Elucidating three-way interactions between soil, pasture and animals that regulate nitrous oxide emissions from temperate grazing systems.

Pasture-based livestock farming contributes considerably to global emissions of nitrous oxide (N2O), a powerful greenhouse gas approximately 265 times more potent than carbon dioxide. Traditionally, the estimation of N2O emissions from grasslands is carried out by means of plot-scale experiments, where externally sourced animal excreta are applied to soils to simulate grazing conditions. This approach, however, fails to account for the impact of different sward types on the composition of excreta and thus the functionality of soil microbiomes, creating unrealistic situations that are seldom observed under commercial agriculture. Using three farming systems under contrasting pasture management strategies at the North Wyke Farm Platform, an instrumented ruminant grazing trial in Devon, UK, this study measured N2O emissions from soils treated with cattle urine and dung collected within each system as well as standard synthetic urine shared across all systems, and compared these values against those from two forms of controls with and without inorganic nitrogen fertiliser applications. Soil microbial activity was regularly monitored through gene abundance to evaluate interactions between sward types, soil amendments, soil microbiomes and, ultimately, N2O production. Across all systems, N2O emissions attributable to cattle urine and standard synthetic urine were found to be inconsistent with one another due to discrepancy in nitrogen content. Despite previous findings that grasses with elevated levels of water-soluble carbohydrates tend to generate lower levels of N2O, the soil under high sugar grass monoculture in this study recorded higher emissions when receiving excreta from cattle fed the same grass. Combined together, our results demonstrate the importance of evaluating environmental impacts of agriculture at a system scale, so that the feedback mechanisms linking soil, pasture, animals and microbiomes are appropriately considered.

Smart forage selection could significantly improve soil health in the tropics.

The use of tropical grasslands to graze livestock is of high economic importance. Declining grassland soil health leads to reduced sustainability of livestock systems. There are high levels of phenotypic diversity amongst tropical forage grasses. We hypothesise that this variation could lead to significant differences in soil health and that selection of forage cultivars to improve soil health could improve the sustainability of livestock production. We measured and compared key soil health metrics (soil organic carbon (SOC) concentration and sugar / alkane composition, aggregate stability, friability, litter decomposition rates, microbial community composition) under four tropical forage varieties (Brachiaria hybrid cv Mulato (BhMulato), B. humidicola cv Tully (CIAT679; Bh679), B. humidicola cv CIAT16888 (Bh16888), and Panicum maximum CIAT 6962 (Pmax)) and a bare soil control, there was a significant difference in soil aggregate stability, friability and SOC concentration between the forage varieties with soil under Bh679 and Bh16888 tending to have greater aggregate stability, friability and SOC concentrations compared to the soil under BhMulato and Pmax. We identified significant spatial variation in soils under BhMulato and Pmax due to their tussock forming growth habit; when compared to soil from adjacent to the tussocks, soil from the gaps between tussocks had significantly reduced aggregate stability under both species, significantly reduced friability under Pmax and significantly reduced SOC under BhMulato. We found limited impact of forage variety on soil microbial community composition, litter decomposition rates or soil alkane and sugar concentrations.

Primary isolation of the bovine spongiform encephalopathy agent in mice: agent definition based on a review of 150 transmissions.

In the epizootic of bovine spongiform encephalopathy (BSE) in Great Britain, the cattle in which a positive diagnosis was made numbered almost 180 000, but strain characterization was performed on only a very small sample of these cases. This report describes the results of BSE transmission to Prnp(a) mice from 150 transmission experiments at the Veterinary Laboratories Agency (VLA) over the last decade. These data, derived from a large sample of BSE-affected cattle, confirmed previous reports that show no evidence for diversity in BSE isolates. The agent was readily transmitted to mice, with a mean incubation period of 408 days in the RIII strain. Because the incubation period was related to the titre of the inoculum, it is not a reliable characteristic of strain type on primary isolation. Consistent neuropathological changes associated with infection by the BSE agent in RIII and C57Bl mice included focal vacuolation in the dorsal cochlear nuclei, vacuolation of the granule cell layer of the cerebellum, absence of lesions in the hippocampus and in the molecular layer of the cerebellum, and a fine particulate distribution of disease-specific PrP (demonstrated immunohistochemically), with few or no amyloid plaques. These features, together with the conventional lesion profile, will be of use in distinguishing the agents of BSE and scrapie in sheep.

