Increased vascular density is a persistent feature of airway remodeling in a sheep model of chronic asthma.

BACKGROUND: Increases in blood vessel density and vascular area are now recognized as important features of remodeled airways in asthma. However, the time sequence for these vascular changes and whether they resolve in the absence of continued antigenic exposure is not well elucidated. The aim of the present study was to correlate progressive changes in airway vascularity with changes in functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen, and to examine the resolution of vascular remodeling following allergen withdrawal. METHODS: Progressive changes in vascular indices were examined in four spatially separate lung segments that received weekly challenges with HDM allergen for 0, 8, 16, or 24 weeks. Reversibility of these changes was assessed in a separate experiment in which two lung segments received 24 weeks of HDM challenges and either no rest or 12 weeks rest. Lung tissue was collected from each segment 7 days following the final challenge and vascular changes assessed by a morphometric analysis of airways immunohistochemically stained with an antibody against type IV collagen. RESULTS: Blood vessel density and percent airway vascularity were significantly increased in bronchi following 24 weeks of HDM challenges compared to untreated controls (P < .05), but not at any of the other time-points. There was no significant correlation between vascular indices and airway responses to allergic or nonspecific stimuli. The increase in blood vessel density induced by repeated allergen exposures did not return to baseline levels following a 12-week withdrawal period from allergen. CONCLUSIONS: Our results show for the first time that the airways of sheep chronically exposed to HDM allergen undergo vascular remodeling. These findings show the potential of this large animal model for investigating airway angiogenesis in asthma.

Exploration of Gender-Sensitive Care in Vocational Rehabilitation Providers Working With Youth With Disabilities: Codevelopment of an Educational Simulation.

BACKGROUND: Although research shows that there is a need for gender-specific vocational support to help youth with disabilities find employment, health care providers often report needing more training in this area. Currently, there are no existing educational simulations of gender-sensitive care within vocational rehabilitation for clinicians who provide care to youth with disabilities. Therefore, developing further educational tools that address gender-sensitive care could help them enhance the care they provide while optimizing patient outcomes. OBJECTIVE: This study aims to codevelop an educational simulation and identify issues relevant to providing gender-sensitive care within the context of vocational rehabilitation for youth with disabilities. METHODS: We used a qualitative co-design approach with a purposive sampling strategy that involved focus group discussions and journal reflections to understand and address issues relevant to gender-sensitive care within vocational rehabilitation for those working with youth with disabilities. A total of 10 rehabilitation providers participated in two sessions (5 participants per session) to design the web-based simulation tool. The sessions (2.5 hours each) were audio recorded, transcribed, and analyzed thematically. RESULTS: Two main themes arose from our analysis of codeveloping a simulation focusing on gender-sensitive care. The first theme involved the relevance of gender within clinical practice; responses varied from hesitance to acknowledging but not talking about it to those who incorporated gender into their practice. The second theme focused on creating a comfortable and safe space to enable gender-sensitive care (ie, included patient-centered care, effective communication and rapport building, appropriate language and pronoun use, respecting gender identity, awareness of stereotypes, and responding to therapeutic ruptures). CONCLUSIONS: Our web-based gender-sensitive care simulation that addressed vocational rehabilitation among youth with disabilities was cocreated with clinicians. The simulation highlights many issues relevant to clinical practice and has potential as an educational tool for those working with young people with disabilities.

Identification of genes associated with regenerative success of Xenopus laevis hindlimbs.

BACKGROUND: Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles. RESULTS: In the current study, we have taken advantage of the N1 transgenic line to directly compare morphology and gene expression in same stage regenerating vs. BMP signalling deficient non-regenerating hindlimb buds. The wound epithelium of N1 transgenic hindlimb buds, which forms over the cut surface of the limb bud after amputation, does not transition normally into the distal thickened apical epithelial cap. Instead, a basement membrane and dermis form, indicative of mature skin. Furthermore, the underlying mesenchyme remains rounded and does not expand to form a cone shaped blastema, a normal feature of successful regeneration. Using Affymetrix Gene Chip analysis, we have identified genes linked to regenerative success downstream of BMP signalling, including the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Gene Ontology analysis showed that genes involved in embryonic development and growth are significantly over-represented in regenerating early hindlimb buds and that successful regeneration in the Xenopus hindlimb correlates with the induction of stress response pathways. CONCLUSION: N1 transgenic hindlimbs, which do not regenerate, do not form an apical epithelial cap or cone shaped blastema following amputation. Comparison of gene expression in stage matched N1 vs. wild type hindlimb buds has revealed several new targets for regeneration research.

Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles.

Bone morphogenetic protein (BMP) signalling is necessary for both the development of the tail bud and for tail regeneration in Xenopus laevis tadpoles. Using a stable transgenic line in which expression of the soluble BMP inhibitor noggin is under the control of the temperature inducible hsp70 promoter, we have investigated the timing of the requirement for BMP signalling during tail regeneration. If noggin expression is induced followed by partial amputation of the tail, then wound closure and the formation of the neural ampulla occur normally but outgrowth of the regeneration bud is inhibited. Furthermore, we show that BMP signalling is also necessary for limb bud regeneration, which occurs in Xenopus tadpoles prior to differentiation. When noggin expression is induced, limb bud regeneration fails at an early stage and a stump is formed. The situation appears similar to the tail, with formation of the limb bud blastema occurring but renewed outgrowth inhibited. The transcriptional repressor Msx1, a direct target of BMP signalling with known roles in vertebrate appendage regeneration, fails to be re-expressed in both tail and limb in the presence of noggin. DNA labelling studies show that proliferation in the notochord and spinal cord of the tail, and of the blastema in the limb bud, is significantly inhibited by noggin induction, suggesting that in the context of these regenerating appendages BMP is mainly required, directly or indirectly, as a mitogenic factor.

