APPA (apocynin and paeonol) modulates pathological aspects of human neutrophil function, without supressing antimicrobial ability, and inhibits TNFα expression and signalling.

Neutrophils are key players in the pathophysiological process underlying inflammatory conditions not only by release of tissue-damaging cytotoxic enzymes, reactive oxygen species (ROS) but also by secretion of important immunomodulatory chemokines and cytokines. Here, we report the effects of the novel agent APPA, undergoing formal clinical development for treatment of osteoarthritis, and its constituent components, apocynin (AP) and paeonol (PA) on a number of neutrophil functions, including effects on TNFα- expression and signalling. Neutrophils were treated with APPA (10-1000 µg/mL) prior to the measurement of cell functions, including ROS production, chemotaxis, apoptosis and surface receptor expression. Expression levels of several key genes and proteins were measured after incubation with APPA and the chromatin re-modelling agent, R848. APPA did not significantly affect phagocytosis, bacterial killing or expression of surface receptors, while chemotactic migration was affected only at the highest concentrations. However, APPA down-regulated neutrophil degranulation and ROS levels, and decreased the formation of neutrophil extracellular traps. APPA also decreased cytokine-stimulated gene expression, inhibiting both TNFα- and GM-CSF-induced cell signalling. APPA was as effective as infliximab in down-regulating chemokine and IL-6 expression following incubation with R848. Whilst APPA does not interfere with neutrophil host defence against infections, it does inhibit neutrophil degranulation, and cytokine-driven signalling pathways (e.g. autocrine signalling and NF-κB activation), processes that are associated with inflammation. These observations may explain the mechanisms by which APPA exerts anti-inflammatory effects and suggests a potential therapeutic role in inflammatory diseases in which neutrophils and TNFα signalling are important in pathology, such as rheumatoid arthritis.

Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry.

This paper investigates the relationship between apparent size distribution and molecular complexity of dissolved organic matter from the natural environment. We used a high pressure size exclusion chromatography (HPSEC) method coupled to UV-Vis diode array detection (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) in order to compare the apparent size of natural organic matter, determined by HPSEC-UV and the molecular mass determined online by ESI-MS. We found that there was a clear discrepancy between the two methods, and found evidence for an important pool of organic matter that has a strong UV absorbance and no ESI-MS signal. Contrary to some previous research, we found no evidence that apparently high molecular weight organic matter is constituted by aggregates of low molecular weight (<1000 Da) material. Furthermore, our results suggest that the majority of apparent size variability within the ESI ionisable pool of organic matter is due to secondary interaction and exclusion effects on the HPSEC column, and not true differences in hydrodynamic size or intermolecular aggregation.

Inter-rater reliability of phenotypes and exploratory genotype-phenotype analysis in inherited hidradenitis suppurativa.

BACKGROUND: Genotype-phenotype correlation measures the correlation between the presence of a physical trait with a group of similar mutations but is dependent on reliable phenotyping. It can provide information on disease pathogenesis, future disease progression, severity or activity. Such indicators would be valuable in hidradenitis suppurativa (HS). OBJECTIVES: To assess inter-rater reliability (IRR) of HS clinical phenotypes and perform exploratory genotype-phenotype correlation in cases of HS with identified sequence variants. METHODS: Linkage disequilibrium between variants was assessed. Genotype-phenotype correlations were explored using Spearman correlation coefficients. IRR was calculated using Cohen's κ. Correlation between phenotype classifications was assessed using the χ2 statistic. RESULTS: Forty-three sequence variants with clinical information were identified. Clinical phenotypes were classified as LC2 (n = 29; 67%), scarring folliculitis (n = 18; 42%), atypical (n = 38; 88%) and nodular (n = 26; 60%). LC1 phenotype was associated with regular (χ2 = 41·289, P < 0·001) and typical (χ2 = 29·013, P < 0·001) phenotypes. Cohen's κ was highest for van der Zee and Jemec (0·815), followed by Martorell-Calatayud et al. (0·813), Naasan and Affleck (0·774) and Canoui-Poitrine et al. (0·435) classifications. High linkage disequilibrium was seen between variants of Han Chinese pedigrees. No significant genotype-phenotype correlations were identified. CONCLUSIONS: These findings may be influenced by selection, publication bias and the assumption that HS is a monogenic disorder. The poor IRR of existing phenotype measures suggests limited utility of existing measures. Further investigations into the correlation of clinical phenotypes with inflammatory biomarkers may aid in prognostic efforts for this disease. What's already known about this topic? Genotype-phenotype correlation can provide information regarding disease pathogenesis and predictions for future disease progression, severity or activity. The identification of such indicators in hidradenitis suppurativa (HS) would be valuable for patients and clinicians alike, given the lack of biomarkers or clinical predictors of disease. What does this study add? Sixty-five sequence variants across 20 separate genes were identified. There was no significant correlation between phenotype classification in four separate classification schema and gene, mutation type or impact on Notch signalling. Utility of current phenotype measurements are limited. The lack of genotype-phenotype correlation in HS is suggestive that the underlying assumption of inherited HS as a monogenic disorder may need revision.