Cell separation improves the sensitivity of detecting rare human normal and leukemic hematopoietic cells in vivo in NOD/SCID mice.

BACKGROUND: This report describes a novel cell-separation procedure developed to improve detection and analysis of rare human hematopoietic populations, obtained from NOD/SCID mice engrafted with normal and/or leukemic stem cells. METHODS: In preliminary experiments, artificial mixtures of murine and human BM cells were labeled with a combination of Abs specific for murine hematopoietic cells, prior to immunomagnetic negative selection using StemSep. In subsequent experiments, BM was harvested from individual NOD/SCID mice transplanted 6-12 weeks earlier with either human cord blood or primary CML cells and a similar immunomagnetic selection procedure was applied to enrich human cells present. RESULTS: Application of this selection procedure to mixtures of murine and human hematopoietic cells using anti-mouse CD45 and Ter-119 allowed a > 1000-fold depletion of murine cells with > 50% recovery of human cells, including progenitors. This level of depletion and recovery were found to be reproducible for NOD/SCID mice transplanted and engrafted with human cord blood stem cells, thus facilitating detection of human progenitors, including colony-forming cells (CFC) and LTCIC. For NOD/SCID mice previously transplanted with CML cells, this procedure increased the sensitivity of detecting rare human cell subsets by up to > 100-fold. This, in turn, improved the sensitivity of RT-PCR for BCR-ABL and made possible the identification by FACS of various minor subsets of human cells, including CD34(-)CD19/20(+) B-lineage cells, CD34(+) progenitors, mature CD15(+) myeloid cells and CD3(+) T cells present in the mice. DISCUSSION: This simple cell-depletion procedure should facilitate future investigations of normal and CML stem cell populations in vitro and in NOD/SCID mice.

Selective amplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping.

Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.

Antigen-independent expansion of T cells from psoriatic skin lesions: phenotypic characterization and antigen reactivity.

The pathogenesis of psoriasis appears to depend on T cells, which have been proposed to mediate the disease through an autoimmune process. To test this hypothesis we have propagated four T-cell lines from biopsies of psoriatic skin lesions by antigen-independent methods. Flow cytometric immunophenotyping showed the lines to be composed mainly of CD4-positive, alpha beta T-cell receptor (TCR)-positive cells, which secreted a cytokine profile suggestive of predominant T-helper type 1 (Th1) status. Analysis of TCR variable region (V beta) usage revealed two- to eight-fold increases in the expression of certain V beta species in lesional lines as compared with autologous peripheral blood mononuclear cells (PBMC), with the increased V beta species being expressed on more than 5% of cells in two of the lines. Lines were also used to test for responses to a range of epidermal antigen preparations in the presence of irradiated autologous PBMC as antigen-presenting cells. The lines failed--to proliferate in response to psoriatic lesional stratum corneum extracts, dispase-separated normal human epidermal extracts, and an epidermal keratin preparation before and after trypsinization, in spite of good proliferative responses to anti-CD3 which indicated that the lines were not anergic. In addition, the lines and PBMC from normal volunteers and the patients with psoriasis gave little or no response to recombinant streptococcal M protein. Thus, in spite of accumulating evidence for selective expansion of certain V beta-expressing T cells in psoriatic lesions, epidermal autoantigens have not been identified by using a bioassay which depended largely on the proliferation of lesional CD4-positive cells. The role of streptococcal M protein, which bears some homology with epidermal keratin is also open to question, at least in chronic plaque psoriasis. Further work is therefore required to obtain direct evidence that autoimmune processes are important in the pathogenesis of chronic plaque psoriasis.

