adolescents (11-18 years) in the UK National Diet and Nutrition Survey (NDNS) were included. The Diet Quality Index for Adolescents (DQI-A) was implemented. The total number of eating occasions and snacks was frequency of food or beverages consumed over 24 h and frequency of foods or beverages consumed outside of the three mealtimes, respectively. Results were generated with and without low-energy food under 210 kJ (50 kcal). Regression models were generated with DQ score as the outcome variable and number of eating occasions and snacks as predictors. RESULTS: The mean (95 % CI) DQ score was 31.1 % (30.2, 32.0). The mean number of eating occasions and snacks was 7.5 (7.3, 7.7) and 2.6 (2.6, 2.7) times/day, respectively. When low-energy events were excluded, the mean number of eating occasions and snacks reduced to 6.2 (6.1, 6.4) and 2.0 (2.0, 2.1) times/day, respectively. DQ score increased by 0.74 points (0.42, 1.05; p < 0.01) and 0.55 points (-0.08, 0.69; p = 0.17) for total eating occasions and snacks, respectively. When low-energy events were excluded, DQ score increased by 0.30 points (-0.84, 0.69; p = 0.13) for each eating occasion and decreased by 1.20 points (-2.1, -0.3; p < 0.01) for each snack. CONCLUSION: Eating more frequently improves dietary quality especially if some eating occasions are low in energy. A focus on replacing high-energy snacks with low-energy alternatives rather than reducing the number of eating occasions may result in improved dietary quality in adolescents.

Measuring diet in primary school children aged 8-11 years: validation of the Child and Diet Evaluation Tool (CADET) with an emphasis on fruit and vegetable intake.

BACKGROUND/OBJECTIVES: The Child And Diet Evaluation Tool (CADET) is a 24-h food diary that measures the nutrition intake of children aged 3-7 years, with a focus on fruit and vegetable consumption. Until now CADET has not been used to measure nutrient intake of children aged 8-11 years. To ensure that newly assigned portion sizes for this older age group were valid, participants were asked to complete the CADET diary (the school and home food diary) concurrently with a 1-day weighed record. SUBJECTS/METHODS: A total of 67 children with a mean age of 9.3 years (s.d.: ± 1.4, 51% girls) participated in the study. Total fruit and vegetable intake in grams and other nutrients were extracted to compare the mean intakes from the CADET diary and Weighed record using t-tests and Pearson's r correlations. Bland-Altman analysis was also conducted to assess the agreement between the two methods. RESULTS: Correlations comparing the CADET diary to the weighed record were high for fruit, vegetables and combined fruit and vegetables (r=0.7). The results from the Bland-Altman plots revealed a mean difference of 54 g (95% confidence interval: -88, 152) for combined fruit and vegetables intake. CADET is the only tool recommended by the National Obesity Observatory that has been validated in a U.K. population and provides nutrient level data on children's diets. CONCLUSIONS: The results from this study conclude that CADET can provide high-quality nutrient data suitable for evaluating intervention studies now for children aged 3-11 years with a focus on fruit and vegetable intake.

Association between sugar-sweetened and artificially sweetened soft drinks and type 2 diabetes: systematic review and dose-response meta-analysis of prospective studies.

The intake of sugar-sweetened soft drinks has been reported to be associated with an increased risk of type 2 diabetes, but it is unclear whether this is because of the sugar content or related lifestyle factors, whether similar associations hold for artificially sweetened soft drinks, and how these associations are related to BMI. We aimed to conduct a systematic literature review and dose-response meta-analysis of evidence from prospective cohorts to explore these issues. We searched multiple sources for prospective studies on sugar-sweetened and artificially sweetened soft drinks in relation to the risk of type 2 diabetes. Data were extracted from eleven publications on nine cohorts. Consumption values were converted to ml/d, permitting the exploration of linear and non-linear dose-response trends. Summary relative risks (RR) were estimated using a random-effects meta-analysis. The summary RR for sugar-sweetened and artificially sweetened soft drinks were 1·20/330 ml per d (95 % CI 1·12, 1·29, P< 0·001) and 1·13/330 ml per d (95 % CI 1·02, 1·25, P= 0·02), respectively. The association with sugar-sweetened soft drinks was slightly lower in studies adjusting for BMI, consistent with BMI being involved in the causal pathway. There was no evidence of effect modification, though both these comparisons lacked power. Overall between-study heterogeneity was high. The included studies were observational, so their results should be interpreted cautiously, but findings indicate a positive association between sugar-sweetened soft drink intake and type 2 diabetes risk, attenuated by adjustment for BMI. The trend was less consistent for artificially sweetened soft drinks. This may indicate an alternative explanation, such as lifestyle factors or reverse causality. Future research should focus on the temporal nature of the association and whether BMI modifies or mediates the association.

