Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid.

Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.     

Sustained effects of biofeedback-assisted relaxation therapy in essential hypertension

The usefulness of biofeedback-assisted relaxation as an adjunct or substitute for pharmacotherapy in essential hypertension can be enhanced if the effects are shown to persist after formal treatment has ended. Patients with essential hypertension successfully treated with biofeedback-assisted relaxation were recalled for follow-up yearly after the termination of treatment. Twenty-six of 40 patients met the BP criterion for success. At one-, two-, and three-year follow-up, 31%, 38%, and 27% of the successful completers continued to meet the criterion for success. The pretreatment-posttreatment decreases in BP were accompanied by decreases in forehead muscle tension and urinary cortisol. Forehead muscle tension, urinary cortisol, and anxiety levels were significantly lower than pretreatment one year after the end of treatment. Self-report data were used to assess continued relaxation practice. No relationship was found between practice and any other dependent measure. It appears that some patients trained in biofeedback-assisted relaxation can maintain lowered blood pressure, muscle tension, anxiety, and cortisol levels over the long term; however, the role of relaxation practice in maintaining these lowered levels remains unclear.     

Neutralization of native human gamma interferon (HuIFN gamma) by antibodies to a synthetic peptide encoded by the 5' end of HuIFN gamma cDNA

A synthetic peptide corresponding to the N-terminal amino acid sequence of human gamma-interferon (HuIFN gamma), based on the cDNA sequence, was used to produce antibodies in rabbits that were reactive with native HuIFN gamma. Antibodies from all immunized rabbits neutralized the antiviral activity of HuIFN gamma. Significant neutralization of other HuIFN and mouse IFN was not observed. The peptide had the sequence Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala ,and was coupled to keyhole limpet hemocyanin by disulfide linkage with the use of cystamine. The specificity of the antibodies produced to the peptide was compared to that of antibodies produced to native HuIFN gamma by neutralization of HuIFN gamma and by reactivity with peptide in the enzyme-linked immunosorbent assay (ELISA). The ratio of anti-peptide antibody neutralization of HuIFN gamma vs reactivity with peptide in the ELISA was at least 28-fold lower than for anti-HuIFN gamma antibody. Thus the antibodies to peptide and to HuIFN gamma were directed primarily against different determinants on native HuIFN gamma or the anti-HuIFN gamma antiserum probably contained antibodies to additional determinants. The anti-peptide antibodies should be useful for further characterization and purification of HuIFN gamma.     

Alleviation of Both Water and Nutrient Limitations is Necessary to Accelerate Ecological Restoration of Waste Rock Tips

Hard rock quarries are commonly located close to national parks and special areas of conservation and are generally regarded as visually intrusive. Consequently, restoration strategies that effectively accelerate natural plant regeneration processes are required. Slate waste tips present extreme conditions for plant establishment with multiple potential limiting factors (e.g., lack of organic matter, nutrients, and poor water retention). In this study, we investigated ecological strategies to accelerate natural regeneration at the largest slate quarry in Europe. A field experiment was conducted to assess ecosystem restoration using a contrasting set of native woody species. Treatments included amendments of waste tips with: polyacrylamide gel to increase water‐holding capacity; mineral fertilizer to increase nutrient supply; and two treatments that increased both (organic waste or boulder clay addition). Ecosystem recovery was evaluated through above‐ and below‐ground productivity (plant and microbial, respectively) and soil analyses. Neither increasing nutrient supply (with mineral fertilizer) nor water‐holding capacity (with polyacrylamide gel) was sufficient, alone, to improve plant establishment. However, both boulder clay and organic waste amendment significantly enhanced plant growth. There was a marked positive interaction in the effects on tree growth of the amendment with organic waste and boulder clay. Large interactions occurred between tree species and substrate amendments. The growth of N₂‐fixing species was strongly favored over non‐fixers where there was no addition of material increasing soil nitrogen supply, whereas the growth advantage of pioneer species over non‐pioneers was greatest with fertilizer, organic waste, or clay additions. Organic waste addition had the greatest positive impact on soil processes.     

Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma. Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.     

Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Preferential vastus medialis oblique activation achieved as a treatment for knee disorders

The purpose of this study was to compare the effect of an open-stance cycling protocol (OSCP) with the traditional cycling foot position (TCFP) for preferential vastus medialis oblique (VMO) muscle activation, measured by surface electromyography (SEMG), and preferential VMO activation as defined by achieving significantly increased VMO/VL (vastus lateralis muscle) ratio values. Forty subjects of both sexes participated, 18 symptomatic with patellofemoral pain and 22 control subjects; ages ranged from 18 to 60 years (mean = 28.7 +/- 8 years). The OSCP and TCFP were ridden in randomized order while SEMG recordings were taken of the VMO and VL muscles, collecting the mean of peak amplitudes to calculate VMO/VL ratio values. The SEMG readings were taken 4 times per testing session with randomized resistance and a consistent cycling cadence of 85 rpm. The OSCP displayed preferential VMO activation for all subject groups (F = 40.47, p = 0.0001), and this study revealed a protocol that effectively treats patellofemoral pain.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.