Evidence for episodic acidification effects on migrating Atlantic salmon Salmo salar smolts.

Field studies were conducted to determine levels of gill aluminium as an index of acidification effects on migrating Atlantic salmon Salmo salar smolts in the north-eastern U.S.A. along mainstem river migration corridors in several major river basins. Smolts emigrating from the Connecticut River, where most (but not all) tributaries were well buffered, had low or undetectable levels of gill aluminium and high gill Na(+) /K(+) -ATPase (NKA) activity. In contrast, smolts emigrating from the upper Merrimack River basin where most tributaries are characterized by low pH and high inorganic aluminium had consistently elevated gill aluminium and lower gill NKA activity, which may explain the low adult return rates of S. salar stocked into the upper Merrimack catchment. In the Sheepscot, Narraguagus and Penobscot Rivers in Maine, river and year-specific effects on gill aluminium were detected that appeared to be driven by underlying geology and high spring discharge. The results indicate that episodic acidification is affecting S. salar smolts in poorly buffered streams in New England and may help explain variation in S. salar survival and abundance among rivers and among years, with implications for the conservation and recovery of S. salar in the north-eastern U.S.A. These results suggest that the physiological condition of outmigrating smolts may serve as a large-scale sentinel of landscape-level recovery of atmospheric pollution in this and other parts of the North Atlantic region.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Effect of dietary nucleotide supplementation on growth and immune function in term infants: a randomized controlled trial.

OBJECTIVE: To examine the effect of nucleotide (NT)-supplemented cow's milk-based formula on growth and biochemical indices of immune function in healthy infants. DESIGN: Randomized controlled trial (RCT) of formula-fed term infants allocated to control formula with an innate level of NT at 10 mg/l (n = 102), or formula fortified with NT at 33.5 mg/l (n = 98). A parallel group of 125 breastfed infants followed the same protocol as a reference. OUTCOME MEASURES: Growth was assessed at enrolment, 7 weeks, 4 months and 7 months of age. Natural killer cell activity, cytokine production and lymphocyte subpopulations were assessed at 7 weeks of age. Antibody responses to diphtheria toxoid, tetanus toxoid and Haemophilus influenzae type b (Hib) immunizations were measured at 7 months of age. RESULTS: NT supplementation did not influence the growth of formula fed infants or any markers of immunity measured at 7 weeks of age. Antibody responses to tetanus toxoid were higher in the NT-supplemented group (n = 68) compared with the control group (n = 70) at 7 months of age (median (5th, 95% percentile): 1.57(0.42, 3.43) vs 1.01(0.41, 4.66) IU/ml, P < 0.03). A difference between treatments was seen in response to diphtheria toxoid but this effect disappeared when adjusted for hepatitis B immunization at birth. There was no effect of treatment on antibody responses to Hib immunization. CONCLUSIONS: Supplementation of formulas with NT at 33.5 mg/l resulted in a modest improvement in antibody response consistent with RCTs that used higher levels of NT supplementation. Whether this translates to clinical benefits in well-nourished infants requires further study. SPONSORSHIP: Supported by a grant from Wyeth Nutrition. Dr Makrides was supported by an RD Wright Fellowship from the National Health and Medical Research Council of Australia and Dr Gibson was partially supported by the MS McLeod Research Trust and a Senior Research Fellowship from the National Health and Medical Research Council of Australia.

