Farm Exposures and Allergic Disease Among Children Living in a Rural Setting.

OBJECTIVES: The purpose of this study was to examine the association between farm exposures and asthma and allergic disease in children while also highlighting the experiences of non-farm rural children. METHODS: This was a cross-sectional analysis of data collected from across the province of Saskatchewan, Canada in 2014. Surveys were completed by parents of 2275 rural dwelling children (farm and non-farm) aged 0 to 17 years within 46 rural schools. Questionnaires were distributed through schools for parents to complete. RESULTS: Asthma prevalence was 7.6%, of which 29.5% of cases were allergic. After adjustment for potential confounders, home location (farm vs non-farm) and other farm exposures were not associated with asthma and asthma phenotypes. Those who completed farm safety education were more likely to have asthma (11.7% vs. 6.7%; p = .001) compared to children without asthma. In sub-analyses among 6-12-year-old children, boys were more likely to have asthma (non-allergic) and use short-acting beta-agonists compared to girls. Doing farm work in the summer was associated with an increased risk of asthma [adjusted OR (aOR) = 1.71 (1.02-2.88); p = .041]. Doing routine chores with large animals was associated with an increased risk of asthma [aOR = 1.83 (1.07-3.15); p = .027] and allergic asthma [aOR = 2.37 (95%CI = 1.04-5.40); p = .04]. CONCLUSION: The present study showed that the prevalence of asthma and asthma phenotypes were similar between farm and non-farm rural children. There did not appear to be differential involvement in farming activities between those with and without asthma although those with asthma had more training suggesting possible attempts to mitigate harm from farm exposures.

Field Evaluations of Sulfuryl Fluoride Fumigation for Control of the Common Bed Bug (Hemiptera: Cimicidae), Using a 1.9× Dosage Factor in Motor Vehicles and Filled Cargo Trailers.

This study investigated the efficacy of using Vikane gas fumigant (sulfuryl fluoride) at the 1.9× dosage rate for eliminating bed bugs (Cimex lectularius L.) in two challenging infestation situations: personal vehicles, and confined spaces densely packed with personal belongings. The vehicles used in this study were large minivans with seating that folded into the floor. The confined spaces were cargo trailers filled to 85% capacity with books, furniture, and other household items. Each van and trailer was equipped with ~90 sentinel bed bugs consisting of three groups of 9-11 bed bug eggs, 10 nymphs, and 10 adults. The Vikane Fumiguide calculator was used to determine the target dosage (g-h/m3) to apply in each replicate (e.g., one van or trailer). Sulfuryl fluoride concentrations were measured throughout the fumigation process using a Spectros SF-ReportIR. Concentration readings were input into the Fumiguide to determine when the accumulated dosage (g-h/m3) was achieved, and when aeration should be initiated. After aeration was complete, the sentinel bed bugs were removed from the replicates and bed bug nymph and adult mortality was recorded. Bed bug eggs were monitored for 23 d to determine latent mortality. Fumigated bed bug mortality for each replication was 100% regardless of life stage. Latent mortality was observed in a single bed bug egg, but the first instar never fully eclosed. This study determined that fumigation with sulfuryl fluoride at the 1.9× dosage factor is an effective method for eliminating resistant bed bugs from vehicles and personal belongings in densely packed situations.

CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks lung inflammation in swine barn dust-instilled mice.

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCε was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

Proteinase-activated receptor-2 activation participates in allergic sensitization to house dust mite allergens in a murine model.

