Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.     

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

In-vitro formation of a collagenous matrix upon previously diseased and experimentally treated cemental root surfaces

Summary A diseased and mechanically treated surface of root cementum is known, clinically, to favor periodontal regeneration. The present investigation was undertaken to test whether previously diseased and experimentally treated root surfaces can support the in-vitro formation of a new collagenous matrix. Three teeth extracted for advanced periodontitis were treated first with 5% sodium hypochlorite for 2 h to remove all organic material from the root surface. After the healthy, apical one third of the root was cut off, the roots were scaled with moderate pressure to remove visible calculus. Non-demineralized root discs were cut and placed on a co-culture of periodontal ligament- and alveolar bone-derived cells. After 7 weeks in culture, either one of two matrix types was found along the root surface. The most frequent matrix consisted of clusters of cells layered within densely aggregated collagen fibrils. The other, less frequent matrix consisted of loosely arranged collagen fibrils adjacent to the cemental surface. The findings support the notion that, in vitro, a collagenous matrix is formed in contact to diseased and experimentally treated root surfaces. However, the smooth, non-demineralized and scaled cemental surface does not appear to be a suitable substrate for interdigitation with newly produced collagen fibrils.     

Elektronenmikroskopische Untersuchungen an der Froschlunge

Zusammenfassung Das Alveolarepithel der Froschlunge weist nur einen einzigen Zelltyp auf. Die Zellkörper sitzen in den Nischen zwischen den Kapillaren, die sie mit Zytoplasmaausläufern überdecken. Die Epithelzellen enthalten große Zytosomen mit osmiophilen Lamellen mit einer Periode von 40–42 Å. Sie sind den Typ II-Pneumozyten der Säugerlunge vergleichbar.Das Alveolarepithel der Froschlunge ist mit einer Grenzschicht bedeckt, die in Abhängigkeit von der Fixierung eine 40–42 Å-Periode aufweist oder aus einer oder mehreren Doppelmembranen zusammengesetzt ist. Gittermuster und Myelinfiguren sind vorhanden. Das bedeutet, daß Surfactant in der Froschlunge in gleicher Weise wie in der Säugerlunge dargestellt werden kann.

---

Electron microscopic studies on the lung of the frogI. Demonstration of the alveolar lining layer (surfactant)

Summary The alveolar epithelium of the frog exhibits only one type of cells. The cell-bodies are situated in the spaces among the capillaries, which they cover with cytoplasmic extensions. The epithelial cells contain large bodies (cytosomes) with osmiophilic lamellae having a period of 40–42 Å. The alveolar cells are considered to be similar to the type II-pneumocytes of mammalian lungs.The alveolar epithelium of the lung of the frog is covered with a lining layer, which depending on the method of fixation consists of periods of 40–42 Å or of one or more double membranes. Lattice formations and myelin figures are seen. This means that the surfactant in the lung of the frog can be demonstrated in the same way as in mammalian lungs.

     

重症肺炎新生儿支气管肺泡灌洗液 病原菌分布及耐药性

目的研究重症肺炎新生儿支气管肺泡灌洗液的病原菌分布和耐药性。方法选择2016年4月至2018年4月在本院呼吸科治疗的新生儿268例,其中符合重症肺炎诊断标准的患儿142例,归为重症肺炎组;不符合重症肺炎诊断标准的患儿126例,归为对照组。检测患儿肺泡灌洗液病原菌分布情况和耐药情况。结果重症肺炎组患儿肺炎克雷伯菌、流感嗜血菌、铜绿假单胞菌、阴沟肠杆菌、大肠埃希菌、金黄葡萄球菌、溶血葡萄球菌、表皮葡萄球菌、肺炎链球菌、草绿链球菌检出率明显高于对照组。肺炎克雷伯菌对亚胺培南,美罗培南的耐药性为0.0%,大肠埃希菌对亚胺培南,美罗培南,阿米卡星的耐药性为0.0%,阴沟肠杆菌对亚胺培南,美罗培南,左氧氟沙星的耐药性为0.0%,肺炎链球菌对万古霉素的耐药性为0.0%,金黄葡萄球菌对万古霉素的耐药性为0.0%。结论新生儿重症肺炎患者病原菌以革兰阴性菌为主,亚胺培南、美罗培南、万古霉素可以用于治疗新生儿重症肺炎,但由于其毒副作用较大,应严格把握适应症。     