Validation of the combined olfactory test.

A new test of olfactory function, the combined olfactory test, has been designed to assess odours easily recognizable by the test population. The test consists of an odour recognition test of nine odours, where an odour in a bottle is chosen from a list of four possible odours in a forced choice manner. This is followed by a threshold test using a series of three-fold dilutions of 1-butanol. The mean of the two scores is the combined olfactory score. The test was subjected to a validation study. It was performed on 133 participants with a normal sense of smell and a normal rhinological examination and on 94 participants who said that they did not have a sense of smell. There was a highly significant difference between the combined olfactory score in the normal and 'anosmic' groups (P < 0.001). This significant difference was the same between the two groups for the threshold and odour recognition arms of the test. There was a highly significant difference (P < 0.001) between the two subgroups of 'completely anosmic' and 'almost anosmic' participants, indicating that the test could grade the degree of olfactory dysfunction.

Hazards of microwave cooking: direct thermal damage to the pharynx and larynx.

A case of a direct burn to the pharynx and larynx is described. This occurred after a patient swallowed a microwaved treacle tart. The potential danger of excessive heating of food in microwave ovens is discussed.

Soluble IL-2 receptor and CD25 cells in psoriasis: effects of cyclosporin A and PUVA therapy.

A study was conducted to quantify soluble IL-2 receptor (sIL-2R) levels in sera of 57 chronic plaque psoriasis patients and correlate these measurements with disease activity and the number of IL-2R-positive (CD25+) lymphocytes in lesional biopsies of 11 cyclosporin A (CsA) and 13 psoralen plus ultraviolet radiation (PUVA) treated patients. Levels of sIL-2R showed a strong correlation with the psoriasis area and severity index (PASI). CsA and PUVA significantly reduced the PASI and sIL-2R levels to a similar degree after 4 weeks of treatment. Although the majority of CsA-treated patients who were biopsied showed reductions in lesional CD25+ cells, these did not reach statistical significance; in five patients biopsied who had PUVA treatment, no consistent effect on the numbers of CD25+ cells was observed. A significant correlation was found between CD25+ cells in lesional biopsies and the PASI score.

Adhesion molecule expression in psoriatic skin lesions and the influence of cyclosporin A.

Normal skin of healthy individuals and both lesional and uninvolved skin from patients with psoriasis before and after receiving cyclosporin A (CsA; 2.5 or 5 mg/kg per day) was examined by immunocytochemistry for differences in expression of adhesion-relevant epitopes. Normal, lesional and uninvolved skin all showed staining of basal keratinocytes for CD29 (the common beta chain of the beta 1-integrin family). No other adhesion molecule investigated was detected on structural components of normal skin. In uninvolved skin, weak expression of CD54 (intercellular adhesion molecule 1, ICAM-1) was noted on vascular endothelium. Uninvolved keratinocytes were found to stain with anti-CD58 (leucocyte function-associated antigen 3, LFA-3) and there was weak expression of CD11b (alpha chain of complement C3bi receptor) and CD11c (alpha chain of p150, 95 molecule) but not CD11a (leucocyte function-associated antigen 1, LFA-1, alpha chain) on those cells. In lesional skin, in addition to expression of CD58, there was also enhanced expression of CD11c. Weak expression of CD54 on keratinocytes was also observed. Lesional blood vessels were found to stain strongly with anti-CD54, CD29 and CD58. CD11a was expressed only on infiltrating mononuclear cells. CsA treatment produced marked clinical improvement, accompanied by the loss of CD54 expression on keratinocytes. However, despite the loss of T cells from lesional skin with CsA treatment, CD54 persisted on blood vessels. CsA was found to have no effect on keratinocyte expression of CD29, CD58 or CD11b and c. The persistence of CD54 on vascular endothelium and of adhesion molecule expression on keratinocytes, despite resolution of the skin lesions, may explain the universal and rapid recurrence of psoriasis on cessation of CsA administration.