BACKGROUND: Many aeroallergens contain proteinase activity and are able to induce allergic sensitization when presented to mucosal surfaces. Some of these allergens activate proteinase-activated receptor-2 (PAR2 ). OBJECTIVE: To determine the role of PAR2 activation in a murine house dust mite (HDM) allergy model. METHODS: We sensitized and challenged PAR2 -deficient mice with HDM, and examined allergic outcomes compared to wild-type animals. To focus on the role of PAR2 in allergic sensitization, we administered a PAR2 blocking antibody to wild-type animals during the sensitization phase and examined the outcomes immediately after sensitization or following subsequent allergen challenge. RESULTS: We found PAR2 -deficient mice sensitized and challenged with HDM failed to develop airway inflammation, did not produce HDM-specific IgG1 and had less IL-4 mRNA in the lungs than wild-type animals. Prevention of PAR2 activation during sensitization in wild-type mice diminished the levels of Th2 mediators, including IL-4, IL-5 and IL-13, in the lungs. Blocking PAR2 during the sensitization phase also led to decreased manifestations of allergic disease, including airway hyperresponsiveness (AHR) and airway inflammation following subsequent allergen challenge. HDM-induced proliferation of splenocytes obtained from animals sensitized in the presence of PAR2 antibody was reduced relative to those that did not receive antibody. The effect of PAR2 blockade could be transferred to naïve mice through splenic CD4(+) T cells from sensitized mice. CONCLUSIONS AND CLINICAL RELEVANCE: PAR2 activation plays a key role during the sensitization phase of our HDM allergy model, leading to increased lung cytokine production and augmented lung reactivity. PAR2 activation is a common mechanism for sensitization to a wide variety of allergens and is therefore a potential pharmacological target to prevent allergy.

Regulatory dendritic cell expression of MHCII and IL-10 are jointly requisite for induction of tolerance in a murine model of OVA-asthma.

BACKGROUND: Allergen-presenting dendritic cells differentiated with IL-10 (DC10) reverse the asthma phenotype in mice by converting their Th2 cells to regulatory T cells (Tregs). DC10 express elevated levels of IL-10, but substantially reduced levels of MHCII and costimulatory molecules, so the relationships between these factors with each other and tolerogenicity have not been clearly elucidated. METHODS: We assessed the roles of these inputs in DC10 reversal of OVA-associated asthma-like disease by treating affected mice with OVA-pulsed DC10 generated from wild-type or IL-10-sufficient MHCII(-/-) or CD80/CD86(-/-) mice, or with MHCII-intact IL-10-silenced DC10. RESULTS: IL-10 silencing did not discernibly affect the cells' immunobiology (e.g., costimulatory molecules, chemokines), but it eliminated IL-10 secretion and the cell's abilities to induce tolerance, as determined by assessments of airway hyper-responsiveness, eosinophilia, and Th2 responses to recall OVA challenge. MHCII(-/-) DC10 expressed normal levels of IL-10, but, nevertheless, were unable to induce allergen tolerance in asthma phenotype mice, while tolerance induced by CD80/CD86(-/-) DC10 was attenuated but not eliminated. We also assessed the induction of multiple Treg cell markers (e.g., ICOS, PD-1, GITR) on pulmonary CD25(+) Foxp3(+) cells in the treated mice. Wild-type DC10 treatments upregulated expression of each marker, while neither IL-10-silenced nor MHCII(-/-) DC10 did so, and the CD80/86(-/-) DC10 induced an intermediate Treg cell activation phenotype. CONCLUSION: Both IL-10 and MCHII expression by DC10 are requisite, but not sufficient for tolerance induction, suggesting that DC10 and Th2 effector T cells must be brought together in a cognate fashion in order for their IL-10 to induce tolerance.

Therapeutic induction of tolerance by IL-10-differentiated dendritic cells in a mouse model of house dust mite-asthma.

