Consumption of apple-boysenberry beverage decreases salivary Actinomyces naeslundii and their adhesion in a multi-species biofilm model.

We hypothesised that consumption of beverage rich in both fibre and polyphenols, rather than each bioactive alone, will modulate populations of selected salivary bacteria, and their adhesion characteristics and that some of these effects may be due to the anti-microbial activity of the beverage bioactives. We investigated the effect of 4 weeks' consumption of beverages, rich in apple fibre, boysenberry polyphenols, or both on salivary bacteria in healthy subjects. In this placebo-controlled crossover study, saliva samples were collected at the beginning and end of each treatment period, and used for qPCR quantitation of Lactobacillus spp., Actinomyces naeslundii and Streptococcus mutans. The counts of salivary A. naeslundii decreased after the consumption of the apple-boysenberry beverage (P<0.05, Student's t-test). We also examined the effect of the subjects' saliva on bacterial adhesion using a mixed species biofilm model. The salivary pellicles prepared before and after each treatment were inoculated with laboratory strains of A. naeslundii, Lactobacillus rhamnosus and S. mutans and tested for biofilm formation. The post appleboysenberry beverage salivary pellicle significantly decreased the adhesion of A. naeslundii at the end of both 3 and 24 h, in the in vitro biofilm. A 1/16 dilution of the apple-boysenberry beverage itself decreased the proliferation of test strains of A. naeslundii and S. mutans by 51 and 55%, respectively (P<0.005), indicating the antimicrobial activity of its bioactives. This study demonstrated that consumption of apple-boysenberry beverage, rather than apple or the boysenberry beverage alone or the placebo, decreased salivary A. naeslundii and their adhesion under laboratory conditions. These changes are factors that influence oral microecology and potentially oral health.

Adjusting worm egg counts for faecal moisture in sheep.

The number of eggs from gastrointestinal nematodes per gram of faeces (worm egg count WEC) is commonly used to determine the need for anti-parasite treatments and the breeding value of animals when selecting for worm resistance. Diarrhoea increases faecal moisture and may dilute the number of worm eggs observed. To quantify this effect, egg counts in sheep at pasture were simulated by dosing 15 animals with chromic oxide particles. The simulated WEC diminished as faecal moisture increased. When faeces were dried, simulated WEC per unit dry matter was not influenced by the amount of faecal moisture present prior to drying. The results suggest that adjustment for faecal moisture may provide an improved estimate of FEC. Drying faeces to calculate the WEC per unit dry matter would provide such an adjustment but may not be practical for industry application. In the past, the CSIRO McMaster Laboratory has used an adjustment factor developed by Gordon based on the classification of faecal consistency derived from the morphology of faeces. To examine the utility of an adjustment factor based on faecal consistency score (FCS), the relationships between FCS and simulated WEC and dry matter were examined. Dry matter and simulated WEC exhibited an exponential decline as FCS increased. The relationship between FCS and dry matter was further examined in 368 samples collected over 12 months from sheep at pasture, where it was observed that dry matter showed a linear decline as FCS increased. Adjustment factors based on dry matter were similar to those proposed by Gordon however adjustment factors predicted from simulated WEC diverged from the remainder for FCS>4. As no samples scored FCS 5 in the study of simulated FEC, the adjustment factors based on the larger study that included samples with FCS 5 was therefore considered more robust. Adjustment factors were given by the equation: WEC(estimated)=(WEC(observed)/(34.21-5.15 FCS))x29.06. This equation estimates for samples with FCS>1 the WEC that would be expected if the samples were FCS 1, the faecal consistency score for normal faeces. The impact of adjustment of observed WEC for faecal moisture predicted by FCS on decision points for treatment and on estimated breeding values requires further examination.

Genetic parameters for indicators of host resistance to parasites from weaning to hogget age in Merino sheep.

