Allergic rhinitis is a local disease: the role of local IgE production, basophils and mast cells

The introduction to this thesis summarizes the literature which indicates that there is a discrepancy between sensitisation and allergic disease. Two aspects which might play a role in this discrepancy are the differences between production and funtion of local versus systemic lgE and the differences in mast cell and basophil function in the blood compared to in the tissues. Mast cells, basophils and IgE are key players in the allergic inflammation. The aim of the studies described in this thesis was to focus on these differences between local and systemic function of these key factors. The research questions addressed in this thesis are: Mast cells and basophils seem to play an important role in the pathogenesis of allergic rhinitis. Do the phenotypes of mast cells and basophilic cells changed by allergen provocation in the nasal mucosa of allergic rhinitis patients (Chapter 3)? The developmental relationship bet\veen mast cells and basophils has not yet been totally resolved. What is the relation bet\veen basophil progenitors, mast cell progenitors, basophils and mast cells in the circulation and in the nasal mucosa (Chapter 3)? Allergic mucosa inflammation is regulated by the local production and release of several Th2 cytokines. Which increase in cytokines and chemokines is correlated to inflammatory cells and symptomatology of the patient? What is the time line of the various cytokines and chemokines after allergen provocation (Chapter 4)? Is it possible to develop a method to detect specific lgE in tissues. Does production of specific IgE take place locally in the nasal mucosa (Chapter 5)? Where do basophils and mast-cell of allergi

Psychometric properties of the Dutch version of the Evidence-Based Practice Attitude Scale (EBPAS).

BackgroundThe Evidence-Based Practice Attitude Scale (EBPAS) was developed in the United States to assess attitudes of mental health and welfare professionals toward evidence-based interventions. Although the EBPAS has been translated in different languages and is being used in several countries, all research on the psychometric properties of the EBPAS within youth care has been carried out in the United States. The purpose of this study was to investigate the psychometric properties of the Dutch version of the EBPAS.MethodsAfter translation into Dutch, the Dutch version of the EBPAS was examined in a diverse sample of 270 youth care professionals working in five institutions in the Netherlands. We examined the factor structure with both exploratory and confirmatory factor analyses and the internal consistency reliability. We also conducted multiple linear regression analyses to examine the association of EBPAS scores with professionals' characteristics. It was hypothesized that responses to the EBPAS items could be explained by one general factor plus four specific factors, good to excellent internal consistency reliability would be found, and EBPAS scores would vary by age, sex, and educational level.ResultsThe exploratory factor analysis suggested a four-factor solution according to the hypothesized dimensions: Requirements, Appeal, Openness, and Divergence. Cronbach's alphas ranged from 0.67 to 0.89, and the overall scale alpha was 0.72. The confirmatory factor analyses confirmed the factor structure and suggested that the lower order EBPAS factors are indicators of a higher order construct. However, Divergence was not significantly correlated with any of the subscales or the total score. The confirmatory bifactor analysis endorsed that variance was explained both by a general attitude towards evidence-based interventions and by four specific factors. The regression analyses showed an association between EBPAS scores and youth care professionals' age, sex, and educational level.ConclusionsThe present study provides strong support for a structure with a general factor plus four specific factors and internal consistency reliability of the Dutch version of the EBPAS in a diverse sample of youth care professionals. Hence, the factor structure and reliability of the original version of the EBPAS seem generalizable to the Dutch version of the EBPAS

Growth and Fecundity of Several Weed Species in Corn and Soybean

Do weeds that emerge later in the season justify additional control costs\u27? If crop yield is not reduced or few or no seeds arc added to the soil seed hank, then no control may he needed. Eight weed species were sown in corn (Zea mays L.) and soybean I Glycine max (L.) Mcrr.l (i) before crop emergence, (ii) at crop emergence, (iii) at V-1, and (iv) at V-2 stages of crop growth in 2002 and 2003. Weed seed was sown close to the crop row and thinned to 1.3 plants m 2• Weed growth and fecundity were influenced by species, time of planting, and year. Only barnyarclgrass (Echinochloa crus-galli L.), rcclroot pigwced (Amaranthus retniflexus L.), and vclvetlcaf (Abutilon theophrasti L.) survived to produce seed. Plants from the pre-emergence seeding had the largest canopy and produced the most seeds. Harnyardgrass had maximum canopy cover in early .July in corn and late .Inly in soybean hut only produced seed in corn. Rcclroot pigweecl and vclvctleaf had maximum canopy cover in late August or midSeptember, and some plants from most seeding elates survived and produced seed in both corn and soybean. However, plants that grew from seed sown at V-1 and V-2 crnp grnwth stages did not reduce yield or biomass of adjacent crop plants, had low fecundity, and may not warrant treatment. Control may be necessary, however, to prevent yield losses if weeds arc present at high densities or to prevent establishment of uncommon species

Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

<p>Abstract</p> <p>Background</p> <p>The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.</p> <p>Results</p> <p>The initial pattern of corneal epithelial patches in <it>XLacZ</it>^+/-X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of <it>XLacZ</it>^+/-eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.</p> <p>Conclusion</p> <p>Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.</p

Differences in nasal cellular infiltrates between allergic children and age-matched controls