Characterization of thoracic duct cells that transfer polyarthritis.

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Transforming growth factor beta in human milk does not change in response to modest intakes of docosahexaenoic acid.

Long-chain polyunsaturated fatty acids have been associated with aspects of immune regulation including cytokine production. The purpose of this study was to investigate the effect of maternal dietary supplementation with tuna oil, rich in docosahexaenoic acid (DHA), on the concentration of transforming growth factor beta 1 (TGFbeta1) and TGFbeta2 in breast milk. In this randomized, dietary intervention trial, mothers of term infants consumed a daily supplement of 2000 mg oil containing either placebo (n = 40), 300 mg DHA (n = 40), or 600 mg DHA (n = 40). The DHA increase in milk and plasma was proportional to dietary DHA. There was no relationship between milk DHA status and TGFbeta1 and TGFbeta2 levels.

Lymphocyte subpopulations in breast-fed and formula-fed infants at six months of age.

UNLABELLED: Factors such as cytokines and lymphocytes present in human milk can influence the developing immune system. This suggests an immunoregulatory role for human milk that is absent in infants consuming formula. There are very few data available from well-defined groups of breast-fed and formula-fed infants with regard to their immune status as reflected by lymphocyte immunophenotypic values. The aim of this study was to investigate the potential difference in lymphocyte subsets between breast-fed and formula-fed infants at 6 months of age. Blood samples were taken by venipuncture. Lymphocytes were analyzed by 2-color direct immunofluorescence with Becton Dickinson Immunocytometry Systems SimulSET reagents (BD, Franklin Lakes NJ). There were 73 breast-fed infants and 55 formula-fed infants at 6 months of age. The frequency of natural killer (NK) cells (CD3-/CD16+ + CD56+) was greater in breast-fed infants (9.2%) than in formula-fed infants (6.6%, P < 0.001), while the CD4 to CD8 ratio was 2.8 in breast-fed infants compared with 3.4 in formula-fed infants (P < 0.001). CONCLUSION: Breast-fed infants (<250mL formula/bovine milk per week) had a greater proportion of NK cells and a lower CD4 to CD8 ratio than formula-fed infants at 6 months of age.

Plasma preparation from whole blood using ultrasound.

A technique to efficiently separate plasma from human whole blood is described. Essentially, 3-mL samples are held on the axis of a tubular transducer and exposed for 5.7 min to an ultrasonic standing wave. The cells concentrate into clumps at radial separations of half wavelength. The clumps grow in size and sediment under gravity. A distinct plasma/cell interface forms as the cells sediment. The volume of clarified plasma increases with time. The separation efficiencies of transducers of 29-mm and 23-mm internal diameters driven, by test equipment, at radial resonances of 3.4 and 1.5 MHz, respectively, were compared. The average efficiency of separation was 99.6% at 1.5 MHz and 99.4% with the 3.4-MHz system. The cleared plasma constituted 30% of the sample volume at 1.5 MHz and 25% at 3. 4 MHz. There was no measurable release of haemoglobin or potassium into the suspending phase, indicating that there was no mechanical damage to cells at either frequency. A total of 114 samples from volunteers and patients were subsequently clarified in a 1.5-MHz system driven by an integrated generator. The average efficiency of clarification of blood was 99.76% for the latter samples. The clarification achieved is a significant improvement on that previously reported (98.5%) for whole blood exposed to a planar ultrasonic standing wave field (Peterson et al. 1986). We have, therefore, now achieved a six-fold reduction of cells in plasma compared to previous results.

Analytical scale ultrasonic standing wave manipulation of cells and microparticles.

The ultrasonic standing-wave manipulation of suspended eukaryotic cells, bacteria and submicron latex or silica particles has been examined here. The different systems, involving plane or tubular ultrasonic transducers and a range of acoustic pathlengths, have been designed to treat suspension volumes of analytical scale i.e. 5 ml to 50 microliters for both sample batch and 'on-line' situations. Frequencies range from 1 to 12 MHz. The influence of secondary cell-cell interaction forces in determining the cell concentration dependence of harvesting efficiency in batch sedimentation systems is considered. Applications of standing wave radiation forces to (1) clarify cell suspensions, (2) enhance particle agglutination immunoassay detection of cells or cellular products and (3) examine and enhance cell-cell interactions in suspension are described.