Combating child obesity: impact of HENRY on parenting and family lifestyle.

BACKGROUND: One-quarter of children in England are overweight/obese at school entry. We investigated the impact of a programme designed to provide parents of infants and preschool children with the skills required for a healthier family lifestyle. METHOD: A cohort of families was followed across the 8-week HENRY (Health Exercise Nutrition for the Really Young) parent course at nine locations in England. Seventy-seven parents enrolled on the course, of which 71 agreed to complete questionnaires addressing eating behaviours, dietary intake and parental self-efficacy. Pre- and post-course data was available from 60 (84.5%) parents (8-week follow-up data from 58 parents) and was analysed using repeated measures analyses. RESULTS: Significant changes were observed, with most sustained at follow-up. Parents reported increased self-efficacy and ability to encourage good behaviour (P < 0.001). Increased consumption of fruits and vegetables was reported in both children and adults, together with reduced consumption of sweets, cakes and fizzy drinks in adults (all P < 0.01). There were also positive changes in eating behaviours (e.g., frequency of family mealtimes and eating while watching television or in response to negative emotion [P < 0.01] ) and reduced screen time in adults (P < 0.001). DISCUSSION: The results build upon earlier evaluation, indicating that the HENRY intervention has a beneficial impact upon the families of infants and preschool children. Furthermore, the findings suggest that positive changes inspired by the programme can be maintained beyond its completion. Such changes may serve to protect against later obesity.

Techniques of assessing hypoxia at the bench and bedside.

Tissues require an adequate supply of oxygen in order to maintain normal cell function. Low oxygen tension (hypoxia) is characteristic of a number of conditions, including cancer, atherosclerosis, rheumatoid arthritis, critical limb ischaemia, peripheral vascular disease, and ischaemic heart disease. Tissue hypoxia is found in tumours, atherosclerotic plaque, and ischaemic myocardium. There is a growing interest in methods to detect and assess hypoxia, given that hypoxia is important in the progression of these diseases. Hypoxia can be assessed at the level of the whole organ, tissue, or cell, using both invasive and non-invasive methods, and by a range of immunohistochemical, biochemical, or imaging techniques. This review describes and critiques current methods of assessing hypoxia that are used at the bench and in clinical practice.

SMART lunch box intervention to improve the food and nutrient content of children's packed lunches: UK wide cluster randomised controlled trial.

BACKGROUND: Government standards are now in place for children's school meals but not for lunches prepared at home. The aim of this trial is to improve the content of children's packed lunches. METHODS: A cluster randomised controlled trial in 89 primary schools across the UK involving 1291 children, age 8-9 years at baseline. Follow-up was 12 months after baseline. A "SMART" lunch box intervention programme consisted of food boxes, bag and supporting materials. The main outcome measures were weights of foods and proportion of children provided with sandwiches, fruit, vegetables, dairy food, savoury snacks and confectionery in each packed lunch. Levels of nutrients provided including energy, total fat, saturated fat, protein, non-milk extrinsic sugar, sodium, calcium, iron, folate, zinc, vitamin A and vitamin C. RESULTS: Moderately higher weights of fruit, vegetables, dairy and starchy food and lower weights of savoury snacks were provided to children in the intervention group. Children in the intervention group were provided with slightly higher levels of vitamin A and folate. 11% more children were provided with vegetables/salad in their packed lunch, and 13% fewer children were provided with savoury snacks (crisps). Children in the intervention group were more likely to be provided with packed lunches meeting the government school meal standards. CONCLUSIONS: The SMART lunch box intervention, targeting parents and children, led to small improvements in the food and nutrient content of children's packed lunches. Further interventions are required to bring packed lunches in line with the new government standards for school meals. Current controlled trials ISRCTN77710993.

A cross-sectional survey of children's packed lunches in the UK: food- and nutrient-based results.