Fecal egg count (FEC) has been widely used as an indicator of host resistance to gastrointestinal parasites in sheep and has been shown to be a heritable trait. Two other possible indicators of parasites, dag score (DS; accumulated fecal material) and fecal consistency score (FCS), were investigated in this study, along with BW. All four traits were studied to see how heritability and genetic correlations varied with age from weaning (4 mo) to hogget age (approximately 400 d). More than 1,100 lambs, the offspring of 37 rams, were recorded eight times between weaning (3 to 5 mo of age) and hogget age (13 to 18 mo of age) on two farms. Sire models were fitted to the data from each trait at each recording and in a repeatability model involving the whole data set. Overall, the heritabilities were 0.28+/-0.072 (FEC), 0.11+/-0.036 (DS), 0.12+/-0.036 (FCS), and 0.23+/-0.070 (BW). By fitting random regression models to the time-series data, it was possible to see how these heritability values varied as the lambs aged, from weaning to hogget age. The heritability of FEC rose from 0.2 at weaning to 0.65 at 400 d. Dag score had a higher heritability (0.25) in the middle of the age range and a low value at weaning (<0.1) and hogget age (0.16). The heritability of FCS was low, with a value of 0.2 at weaning reducing to 0.05 as the animals aged. Body weight had zero heritability at weaning, which rose to greater than 0.6 at hogget age. Most traits had low genetic correlations between them, the only exception being that between FCS and DS (0.63). Most genetic correlations varied little over the age range with the exception of FEC and BW, which fell from 0 at weaning to -0.63 at hogget age. Whereas FCS and DS may be good indicators of scouring, they are very different from FEC as an indicator of host resistance to gastrointestinal parasites.

Some relationships between age, immune responsiveness and resistance to parasites in ruminants.

In the Australian livestock industries, susceptibility to infectious diseases is generally greater in young than in mature ruminants. The increased susceptibility is manifest as respiratory and intestinal infections (viral and bacterial) of calves, as well as fleece rot, flystrike and, especially, gastrointestinal parasitic infestations of young sheep. Lower resistance to infectious disease in young ruminants appears to be due largely to immunological hyporesponsiveness, and is not simply a consequence of their not having been exposed sufficiently to pathogens to develop active immunity. Young sheep have significantly lower proportions of CD4+ and CD8+ lymphocytes, but similar proportions of T19+ and B lymphocytes in blood, lymph and skin compared with mature sheep. Blood lymphocytes from young sheep produce less interferon-gamma in culture and young sheep invariably mount smaller antibody responses than do mature animals. Taken together, these findings begin to explain why young ruminants are more susceptible to infectious diseases in general, and to gastrointestinal parasites in particular, when compared to mature animals. Haematological markers of disease resistance, the prevalence of non-selected diseases and immune responses to vaccination were examined in the internal parasite-resistance flocks in Armidale NSW and the fleece rot/flystrike selection flocks at Trangie NSW. Any programme that seeks to improve resistance to parasitic or any other disease should have the capacity to make contemporary measurements of resistance to other diseases which are important in, or threaten, the production system.

The value of circulating eosinophil count as a selection criteria for resistance of sheep to trichostrongyle parasites.

In merino sheep bred for either increased or decreased resistance to Haemonchus contortus, faecal worm egg counts (FEC) were lower in the resistant line (6,831 vs 17,645 epg, P < 0.01), and circulating eosinophils (EOS) were higher, but not significantly so (3.40 x 10(4) ml(-1) vs 1.40 x 10(4) ml(-1), P = 0.1 1). Another flock was artificially infected with Trichostrongylus colubriformis and significant genetic variation was found in both FEC (heritability 0.40 +/- 0.11) and EOS (0.19 +/- 0.08). In a third flock comprising tropical sheep breeds, a natural challenge with T. colubriformis resulted in significant sire effects on FEC (heritability 0.20 +/- 0.10) but not EOS (heritability inestimable). We conclude that EOS offers no advantage over FEC as it selection criterion for resistance.

Immunomodulation of cultured vascular endothelial cells by serial cell passage.