Differential effects of cyclosporine A on Langerhans cells and regulatory T-cell populations in severe psoriasis: an immunohistochemical and flow cytometric analysis.

Systemic administration of cyclosporine A (Cy-A; initial dose 5 or 2.5 mg/kg/day) to patients with severe chronic plaque psoriasis produced marked reductions in psoriasis area and severity index within 4 weeks. The clinical response was accompanied, within 1 week, by progressive reductions in T-cell subpopulations (CD3+ and CD4+) and in numbers of interleukin-2 receptor (IL-2-R)-positive (CD25+) cells within lesional skin. Over the first 4 weeks of treatment, these changes were accompanied by reductions in DR+ cells within the epidermis (minor) and dermis (substantial). In contrast, numbers of epidermal CD1+ cells increased substantially during resolution of the skin lesions. Unlike lesional skin, however, no significant changes in absolute numbers of circulating immunoregulatory T-cell populations, including helper/inducer (CD45R) and suppressor/inducer (CD29W) subsets, quantified by dual immunofluorescence labelling, were detected. Moreover, numbers of blood-borne HLA-DR, IL-2-R and transferrin receptor (CD71) positive lymphocytes were unaffected by Cy-A therapy, nor were any differences detected between psoriatic patients and normal controls using these cell markers. Our data suggest that the immunoregulatory effects of Cy-A in psoriasis are mediated via lesional T lymphocytes and that epidermal CD1+ DR- dendritic cells may play an influential role in the regulation of T-cell function and keratinocyte growth during resolution of the skin lesions.

Flow cytometric and histological analysis of ductal carcinoma in situ of the breast.

Using archival paraffin wax embedded tumour we have investigated histological grade, DNA ploidy, S phase fraction and proliferative index in 74 patients with symptomatic ductal carcinoma in situ (DCIS) of the breast. Nine patients developed local recurrence, six invasive in character. No patients with the cribriform subtype of DCIS developed local recurrence. The cribriform subtype showed a significantly lower rate of DNA aneuploidy and a lower proliferative index than the other subtypes. Cribriform tumours were almost exclusively well differentiated in contrast with the comedo and solid variants. Our results suggest the cribriform variant is less aggressive than other subtypes of DCIS. This has possible implications for management of these lesions.

Vaginal candidosis: relation between yeast counts and symptoms and clinical signs in non-pregnant women.

Of 277 non-pregnant women, 67 (24%) harboured Candida albicans in the genital tract. Of 56 women yielding C albicans who had no other infection diagnosed, 14 were symptomless and 10 had no clinical signs of vulval redness, vaginitis, or discharge. Symptoms and signs tended to be more severe in women with higher yeast counts. Of 22 women with moderate or severe symptoms (pruritus with or without discharge) and signs, 15 had yeast counts of more than 10(3) colony forming units (cfu)/ml, whereas six of nine women with no symptoms or signs had counts of fewer than 10(3) cfu/ml.

Evaluation of a new slide latex agglutination test for diagnosis of vaginal candidosis.

A new commercial slide latex particle agglutination test for rapid (2 min) diagnosis of vaginal candidosis was evaluated and compared with conventional methods. Of the 263 women studied, 63 (23.9%) had yeasts in the vagina. Clinical signs of vulvitis or vaginitis were seen in 23 women (8.8%) and 40 (15.2%) were harbouring yeasts without clinical signs. Yeast counts were generally higher in women with clinical signs of vaginal candidosis than in those without. The test was positive in 15 of the 23 women (65.2%) with clinical signs, the incidence of a positive test increasing in direct proportion to the amount of yeasts isolated. The test's sensitivity, specificity and predictive values were comparable to those of microscopy and culture. Being both rapid and simple to perform, this new test offers a useful alternative to conventional methods for the diagnosis of vaginal candidosis.