BACKGROUND: Standards for school meals were recently introduced in the UK; however, no such standards exist for packed lunches. This study measures the provision and consumption of a range of food types and nutrients in British children's packed lunches and compares the results with the prevailing school meal standards in England. METHODS: Cross-sectional survey data was collected from 1294 children, age 8-9 years, attending 89 British primary schools. Eighty-seven primary schools declined to take part. The outcomes were the weight of food types and nutrients, provided and consumed in packed lunches and the proportion meeting the government food and nutrient school meal standards for England. RESULTS: Fourteen out of 1294 (1.1%) of packed lunches met all the food-based standards for school meals in England. Eighty-five per cent of children were provided with a sandwich, 19% with vegetables, 54% with fruit, 17% with cheese, 44% with a milk-based dessert, 82% with restricted snacks (crisps or confectionery) and 61% with a sweetened drink. The nutrient standards most likely to be met were protein and vitamin C. The nutrient standards least likely to be met were non-milk extrinsic sugars (NMES) and sodium. Girls were provided with, and consumed, more fruit, vegetables and milk-based desserts. Children at schools with lower percentage free school meals eligibility (% FSME) were provided with, and consumed, more vegetables. CONCLUSION: Few packed lunches meet the school meal standards. Future research should address policy, interventions, and programmes to educate parents about the nutritional content of packed lunches.

Alterations in corticostriatal synaptic plasticity in mice overexpressing human alpha-synuclein.

Most forms of Parkinson's disease (PD) are sporadic in nature, but some have genetic causes as first described for the alpha-synuclein gene. The alpha-synuclein protein also accumulates as insoluble aggregates in Lewy bodies in sporadic PD as well as in most inherited forms of PD. The focus of the present study is the modulation of synaptic plasticity in the corticostriatal pathway of transgenic (Tg) mice that overexpress the human alpha-synuclein protein throughout the brain (ASOTg). Paired-pulse facilitation was detected in vitro by activation of corticostriatal afferents in ASOTg mice, consistent with a presynaptic effect of elevated human alpha-synuclein. However basal synaptic transmission was unchanged in ASOTg, suggesting that human alpha-synuclein could impact paired-pulse facilitation via a presynaptic mechanism not directly related to the probability of neurotransmitter release. Mice lacking alpha-synuclein or those expressing normal and A53T human alpha-synuclein in tyrosine hydroxylase-containing neurons showed, instead, paired-pulse depression. High-frequency stimulation induced a presynaptic form of long-term depression solely in ASOTg striatum. A presynaptic, N-methyl-d-aspartate receptor-independent form of chemical long-term potentiation induced by forskolin (FSK) was enhanced in ASOTg striatum, while FSK-induced cAMP levels were reduced in ASOTg synaptoneurosome fractions. Overall the results suggest that elevated human alpha-synuclein alters presynaptic plasticity in the corticostriatal pathway, possibly reflecting a reduction in glutamate at corticostriatal synapses by modulation of adenylyl cyclase signaling pathways. ASOTg mice may recapitulate an early stage in PD during which overexpressed alpha-synuclein dampens corticostriatal synaptic transmission and reduces movement.

A history and review of school meal standards in the UK.

This review details the history of school meals in the UK, from their origin in the mid-19th Century, to the present day, and provides a summary of how each country has independently developed its own food and or nutrient-based standards for school meals. The standards in place in the UK are amongst some of the most detailed and comprehensive in the world. Regular monitoring to ensure that these standards are being met and that schools are improving healthy eating is essential to their success. Of no lesser importance are assessments to determine whether changes to school meals are having an impact on the diets of school children. It is early days in terms of evaluation because food-based standards have only recently been introduced and nutrient-based standards are in the process of being developed and implemented. Studies in England provide some evidence that the re-introduction of standards for school food is having a positive impact on both pupil's food choices and the nutritional profile of school lunches. At present, there does not appear to be a pattern between current obesity levels and the types of school meals provided, although it is anticipated that, in the long term, these comprehensive standards may contribute to a less obesogenic environment.

Cyclic hydrostatic pressure and particles increase synthesis of 1,25-dihydroxyvitamin D3 by human macrophages in vitro.

1,25-Dihydroxyvitamin D(3) has a pivotal role in bone resorption and osteoclast activity. As activated macrophages are known to synthesise 1,25-dihydroxyvitamin D(3), this study examined whether pressure modulated its synthesis. Pressure and particles have been shown to increase synthesis of pro-resorptive cytokines and other factors by cultured macrophages. Human peripheral blood macrophages were isolated, cultured and exposed to pressure (similar to that found in the human joint) and/or particles. Synthesis of 1,25-dihydroxyvitamin D(3) by macrophages was assayed using high pressure liquid chromatography and in situ hybridization. Synthesis of 1,25-dihydroxyvitamin D(3) but not 24,25-dihydroxyvitamin D(3) was increased in macrophages under pressure. In situ hybridization demonstrated an increase in 1alpha-hydroxylase expression in response to pressure or particles and simultaneous exposure to both stimuli generated higher expression of 1alpha-hydroxylase. In conclusion, this is the first study to demonstrate that mechanical loading, in the form of pressure, stimulates 1,25-dihydroxyvitamin D(3) synthesis in human macrophages. These findings have implications for the in vivo situation, as they suggest that 1,25-dihydroxyvitamin D(3) could be one factor stimulating osteoclastic bone resorption in pathologies, such as arthritis or implant loosening, where intra-articular or intra-osseous pressure is raised or where wear particles interact with macrophages.

Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK.

BACKGROUND: Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa beta ligand /receptor activator of NF-kappa beta (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK. METHODS: In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics. RESULTS: In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients > or = 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls. CONCLUSION: These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types. These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.

Retention characteristics of a new butylimidazolium-based stationary phase.

A new HPLC stationary phase has been synthesized based on the ionic liquid n-butylimidazolium bromide. Imidazolium was covalently immobilized on a silica substrate through an n-alkyl tether and the retention characteristics of the resulting stationary phase were evaluated systematically. Using 28 small aromatic test solutes and reversed phase conditions, the linear solvation energy relationship approach was successfully used to characterize this new phase. The retention characteristics of the test solutes show remarkable similarity with phenyl stationary phases, despite the presence of a positive charge on the new imidazolium phase. Operated in the reversed phase mode, this new stationary phase shows considerable promise for the separation of neutral solutes and points to the potential for a truly multi-modal stationary phase.

The influence on human osteoblasts in vitro of non-steroidal anti-inflammatory drugs which act on different cyclooxygenase enzymes.

There is increasing evidence that non-steroidal anti-inflammatory drugs (NSAIDs) can adversely affect bone repair. We have, therefore, studied the in vitro effects of NSAIDs, which differentially inhibit cyclooxygenases (COX), the prostaglandin/thromboxane synthesising enzymes, on human osteoblasts. Indomethacin and the new nitric oxide (NO)-donating NSAIDs block the activity of both COX-1 and COX-2. Indomethacin and 5,5-dimethyl-3-(3 fluorophenyl)-4-(4 methylsulphonal) phenyl-2 (5H)-furanone (DFU) reduced osteoblast numbers in a dose-dependant manner and increased collagen synthesis and alkaline phosphatase activity. The reduction in osteoblast numbers was not caused by loss of adhesion and was reversible. Neither NSAID influenced DNA synthesis. There was no difference between the effects of indomethacin and DFU. NO-NSAIDs did not affect cell numbers. These results suggest that care should be taken when administering NSAIDs to patients with existing skeletal problems and that NO-NSAIDs may be safer.

Role of cyclical pressure and particles in the release of M-CSF, chemokines, and PGE2 and their role in loosening of implants.

A septic loosening of orthopaedic implants is usually attributed to the action of wear debris from the prosthesis. Recent studies, however, have also implicated physical pressures in the joint as a further cause of loosening. We have examined the role of both wear debris and pressure on the secretion of two chemokines, MIP-1alpha and MCP-1, together with M-CSF and PGE2, by human macrophages in vitro. The results show that pressure alone stimulated the secretion of more M-CSF and PGE2 when compared with control cultures. Particles alone stimulated the secretion of M-CSF and PGE2, when compared with unstimulated control cultures, but did not stimulate the secretion of the two chemokines. Exposure of macrophages to both stimuli simultaneously had no synergistic effect on the secretion of the chemokines, but both M-CSF and PGE2 were increased in a synergistic manner. Our findings suggest that pressure may be an initiating factor for the recruitment of cells into the periprosthetic tissue.

Bisphosphonates modulate the effect of macrophage-like cells on osteoblast.

Macrophages (MPs) are present in many tissues and have been implicated in the excessive bone resorption seen in patients with skeletal disorders. Our previous studies showed that macrophage-like cells influenced osteoblasts (OB) in co-culture, as number and activity of osteoblasts were decreased in co-cultures compared with controls. Macrophages are probable precursors of osteoblasts which have been shown to be inhibited by bisphosphonates (BPs). Bisphosphonates also modulate macrophage and osteoblasts activity. This study investigated whether addition of bisphosphonates to co-cultures of osteoblast and macrophages could reduce or block the adverse effects of macrophages on osteoblasts. The results showed that, compared to controls, fewer osteoblasts were present over time in macrophage/osteoblast co-cultures (at day 12, 15.5 x 10(4) and 8.8 x 10(4); P<0.0001) and that addition of bisphosphonates (10(-9)-10(-5)M) to the co-cultures prevented this reduction (P<0.001). Bisphosphonates also elicited an increase in numbers of osteoblast (82%) and restored alkaline phosphatase (ALP) activity, which was reduced by 15% (P approximately equal to 0.05) compared to control levels. The number of macrophages in co-cultures was reduced when bisphosphonates were added (P<0.001) and release of lactate dehydrogenase (LDH) was seen, which was not detectable in control cultures. It therefore, appears that bisphosphonates initiated macrophage death. These results demonstrated that the inhibitory effect of macrophages on osteoblasts in vitro could be overcome by the action of bisphosphonates. These findings have implications for the treatment of skeletal conditions where macrophage-derived cytokines are important, such as arthritis and implant loosening, although it is clearly important to distinguish between those bisphosphonates which enhance synthesis of pro-inflammatory cytokines and those which inhibit such synthesis.