UNLABELLED: Endothelial cell seeding has been successful in reducing the thrombogenecity of prosthetic vascular grafts in animal models, but results from clinical trials have been largely disappointing. These poor results have been associated with poor graft coverage in immediate seeding trials, and failure of cell culture in staged procedures. These problems could be largely overcome by utilising a bank of allogeneic endothelial cells, providing an ever ready supply. However, one potential pitfall with this technique would be the possibility of a rejection response following transplantation. AIM: To study the effects of prolonged tissue culture, on the ability of endothelial cells to generate an immune response. METHODS AND RESULTS: The immunogenecity of human umbilical vein endothelial cells was measured using the mixed lymphocyte endothelial reaction. It was demonstrated that prolonged tissue culture significantly reduced the immunogenecity of the cells, from a mean of 7261 cpm (S.E. +/- 243, n = 3) for cells of subculture 3, to 5478 cpm (+/- 156, p = 0.04) for cells of subculture 7 (p = 0.04, Wilcoxon paired rank test), but did not significantly impair morphology or antithrombotic function. CONCLUSION: This study provides evidence that prolonged tissue culture provides morphologically and functionally intact, immunomodified endothelial cells which may potentially be used in seeding prosthetic vascular grafts.

Attachment, replication and thrombogenicity of genetically modified endothelial cells.

UNLABELLED: Endothelial cell seeding of prosthetic surfaces has been proposed as a technique to improve the patency of vascular grafts following arterial reconstruction. The introduction of specific recombinant DNA into seeded endothelial cells may enhance the anti-thrombogenic nature of the endothelial-blood interface with a consequent reduction in graft thrombosis. However, the successful use of genetically modified endothelial cells in the seeding process relies on the cells retaining normal function in terms of cellular replication, attachment and secretion of anti-thrombotic mediators. Successful genetic manipulation of human endothelial cells has been accomplished by viral and chemical methods. AIM: To study the functional characteristics of electrontransfected endothelial cells. METHODS AND RESULTS: Endothelial cells were electro-transfected with the test plasmid pTCF at a transfection efficiency of 10% utilising a single electric pulse with an electric field of 1000 volts/cm and a time constant of 12.8ms. The functional status of transfected endothelial cells was then compared with a control endothelial cell population. There were no significant differences in replication (p = 0.76), attachment (p = 0.43), basal (p = 0.89) or stimulated (p = 0.11) prostacyclin release between transfected cells as compared with control endothelial cells. CONCLUSIONS: Genetically modified cells are functionally normal, and may be used in endothelial cell seeding of prosthetic vascular surfaces.

Optimisation of gene transfer into vascular endothelial cells using electroporation.

OBJECTIVES: We have examined the conditions required to obtain optimum transfection efficiencies for human umbilical vein endothelial cells by transduction with a plasmid conferring neomycin resistance. MATERIALS AND METHODS: Preliminary studies examined the effects of electric discharges using the Biorad Gene Pulser on endothelial cells. Post-electroporation, there was a significant decrease in cell survival with increasing voltages (100-400 volts; p = 0.03), capacitances [125-960 microFarads (microF); p = 0.02], number of electric pulses (1-2; p = 0.03) and decreasing cell concentrations (p = 0.01). The optimal cell concentration was 3 x 10(6) cells/ml. Transfection studies utilised the neomycin resistance expressing plasmid, pTCF; transfectants were selected with the neomycin analogue G-148. RESULTS: Electro-transfection was optimised with increasing voltages (p = 0.02) and capacitances (p = 0.01) using a single pulse. Optimal transfection was obtained using 400 volts with a capacitance of 960 microF using a single pulse; the median transfection efficiency was 10%. Transduced endothelial cells stably expressed the plasmid for 12 days and at least two cell passages. CONCLUSIONS: The results indicate that endothelial cells can be efficiently transduced by electroporation to stably express an introduced gene. This may have important implications in vascular surgery.

Effect of pulsatile shear stress on endothelial attachment to native vascular surfaces.

An in vitro model of vascular damage was used to investigate the ability of seeded endothelial cells to resist shear stresses generated in a perfusion circuit. At perfusion rates of 100 ml/min the maximum shear stress reached 16.5 dyn/cm2. At this level the rate of cell detachment from the damaged vascular surface was 88 per cent per h for the first 20 min of flow but gradually decreased to 5 per cent per h after 90 min. These findings suggest that endothelial cells may be retained on damaged vascular surfaces in conditions that approximate to arterial flow.

The effect of transluminal endothelial seeding on myointimal hyperplasia following angioplasty.