BACKGROUND: It has been reported that retrovirally transduced IL-10-expressing dendritic cells can reverse the asthma phenotype in mice, but that i.v. delivery of dendritic cells differentiated with IL-10 alone (DC10) does not. We report herein DC10 can be highly effective therapeutically in experimental asthma. METHODS: BALB/c mice were sensitized by airway exposure to house dust mite (HDM) without use of adjuvants, then treated with 106 allergen-presenting DC10. We assessed the airway hyperresponsiveness (AHR) to methacholine, circulating levels of IgE and IgG1, and airway recall responses to HDM allergen, including eosinophilia and Th2 cytokines. We also asked whether the DC10 treatments induced tolerance through activation of pulmonary regulatory T cell activities. RESULTS: In vitro, cognate-, but not irrelevant-, allergen-presenting DC10 productively engaged pulmonary Th2-phenotype CD4(+) cells magnetically sorted from HDM-asthmatic mice in Forster (or fluorescence) resonance energy transfer assays. In vivo, treatment of HDM-asthmatic mice with HDM, but not ovalbumin-presenting DC10 abrogated AHR within 4 weeks, and significantly reduced airway eosinophilia, IL-4, IL-5, and IL-13 responses, and circulating HDM-specific IgE and IgG1 levels (each, P ≤ 0.01 versus control mice). CD4(+) CD25(+) Foxp3(+) cells from the lungs of the DC10-treated mice, but not those from asthmatic animals, up-regulated expression of the activated regulatory T cell markers CTLA4 and LAG3, and passive transfer of pulmonary CD4(+) T cells from these mice induced allergen tolerance in HDM-asthmatic recipients. CONCLUSIONS: These findings indicate that allergen-presenting DC10 treatments up-regulate T cell regulatory activities and thereby induce allergen-specific tolerance in a relevant model of human asthma.

Measured concentrations of combustion gases from the use of unvented gas fireplaces.

UNLABELLED: Measurements of combustion product concentrations were taken in 30 homes where unvented gas fireplaces were used. Measurements of CO, CO(2), NO(x), NO(2) , O(2) (depletion), and water vapor were taken at 1-min interval. The analyzers were calibrated with certified calibration gases for each placement and were in operation for 3-4 days at each home. Measured concentrations were compared to published health-based standards and guidelines. The two combustion gases that exceeded published values were NO(2) and CO. For NO(2) , the Health Canada guideline of 250 ppb (1-h average) was exceeded in about 43% of the sample and the World Health Organization (WHO) guideline of 110 ppb (1-h average) was exceeded in 80% of the sample. Carbon monoxide levels exceeded the U.S. EPA 8-h average standard of 9 ppm in 20% of the sample. Moisture problems were not evident in the test homes. An analysis of the distribution of CO showed that the CO is dispersed throughout the home almost immediately upon operation of the fireplace and that the concentrations throughout the home away from the immediate vicinity of the fireplace are 70-80% of the level near the fireplace. Decay analysis of the combustion gases showed that NO was similarly stable to CO and CO(2) in the indoor environment but that both NO(2) and water vapor were removed from the air at much greater rates. PRACTICAL IMPLICATIONS: Previous studies on unvented gas fireplaces have made assumptions of how they are operated by users. This article presents the results of field monitoring of 30 unvented gas fireplaces under normal operation, regardless of whether users follow industry recommendations regarding installation, usage patterns, and maintenance. The monitoring found that health-based standards and guidelines were exceeded for CO in 20% of homes and for NO(2) in most homes. There were no identified moisture problems in these homes. Nearly, half of the fireplaces were used at least once for longer than 2 h, counter to manufacturers' intended usage as supplemental heating. This demonstrates that given actual usage patterns and compared to current health-based thresholds, these appliances can produce indoor air concentrations considered to be unhealthy to at least sensitive or at-risk individuals.

Postnatal changes in testicular concentrations of transforming growth factors-alpha and-beta 1, 2 and 3 and serum concentrations of insulin like growth factor I in bulls.

Based on work largely in laboratory animals, transforming growth factors (TGF) and insulin like growth factors (IGF) could be regulators of testicular development. The aim of this study was to see if TGF-alpha and -beta 1, 2 and 3 are present in the bovine testis and to monitor concentrations of these factors in the testis and IGF-I in serum during reproductive development. Separate groups of Hereford x Charolais calves (n = 6) were castrated every 4 weeks from 5 to 33 weeks of age and at 56 weeks of age. A week prior to castration, from 5 to 33 weeks of age, blood was collected every 15 min for 10 h. Serum IGF-I concentrations increased from 8 to 12 weeks of age, decreased from 24 to 28 weeks and increased to 32 weeks of age (p < 0.05). Testicular TGF-alpha concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 33 to 56 weeks of age (p < 0.05). Testicular TGF-beta 1 concentrations decreased from 17 to 21 weeks of age, increased to 25 weeks and decreased from 25 to 33 weeks of age (p < 0.05). Testicular TGF-beta 2 concentrations increased from 5 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks and decreased at 29 weeks of age (p < 0.05). Testicular TGF-beta 3 concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 25 to 29 weeks of age (p < 0.05). We concluded that TGF-alpha and TGF-beta 1, 2 and 3 are present in the testis of the bull calf, and changes in concentrations with age suggest a functional role in the development of the testis.

