Development of an inhaled endotoxin challenge protocol for characterizing evoked cell surface phenotype and genomic responses of airway cells in allergic individuals.

BACKGROUND: Environmental exposure to endotoxin is a known cause of exacerbation of asthma. Inhaled endotoxin protocols have been used to evaluate airway cell surface phenotypes associated with antigen presentation and innate immunity in healthy volunteers, but not in allergic volunteers. OBJECTIVES: To establish the safety of challenge with low-dose endotoxin (10,000 endotoxin units) (lipopolysaccharide [LPS]) inhalation in allergic individuals, to measure airway cell surface phenotypes associated with antigen presentation and innate immunity in induced sputum (IS) after LPS challenge, and to conduct gene expression profiling in IS cells to determine which host genetic networks are modified by LPS inhalation. METHODS: Induced sputum was obtained before and 6 hours after LPS inhalation in 10 allergic volunteers (8 with asthma and 2 with rhinitis). Flow cytometry was used to examine cell surface phenotypes on IS cells. Genomic expression was analyzed on a subset of IS samples (n = 10) using microarray and ingenuity pathway analysis. RESULTS: A total of 10,000 endotoxin units of LPS induced significant up-regulation of membrane CD14, CD11b, CD16, HLA-DR, CD86, and Fcepsilon receptor 1 on sputum phagocytes and increased expression of genes that influence antigen-presenting surface molecules (HLA-DR, chemokine ligand 2 or monocyte chemoattractant protein 1, v-rel reticuloendotheliosis viral oncogene homolog, prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2, and transforming growth factor beta), immune activation (CD14, interleukin 1beta, and regulated upon activation, normal T cell expressed and secreted), and inflammation (intracellular adhesion molecule 1 and inhibitory kappaBalpha). Gene profiles for nuclear factor kappaB, interleukin 1, and tumor necrosis factor pathways were also significantly affected. CONCLUSIONS: Low-dose inhaled endotoxin challenge is safe in allergic individuals with mild to moderate disease. It enhances airway cell surface phenotypes and expression of genes associated with antigen presentation, innate immunity, and inflammation. Microarray with ingenuity pathway analysis can be successfully applied to sputum cells to characterize genetic responses to inhaled exacerbants.     

Sex differences in the oxygen uptake kinetic response to heavy-intensity exercise in prepubertal children

It has been demonstrated that there are no sex differences in the oxygen uptake (O₂) kinetic response to moderate intensity exercise. However, sex differences in the response to maximal exercise are readily apparent even in the prepubertal years. The purpose of this study was therefore to investigate if sex differences exist in the O₂ kinetic response to heavy-intensity exercise. Forty-eight prepubertal children (25 male, 23 female) completed four transitions from baseline to 40% of the difference between their previously determined T_V-slope (ventilatory threshold determined by the V-slope method) and peak O₂ on an electronically braked cycle ergometer. Each subjects breath-by-breath responses were interpolated to 1 s intervals, time aligned and averaged. The data following phase 1 were fit with: (1) a double exponential model and (2) a single exponential model within a fitting window that was previously identified to exclude the slow component. There were no significant differences in the parameters of the primary component between each model. Subsequent analysis was carried out using model 2. The primary time constant (₁) was significantly (P<0.05) faster in boys [17.6 (5.8) s] than girls [21.9 (8.2) s], and the slow component contribution to the total change in amplitude after 9 min was significantly greater in girls [11.8 (5.5)%] than boys [8.9 (3.7)%]. Sex differences in the kinetic response to heavy-intensity exercise were identified and suggest that during the prepubertal years, sex differences exist in the ability to deliver and/or utilise oxygen in children.     

Characterization of three rabbit oral papillomavirus oncogenes

Rabbit oral papillomavirus (ROPV) induces warts in mucosal tissues, and represents a useful model for understanding host-virus interactions that are reflected in mucosal/HPV infections. ROPV induces benign papillomas that regress in 100% of infected rabbits. We previously reported the complete genome sequence of ROPV. However, the oncogenic potential of this virus is unknown because of immunologically mediated regression. The purpose of this study was to characterize the transforming proteins of E6, E7, and E8 genes of ROPV. E6, E7, and E8 genes of ROPV were cloned into the expression vector PCR3. Two hybrid CRPV-ROPV E6 genes were also constructed and tested together with the three wild-type ROPV genes. Each construct was transfected into NIH3T3 cells and stable transfected cell lines were established. Transforming properties of ROPV E6, E7, and E8 were tested via anchorage-independent growth of cells in agar plates and tumor growth in athymic mice. Cells with ROPV E6, E7, or E8 formed colonies in agar and tumors in athymic mice. These observations suggest that ROPV E6, E7, and E8 are oncogenic.     