Myointimal hyperplasia develops as a generalised response to vascular injury, and may cause stenoses in 40% of all peripheral arterial reconstructions. Disruption of the endothelial monolayer is a prerequisite for the development of intimal hyperplasia, and may be the initiating event in this process. This study examined the hypothesis that restenosis following balloon angioplasty may be reduced by rapid restoration of the endothelial monolayer, achieved by endothelial seeding. Bilateral iliac angioplasties were performed in 11 rabbits. A double balloon catheter was used to seed one angioplasty site with autogenous endothelial cells; the contralateral site was sham seeded with culture medium and acted as a control. Arterial patency rates, the degree of intimal hyperplasia (IH/IEL), and the extent of endothelialisation were quantified at 1 (n = 5) and 3 (n = 6) weeks following balloon dilatation. The results suggest that transluminal endothelial seeding may be a therapeutically applicable technique as it decreases myointimal hyperplasia, and increases patency following angioplasty. This study also illustrates the protective effect of the vascular endothelium following arterial injury, and indicates that intensive efforts should be made to preserve the endothelium during vascular reconstruction.

Platelet deposition after angioplasty is abolished by restoration of the endothelial cell monolayer.

PURPOSE: Percutaneous transluminal angioplasty of an atheromatous plaque causes endothelial desquamation and intimal dissection with the consequent formation of a thrombogenic flow surface. In this study we investigated the hypothesis that platelet deposition after balloon angioplasty may be decreased by rapid restoration of the endothelial cell monolayer, achieved by transluminally seeding angioplasty sites with endothelial cells. METHODS: Bilateral external iliac angioplasty was performed in eight New Zealand white rabbits. One angioplasty site was isolated from the circulation and incubated with a supraconfluent endothelial cell suspension with a double balloon catheter; the contralateral angioplasty site was sham seeded with culture medium. The deposition of autologous indium 111-labeled platelets on the angioplasty sites was quantified 30 minutes after restoration of flow and was referenced to an undamaged segment of aorta that acted as a negative control. RESULTS: Platelet deposition on the nonseeded angioplasty site (13.1 x 10(4) platelets/mm2) was significantly higher than on nondilated segments (3.4 x 10(4) platelets/mm2; p = 0.014). Restorationof endothelial cell coverage by endothelial seeding significantly reduced platelet deposition on dilated arterial segments to levels not significantly higher than in controls (3.6 x 10(4) platelets/mm2; p = 0.014). CONCLUSIONS: These results illustrate that rapid reendothelialization of angioplasty sites decreases subsequent platelet deposition and may reduce the rate of acute arterial reocclusion complicating endovascular techniques.

A method to transluminally seed angioplasty sites with endothelial cells using a double balloon catheter.

Acute arterial reocclusion and late restenosis following angioplasty may be partially caused by loss of the endothelial cell monolayer during balloon dilatation. Rapid restoration of the endothelial cell monolayer by endothelial cell transplantation has the potential to increase the antithrombotic nature of the angioplasty site and also to prevent myointimal hyperplasia which is the cause of late restenosis. We have investigated a method to transluminally deliver endothelial cells to angioplasty sites using a double balloon catheter with a central instillation port. Inflation of the balloons allows a segment of artery to be isolated from the circulation which may then be incubated with infused endothelial cells. The external iliac arteries of New Zealand white rabbits were dilated for 30 s at 8 atm pressure using a 3 mm balloon angioplasty catheter. Indium-111 labelled allogeneic rabbit endothelial cells were seeded onto the angioplasty site and retained in contact with the vascular surface for 30 min by continued inflation of the isolation balloons. After 30 min, the balloons were deflated and flow restored. Percentage cell retention was calculated by determining the gamma activity of the seeded angioplasty site, and referencing this to the contralateral sham seeded site. Results are expressed as a median with 95% confidence intervals and suggest that endothelial cells may be successfully delivered and retained on damaged vascular surfaces [table: see text].

Effect of seeding time and density on endothelial cell attachment to damaged vascular surfaces.

An in vitro model to facilitate the study of endothelial cell seeding of damaged vascular surfaces has been developed. This may have applications in the study of endothelial seeding of angioplasty and endarterectomy sites. Using this model, the optimum endothelial seeding time for attachment to damaged vascular surfaces should not exceed 30 min and, to achieve confluent cell attachment, a seeding density > 5 x 10(5) cells/cm2 should be used.

