Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Fatal-Mixed Cutaneous Zygomycosis–Aspergillosis: A Case Report

Mucorales and Aspergillus are molds responsible for infections in immunocompromised patients. In this report, we describe a case of a rare extensively mixed cutaneous infection caused by Lichtheimia ramosa, Aspergillus fumigatus and Aspergillus terreus in a neutropenic patient suffering from an acute leukemia. The fatal outcome of this patient can be attributed to its hematologic malignancy, the extensive nature of the lesions and the resistance of the strains to antifungals.     

Lion,Ungulate, and Visitor Reactions to Playbacks of Lion Roars at Zoo Atlanta

Felids in captivity are often inactive and elusive in zoos, leading to a frustrating visitor experience. Eight roars were recorded from an adult male lion and played back over speakers as auditory enrichment to benefit the lions while simultaneously enhancing the zoo visitor experience. In addition, ungulates in an adjacent exhibit were observed to ensure that the novel location and increased frequency of roars did not lead to a stress or fear response. The male lion in this study roared more in the playback phase than in the baseline phases while not increasing any behaviors that would indicate compromised welfare. In addition, zoo visitors remained at the lion exhibit longer during playback. The nearby ungulates never exhibited any reactions stronger than orienting to playbacks, identical to their reactions to live roars. Therefore, naturalistic playbacks of lion roars are a potential form of auditory enrichment that leads to more instances of live lion roars and enhances the visitor experience without increasing the stress levels of nearby ungulates or the lion themselves, who might interpret the roar as that of an intruder.     

Transferability,development of simple sequence repeat (SSR) markers and application to the analysis of genetic diversity and population structure of the African fan palm (Borassus aethiopum Mart.) in Benin

Biological pathways play an important role in the occurrence, development and recovery of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways. We propose a path-specific effect statistic (PSE) to detect the differential specific paths under two conditions (e.g. case VS. control groups, exposure Vs. nonexposure groups). In observational studies, the path-specific effect can be obtained by separately calculating the average causal effect of each directed edge through adjusting for the parent nodes of nodes in the specific path and multiplying them under each condition. Theoretical proofs and a series of simulations are conducted to validate the path-specific effect statistic. Applications are also performed to evaluate its practical performances. A series of simulation studies show that the Type I error rates of PSE with Permutation tests are more stable at the nominal level 0.05 and can accurately detect the differential specific paths when comparing with other methods. Specifically, the power reveals an increasing trends with the enlargement of path-specific effects and its effect differences under two conditions. Besides, the power of PSE is robust to the variation of parent or child node of the nodes on specific paths. Application to real data of Glioblastoma Multiforme (GBM), we successfully identified 14 positive specific pathways in mTOR pathway contributing to survival time of patients with GBM. All codes for automatic searching specific paths linking two continuous variables and adjusting set as well as PSE statistic can be found in supplementary materials.  The proposed PSE statistic can accurately detect the differential specific pathways contributing to complex disease and thus potentially provides new insights and ways to unlock the black box of disease mechanisms.     

Regulation of the high affinity IgE receptor (Fc epsilonRI) in human neutrophils: role of seasonal allergen exposure and Th-2 cytokines

The high affinity IgE receptor, Fc epsilonRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional Fc epsilonRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of Fc epsilonRI-alpha chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, Fc epsilonRI-alpha chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/Fc gammaRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced Fc epsilonRI-alpha chain surface expression. In conclusion, these results suggest that enhanced Fc epsilonRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.