The experience of senior stroke survivors: factors in community participation among wheelchair users.

PURPOSE: This qualitative study investigated the lived experience of senior stroke survivors who used prescribed wheelchairs in their home and community. METHODS: The study involved semi-structured, in-depth interviews that were conducted with 10 participants, aged 70 to 80, who had used a wheelchair for a mean of 5.6 years. All participants used manual wheelchairs and two additionally used a power wheelchair and a scooter. RESULTS: A constant comparative inductive method of analysis of the data yielded several themes which are examined within the International Classification of Functioning, Disability and Health framework of body functions and structure, activity limitations, participation, and contextual factors. Overall, the wheelchair, manual or power, was an enabler of community participation among the participants. However, the wheelchair's use also created problems, such as limiting destinations and creating increased dependence on others. PRACTICE IMPLICATIONS: A balance scale model was proposed to facilitate an understanding of how the factors affecting community participation interact in this population. This model can assist occupational therapists who prescribe wheelchairs for stroke clients for use in the community.

The Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma.

Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 µm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.

Expression of the sweet protein brazzein in maize for production of a new commercial sweetener.

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.

Delivery of subunit vaccines in maize seed.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

Acceptance and meanings of wheelchair use in senior stroke survivors.

The purpose of this qualitative study was to gain understanding of the lived experience of senior stroke survivors who used prescribed wheelchairs in their homes and communities. The study involved semistructured, in-depth interviews that were conducted with 10 participants, ages 70 to 80 years old, who had used a wheelchair for a mean of 5.6 years. A constant comparative inductive method of analysis was performed. Three different categories of acceptance of wheelchair use were identified; reluctant acceptance, grateful acceptance, and internal acceptance. Increased mobility, varied social response, and loss of some valued roles were common to all three wheelchair acceptance categories. Aspects of level of burden, freedom, and spontaneity varied in degree among the three acceptance categories. As the wheelchair provided opportunity for increased continuity in the lives of these stroke survivors, it appeared to be accepted more fully and viewed more positively. Prestroke lifestyle and values need to be carefully considered in order to maximize acceptance of wheelchair use among senior stroke survivors.

K(Ca)3.1 channel-blockade attenuates airway pathophysiology in a sheep model of chronic asthma.

BACKGROUND: The Ca(2+)-activated K(+) channel K(Ca)3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K(Ca)3.1 modifies experimental asthma in sheep. METHODOLOGY AND PRINCIPAL FINDINGS: Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K(Ca)3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling. CONCLUSIONS: These findings suggest that K(Ca)3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K(Ca)3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Increased mast cell density and airway responses to allergic and non-allergic stimuli in a sheep model of chronic asthma.

BACKGROUND: Increased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: MC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MC(T) and MC(TC) respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase. MC(T) and MC(TC) density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MC(TC)/MC(T) ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MC(T) and MC(TC) density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MC(T) density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01). CONCLUSIONS: MC(T) and MC(TC) density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation.

Assessment of peripheral airway function following chronic allergen challenge in a sheep model of asthma.

BACKGROUND: There is increasing evidence that the small airways contribute significantly to the pathophysiology of asthma. However, due to the difficulty in accessing distal lung regions in clinical settings, functional changes in the peripheral airways are often overlooked in studies of asthmatic patients. The aim of the current study was to characterize progressive changes in small airway function in sheep repeatedly challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: Four spatially separate lung segments were utilized for HDM challenges. The right apical, right medial, right caudal and left caudal lung segments received 0, 8, 16 and 24 weekly challenges with HDM respectively. A wedged-bronchoscope technique was used to assess changes in peripheral resistance (R(p)) at rest, and in response to specific and non-specific stimuli throughout the trial. Allergen induced inflammatory cell infiltration into bronchoalveolar lavage and increases in R(p) in response to HDM and methacholine were localized to treated lung segments, with no changes observed in adjacent lung segments. The acute response to HDM was variable between sheep, and was significantly correlated to airway responsiveness to methacholine (r(s) = 0.095, P<0.01). There was no correlation between resting R(p) and the number of weeks of HDM exposure. Nor was there a correlation between the magnitude of early-phase airway response and the number of HDM-challenges. CONCLUSIONS: Our findings indicate that airway responses to allergic and non-allergic stimuli are localized to specific treated areas of the lung. Furthermore, while there was a decline in peripheral airway function with HDM exposure, this decrease was not correlated with the length of allergen challenge.

Overexpression of the transcription factor Msx1 is insufficient to drive complete regeneration of refractory stage Xenopus laevis hindlimbs.

Xenopus laevis tadpoles are capable of hindlimb regeneration, although this ability declines with age. Bmp signaling is one pathway known to be necessary for successful regeneration to occur. Using an inducible transgenic line containing an activated version of the Bmp target Msx1, we assessed the ability of this transcription factor to enhance regeneration in older limbs. Despite considerable evidence correlating msx1 expression with regenerative success in vertebrate regeneration models, we show that induction of msx1 during hindlimb regeneration fails to induce complete regeneration. However, we did observe some improvement in regenerative outcome, linked to morphological changes in the early wound epithelium and a corresponding increase in proliferation in the underlying distal mesenchyme, neither of which are maintained later. Additionally, we show that Msx1 is not able to rescue limb regeneration in a Bmp signalling-deficient background, indicating that additional Bmp targets are required for regeneration in anuran limbs. Developmental Dynamics 238:1366-1378, 2009. (c) 2009 Wiley-Liss, Inc.

Corn as a production system for human and animal vaccines.

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.

Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.