Post-natal changes in testicular concentrations of interleukin-1 alpha and beta and interleukin-6 during sexual maturation in bulls.

Based on observations in laboratory animals interleukins could be regulators of testicular development. The objects of this study were to see if interleukins (IL-1 and IL-6) are present in the developing bull testis and to establish the temporal patterns of concentrations of IL-1 and IL-6 in the bovine testis during development. Separate groups of six bull calves were castrated every 4 weeks from 5 to 33 weeks of age, and at 56 weeks of age. Mean testicular IL-1 alpha concentrations decreased (p < 0.01) from 5 to 9 weeks of age and 13 to 21 weeks of age. Mean testicular IL-1 beta concentrations decreased (p < 0.01) from 13 to 17 weeks of age and from 29 to 33 weeks of age. Mean IL-1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (p < 0.05). Mean testicular IL-6 concentrations decreased (p < 0.05) from 9 to 13 weeks of age, increased (p < 0.05) to 21 weeks, decreased (p < 0.05) to 25 weeks, increased (p < 0.05) to 29 weeks and decreased (p < 0.01) to 56 weeks of age. In conclusion, testicular IL-1 alpha, IL-1 beta and IL-6 were found in the bovine testis and concentrations were age dependent. Testicular IL-1 alpha and IL-1 beta concentrations were highest in the early post-natal period; however, IL-1 bioactivity and IL-6 concentrations were greatest in the immediate pre-pubertal period. These findings suggest a functional role for interleukins in testicular development in the bull.

Regular salbutamol use increases CXCL8 responses in asthma: relationship to the eosinophil response.

Regular salbutamol use can exacerbate allergen-induced airway eosinophilia in asthmatics, but its effect on airway eosinophil chemokine responses is unknown. Asthmatic subjects (n=14) were treated for 10 days with placebo or salbutamol in a double-blind, cross-over study, then given same-dose allergen challenges. Their sputa were then analysed 1 and 7 h later for a panel of eosinophil-related cytokines. Eosinophils from five test and three control subjects were tested for expression of CXCL8/interleukin (IL)-8, and its receptors and responsiveness to CCL11/eotaxin and CXCL8/IL-8. Sputum CXCL8/IL-8, but not IL-5, CCL5/regulated on activation, T-cell expressed and secreted, CCL7/monocyte chemotactic protein-3, CCL11/eotaxin, granulocyte-macrophage colony-stimulating factor or tumour necrosis factor levels, were increased (42%) by the salbutamol treatments. The CXCL8/IL-8 levels correlated with the proportions of sputum eosinophils and these cells, but not other sputum cells, stained strongly for CXCL8/IL-8. The circulating eosinophils of the tested subjects (n=5) expressed CXCL8/IL-8 receptors and secreted high levels of this chemokine. Neutralisation of sputum CXCL8/IL-8 reduced eosinophil chemotactic responses to these samples by 19 +/- 5%. These data suggest that regular use of salbutamol can augment airway CXCL8/interleukin-8 responses to allergen challenge and that this CXCL8/interleukin-8 could contribute to the airway inflammatory response.

Psychosocial issues near the end of life.