Synergistic effect of particles and cyclic pressure on cytokine production in human monocyte/macrophages: proposed role in periprosthetic osteolysis.

Macrophages, activated by particulate wear debris, are important in the process of osteolysis, which occurs during joint implant loosening. We previously found increased levels of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha in cultured macrophages subjected to cyclical pressure of 0.138 MPa, suggesting that cyclic pressure may be another relevant cause of macrophage activation. The current study first investigated the effects of a range of cyclic pressures on cultured macrophages, including an investigation of the time course of cytokine expression. At 0.138 MPa, supernatant levels of TNF-alpha were maximal at 12 h, whereas IL-6 and IL-1beta were maximal at 24 h. All four cyclic pressure levels tested (without particles) resulted in increased production of all three cytokines relative to control. These increases were most marked at 0.069 and 0.035 MPa, and the increase in cytokine production at 0.017 MPa was not statistically significant. Further studies demonstrated that conditioned media from cyclically pressurized macrophages stimulated bone resorption in a neonatal mouse calvarial assay system. There were increased levels of calcium released from calvaria cultured in conditioned media from pressurised monocytes, and an increase in tartate-resistant acid phosphatase-positive osteoclasts was observed microscopically. As particulate wear debris is important in implant loosening, ultra high molecular weight polyethylene particles were also added to the pressurized cell cultures. The experiments compared the effect of atmospheric pressure, cyclic pressure alone, particles alone, and particles and cyclic pressure combined. A combination of ultra high molecular weight polyethylene particles and cyclic pressure at 0.017 MPa resulted in a dramatic synergistic elevation of levels of all three cytokines compared with the levels found with either pressure or particles alone. We propose that monocyte/macrophage activation by cyclic pressure plays a major role in the osteolysis seen in aseptic loosening of implants. The synergistic effect observed between particles and pressure could accelerate implant loosening, and implies that reduction in either cyclic pressure (by improving implant fixation) or wear debris load would reduce osteolysis.

An in vitro comparison of human flexor and extensor tendon cells.

This study examined the in vitro phenotype of cells cultured from both flexor and extensor tendons. Matrix proteins secreted by tendon cells were examined, together with their response to mechanical strain, using cells from the two types of tendon. Immunocytochemical staining using specific antibodies to matrix proteins demonstrated that flexor tendon cells synthesised only three of the five proteins stained for, whilst extensor tendon cells synthesised all five. Gel electrophoresis (used to separate mixtures of proteins) demonstrated that protein secretion was similar for both cell types. Both cell types showed similar patterns of synthesis for total collagen and total protein over time. No difference was found in the response to cyclical strain of cells from the two types of tendon; both cell proliferation and collagen synthesis were stimulated. The study demonstrated that cells obtained from two different types of tendon behave similarly when exposed to identical environmental conditions in vitro.

In-line valve injection for capillary electrophoresis.

Direct in-line injection is successfully demonstrated for capillary electrophoresis using a commercially available injection valve designed for liquid chromatographic applications. The internal, fluid-contacting materials in this valve injector are composed of ceramics and PEEK (polyetheretherketone). In studies up to 20 kV, this materials design provides a sufficient dielectric interface to insulate the high-voltage buffer from the metal valve body. Partial-loop injections from 6 to > 60 nL are shown to be highly reproducible and generally consistent with direct electrokinetic injections under the same experimental conditions. The small extracolumn variance contributed by the valve injection system is symmetrical, and the measured theoretical plates for 75-microm- and 100-microm-i.d. separation capillaries are 1.6 x 10(5) and 2.5 x 10(5), respectively. As a result, the separation performance is quite good, demonstrating the viability of in-line valve injection for capillary electrophoresis. This development in capillary electrophoretic instrumentation has important implications for the advancement of electrophoretic applications as well as for the design of completely integrated analysis systems.