The canals of hering might represent a target of methotrexate hepatic toxicity

Methotrexate treatment for psoriasis is known to cause hepatic fibrosis in some patients, which might progress to cirrhosis. The fine, radiating, fibrous septa developing in this setting have a distribution that is reminiscent of the location of the canals of Hering (coH). To assess the possibility of fibrous obliteration of the coH in patients receiving methotrexate, we developed a staining technique by combining an immunohistochemical stain for cytokeratin 7 with a modified Masson trichrome. Sixteen biopsy specimens from 7 patients were evaluated. The biopsies had a variety of histologic changes, including steatosis, anisonucleosis, multinucleation, chronic inflammation, bile duct damage, and ductular reaction. Fibrosis was present in 13 biopsy specimens (81%) and was mild in 7, moderate in 3, and severe in 3 specimens. Compared with normal (control) liver specimens, biopsy specimensfrom patients receiving methotrexate had decreased numbers of coH (1.9 +/- 0.8 vs 5.2 +/- 1.7; P < .025). In specimens with moderate or severe fibrosis, fibrous septa sometimes extended along the coH. These findings suggest that scarring of the coH might be a consequence of the toxic effects of methotrexate.     

Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori

Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme. Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity. This finding prompted us to disrupt the two remaining unstudied hyp genes of H. pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed. In both mutants hydrogenase activity was abolished but urease activity remained unaffected. Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant. In addition, we also disrupted the genes HP0634 (referred as hydD in the H. pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process. In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.     

Effect of inhaled corticosteroids on symptom severity and sputum mediator levels in chronic persistent cough

BACKGROUND: Chronic cough often lasts for more than 1 year and is associated with airway inflammation. The effect of inhaled corticosteroids on symptom severity and inflammatory mediator levels in these patients is unknown. OBJECTIVE: We sought to determine whether inhaled corticosteroids reduce cough severity and sputum mediator concentrations in patients with chronic persistent cough. METHODS: We performed a double-blind, randomized, placebo-controlled crossover study with inhaled fluticasone, 500 microg twice daily, and placebo for 14 days in 88 patients with cough for more than 1 year, with normal chest radiography and spirometry results. Outcome measures were a daily cough visual analogue scale and induced sputum concentrations of eosinophilic cationic protein (ECP), myeloperoxidase, leukotriene B(4) (LTB(4)), leukotrienes C(4)/D(4)/E(4) (cysteinyl leukotrienes [Cys-LTs]), prostaglandin E(2) (PGE(2)), IL-8, and TNF-alpha. Sputum cell counts, exhaled nitric oxide levels, and carbon monoxide levels were also measured. RESULTS: There was a significant improvement in the cough visual analogue scale after inhaled fluticasone compared with placebo (mean difference, 1.0; 95% CI, 0.4-1.5; P <.001). LTB(4), Cys-LT, and PGE(2) levels were increased in all causes of cough. Sputum ECP counts, exhaled nitric oxide levels, and carbon monoxide levels decreased significantly after inhaled fluticasone. There was no change in sputum cell counts and other mediator concentrations. CONCLUSION: Cough severity and sputum ECP levels are modestly reduced by inhaled corticosteroids in patients with chronic cough persisting for more than 1 year. LTB(4), Cys-LT, PGE(2), IL-8, myeloperoxidase, and TNF-alpha levels are unaltered by this therapy. This raises the possibility that drugs targeted to reduce the effects of these mediators might be of benefit in chronic persistent cough.     