End-of-life care has received increasing attention in the last decade; however, the focus continues to be on the physical aspects of suffering and care to the virtual exclusion of psychosocial areas. This paper provides an overview of the literature on the intra- and interpersonal aspects of dying, including the effects that psychosocial variables have on end-of-life decision-making; common diagnosable mental disorders (e.g., clinical depression, delirium); other types of personal considerations (e.g., autonomy/control, grief); and interpersonal/environmental issues (e.g., cultural factors, financial variables). Six roles that qualified mental health professionals can play (i.e., advocate, counselor, educator, evaluator, multidisciplinary team member, and researcher) are also outlined. Because psychosocial issues are ubiquitous and can have enormous impact near the end of life, properly trained mental health professionals can play vital roles in alleviating suffering and improving the quality of life of people who are dying.

Induction of pulmonary allergen-specific IgA responses or airway hyperresponsiveness in the absence of allergic lung disease following sensitization with limiting doses of ovalbumin-alum.

Respiratory allergies represent a failure to generate nonpathogenic responses to innocuous foreign materials. Herein we assessed the role of the sensitizing dose of allergen in this response/nonresponse paradigm, sensitizing BALB/c mice with 5 ng-2 microg of OVA-alum and assessing their responses to repeated OVA aerosol challenge. Mice sensitized with < or = 25 ng of OVA-alum did not develop atopic antibodies, airway hyperresponsiveness (AHR), eosinophilia, or pulmonary Th2 responses, but the 25-ng group animals did develop significant IgA responses. The mice sensitized with 100 ng of OVA-alum developed AHR in the absence of detectable allergic disease, while the mice sensitized with 250 ng-2 microg of OVA/alum developed full-spectrum allergic disease (i.e., eosinophilia, IgE, IgG1, pulmonary Th2 cytokine responses, and AHR). These data indicate that limiting doses of allergen can differentially induce IgA or AHR in the absence of atopic disease in mice.

Il-8((3-73))K11R is a high affinity agonist of the neutrophil CXCR1 and CXCR2.

In studies aimed at developing a high affinity IL-8 antagonist, our first objective was to generate a high-affinity IL-8 analogue. We targeted amino acids within the receptor-binding domain and found that IL-8((3-73))K11R induced significantly more neutrophil beta-glucuronidase release than either IL-8 or the alternate analogues and, in chemotaxis assays, induced 2-3-fold greater neutrophil responses than IL-8. Furthermore, in competitive radio- or biotinylated-ligand binding assays, IL-8((3-73))K11R was more effective than IL-8, IL-8((3-73)), or its T12S, H13F, and K11R/T12S/H13F analogues in blocking IL-8 binding to neutrophils; 1.8 pmol IL-8((3-73))K11R inhibited by 50% the binding of approximately 20 pmol (125)I-IL-8 to neutrophils. Both IL-8 (a CXCR1/CXCR2 ligand) and the CXCR2-specific ligand GROalpha differentially inhibited binding of (125)I-IL-8((3-73))K11R to neutrophils, albeit weakly, suggesting that IL-8((3-73))K11R is a high affinity ligand for both the CXCR1 and CXCR2. Thus IL-8((3-73))K11R is an excellent candidate for further studies aimed at generating a high affinity IL-8 antagonist.

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

Expression of transforming growth factor beta(1), beta(3), and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax.

OBJECTIVE: To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN: Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS: Four 2- to 4-year-old horses. METHODS: Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS: TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS: Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE: The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.

Neutrophils and B cells express XCR1 receptor and chemotactically respond to lymphotactin.

The C chemokine lymphotactin (Lptn) has been reported to act specifically on CD4(+) and CD8(+) T lymphocytes and natural killer (NK) cells, but not monocytes. However, the chemotactic effect of Lptn on other types of hematopoietic cells has not been well studied. In this study we investigated (i) the chemotactic influences of Lptn on T and B lymphocytes, neutrophils, monocytes, and dendritic cells, and (ii) the expression of the Lptn receptor (XCR1) by these cells, using RT-PCR. Our data showed that Lptn is chemotactic for B lymphocytes and neutrophils as well as T lymphocytes, but not for monocytes or dendritic cells, and that XCR1 expression is found only in association with T and B lymphocytes and neutrophils, but not monocytes or dendritic cells. Thus, this study is the first demonstration of a chemotactic effect of Lptn on neutrophils and confirms the association of this effect with expression of the XCR1 receptor on these cells. These data suggest that Lptn could potentially be an important protein in the regulation of T and B lymphocytes and neutrophil trafficking, and thereby also their roles in inflammatory and immunological responses.