Clarification of plasma from whole human blood using ultrasound.

There has been interest for a number of years in the possibility of separating blood into cells and plasma by methods other than centrifugation, so that the plasma can be analysed on-line. Cells in whole blood normally occupy about 45% of the suspension volume. It has been shown with a number of different cell types, such as yeast and bacteria, that for concentrations of this order the cells are not as efficiently harvested by ultrasound as those for lower concentrations. In this study, removal of cells from 3-4 ml whole blood volumes has been examined in ultrasonic standing wave fields from tubular transducers driven at a frequency of 1.6 MHz. Samples of whole human blood (n = 11) from two volunteers have been processed by three tubular transducers where high levels of cell removal, 99.7% on average, have been demonstrated with high reproducibility between samples as well as for different transducers.

Observation of yeast cell movement and aggregation in a small-scale MHz-ultrasonic standing wave field.

Aggregation of suspended yeast cells in a small-scale ultrasonic standing wave field has been monitored and quantified. The aggregation effect is based on the acoustic radiation force, which concentrates the cells in clumps. The ultrasonic chamber employed (1.9 MHz, one wavelength pathlength) had a sonication volume of 60 microl. The aggregation process was observed from above the transducer through a transparent glass reflector. A distinct, reproducible, pattern of clumps formed rapidly in the sound field. The sound pressure was estimated experimentally to be of the order of 1 MPa. Microscopic observations of the formation of a single clump were recorded onto a PC. The time dependent movement patterns and travelling velocities of the cells during the aggregation process were extracted by particle image velocimetry analysis. A time dependent change was seen in the particle motion pattern during approach to its completion of clump formation after 45 s. Streaming eddies were set-up during the first couple of seconds. The scale of the eddies was consistent with Rayleigh micro-streaming theory. An increase in the travelling velocity of the cells was observed after 30 s from initially about 400 microm s(-1) to about 1 mm s(-1). The influence of a number of mechanisms on particle behaviour (e.g. micro-streaming, particle interactions and convective flow) is considered. The experimental set-up introduced here is a powerful tool for aggregation studies in ultrasonic standing waves and lays the foundation for future quantitative experiments on the individual contributions of the different mechanisms.

Development of an automated on-line analysis system using flow injection, ultrasound filtration and CCD detection.

The design and construction of an automated on-line analysis system is described with reference to applications in bioprocess control, clinical, and environmental analysis. The new system is built around three main elements: ultrasound filtration, a micro flow injection analysis (FIA) system, and direct readout spectrophotometry. The advantages of three on-line ultrasound filtration devices, developed for clarification of water, human blood and mammalian cell culture samples, are described. The filters avoid off-line centrifugation and do not suffer from the blockage problems associated with conventional filters. The separation efficiency of the ultrasound filters is also discussed. The delivery system is based on a gas driven FIA technique, using helium to avoid bubble formation, with the carrier and reagent flow being controlled by computer switched solenoid valves. Direct readout spectrometers are used, based on charge coupled devices (CCDs) covering the wavelength range 200-900 nm. These detectors provide near instantaneous capture of full spectra, allowing several analytes to be monitored simultaneously, and are much smaller than conventional spectrophotometers. Optical fibres are used to link the light source to the detector via a flow cell in the FIA system. Software to run the entire system was developed using the LabVIEWtrade mark graphical programming environment, enabling rapid development of the control system and user interface. The integration of these components has shown significant improvement in the application of FIA techniques to on-line analysis.

Cytokines (IL-1beta, IL-6, TNF-alpha, TGF-beta1, and TGF-beta2) and prostaglandin E2 in human milk during the first three months postpartum.