Little is known about the cellular infiltrates in the nasal mucosa of children. This study was set up to compare the nasal cellular infiltrates in biopsy specimens from allergic children and controls. Atopic children were distinguished from controls on the basis of symptoms of allergic rhinitis and/or asthma, total serum immunoglobulin (Ig)E, family history and specific serum IgE to food and aeroallergens. Fifteen allergic patients (median age 4.3 yrs) and 15 age-matched nonallergic control subjects were evaluated. The number of cells positive for CD1a, CD4, CD8, CD19, CD68, chymase, tryptase, IgE and major basic protein was determined in the mucosa of the inferior turbinate. A significantly higher number of IgE-positive cells and mast cells was found in the epithelia of the allergic group. In the lamina propria, higher numbers of IgE-positive cells and eosinophils were found. Langerhans' cells positive for IgE were only seen in allergic children with specific serum IgE against aeroallergens. These children also had a higher number of IgE-positive mast cells compared to controls and atopic children without specific serum IgE. These results show that the nasal cellular infiltrates of allergic children differ from nonallergic control subjects. Prior to the detection of specific serum immunoglobulin E, cellular changes can be found in the nasal mucosa of atopic children

Spatial Distribution, Temporal Stability, and Yield Loss Estimates for Annual Grasses and Common Ragweed (Ambrosia artimisiifolia) in a Corn/Soybean Production Field Over Nine Years

Weeds generally occur in patches in production fields. Are these patches spatially and temporally stable? Do management recommendations change on the basis of these data? The population density and location of annual grass weeds and common ragweed were examined in a 65-ha corn/soybean production field from 1995 to 2004. Yearly treatment recommendations were developed from field means, medians, and kriging grid cell densities, using the hyperbolic yield loss (YL) equation and published incremental YL values (I ), maximum YL values (A), and YL limits of 5, 10, or 15%. Mean plant densities ranged from 12 to 131 annual grasses m22 and , 1 to 37 common ragweed m22. Median weed densities ranged from 0 to 40 annual grasses m22 and were 0 for common ragweed. The grass I values used to estimate corn YL were 0.1 and 2% and treatment was recommended in only 1 yr when the high I value and either the mean or median density was used. The grass I values used for soybean were 0.7 and 10% and estimated YL was over 10% all years, regardless of I value. The common ragweed I values were 4.5 and 6% for corn and 5.1 and 15.6% for soybean. On the basis of mean densities, fieldwide treatment would have been recommended in 6 of 9 yr but in no years when the median density was used. Recommendations on the basis of grid cell weed density and kriging ranged from . 80% of the field treated for grass weeds in 3 of 4 yr in soybean to , 20% of the field treated for common ragweed in 2002 and 2004 (corn). Grass patches were more stable in time, space, and density than common ragweed patches. Population densities and spatial distribution generally were variable enough so that site-specific information within this field would improve weed management decisions

Functional conservation of Pax6 regulatory elements in humans and mice demonstrated with a novel transgenic reporter mouse

<p>Abstract</p> <p>Background</p> <p>The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the <it>Pax6 </it>gene model closely humans with loss-of-function mutations in the <it>PAX6 </it>locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown <it>structural </it>conservation of the gene's regulatory regions.</p> <p>Results</p> <p>We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human <it>PAX6 </it>regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry.</p> <p>Conclusion</p> <p>This work demonstrates the <it>functional </it>conservation of the regulatory elements controlling <it>Pax6/PAX6 </it>expression in mice and humans. The transgene provides an excellent tool for studying the functions of different <it>Pax6/PAX6 </it>regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which <it>Pax6 </it>is activated, irrespective of the status of the endogenous locus.</p

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

Allogeneic chondrogenically differentiated human bone marrow stromal cells do not induce dendritic cell maturation

Bone marrow stromal cell (BMSC)-mediated endochondral bone formation may be a promising alternative to the current gold standards of autologous bone transplantation, in the development of novel methods for bone repair. Implantation of chondrogenically differentiated BMSCs leads to bone formation in vivo via endochondral ossification. The success of this bone formation in an allogeneic system depends upon the interaction between the implanted constructs and the host immune system. The current study investigated the effect of chondrogenically differentiated human bone marrow stromal cell (hBMSC) pellets on the maturation and function of dendritic cells (DCs) by directly coculturing bone forming chondrogenic hBMSC pellets and immature or lipopolysaccharide (LPS)-matured DCs in vitro. Allogeneic chondrogenic hBMSC pellets did not affect the expression of CD80, CD86, or HLADR on immature or LPS-matured DCs following 24, 48, or 72 hr of coculture. Furthermore, they did not induce or inhibit antigen uptake or migration of the DCs over time. IL-6 was secreted by allogeneic chondrogenic hBMSC pellets in response to LPS-matured DCs. Overall, this study has demonstrated that maturation of immature DCs was not influenced by allogeneic chondrogenic hBMSC pellets. This suggests that allogeneic chondrogenic hBMSC pellets do not stimulate immunogenic responses from DCs in vitro and are not expected to indirectly activate T cells via DCs. For this reason, allogeneic chondrogenic bone marrow stromal cell pellets are promising candidates for future tissue engineering strategies utilising allogeneic cells for bone repair