DNA array and biological characterization of the impact of the maturation status of mouse dendritic cells on their phenotype and antitumor vaccination efficacy.

We systematically investigated the impact of the relative maturation levels of dendritic cells (DCs) on their cell surface phenotype, expression of cytokines and chemokines/chemokine receptors (by DNA array and RNase protection analyses), biological activities, and abilities to induce tumor immunity. Mature DCs expressed significantly heightened levels of their antigen-presenting machinery (e.g., CD54, CD80, CD86) and numerous cytokines and chemokines/chemokine receptors (i.e., Flt-3L, G-CSF, IL-1alpha and -1beta, IL-6, IL-12, CCL-2, -3, -4, -5, -17, and -22, MIP-2, and CCR7) and were significantly better at inducing effector T cell responses in vitro. Furthermore, mice vaccinated with tumor peptide-pulsed mature DCs better survived challenge with a weakly immunogenic tumor (8 of 8 survivors) than did mice vaccinated with less mature (3 of 8 survived) or immature (0 of 8 survivors) DCs. Nevertheless, intermediate-maturity DCs expressed substantial levels of Flt-3L, IGF-1, IL-1alpha and -1beta, IL-6, CCL-2, -3, -4, -9/10, -17, and -22, MIP-2, osteopontin, CCR-1, -2, -5, and -7, and CXCR-4. Taken together, our data clearly underscore the critical nature of employing DCs of full maturity for DC-based antitumor vaccination strategies.

The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions.

Thrombin induces IL-6 but not TNFalpha secretion by mouse mast cells: threshold-level thrombin receptor and very low level FcepsilonRI signaling synergistically enhance IL-6 secretion.

Mast cells become activated in multiple diseases wherein thrombin generation is often clinically apparent, but the effect of thrombin on cytokine release by mast cells remains unexplored. Thus, we examined IL-6 and TNFalpha release by thrombin-challenged mast cells. Thrombin and the protease-activated receptor (PAR)-1 peptide TRAP(14) induced these cells to secrete IL-6 in a dose-dependent fashion. Mast cells secreted > or =2800 pg IL-6/10(6) cells over 24 h, but only low levels of serotonin and no significant TNFalpha. Furthermore, at near-background levels of allergen, threshold doses of alpha-thrombin synergistically enhanced the IL-6 response (by up to 100-fold), but high-dose costimulation led to a simple additive response. Both the PI(3)- and sphingosine-kinase signaling pathways contributed importantly to the thrombin response. Our data thus clearly demonstrate that low-level thrombin and FcepsilonRI signaling can synergize to augment mast cell IL-6 responses, and that thrombin also differentially induces cytokine secretion by mast cells.

Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes.

Attempts to attribute ileal brush-border chloride conductance to specific proteins were pursued by screening a porcine intestinal cDNA library. A 0.94-kb clone was identified on expression screening with a monoclonal antibody that inhibited enterocyte brush-border chloride conductance. Further screening approaches led to the isolation of a 3.1-kb full-length sequence called pCLCA1, consistent with the identification of a 2.9-kb transcript through Northern analysis. This sequence had significant homology to the CLCA gene family of calcium-regulated chloride channels, especially to hCLCA1. However, a strong A-kinase consensus phosphorylation site in a predicted cytoplasmic loop of the protein was a notable difference from the hCLCA1 gene product. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization studies localized the expression of pCLCA1 mRNA to the crypt and villus epithelia of porcine ileum, whereas tracheal expression was observed in both surface epithelium and submucosal glands. In situ expression of pCLCA1 in mouse 3T3 cells induces an ionomycin-dependent chloride conductance activity in these cells.