Postpartum changes in the concentrations of IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, and prostaglandin E2 in 257 human milk samples collected longitudinally from 49 healthy mothers during the first 12 wk of lactation were determined by ELISA or RIA. The proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha were present in only a proportion of samples, and there was a wide range of concentrations detected at each time in the present study (IL-1beta, <15-400 pg/mL; IL-6, <15-1032 pg/mL; TNF-alpha, <15-2933 pg/mL). Concentrations of prostaglandin E2 increased after the first week and remained elevated for the remainder of the study (range, < 10-9966 pg/mL). The antiinflammatory cytokines TGF-beta1 (range, 43-7108 pg/mL) and TGF-beta2 (range, 208-57935 pg/mL) were present in substantial quantities in all samples, and there was little change in the mean concentration during 12 wk of lactation. The present study shows that immunomodulating agents are normally present in human milk in physiologically relevant quantities for at least the first 3 mo of the breast-fed infant's life.

Interleukin-12 in human milk.

The presence of interleukin-12 (IL-12) in 39 samples of human milk was investigated using the enzyme-linked immunosorbent assay. IL-12 (>40 pg/mL) was detected in 24 of the 39 samples collected (1408 +/- 2256 pg/mL, mean +/- SD, n = 24). A range of concentrations of IL-12 was observed in colostrum, transitional, and mature milk, with an apparent decrease in the mean concentration over time postpartum.

The effect of breast feeding on lymphocyte subpopulations in healthy term infants at 6 months of age.

Breast milk contains many immunologically active components that influence the development of the immune system of the breast-fed infant. The purpose of this study was to investigate the difference in specific lymphocyte subsets between breast-fed and formula-fed 6-mo-old infants. Peripheral blood samples were collected from 79 breast-fed (< 120 mL formula/wk) and 69 formula-fed (breast-fed < 4 wk) infants at 6 mo. All infants had been born at term and had no known illness at the time of blood collection. Packed cells from whole blood were incubated with fluorochrome-labeled monoclonal antibodies, followed by erythrocyte lysis. Washed lymphocytes were analyzed by two-color direct immunofluorescence on a flow cytometer. The percentage of T and B lymphocytes in the peripheral blood of 6-mo-old infants was the same, regardless of feeding regimen. However, the relative frequency of natural killer (NK) cells was greater in breast-fed infants than in formula-fed infants (9.7% vs 7.1%; p < 0.001). The percentage of cells expressing CD4 was lower in breast-fed infants than in formula-fed infants (47.3% vs 50.9%; p < 0.005), and that of cells expressing CD8 was greater (18.0% vs 16.4%; p < 0.05). As a result, the CD4:CD8 ratio in breast-fed infants was lower than that in formula-fed infants (2.8 vs 3.3; p < 0.005). The absolute size of the lymphocyte subpopulations T, B, and CD8+ was the same for each of the two populations of infants. However, breast-fed infants had fewer CD4+ T cells (p < 0.05) and a greater number of NK cells (p < 0.01) than the age-matched formula-fed infants. The immunophenotypic differences between breast-fed and formula-fed infants are consistent with reported age-related changes, suggesting greater maturity in the development of the immune system of breast-fed infants.

Microparticle manipulation in millimetre scale ultrasonic standing wave chambers.

Ultrasonic standing wave chambers with acoustic pathlengths of 1.1 and 0.62 mm have been constructed. The chambers were driven at frequencies over the range 0.66-12.2 MHz. The behaviour of 2 microns diameter latex microparticles and 5 microns diameter yeast in the chambers has been elucidated. One (flow) chamber had a downstream laminar flow expansion section to facilitate observation of concentrated particle bands formed in the ultrasonic field. A second (microscopy) chamber allowed direct observation of band formation in the field and their characterisation by confocal scanning laser microscopy. Clear band formation occurs when the chamber pathlength is a multiple of half wavelengths at the driving frequency, so that the chamber rather than the transducer resonance has the most influence on band formation in this system. Band formation occurred in half-wavelength steps from a position one quarter of a wavelength off the transducer to a band at a similar distance from the reflector. Ordered band formation was preserved by the laminar flow in the expansion chamber, although bands that formed very close to the wall were dissipated downstream. The microscopy chamber provided evidence of significant lateral particle concentration within bands in the pressure nodal planes. The approaches described will be applicable to the manipulation of smaller particles in narrower chambers at higher ultrasonic frequencies.