Endothelial cell seeding of damaged native vascular surfaces: prostacyclin production.

Endothelial cell seeding has been successful in reducing the thrombogenicity of prosthetic vascular grafts in animal and clinical studies. The reduction in thrombogenicity may be attributed to the intrinsic properties of endothelial cells themselves, and their ability to produce anti-thrombogenic mediators such as prostacyclin, and endothelium-derived relaxing factor. Endothelial seeding of damaged vascular surfaces produced during percutaneous transluminal angioplasty and endarterectomy is an attractive possibility due to the excellent attachment characteristics of the sub-endothelial tissue exposed during these procedures. The ability of endothelial seeded damaged vascular surfaces to produce prostacyclin was measured in an in vitro model of vascular injury. Endothelial-seeded damaged surfaces produced significantly higher prostacyclin release than did vessels damaged by balloon dilatation (265.5 pg cm-2 min-1 and 87.5 pg cm-2 min-1 respectively). This study provides evidence that endothelial seeding of damaged native vascular surfaces is technically feasible and that seeding may reduce the thrombogenicity of vascular surfaces following balloon dilatation.

There are two major types of skeletal keratan sulphates.

High-field 1H-n.m.r.-spectroscopic studies supported by chemical carbohydrate analyses show that skeletal keratan sulphates (KS-II) of bovine origin may be sub-classified into two groups. Keratan sulphate chains from articular and intervertebral-disc cartilage (KS-II-A) contain two structural features, namely alpha(1----3)-fucose and alpha(2----6)-linked N-acetyl-neuraminic acid residues, that are absent from keratan sulphates from tracheal or nasal-septum cartilage (KS-II-B).

Speech intonation and focus location in matched statements and questions.

An acoustical study of speech production was conducted to determine the manner in which the location of linguistic focus influences intonational attributes of duration and fundamental voice frequency (F0) in matched statements and questions. Speakers orally read sentences that were preceded by aurally presented stimuli designed to elicit either no focus or focus on the first or last noun phrase of the target sentences. Computer-aided acoustical analysis of word durations showed a localized, large magnitude increase in the duration of the focused word for both statements and questions. Analysis of F0 revealed a more complex pattern of results, with the shape of the F0 topline dependent on sentence type and focus location. For sentences with neutral or sentence-final focus, the difference in the F0 topline between questions and statements was evident only on the last key word, where the F0 peak of questions was considerably higher than that of statements. For sentences with focus on the first key word, there was no difference in peak F0 on the focused item itself, but the F0 toplines of questions and statements diverged quite dramatically following the initial word. The statement contour dropped to a low F0 value for the remainder of the sentence, whereas the question remained quite high in F0 for all subsequent words. In addition, the F0 contour on the focused word was rising in questions and falling in statements, regardless of focus location. The results provide a basis for work on the perception of linguistic focus.

Acoustical aspects of contrastive stress in question-answer contexts.

A series of experiments was conducted to examine the influence of contrastive stress on intonational patterns of duration and fundamental voice frequency (F0). In a preliminary study, intonational patterns in spontaneous speech were compared with the patterns in oral reading of the transcribed spontaneous material. In both circumstances, a general declination of F0 peaks throughout the course of a sentence was not regularly observed, contrary to earlier findings with oral reading of isolated sentences. Two experiments were then conducted to examine the possibility that the general absence of declination observed here might be due to the influence of sentence focus, following previous results from Swedish showing that declination was not observed consistently in isolated sentences until after the site of focus. In experiment 1, we found no evidence of this restriction on declination in the oral reading of English declaratives. Declination was observed both before and after the focused item in a sentence. Furthermore, our analyses revealed that focus (as manifested by contrastive stress) is generally accompanied by an increase in duration on the focused word and by a sharp drop in F0 following the focused item. In experiment 2, these effects were replicated and extended using longer sentences. In both experiments, the influence of focus was strictly localized to the site of focus in the case of duration, but not F0. The results for both duration and F0 indicate that the influence of focus assignment on these attributes varies systematically as a function of the focus location in the sentence. Finally, the combined elongation effects for focus and utterance-final lengthening were less than would be predicted by an additive model, suggesting the operation of an expandability constraint on segmental lengthening in speech production.