In vivo interaction between alveolar macrophages and Cryptococcus neoformans

In vivo interactions of rabbit alveolar macrophages (AM) and Cryptococcus neoformans, a yeast pathogenic for humans, were studied. As a control, inert silica particles of a similar diameter (5–6 μm) were used. Of 16 rabbits, 6 were instilled intratracheally with fluorescein-labelled heat-killed C. neoformans, 6 with fluorescein-labelled silica particles and 4 with saline only. After 24 h, the AM were collected by lung lavage, and phagocytosis, oxidative metabolism, phagolysosomal pH and morphology were studied. The accumulated number of yeasts attached to the AM was almost the same for C. neoformans as for the silica particles. The ingested fraction of C. neoformans was even higher than that of the silica particles. Quantitative NBT reduction by the AM, reflecting their oxidative metabolism, was markedly increased by exposure to C. neoformans for 24 h. The phagolysosomal pH was on the average lower in phagolysosomes with C. neoformans than with the silica particles, although approximately 2% of the phagolysosomes with C. neoformans had neutral pH. Phagolysosomes with neutral pH was not observed for silica particles. Electron microscopy showed presence of C. neoformans in phagolysosomes of AM. The conclusion of this study is that the phagocytic activity, oxidative metabolism and phagolysosomal pH AM against C. neoformans are significant 24 h after the exposure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron microscopic study of the in vivo erythrophagocytosis by alveolar macrophages of the cat

Summary Erythrophagoeytosis in vivo by cat alveolar macrophages was studied under the electron microscope by collecting the macrophages at 2 hours and 48 hours following the intratracheal injection of autologous blood. Considering the progressive ultrastructural modifications of the red blood cell plasma membrane, different successive stages were observed, corresponding to the hemolysis of the erythrocytes: 1. A recently engulfed erythrocyte appears unaltered within the phagocytic vacuole. 2. A dense layer, surrounding the plasma membrane of the red cell, is observed within the phagocytic vacuole. 3. The content of the vacuole is uniformly dense and the plasma membrane of the red cell exhibits discontinuous thickenings. 4. The whole vacuole appears very dense (hyperdense stage) and the plasma membrane is shown altered. The whole process of erythrophagocytosis is accompanied by an active fusion of the phagocytic vacuole with typical lysosomes and lysosomes containing crystal-like material. It is suggested that hemolysis may be explained in terms of enzymic digestion of the proteinic part of the plasma membrane of the erythrocyte.The authors wish to thank Miss Gabrielle Audet for her technical assistance, and Mr. Gaston Chevalier for revision of the English text.     

TLR-7 agonist attenuates airway reactivity and inflammation through Nrf2-mediated antioxidant protection in a murine model of allergic asthma

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant–antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.     

A non‐destructive sampling protocol for field studies of seed dispersal by fishes

This paper presents a standardized protocol for the non‐lethal capture of fishes, sampling of stomach contents and quantification of seed dispersal efficiency by frugivorous fishes. Neotropical pacu Piaractus mesopotamicus individuals were collected with fruit‐baited hooks. The diets of 110 fish were sampled using a lavage method, which retrieved >90% of stomach contents of both juveniles and adults and allowed individuals to recover within 5 min of treatment. The proportional volume of six food categories was similar for stomachs and whole digestive tracts retrieved by dissection. Fruit pulp was proportionally lower in the stomach. The abundance and species richness of intact seeds increased with fish size independent of whether only stomachs or whole digestive tracts were analysed. The analysis of stomach contents accounted for 62·5% of the total species richness of seeds dispersed by P. mesopotamicus and 96% of common seeds (seed species retrieved from more than one fish). Germination trials revealed that seed viability was similar for seeds collected from the stomach via lavage and seeds that passed through the entire digestive tract. Therefore, stomach contents provide an unbiased representation of the dietary patterns and seed dispersal of frugivorous fishes.