An examination of hardiness throughout the sport-injury process: A qualitative follow-up study

Objectives. This qualitative follow-up study aimed to enhance the interpretability and meaningfulness of the findings that emerged from a quantitative study that explored the effect of hardiness on the prediction of, and response to, sport injury (i.e., Wadey, Evans, Hanton, & Neil, 2012). Design. Using theory-based and maximum-variation sampling to contextualize and provide an in-depth understanding of the previous findings, the participants comprised a purposeful sample of 10 athletes from the quantitative study (M age = 21.7; SD= 1.06). Methods. Data were derived through semi-structured interviews, and analysed and displayed using composite sequence analysis (Miles & Huberman, 1994). Results. The findings extended Wadey et al.'s (2012) study by identifying the perceived mechanisms by which athletes high and low in hardiness exacerbated or attenuated the impact of pre-injury negative major life events (i.e., a significant predictor of sport injury) and post-injury responses. Specifically, the findings demonstrate that athletes high in hardiness possessed a refined repertoire of problem- and emotion-focused coping strategies that they used pre- and post-injury. Those athletes low in hardiness used avoidance coping strategies that had long-term negative implications. Conclusions. These findings have important implications for the structure, timing, and content of hardiness interventions that aim to reduce rates of injury occurrence and expedite injured athletes' return to competitive sport. STATEMENT OF CONTRIBUTION: WHAT IS ALREADY KNOWN ON THIS SUBJECT??: Although the personality trait of hardiness has received limited research attention in the context of sport injury, a recent study demonstrated that hardiness lowers the risk of, and promotes recovery from, injury (Wadey, Evans, Hanton, & Neil, 2012). Despite these encouraging findings, they fail to contextualize and provide an in-depth understanding of the mechanisms by which hardiness operates. WHAT DOES THIS STUDY ADD??: ? A number of personal and situational factors affected athletes' responses pre- and post-injury. ? Athletes high and low in hardiness used different coping strategies throughout the sport-injury process. ? These findings have implications for the structure, timing, and content of hardiness interventions.     

Common genetic variants in complement genes other than CFH, CD46 and the CFHRs are not associated with aHUS

It is well established that common genetic variants in CFH, CD46 and the CFHRs are additional risk factors for the development of aHUS. To examine the hypothesis that common variants in other complement genes have a similar effect we genotyped 501 SNPs in 47 complement genes in 94 aHUS patients from Newcastle, 126 aHUS patients from Paris, 374 UK controls and 165 French controls. We replicated the associations in CFH, CD46 and the CFHRs but found no association with any other complement gene. The strongest associations replicated in both cohorts were found for four SNPs within CD46 (p-value<10(-3)) and five SNPs within CFH (p-value<5×10(-3)). Significant replicable associations with single SNPs in CFHR2, CFHR4 and an intergenic SNP (CR1-CD46) were also found. Analysis of the Paris cohort showed that the association with CD46 SNPs was only present in those patients with complement mutations. Haplotype analysis showed at-risk and protective haplotypes in both CD46 and CFH. The CD46 haplotype was only disease-associated in those patients with mutations.     

Correlation of p63 protein expression with histological grade of meningiomas: an immunohistochemical study

Prediction of tumor behavior in meningiomas based on morphological features alone remains difficult. Several immunohistochemical biomarkers have been proposed to assist conventional methods. However, no single immunohistochemical marker can unequivocally discriminate between benign and aggressive meningiomas. There is only 1 study available in the literature that correlates p63 expression with overall histological grade of the meningioma. The present study is undertaken to assess the correlation between p63 expression and histological grade of meningiomas. For this purpose, the authors studied and analyzed the immunohistochemical expression of p63 in 85 cases of meningioma, including WHO grade I (63), grade II (11), and grade III (11) cases. Correlation between histological grade and nuclear immunoreactivity to p63 antibody was performed. Furthermore, expression of p63 protein was correlated with short clinical follow-up and Ki-67 proliferation index. Among 85 patients analyzed, there were 61 women (71.7 %) and 24 men (28.2%) between 7 and 75 years old. Expression of p63 protein was found in 34.9% of grade I cases, but in grade II and III, 63.6% of cases each were immunoreactive. Correlation between histological grade and p63 immunoreactivity was significant (P = .02). p63-positive grade I meningiomas did not show elevated Ki-67 index. The present study contradicts earlier reports because there are a considerable number of grade I meningiomas that express p63. Although p63 expression is significantly associated with higher histological grade of meningiomas, it may not be considered as a sole biomarker to assess aggressive behavior of the tumor.