Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Alteration of surfactant protein A expression in renal cell carcinoma

Surfactant protein-A (SP-A) belongs to a family of collagen-containing C-type lectins called collectins. SP-A is expressed by renal tubule epithelial cells. We investigated the distribution of SP-A in renal cell carcinomas (RCC) using immunohistochemical techniques and western blotting. We used 35 formalin fixed, paraffin embedded (FFPE) RCC tissue samples. We compared results with clinico-pathological parameters of RCC including age, sex, Fuhrman grade, tumor volume, tumor node metastasis (TNM) and clinical stage. SP-A was localized in the glomerulus and renal tubule epithelium in nontumor tissue and strong SP-A immunoreactivity was observed in tumor tissue. SP-A was expressed in the RCC tumor cells (64%) and nontumor cells (34%) in males and RCC tumor cells (90%) and nontumor cells (30%) in females. There was a significant correlation between SP-A immunoreactivity in tumor cells and gender, age, tumor diameter, Fuhrman grade and tumor diameter. Western blot analysis supported the immunohistochemical findings. We present evidence for involvement of SP-A in RCC and suggest that increased SP-A expression in RCC is associated with favorable prognosis.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Thrombin stimulates the expression of PDGF in lung epithelial cells

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.     

Effect of unilateral and bilateral resistance exercise on maximal voluntary strength,total volume of load lifted,and perceptual and metabolic responses

The present study investigated the effect of unilateral and bilateral resistance exercise (RE) on maximal voluntary strength, total volume of load lifted (TVLL), rating of perceived exertion (RPE) and blood lactate concentration of resistance-trained males. Twelve healthy men were assessed for the leg extension one-repetition maximum (1RM) strength using bilateral and unilateral contractions. Following this assessment, an RE session (3 sets of repetitions to failure) was conducted with bilateral and unilateral (both limbs) contractions using a load of 50% 1RM. The TVLL was calculated by the product of the number of repetitions and the load lifted per repetition. RPE and blood lactate were measured before, during and after each set. Session RPE was measured 30 minutes after RE sessions. There was a significant difference in the bilateral (120.0±11.9) and unilateral (135.0±20.2 kg) 1RM strength (p < 0.05). The TVLL was similar between both RE sessions. Although the repetitions decreased with each successive set, the total number of repetitions completed in the bilateral protocol (48) was superior to the unilateral (40) protocol (p < 0.05). In both bouts, RPE increased with each subsequent set whilst blood lactate increased after set 1 and thereafter remained stable (p < 0.05). The RPE and lactate responses were not significantly different between both sessions. In conclusion, a bilateral deficit in leg extension strength was confirmed, but the TVLL was similar between both RE sessions when exercising to voluntary fatigue. This outcome could be attributed to the number of repetitions completed in the unilateral RE bout. The equal TVLL would also explain the similar perceptual and metabolic responses across each RE session.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.     

三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响

目的：研究三氧化二砷（As2O3）对食管癌细胞增殖和热休克蛋白70（HSP70）表达的影响。方法：通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果：与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70（heat shock protein70）及HSC70(heat shock cognate protein70)表达均增加。结论：As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。     

Persistent Release of IL-1s from Skin Is Associated with Systemic Cardio-Vascular Disease,Emaciation and Systemic Amyloidosis: The Potential of Anti-IL-1 Therapy for Systemic Inflammatory Diseases

The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1β antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.     

Differential Gene Expression in Thrombomodulin (TM; CD141)+ and TM? Dendritic Cell Subsets

Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin⁺ dendritic cells are tolerogenic while thrombomodulin⁻ dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin⁺ and thrombomodulin⁻ dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin⁺ dendritic cells compared to control dendritic cells and thrombomodulin⁻ dendritic cells. Thrombomodulin-treated and thrombomodulin⁺ dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin⁺ dendritic cells produced more lipoxins than thrombomodulin⁻ dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin⁺ dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin⁺ dendritic cells contained more cells actively dividing than those of thrombomodulin⁻ dendritic cells. Production of IL-10 is increased in thrombomodulin⁺ dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin⁺ dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin⁺ dendritic cells, which have significantly altered gene expression compared to thrombomodulin⁻ dendritic cells in key immune function pathways.